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https://openalex.org/W3195026752	http://real.mtak.hu/148655/1/farnesol%20transz.pdf	English	null	Transcriptional profiling of the <i>Candida auris</i> response to exogenous farnesol exposure	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	8,418	Transcriptional Proﬁling of the Candida auris Response to Exogenous Farnesol Exposure Ágnes Jakab,a Noémi Balla,b,c Ágota Ragyák,a,d Fruzsina Nagy,b Fruzsina Kovács,b,c Zsófi Sajtos,d Zoltán Tóth,b Andrew M. Borman,e,f István Pócsi,a Edina Baranyai,d László Majoros,b Renátó Kovácsb,g Ágnes Jakab,a Noémi Balla,b,c Ágota Ragyák,a,d Fruzsina Nagy,b Fruzsina Kovács,b,c Zsófi Sajtos,d Zoltá Andrew M. Borman,e,f István Pócsi,a Edina Baranyai,d László Majoros,b Renátó Kovácsb,g aDepartment of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, D bDepartment of Medical Microbiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary cDoctoral School of Pharmaceutical Sciences, University of Debrecen, Debrecen, Hungary dDepartment of Inorganic and Analytical Chemistry, Agilent Atomic Spectroscopy Partner Laboratory, University of Debrecen, Debrecen, Hungary eUK National Mycology Reference Laboratory, Public Health England, Science Quarter, Southmead Hospital, Bristol, United Kingdom fMedical Research Council Centre for Medical Mycology (MRC CMM), University of Exeter, Exeter, United Kingdom gFaculty of Pharmacy, University of Debrecen, Debrecen, Hungary ABSTRACT The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alter- native treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene transcription analysis was performed using transcriptome sequencing (RNA-Seq). Farnesol exposure resulted in 1,766 differentially expressed genes. Of these genes, 447 and 304 genes with at least 1.5-fold increase or decrease in transcription, respectively, were selected for further investigation. Genes involved in morphogenesis, bioﬁlm events (maturation and dispersion), gluconeogenesis, iron me- tabolism, and regulation of RNA biosynthesis showed downregulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid b-oxidation, and peroxisome processes were upregulated. In addition, farnesol treat- ment increased the transcription of certain efﬂux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by the change in the number of CFU, was signiﬁcantly inhibited within 2 h of the addition of farnesol (5.8 107 6 1.1 107 and 1.1 107 6 0.3 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P , 0.001). RESEARCH ARTICLE September/October 2021 Volume 6 Issue 5 e00710-21 Transcriptional Proﬁling of the Candida auris Response to Exogenous Farnesol Exposure In addition, farnesol treatment caused a signiﬁcant reduction in intracellular iron (152.2 6 21.1 versus 116.0 6 10.0 mg/kg), manganese (67.9 6 5.1 versus 18.6 6 1.8 mg/ kg), and zinc (787.8 6 22.2 versus 245.8 6 34.4 mg/kg) (P , 0.05 to 0.001) compared to untreated control cells, whereas the level of cooper was signiﬁcantly increased (274.6 6 15.7 versus 828.8 6 106.4 mg/kg) (P , 0.001). Our data demonstrate that farne- sol signiﬁcantly inﬂuences the growth, intracellular metal ion contents, and gene transcrip- tion related to fatty acid metabolism, which could open new directions in developing al- ternative therapies against C. auris. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. IMPORTANCE Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with a high mortality rate. As conven- tional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator mole- cule of fungal morphogenesis, inducing phenotypic adaptations and inﬂuencing bio- ﬁlm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, espe- cially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron msphere.asm.org 1 September/October 2021 Volume 6 Issue 5 e00710-21 Jakab et al. metabolism as well as increases fungal intracellular copper content. In addition, me- tabolism was modulated toward b-oxidation. These results provide deﬁnitive explan- ations for the observed antifungal effects. KEYWORDS Candida auris, farnesol, quorum sensing, transcriptome analysis, oxidative stress, metal, iron, zinc, copper A A dramatic increase in resistance to conventional antifungal agents has been reported for Candida auris worldwide, leading to evasion from efﬁcient therapeu- tic options. The current coronavirus disease 2019 (COVID-19) pandemic situation may further promote the spreading of this fungal superbug. Superinfections by C. auris in critically ill COVID-19 patients have been related to high 30-day mortality rates, usually above 50% (1–3). Farnesol is a fungal quorum-sensing molecule inducing hypha-yeast morphological switching in Candida albicans (4). Transcriptional Proﬁling of the Candida auris Response to Exogenous Farnesol Exposure In the past decade, several studies have reported that farnesol can generate oxidative stress and inﬂuence membrane permeability and cellular polarization in certain fungal species, especially at supraphysiological concentrations (5– 7). Although farnesol does not affect the growth rate of C. albicans growing in the plank- tonic form, it signiﬁcantly decreased the growth of C. auris regarding both planktonic cells and also 1-day-old bioﬁlms of this organism (7). Recently, alternative therapeutic approaches designed to disturb quorum sensing have become an attractive treatment strategy (8, 9). The usage of farnesol and traditional antifungal drugs in combination may provide new insights into the management of newly emerged fungal species, such as C. auris, which poses a global threat to the nosocomial environment (7, 10). Different metal ions facilitate numerous essential molecular processes within bacte- rial and fungal pathogens in quorum sensing-related pathways (11–13). Metals play a pivotal role in infection as cofactors in several enzymes related to metabolic activity and virulence, such as metal-dependent superoxide dismutases, metalloproteases, or melanin-producing laccases (14). We hypothesize that therapies interfering with quo- rum sensing may disturb the intracellular ion homeostasis, which may further elucidate the observed supraphysiological quorum-sensing molecule-related antifungal effect. nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Previously, our group has reported the potential therapeutic beneﬁt of farnesol against C. auris (7, 10). However, to date, there are no data that describe the total tran- scriptome changes induced by farnesol. Such data might help reveal the C. auris-spe- ciﬁc response to exogenous farnesol exposure. To gain further insights into previously described physiological consequences of farnesol treatment, we determined genome- wide gene transcription changes induced by farnesol exposure using total transcrip- tome sequencing (RNA-Seq). Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 2 RESULTS Farnesol exposure inhibits the growth of Candida auris. The growth of C. auris was examined following 75 mM farnesol treatment in yeast extract-peptone-dextrose (YPD). Adding farnesol to preincubated cells resulted in a remarkable growth inhibition, starting at 6 h postinoculation, which was conﬁrmed by both absorbance (optical density at 640 nm [OD640]) measurements and CFU determination. Growth was signiﬁcantly inhib- ited within 2 h of the addition of farnesol as assessed both by CFU changes (5.8 107 6 1.1 107 and 1.1 107 6 0.3 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P , 0.001) and observed absorbance values (1.28 6 0.04 and 0.72 6 0.04 for untreated control and farnesol-exposed cells, respectively, at OD640) (P , 0.001) (Fig. 1). The observed growth inhibition was further conﬁrmed by changes in meas- ured dry cell mass (DCM) at 12-h incubation time (5.5 6 0.2 and 1.3 6 0.1 g/liter for untreated control and farnesol-exposed cells, respectively) (P , 0.001). The ratios of nonviable cells were 3.3% 6 1.2%, 1% 6 0%, 1.7% 6 0.6%, and 3.6% 6 0.6% for 0-h cells, 4-h cells, 6-h untreated and 6-h farnesol-exposed cells, respectively (Fig. 2A to D). September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 2 Effects of Farnesol against C. auris FIG 1 Farnesol exposure inhibits the growth of Candida auris. Changes in the growth of C. auris were monitored by measurement of the absorbance (OD640). Following a 4-h incubation time, farnesol (FAR) was added at a ﬁnal concentration of 75 mM to the YPD cultures. Data represent mean values 6 standard deviations (SD) (error bars) calculated from six independent experiments. The asterisks indicate a statistically signiﬁcant difference between control and farnesol-treated cultures calculated by paired Student’s t test (*, P , 0.001). FIG 1 Farnesol exposure inhibits the growth of Candida auris. Changes in the growth of C. auris were monitored by measurement of the absorbance (OD640). Following a 4-h incubation time, farnesol (FAR) was added at a ﬁnal concentration of 75 mM to the YPD cultures. Data represent mean values 6 standard deviations (SD) (error bars) calculated from six independent experiments. The asterisks indicate a statistically signiﬁcant difference between control and farnesol-treated cultures calculated by paired Student’s t test (, P , 0.001). Transcriptional proﬁling and RNA-Seq data validation. September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 3 RESULTS Principal-component analysis (PCA) and hierarchical clustering were performed to provide a visual represen- tation of the transcriptomic similarities between samples treated with farnesol and the untreated controls (Fig. 3A and B). Samples from different conditions (with or without farnesol) clustered separately, whereas those from the same conditions clustered to- gether, indicating a high level of correlation among samples as well as distinctive tran- scriptome proﬁles. Analyses of the RNA sequencing data clearly indicated that farnesol has a remarkable effect on C. auris gene transcription, leading to signiﬁcant alterations in the transcriptome. nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Comparison of the farnesol-exposed C. auris global gene transcription proﬁle with that of unexposed cells revealed 1,766 differentially expressed genes. Among these genes, 447 were upregulated and 304 were downregulated in the farnesol-exposed samples compared to the untreated controls (Fig. 4 and 5; see also Tables S2 and S3 in the supplemental material). Evaluation of farnesol-responsive genes. To identify larger patterns in differential gene transcription and to obtain an overall insight into the impact of farnesol, gene ontology (GO) terms were assigned to all of the genes in the C. auris genome; after- wards, we compared the terms for both the downregulated and upregulated genes to a background of all terms. We found 19 and 22 signiﬁcant gene groups that were underrepresented and overrepresented in this analysis, respectively (Fig. 5 and Tables S2 and S3). (i) Virulence-related genes. Virulence-related genes were signiﬁcantly enriched within the farnesol-responsive downregulated gene group, according to Fisher’s exact test (Table S3). Most of these 11 putative genes are involved in bioﬁlm maturation (RBT1, HWP1, BCR1, EFG1, DSE1, BRG1, UME6, ZAP1, and RLM1) and dispersion (NRG1 and UME6) (Fig. 4 and 5 and Table S3); also, ﬁve downregulated morphogenesis genes (EFG1, HWP1, HGC1, WAL1, and VRP1) are notable (Fig. 4 and Table S3). Downregulation of RBT1 and NRG1 under farnesol treatment was also supported by reverse transcriptase- quantitative PCR (RT-qPCR) data (Fig. 6 and Table S4). (ii) Oxidative stress-related genes. Genes belonging to antioxidative defense- related GO terms were enriched in the farnesol-responsive upregulated gene group September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 3 Jakab et al. FIG 2 Phase-contrast and ﬂuorescence microscopy. Phase-contrast and ﬂuorescence microscopy images of untreated C. auris cells at 0, 4, and 6 h (A, B, and C, respectively) and cells treated with 75 mM farnesol at 6 h (D). September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 4 RESULTS Propidium iodide ﬂuorescent dye was used to stain the nonviable cells (red). Bars, 10 mm. FIG 2 Phase-contrast and ﬂuorescence microscopy. Phase-contrast and ﬂuorescence microscopy images of untreated C. auris cells at 0, 4, and 6 h (A, B, and C, respectively) and cells treated with 75 mM farnesol at 6 h (D). Propidium iodide ﬂuorescent dye was used to stain the nonviable cells (red). Bars, 10 mm. (Fig. 4 and 5 and Table S3). Altogether, eight genes were upregulated after farnesol treatment, namely, CCP1, SOD1, SOD2, SOD6, GPX1, DOT5, PRX1, and AHP1 (Fig. 4 and Table S3). In addition, farnesol exposure increased the transcription of HSP21, YPD1, and HOG1, encoding small heat shock protein, phosphorelay protein, and mitogen-activated protein (MAP) kinase (Fig. 4 and Table S3). Upregulation of CAT1, coding for catalase in farnesol-treated cells, was also conﬁrmed by RT-qPCR (Fig. 6 and Table S4). nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. (iii) Metabolic pathway-related genes. Selected genes involved in glucose catab- olism and fatty acid metabolism were determined with the Candida Genome Database (http://www.candidagenome.org). Farnesol treatment downregulated PCK1 and FBP1, encoding key enzymes speciﬁc to gluconeogenesis, but not glycolysis and tricarboxylic acid cycle genes (Fig. 4 and Table S3). In addition, three genes related to the glyoxylate cycle (ACO2, ICL1, and MDH1-3) were signiﬁcantly enriched in the upregulated gene set (Fig. 4 and Table S3). Signiﬁcant upregulation of ﬁve putative genes encoding fatty acid b-oxidation enzymes was observed (POX1, ECI1, FAT1, FAA21, and POT1) (Fig. 4 and 5 and Tables S2 and S3). In addition, farnesol exposure decreased the transcription of INO1, encoding inositol-1-phosphate synthase (Fig. 4 and Table S3). Genes involved in iron homeostasis, including essential elements of reductive iron uptake (FRE10, FET31, SMF12, and FTR1), siderophore transport (SIT1), and hemoglobin use (RBT5), as well as manganese (SMF1, transporter), copper uptake (CRP1, trans- porter), and zinc metabolism (CSR1, transcription factor), were enriched in the downre- gulated gene set (Fig. 4 and 5 and Table S3). The upregulation of POT1 (3-oxoacyl coenzyme A [CoA] thiolase) and the downreg- ulation of INO1 and FTR1 were supported by RT-qPCR data (Fig. 6 and Table S4). September/October 2021 Volume 6 Issue 5 e00710-21 Effects of Farnesol against C. auris FIG 3 Cluster (A) and principal component (B) analysis of the transcriptome data. Symbols represent untreated control (Cont) and 75 mM farnesol exposure (FAR) cultures. msphere.asm.org September/October 2021 Volume 6 Issue 5 e00710-21 RESULTS The distribution of transcriptome data obtained in three independent series of experiments (I, II, and III). Analyses were performed with the StrandNGS software using default settings. FIG 3 Cluster (A) and principal component (B) analysis of the transcriptome data. Symbols represent untreated control (Cont) and 75 mM farnesol exposure (FAR) cultures. The distribution of transcriptome data obtained in three independent series of experiments (I, II, and III). Analyses were performed with the StrandNGS software using default settings. (iv) Transmembrane transport-related genes. Farnesol treatment led to the increased transcription of numerous genes (60 genes altogether) involved in trans- membrane transport, including 5 putative antifungal drug transporter genes (MDR1, CDR1, CDR4, HOL3, and YOR1), 4 putative carbohydrate transport genes (HGT2, HGT17, HGT19, and HXT5), 13 putative amino acid transport genes, as well as 4 putative phos- phate and sulfate transporter genes (PHO84, PHO89, GIT1, and SUL2) (Fig. 4 and 5 and Table S3). Farnesol exposure also caused a signiﬁcant increase in the transcription of CDR1 and MDR1 (ABC transporters) as well as HGT2 (glucose transmembrane trans- porter) of treated cells, according to the RT-qPCR results (Fig. 6 and Table S4). nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. //journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. FIG 4 Overview of transcriptional changes induced by farnesol in C. auris. Upregulated (red) and downregulated (blue) genes were deﬁned as differentially expressed genes (corrected P value of ,0.05), with more than a 1.5-fold increase or decrease in their transcription (farnesol treated versus untreated). On the sides of the volcano plot are representative genes upregulated or downregulated by farnesol treatment. The data set is available in Table S3 in the supplemental material. loaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122. FIG 4 Overview of transcriptional changes induced by farnesol in C. auris. Upregulated (red) and downregulated (blue) genes were deﬁned as differentially expressed genes (corrected P value of ,0.05), with more than a 1.5-fold increase or decrease in their transcription (farnesol treated versus untreated). On the sides of the volcano plot are representative genes upregulated or downregulated by farnesol treatment. The data set is available in Table S3 in the supplemental material. oaded from h msphere.asm.org September/October 2021 Volume 6 Issue 5 e00710-21 September/October 2021 Volume 6 Issue 5 e00710-21 Jakab et al. Farnesol exposure signiﬁcantly inﬂuences the metal contents of C. auris cells. msphere.asm.org 6 RESULTS FIG 5 Summary of gene enrichment analyses and the number of genes affected by farnesol exposure of C. auris. Downregulated (blue) (A) and upregulated (red) (B) genes were deﬁned as differentially expressed genes (corrected P value of ,0.05). The enrichment of these gene groups was identiﬁed with the Candida Genome Database Gene Ontology Term Finder (http://www.candidagenome.org/cgi-bin/GO/goTermFinder) or was tested by Fisher’s exact test. The data sets for the gene groups are available in Tables S2 and S3 in the supplemental material. nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. FIG 5 Summary of gene enrichment analyses and the number of genes affected by farnesol exposure of C. auris. Downregulated (blue) (A) and upregulated (red) (B) genes were deﬁned as differentially expressed genes (corrected P value of ,0.05). The enrichment of these gene groups was identiﬁed with the Candida Genome Database Gene Ontology Term Finder (http://www.candidagenome.org/cgi-bin/GO/goTermFinder) or was tested by Fisher’s exact test. The data sets for the gene groups are available in Tables S2 and S3 in the supplemental material. Farnesol exposure signiﬁcantly inﬂuences the metal contents of C. auris cells. Farnesol treatment caused a 24%, 73%, and 69% reduction in intracellular iron, manganese, and zinc content, respectively, compared to untreated control cells (152.2 6 21.1 mg/kg versus 116.0 6 10.0 mg/kg, 67.9 6 5.1 mg/kg versus 18.6 6 1.8 mg/kg, and 787.8 6 22.2 mg/kg versus 245.8 6 34.4 mg/kg, for iron, manganese, and zinc, respectively) (P , 0.05 to 0.001), whereas the level of intracellular copper showed a 302% increase (274.6 6 15.7 mg/kg versus 828.8 6 106.4 mg/kg), as shown in Table 1 (P , 0.001). September/October 2021 Volume 6 Issue 5 e00710-21 Effects of Farnesol against C. auris FIG 6 Correlation between RT-qPCR and transcriptome data. The expression patterns of genes related to bioﬁlm formation (RBT5 and NRG1), oxidation-reduction (CAT1), membrane transport (MDR1, CDR1, HGT2, and FTR1), and metabolism (PFK1, PDC12, ADH1, INO1, POT1, and ERG1) were conﬁrmed by the RT-qPCR assays. RNA-Seq data are presented as fold change (FC) values. Relative transcription levels were quantiﬁed as DDCP = DCPcontrol 2 DCPtreated, where DCPtreated = CPtested gene 2 CPreference gene, measured from farnesol-treated cultures, and DCPcontrol = CPtested gene 2 CPreference gene, measured from control cultures. CP values represent the qRT-PCR cycle numbers of crossing points. The ACT1 gene was used as a reference gene. RESULTS DDCP values signiﬁcantly (P , 0.05 by Student’s t test; n = 3) higher or lower than zero (up- or downregulated genes) are indicated in red and blue, respectively. Pearson’s correlation coefﬁcient between the RT-qPCR and RNA-Seq values was 0.87. The data set is available in Table S4. Effects of Farnesol against C. auris FIG 6 Correlation between RT-qPCR and transcriptome data. The expression patterns of genes related to bioﬁlm formation (RBT5 and NRG1), oxidation-reduction (CAT1), membrane transport (MDR1, CDR1, HGT2, and FTR1), and metabolism (PFK1, PDC12, ADH1, INO1, POT1, and ERG1) were conﬁrmed by the RT-qPCR assays. RNA-Seq data are presented as fold change (FC) values. Relative transcription levels were quantiﬁed as DDCP = DCPcontrol 2 DCPtreated, where DCPtreated = CPtested gene 2 CPreference gene, measured from farnesol-treated cultures, and DCPcontrol = CPtested gene 2 CPreference gene, measured from control cultures. CP values represent the qRT-PCR cycle numbers of crossing points. The ACT1 gene was used as a reference gene. DDCP values signiﬁcantly (P , 0.05 by Student’s t test; n = 3) higher or lower than zero (up- or downregulated genes) are indicated in red and blue, respectively. Pearson’s correlation coefﬁcient between the RT-qPCR and RNA-Seq values was 0.87. The data set is available in Table S4. DISCUSSION Alternative treatments interfering with quorum sensing have recently become attrac- tive therapeutic strategies, particularly against difﬁcult-to-treat multidrug-resistant patho- gens such as C. auris (9, 15, 16). Previous studies have reported that fungal quorum-sens- ing molecules may have a remarkable antifungal effect and/or a potent adjuvant effect in combination with traditional antifungal agents (7, 10, 17–20). For example, Nagy et al. reported that supraphysiological farnesol exposure caused a signiﬁcant reduction in the growth rate and metabolic activity of C. auris planktonic cells and bioﬁlms, respectively (7). In addition, 75 mM farnesol treatment signiﬁcantly decreased the fungal kidney bur- den in an immunocompromised systemic mouse model (7). Total transcriptome analysis using RNA-Seq may be an important technique to fully understand the underlying mech- anisms of the observed antifungal effect exerted by these molecules. In C. albicans, the transcription level of several genes has been shown to be affected by supraphysiological farnesol. Cao et al. (21) reported that farnesol exposure caused an increased expression of TUP1 (related to morphogenesis), FCR1 (drug resistance gene), FTR2 (iron transport gene), and CHT2 and CHT3 (chitinase genes). CSH1 (related to cell surface hydrophobicity) had a downregulation response in the presence of farnesol similar to HSP70, HSP90, and SSA2 (encoding heat shock proteins), PDR16 (drug resistance gene), and CRK1 and PDE2 (related to morphogenesis) (8, 21). nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. The downregulated transcription of CSR1, encoding a major transcription factor that stabilizes zinc homeostasis and provides cells with zinc-dependent protection against farnesol-induced oxidative stress (14), is related to the decreased intracellular zinc level observed. Zinc is an essential transition metal in oxidative stress defense because it is a structural component of superoxide dismutase, which is a key enzyme in the neutralization of superoxide radical anions (O22) (14). In contrast to the majority of metals, manganese acts as an antioxidant element at high concentrations rather than a reactive oxygen species producer (14). However, far- nesol inhibited the transcription of SMF1, which is responsible for maintaining the in- tracellular manganese levels for antioxidant actions (14). In addition, the transcription of PMR1 (P , 0.05, fold change [FC] = 1.2) was also inhibited, decreasing the virulence of fungal cells (27). This was associated with our previously published data, where daily farnesol treatment signiﬁcantly decreased the virulence of C. auris (7). nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. The observed downregulation of the copper exclusion system (CRP1 and/or CCC2, encoding P-type ATPases) may be associated with the signiﬁcantly increased copper contents and the remarkable growth inhibition in farnesol-treated cells. Copper regu- lates a variety of cellular processes in fungal pathogens. When it presents in excess, it is associated with the generation of reactive oxygen species via the Fenton reaction and destroys the iron-sulfur cluster reducing the viability of cells (14, 28–31). The ele- vated free copper levels in the farnesol-exposed cells may contribute to the increased redox imbalance quantiﬁed by DCF production (7), which was accompanied by increases in the speciﬁc activity of superoxide dismutase (7). Moreover, recent studies have shown that copper efﬂux pumps may be equally important in fungal defense strategies against phagocytes as for the virulence in C. albicans (14, 28–31). Interestingly, farnesol exposure exerted a signiﬁcant upregulation in several fatty acid b-oxidation-related genes (POX1, ECI1, FAT1, FAA21, and POT1). The elimination of unnecessary membrane lipids and the increased usage of fatty acids may provide a higher metabolic ﬂux, needed for the maintenance of membrane ﬂuidity (32). Jabra- Rizk et al. (5) and Rossignol et al. (6) reported that farnesol inﬂuences the membrane permeability in non-albicans species such as Candida dubliniensis and Candida parapsi- losis. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. On the basis of previous studies, farnesol induces a dose-dependent production of re- active species in C. albicans, especially at supraphysiological concentrations (22, 23). Moreover, farnesol inﬂuences the transcription of CAT1, SOD1, and SOD2, which were TABLE 1 Farnesol exposure signiﬁcantly inﬂuences the metal contents of Candida auris cells Culture Dry cell mass (g/liter)a Metal contents/treatment (mg/kg) (mean ± SDa) Fe Mn Zn Cu Untreated cultures 0.38 6 0.08 152.2 6 21.1 67.9 6 5.1 787.8 6 22.2 274.6 6 15.7 Farnesol-treated cultures 0.09 6 0.01** 116.0 6 10.0* 18.6 6 1.8* 245.8 6 34.4* 828.8 6 106.4*** aMean values 6 standard deviations (SD) calculated from three independent experiments are presented. The asterisks indicate signiﬁcant differences calculated by two-way ANOVA comparing untreated control and farnesol-treated cultures as follows: , P , 0.05; **, P , 0.001. nloaded from https://journals. TABLE 1 Farnesol exposure signiﬁcantly inﬂuences the metal contents of Candida auris cells Jakab et al. linked to the oxidative stress response in C. albicans (8). These ﬁndings coincided with the C. auris-related physiological experiments published by Nagy et al. (7). In this study, sev- eral putative oxidative stress-responsive genes, namely, CAT1 (encoding catalase activity), GPX1 (encoding glutathione peroxidase), and SOD1, SOD2, and SOD6 (encoding superox- ide dismutases), were upregulated following exposure to farnesol. It is noteworthy that farnesol exposure also upregulated HOG1 MAP kinase, which is a critical component of the fungal oxidative stress response, further supporting the farnesol-induced oxidative stress in C. auris (24). This fact is further conﬁrmed by the elevated 29,79-dichloroﬂuores- cein (DCF) and superoxide dismutase levels in farnesol-exposed cultures (7). Recent transcriptomic data have demonstrated that farnesol treatment affected the transcription of iron homeostasis-related genes, as well as the iron, zinc, manganese, and copper contents of C. auris. The downregulation of iron uptake genes was associ- ated with the signiﬁcantly decreased iron content measured in farnesol-exposed cells. Similarly, the menadione sodium bisulﬁte-induced oxidative stress also affected the transcription of iron homeostasis-related genes and the iron content of C. albicans cells (25). It should be noted that this response related to iron decrease may be a part of a general defense mechanism against farnesol and menadione sodium bisulﬁte to mini- mize the damage caused by ferrous ions. According to previous studies, elevated free intracellular iron levels facilitate the formation of reactive oxygen species and mediate iron-dependent cell death in Saccharomyces cerevisiae (25, 26). September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 8 Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. The elevated fatty acid oxidation activity may explain the membrane-related far- nesol effect, which may elucidate the previously observed antifungal effect (7). A fur- ther potential explanation of the antifungal effect can be found in the downregulation of ergosterol biosynthesis-related genes, which alter the membrane permeability and/ or ﬂuidity (33). Dižová et al. reported that the presence of 200 mM farnesol September/October 2021 Volume 6 Issue 5 e00710-21 Effects of Farnesol against C. auris downregulated the ERG20, ERG11, and ERG9 genes in C. albicans (33). Based on these facts, exogenous farnesol has an effect on the synthesis of ergosterol. In our study, the ERG6 gene was downregulated following farnesol exposure, which may enhance the passive diffusion of farnesol across the membrane; furthermore, the decreased Erg6 content may conﬁrm the higher susceptibility of C. auris cells to oxida- tive stress (34, 35). Oliveira et al. (34) showed that the ERG6 mutant Cryptococcus neo- formans displays impaired thermotolerance and increased susceptibility to oxidative stress as well as to different antifungal drugs, explaining, for instance, the previously reported synergizing effect with azoles (7, 34). Furthermore, the ERG6 mutant C. neofor- mans was totally avirulent in an invertebrate model, which may also explain the reduced virulence of C. auris after daily farnesol treatment (7, 34). Beside ERG6, INO1 was also downregulated following farnesol treatment. This gene encodes the inositol- 1-phosphate synthase, a key enzyme in the synthesis of inositol for phosphotidylinosi- tol synthesis. The downregulation of this gene may further explain the synergizing effect of farnesol with azoles against C. auris (7), because INO1 is signiﬁcantly upregu- lated in drug-resistant Candida isolates (21). With respect to the transport efﬂux pump-related genes, farnesol exposure caused a signiﬁcant increase in the transcription of CDR1, CDR4, MDR1, HOL3, and YOR1, whereas the transcription level of SNQ2 was decreased. Previous studies have revealed that these transporters mediate drug resistance for C. auris (36, 37). Srivastava and Ahmad found that CDR1, CDR2, MDR1, MDR2, and SNQ2 are signiﬁcantly downregu- lated in the presence of farnesol (38). Notably, there was a 1,000-fold difference between the farnesol dosages exerting the upregulating effect (125 mM) compared to the concentration used in our study (75 mM). Nevertheless, our data support the hy- pothesis that farnesol, at lower concentrations, may be a potential substrate for the up- regulated transport proteins in order to protect the cells themselves from the oxidative stress induced by farnesol. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. This is the ﬁrst study analyzing the global changes in gene transcription in C. auris following farnesol exposure, providing important insights into the mechanism of anti- fungal action of farnesol and the response of C. auris, facilitating a better understand- ing of farnesol-related antifungal activity. In summary, farnesol exposure enhanced the oxidative stress response and upregulated drug efﬂux pumps, while reducing zinc and manganese intracellular content as well as iron metabolism. Moreover, cellular metab- olism was modulated toward b-oxidation. These ﬁndings reveal the mechanisms underlying the antifungal effect and suggest that farnesol may represent a potent ther- apeutic option against this multiresistant fungal superbug. nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 9 Jakab et al. Microscopy. Farnesol-induced morphological and viability changes were examined at 75 mM after 0, 4, and 6 h of incubation at 37°C. Afterwards, 999 ml of the culture was stained with 1 ml of 20 mM propi- dium iodide (ThermoFisher, Waltham, MA, USA). Fluorescently stained cells were incubated further at 37°C for 30 min; then, 10 ml of medium was mounted on a slide and examined using a Zeiss Axioskop 2 mot microscope coupled with a Zeiss Axiocam HRc camera using the phase-contrast and ﬂuorescent technique to assess cell morphology and the ratio of nonviable cells, respectively. Further picture analy- sis and calculation of the percentage of the dead cells were performed using ImageJ software (version 2.1.0/1.53c) (Fiji, ImageJ; Wayne Rasband, National Institutes of Health) (42). RNA isolation and sequencing. Total RNA was extracted from untreated control cells and 75 mM farnesol-treated cultures in three biological replicates. Brieﬂy, fungal cells were collected at 2 h following farnesol exposure by centrifugation (5 min at a relative centrifugal force [RCF] of 4,000 g at 4°C). The cells were washed three times with phosphate-buffered saline (PBS) and stored at 270°C until use. Total RNA samples were prepared from freeze-dried cells (CHRIST Alpha 1-2 LDplus lyophilizer, Osterode, Germany) derived from untreated and farnesol-treated cultures using TRIzol (Invitrogen, Austria) reagent by the method of Chomczynski et al. (43). To determine the ﬁnal RNA concentration and quality, samples were analyzed on an Agilent BioAnalyzer using the Eukaryotic Total RNA Nano kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer’s protocol. Samples with RNA integrity number (RIN) values of .7 were accepted for the library preparation process. Three independent cultures were used for RNA-Seq experiments and RT-qPCR tests. q p q To obtain global transcriptome data, high-throughput mRNA sequencing was performed. The RNA-Seq libraries were prepared from total RNA using the NEBNext Ultra II RNA sample preparation kit (NEB, USA) according to the manufacturer’s protocol. The single-read 75-bp-long sequencing reads were generated on an Illumina NextSeq500 instrument. Approximately 18 to 22 million reads per samples were generated. The library preparations and the sequencing run were performed by the Genomic Medicine and Bioinformatics Core Facility of the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary. Raw reads were aligned to the reference genome (genome, https://fungi.ensembl.org/ _candida_auris_gca_002759435/Info/Index; features, http://www.candidagenome.org/download/gff/C_auris _B8441/archive/C_auris_B8441_version_s01-m01-r11_features_with_chromosome_sequences.gff.gz), and aligned reads varied between 90 and 95% in each sample. Jakab et al. The DESeq algorithm (StrandNGS software) was used to obtain normalized gene transcription values. Gene transcription differences between farnesol- exposed and control groups were compared by a moderated t test; the Benjamini-Hochberg false discovery rate was used for multiple-testing correction, and a corrected P value of ,0.05 was considered signiﬁcant (differentially expressed genes). Up- and downregulated genes were deﬁned as differentially expressed genes with .1.5-fold change (FC, upregulated genes) or less than 21.5-FC (downregulated genes) values. The FC ratios were calculated from the normalized gene transcription values. Reverse transcriptase-quantitative PCR assays. Changes in the transcription of selected oxidative stress response, membrane transport, virulence, and primary metabolism genes were validated by reverse transcriptase-quantitative PCR (RT-qPCR) (41). The RT-qPCRs with Luna Universal one-step RT- qPCR kit (NEB, USA) were performed according to the protocol of the manufacturer, using 500 ng of DNase (Sigma, Budapest, Hungary)-treated total RNA per reaction. Oligonucleotide primers (see Table S1 in the supplemental material) were designed with the software packages Oligo Explorer (version 1.1.) and Oligo Analyzer (version 1.0.2). Three parallel measurements were performed with each sample in a LightCycler 96 real-time PCR instrument (Roche, Switzerland). Relative transcription levels (DDCP value) were calculated as DCPcontrol 2 DCPtreated, where DCPcontrol = CPtested gene, control 2 CPreference gene, control for untreated control, and DCPtreated = CPtested gene, treated 2 CPreference gene, treated for farnesol-exposed cultures (13). The CP values represent the RT-qPCR cycle numbers of crossing points. The reference gene used was ACT1 (B9J08_000486). The DDCP values are expressed as mean 6 SD calculated from three inde- pendent measurements, and DDCP values signiﬁcantly (P , 0.05) higher or lower than zero were deter- mined using the Student’s t test. nloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. Functional enrichment analysis. Gene set enrichment analyses on the upregulated and downregu- lated gene sets were performed with Candida Genome Database Gene Ontology Term Finder (http:// www.candidagenome.org/cgi-bin/GO/goTermFinder), using function, process, and component gene on- tology (GO) terms. Only hits with a P value of ,0.05 were considered in the evaluation process (Table S2). Besides GO terms, groups of functionally related genes were also generated by extracting data from the Candida Genome Database (http://www.candidagenome.org) unless otherwise indicated. The enrichment of C. auris genes from these gene groups in the upregulated and downregulated gene sets was tested with Fisher’s exact test (P , 0.05). The following gene groups were created. (i) Virulence-related genes. September/October 2021 Volume 6 Issue 5 e00710-21 MATERIALS AND METHODS Fungal strain, media, and culture conditions. The C. auris isolate 12 (NCPF 8973), belonging to the South Asian/Indian lineage, was obtained from the National Mycology Reference Laboratory (United Kingdom) (39). For the tested C. auris isolate, the MICs were 0.125 mg/liter, 1 mg/liter, 0.125 mg/liter, 4 mg/liter, and 0.25 mg/liter for anidulafungin, caspofungin, micafungin, ﬂuconazole, and amphotericin B, respectively. The test strain was maintained and cultured on yeast extract-peptone-dextrose (YPD) agar (1% yeast extract [Alfa Aesar, USA], 2% mycological peptone [Oxoid, UK], 2% dextrose [VWR International Llc., Hungary], with or without 2% agar [VWR International Llc., Hungary] [pH 5.6]) as described previ- ously (40). To study the effect of farnesol on short-term transcriptional response, C. auris precultures were grown in 5 ml YPD medium at 30°C at a shaking frequency of 2.3 Hz for 18 h. Subsequently, the inocu- lum was diluted to an optical density of 0.1 at l = 640 nm (OD640) with YPD (at 0-h incubation time as deﬁned in growth assays), and the cultures were further grown at 37°C and 2.3-Hz shaking frequency. At 4-h incubation time, the cultures were supplemented with 75 mM farnesol, and microbial growth was monitored by measuring changes in OD640 and CFU (7, 41). Farnesol (Merck, Budapest, Hungary) was obtained as a 3 M stock solution that was diluted to a 30 mM working stock solution in 100% methanol. The working concentrations were prepared in YPD. Farnesol-free control ﬂasks contained 1% (vol/vol) methanol. Growth was evaluated in six independent experiments and is presented as the mean 6 stand- ard deviation (SD). Statistical comparison of growth-related data was performed by paired Student’s t test. The differences between values for treated and control cells were considered signiﬁcant at a P value of ,0.05. Jakab et al. Jakab et al. Genes involved in the genetic control of C. albicans virulence were col- lected by the methods of Mayer et al. (44), Höfs et al. (45), and Araújo et al. (46). (ii) Metabolic pathway-related genes. This group contains all genes related to the carbohydrate, ergosterol, and fatty acid biochemical pathways according to the pathway databases (http://pathway .candidagenome.org/). (iii) Antioxidant enzyme genes. This group includes genes encoding functionally veriﬁed and/or putative antioxidant enzymes according to catalases (GOID:4096), SODs (GOID:4784), glutaredoxins (GOID:6749), thioredoxins (GOIDs:8379 and 51920), and peroxidases (GOID:4601) GO terms. (iv) Iron metabolism-related genes. Genes involved in iron acquisition by C. albicans were col- lected by the method of Fourie et al. (47). (v) Zinc, manganese, and copper homeostasis genes. Genes involved in zinc and copper acquisi- tion were collected by the method of Gerwien et al. (14). September/October 2021 Volume 6 Issue 5 e00710-21 msphere.asm.org 10 Effects of Farnesol against C. auris The complete gene lists of the above-mentioned gene groups are available in Table S3. The complete gene lists of the above-mentioned gene groups are available in Table S3. Assays of iron, manganese, zinc, and copper contents of Candida auris cells. C. auris precultures were grown, and farnesol exposure was performed as described above. Yeast cells were collected by centrifugation (5 min, 4,000 g, 4°C) after 2 h of incubation following farnesol exposure. Changes in fungal dry cell mass (DCM) were determined after freeze-drying (25). The metal contents of the dry bio- mass were measured by inductively coupled plasma optical emission spectrometry (ICP-OES; 5110 Agilent Technologies, Santa Clara, CA, USA) following atmospheric wet digestion in 3 ml of 65% (mass percent [M/M]) HNO3 and 1 ml of 30% (M/M) H2O2 in glass beakers. The metal contents of the samples were calculated and expressed in DCM units (in milligrams per kilogram) by the method of Jakab et al. (25). The metal contents of the biomasses were determined in triplicate, and mean 6 SD values were calculated. Statistical signiﬁcance of changes was determined by two-way analysis of variance (ANOVA). Signiﬁcance was deﬁned as a P value of ,0.05. Availability of data. The RNA sequencing data discussed have been deposited in NCBI’s Gene Expression Omnibus (48) (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE180093. REFERENCES 7. Nagy F, Vitális E, Jakab Á, Borman AM, Forgács L, Tóth Z, Majoros L, Kovács R. 2020. In vitro and in vivo effect of exogenous farnesol exposure against Candida auris. Front Microbiol 11:957. https://doi.org/10.3389/ fmicb.2020.00957. 1. Chowdhary A, Tarai B, Singh A, Sharma A. 2020. Multidrug-resistant Can- dida auris infections in critically ill coronavirus disease patients, India, April-July 2020. Emerg Infect Dis 26:2694–2696. https://doi.org/10.3201/ eid2611.203504. 1. Chowdhary A, Tarai B, Singh A, Sharma A. 2020. Multidrug-resistant Can- dida auris infections in critically ill coronavirus disease patients, India, April-July 2020. Emerg Infect Dis 26:2694–2696. https://doi.org/10.3201/ eid2611.203504. 2. Magnasco L, Mikulska M, Giacobbe DR, Taramasso L, Vena A, Dentone C, Dettori S, Tutino S, Labate L, Di Pilato V, Crea F, Coppo E, Codda G, Robba C, Ball L, Patroniti N, Marchese A, Pelosi P, Bassetti M. 2021. Spread of carbape- nem-resistant Gram-negatives and Candida auris during the COVID-19 pan- demic in critically ill patients: one step back in antimicrobial stewardship? Microorganisms 9:95. https://doi.org/10.3390/microorganisms9010095. 8. Rodrigues CF, Cernáková L. 2020. Farnesol and tyrosol: secondary metab- olites with a crucial quorum-sensing role in Candida bioﬁlm development. Genes 11:444. https://doi.org/10.3390/genes11040444. 9. Kovács R, Majoros L. 2020. Fungal quorum-sensing molecules: a review of their antifungal effect against Candida bioﬁlms. J Fungi 6:99. https://doi .org/10.3390/jof6030099. 3. Mulet Bayona JV, Tormo Palop N, Salvador García C, Fuster Escrivá B, Chanzá Aviñó M, Ortega García P, Gimeno Cardona C. 2021. Impact of the SARS-CoV-2 pandemic in candidaemia, invasive aspergillosis and antifun- gal consumption in a tertiary hospital. J Fungi 7:440. https://doi.org/10 .3390/jof7060440. 3. Mulet Bayona JV, Tormo Palop N, Salvador García C, Fuster Escrivá B, Chanzá Aviñó M, Ortega García P, Gimeno Cardona C. 2021. Impact of the SARS-CoV-2 pandemic in candidaemia, invasive aspergillosis and antifun- gal consumption in a tertiary hospital. J Fungi 7:440. https://doi.org/10 .3390/jof7060440. 10. Nagy F, Tóth Z, Daróczi L, Székely A, Borman AM, Majoros L, Kovács R. 2020. Farnesol increases the activity of echinocandins against Candida auris bio- ﬁlms. Med Mycol 58:404–407. https://doi.org/10.1093/mmy/myz057. 11. Simm C, May RC. 2019. Zinc and iron homeostasis: target-based drug screening as new route for antifungal drug development. Front Cell Infect Microbiol 9:181. https://doi.org/10.3389/fcimb.2019.00181. 4. Hornby JM, Jensen EC, Lisec AD, Tasto JJ, Jahnke B, Shoemaker R, Dussault P, Nickerson KW. 2001. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol 67: 2982–2992. https://doi.org/10.1128/AEM.67.7.2982-2992.2001. 12. SUPPLEMENTAL MATERIAL Supplemental material is available online only. TABLE S1, DOCX ﬁle, 0.01 MB. TABLE S2, XLSX ﬁle, 0.02 MB. TABLE S3, XLSX ﬁle, 0.1 MB. TABLE S4, XLSX ﬁle, 0.01 MB. ACKNOWLEDGMENTS This project was supported by the EFOP-3.6.3-VEKOP-16-2017-00009 program. This research was funded by the European Union and the European Social (EFOP-3.6.1-16- 2016-00022) by the Thematic Excellence Programme (TKP2020-IKA-04) of the Ministry for Innovation and Technology in Hungary. This research was funded by the Hungarian National Research, Development and Innovation Ofﬁce (NKFIH FK138462) (R. Kovács). R. Kovács was supported by OTKA Bridging Fund and FEMS Research and Training Grant (FEMS-GO-2019-502). R. Kovács was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. F. Nagy was supported by the ÚNKP-19-3 and ÚNKP-20-3 New National Excellence Program of the Ministry for Innovation and Technology. L. Majoros received conference travel grants from MSD, Astellas, and Pﬁzer. All other authors declare no conﬂicts of interest. Downloaded from https://journals.asm.org/journal/msphere on 13 October 2021 by 95.214.122.2. September/October 2021 Volume 6 Issue 5 e00710-21 Jakab et al. 14. 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https://openalex.org/W3101422573	https://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=5497&context=oapubs	English	null	Compilation and Analysis of Web-Based Orthopedic Personalized Predictive Tools: A Scoping Review	Journal of personalized medicine	2,020	cc-by	10,051	Received: 9 October 2020; Accepted: 10 November 2020; Published: 12 November 2020 Abstract: Web-based personalized predictive tools in orthopedic surgery are becoming more widely available. Despite rising numbers of these tools, many orthopedic surgeons may not know what tools are available, how these tools were developed, and how they can be utilized. The aim of this scoping review is to compile and synthesize the proﬁle of existing web-based orthopedic tools. We conducted two separate PubMed searches—one a broad search and the second a more targeted one involving high impact journals—with the aim of comprehensively identifying all existing tools. These articles were then screened for functional tool URLs, methods regarding the tool’s creation, and general inputs and outputs required for the tool to function. We identiﬁed 57 articles, which yielded 31 unique web-based tools. These tools involved various orthopedic conditions (e.g., fractures, osteoarthritis, musculoskeletal neoplasias); interventions (e.g., fracture ﬁxation, total joint arthroplasty); outcomes (e.g., mortality, clinical outcomes). This scoping review highlights the availability and utility of a vast array of web-based personalized predictive tools for orthopedic surgeons. Increased awareness and access to these tools may allow for better decision support, surgical planning, post-operative expectation management, and improved shared decision-making. Keywords: web-based tools; orthopedics; predictive tools Compilation and Analysis of Web-Based Orthopedic Personalized Predictive Tools: A Scoping Review Patrick Curtin 1, Alexandra Conway 1, Liu Martin 1, Eugenia Lin 2 , Prakash Jayakumar 2 and Eric Swart 1,* Patrick Curtin 1, Alexandra Conway 1, Liu Martin 1, Eugenia Lin 2 , Prakash Jayakumar 2 a Eric Swart 1,* 1 Department of Orthopedics, University of Massachusetts Medical Center, 55 N Lake Avenue, Worcester MA 01655, USA; Patrick.Curtin@umassmemorial.org (P.C.); Alexandra.Conway@umassmed.edu (A.C.) Liu_Martin@branson.org (L.M.) 1 Department of Orthopedics, University of Massachusetts Medical Center, 55 N Lake Avenue, Worcester, MA 01655, USA; Patrick.Curtin@umassmemorial.org (P.C.); Alexandra.Conway@umassmed.edu (A.C.); Liu_Martin@branson.org (L.M.) Liu_Martin@branson.org (L.M.) 2 Department of Surgery and Perioperative Care, University of Texas at Austin Dell Medical School, 1601 Trinity Street, Austin, TX 78712, USA; Eugenia.Lin@austin.utexas.edu (E.L.); Prakash.Jayakumar@austin.utexas.edu (P.J.) * Correspondence: Eric.Swart@umassmemorial.org R i d 9 O t b 2020 A t d 10 N b 2020 P bli h d 12 N b 2020 _ g ( ) 2 Department of Surgery and Perioperative Care, University of Texas at Austin Dell Medical School, 1601 Trinity Street, Austin, TX 78712, USA; Eugenia.Lin@austin.utexas.edu (E.L.); Prakash.Jayakumar@austin.utexas.edu (P.J.) * Correspondence: Eric.Swart@umassmemorial.org g 2 Department of Surgery and Perioperative Care, University of Texas at Austin Dell Medical School, 1601 Trinity Street, Austin, TX 78712, USA; Eugenia.Lin@austin.utexas.edu (E.L.); Prakash.Jayakumar@austin.utexas.edu (P.J.) * Correspondence: Eric.Swart@umassmemorial.org J. Pers. Med. 2020, 10, 223; doi:10.3390/jpm10040223 Compilation and Analysis of Web-Based Orthopedic Personalized Predictive Tools: A Scoping Review Item Type Journal Article Authors Curtin, Patrick; Conway, Alexandra; Martin, Liu; Lin, Eugenia; Jayakumar, Prakash; Swart, Eric F. Citation <p>Curtin P, Conway A, Martin L, Lin E, Jayakumar P, Swart E. Compilation and Analysis of Web-Based Orthopedic Personalized Predictive Tools: A Scoping Review. J Pers Med. 2020 Nov 12;10(4):223. doi: 10.3390/jpm10040223. PMID: 33198106; PMCID: PMC7712817. <a href="https://doi.org/10.3390/ jpm10040223">Link to article on publisher's site</a></p> DOI 10.3390/jpm10040223 Rights Copyright © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/ by/4.0/). Download date 24/10/2024 04:15:25 Item License http://creativecommons.org/licenses/by/4.0/ Link to Item http://hdl.handle.net/20.500.14038/41676 Journal of Personalized Medicine Journal of Personalized Medicine Journal of Personalized Medicine 1. Introduction The ability to provide personalized predictions of clinical outcomes in the ﬁeld of orthopedics is gaining interest [1–5]. Databases encompassing robust and accurate patient-level data [6–8], greater access to patient information via the electronic medical record [9], and the rise of advanced analytical capabilities, such as machine learning [10,11], provide the prospect of great strides in both our understanding of musculoskeletal problems and the outcomes of orthopedic interventions. Unlike simple risk calculations, web-based predictive tools analyze larger amounts of patient data and utilize algorithmic mathematical modeling and prediction analytics using advanced computing. Despite the technological advances in predictive tools, many challenges exist in practical implementation of these solutions in clinical settings. Firstly, there is no common repository or standardized location to access personal predictive tools. These tools span various subspecialties within the ﬁeld of orthopedics and other surgical specialties (e.g., capable of providing general risk calculations). As a result, where to ﬁnd and identify tools appropriate for their clinical needs remains a barrier for orthopedic surgeons in practice. Secondly, once a tool has been identiﬁed for use, it can be J. Pers. Med. 2020, 10, 223; doi:10.3390/jpm10040223 www.mdpi.com/journal/jpm www.mdpi.com/journal/jpm J. Pers. Med. 2020, 10, 223 2 of 20 diﬃcult to discern how the tool was developed (i.e., what data inputs have been used to deﬁne the tool’s algorithm) and how it has been assessed for technical feasibility and validated (i.e., the extent to which a tool can be used in a practice setting to ﬁt a given need). Our overarching goal was to perform a scoping review to comprehensively map and organize the knowledge base around web-based personalized predictive tools in orthopedics. Our primary objective was to map the current range of web-based predictive tools by type of data input, study characteristics and statistical methods used to develop the tool, type of data output, and the function of the tool. Our secondary objective was to qualitatively synthesize this data to generate a set of considerations for disseminating and implementing these tools in routine orthopedic practice. Our ﬁndings aim to provide orthopedic surgeons comprehensive insights into the range of available tools, researchers and technologists a premise to develop further innovative solutions in this ﬁeld, and health systems a framework to integrate these tools to advance facets of orthopedic care. Table 1. Targeted Orthopedic Subspecialties Used in Secondary Search. We deﬁned the data extraction elements by consensus and managed any inconsistencies and disagreements from data screening and extraction via consensus discussions and rounds of voting. There were no disagreements that were unable to be resolved, so no extra party was needed to act as a tiebreaker. Table 2. Types of User Input Data. 2. Methods We drafted a protocol a priori using the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols (PRISMA-SCR), and members of our research team further reﬁned the protocol through a collaborative process. Our protocol was registered through Open Science Framework on 24 September 2020 [12]. (To be included in this review, articles and studies needed to be in English, and include the uniform resource link (URL) for their web-based tool and/or suﬃcient information (references, tool name, researcher(s), institution, etc.) to ﬁnd the tool. The development and validation studies mentioned in these articles needed to describe their patient population, intervention in the form of a personalized web-based predictive tool, methodology and description of their validation and development, outcomes provided by the web-based tool, and type of study. Articles referencing studies for which this information was missing were excluded. Exclusion criteria also included articles without URLs or suﬃcient information to identify the tool, article links with no access to full-text PDFs, and articles written in languages other than English. Articles about tools with no orthopedic relevance, such as a tool predicting cardiovascular disease risk in patients taking statins, were also excluded from this study. For the purposes of this review, prospective cohort studies, retrospective cohort studies, and meta-analyses were included, while case reports were excluded. To identify web-based tools that ﬁt the above inclusion criteria, a comprehensive search of the bibliographic electronic database PubMed (NLM) was conducted using speciﬁc search terms and with no time-period restriction. Search terms were initially drafted by an experienced orthopedic surgeon familiar with web-based orthopedic tools, and further reﬁned through discussion among the research team. Search results were then screened for the above inclusion criteria and included only if all criteria were met. To ensure the comprehensive capture of any additional web-tools, a second more targeted search was performed, focusing on a set of high-impact orthopedic journals within each subspecialty. Targeted subspecialties are listed in Table 1. The list of high-impact orthopedic journals was created by an experienced orthopedic surgeon and the research team. These journals are listed in Appendix A. Duplicate articles were removed for both searches. After identifying all tools, missing original development and validation articles were found and included for any tools that lacked them in the initial searches. The ﬁnal search strategy with both primary and secondary searches is recorded in Appendix B. 3 of 20 J. Pers. Med. 2020, 10, 223 Table 1. Targeted Orthopedic Subspecialties Used in Secondary Search. Table 1. Targeted Orthopedic Subspecialties Used in Secondary Search. Joint Replacement Research Trauma Sports Hand and Upper Extremity Shoulder and Elbow Foot and Ankle Spine Pediatric Screening of the identiﬁed articles was performed by all members of the research team. For articles that passed preliminary screening, we downloaded the full-text versions of the studies, mostly as portable document ﬁles (PDFs). We subsequently extracted and recorded relevant data using an electronic data collection sheet. For each tool we extracted the following parameters: tool name, functional URL link, user input data, tool outputs, type of study used for tool creation and/or validation, number of patients involved in those studies, and statistical methods used for creation of tool. User inputs were categorized into demographic, clinical, and patient reported data. Clinical input data were deﬁned to include medical, biometric, and radiologic ﬁndings. Categorical examples of user input data are listed in Table 2. Tools were then grouped by output category, examples for which are listed in Table 3. Table 2. Types of User Input Data. Category Examples Demographic Age, Sex, Body Mass Index (BMI) Clinical Medical History, Injury Characteristics, Procedure Characteristics Patient Reported Social Information (e.g., housing, sexual activity, recreational activities, etc.), Smoking Status, Current Alcohol Use, Recent Fall History Table 3. Types of Tool Output Data. Category Examples Fracture Prediction Loss of Position, Risk of Fracture Mortality Prediction Survival Rates Clinical Events Prediction Surgical Complications, Post-Operative Pain, Readmission Rates, Treatment Options Processes Prediction Length of Stay, Discharge Disposition Data were checked by multiple team members (P.C./L.M./A.C.) to ensure accurate collection. We deﬁned the data extraction elements by consensus and managed any inconsistencies and disagreements from data screening and extraction via consensus discussions and rounds of voting. There were no disagreements that were unable to be resolved, so no extra party was needed to act as a tiebreaker. Table 2. Types of User Input Data. Category Examples Demographic Age, Sex, Body Mass Index (BMI) Clinical Medical History, Injury Characteristics, Procedure Characteristics Patient Reported Social Information (e.g., housing, sexual activity, recreational activities, etc.), Smoking Status, Current Alcohol Use, Recent Fall History Table 3. Types of Tool Output Data. Category Examples Fracture Prediction Loss of Position, Risk of Fracture Mortality Prediction Survival Rates Clinical Events Prediction Surgical Complications, Post-Operative Pain, Readmission Rates, Treatment Options Processes Prediction Length of Stay, Discharge Disposition Data were checked by multiple team members (P.C./L.M./A.C.) to ensure accurate collection. 3. Results We identiﬁed 358 total records through our search strategy, 57 of which matched our inclusion criteria. From those 57 articles, 31 unique tools were identiﬁed, analyzed, and included for this scoping review (Table 4). There was a higher number of articles identiﬁed compared to tools because some articles were reviews of multiple tools or re-validation studies of already identiﬁed tools. Useable links for each tool are listed in Appendix C. 4 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Tools Categorized by Output. 3. Results Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development Edinburgh Wrist Calculator Journal of Orthopaedic Trauma (2018) [13,14], The Journal of Bone and Joint Surgery (2006) [15] Prospective Cohort 4000 distal radius fractures Age Ulnar Variance, Dorsal Comminution Present, Physical Dependence Fracture prediction: Loss of position Univariate and Multiple Logistic Regression CAROC (Canadian Risk for Osteoporosis Calculator) Journal of Clinical Densitometry (2017) [16], (2010) [17], (2007) [18] Retrospective Cohort 39,603 patients Age, Sex Femoral Neck T-Score, Fragility Fracture after age 40, Recent prolonged glucocorticoid use Fracture prediction: Risk categorization Kaplan–Maier Method FRAX Journal of Clinical Densitometry (2017) [19], Turkish Journal of Urology (2019) [20] Systematic Review 290,000 patients Age, Sex, Height, Weight Previous Fracture, Glucocorticoids, Rheumatoid arthritis, Secondary Osteoporosis, Femoral neck BMD Smoking status, Alcohol use, Parental history of hip fracture Fracture prediction: 10-year risk Multiple Logistic Regression FRS (Fracture Risk Scale) BioMed Central (BMC) Geriatrics (2018) [21], British Medical Journal (BMJ) Open (2017) [22] Retrospective Cohort 29,848 patients Age, BMI Wandering frequency, Walking in corridor, Transfer status, Cognitive performance scale, hip fracture history Fall history Fracture prediction: 1-year hip fracture risk Decision Tree and Logistic Regression Garvan Journal of Clinical Densitometry (2017) [19], Osteoporosis International (2008) [23] Prospective Cohort 2216 patients Age, Sex Fractures and fall history, T-scores, actual BMD Fracture prediction: 5- and 10-year risk Cox’s Proportional Hazards Analysis QFracture Journal of Clinical Densitometry (2017) [16,19], The British Medical Journal (Clinical research ed.) (2012) [24] Prospective Cohort 4,726,046 patients Age, Sex, BMI, Ethnicity Diabetes, osteoporotic fracture history, Dementia, Cancer, Asthma or COPD, Cardiovascular disease, liver disease, kidney disease, Parkinson’s, Rheumatoid arthritis, SLE, GI malabsorption, Endocrine problems, Epilepsy, Hormone Therapy, use of anticonvulsants/ antidepressants/corticosteroids/estrogen Smoking status, Alcohol use, Fall history, Parental history of hip fracture/osteoporosis, Residence Fracture prediction: 10-year risk Multivariate Final Cox Regression NHFS (Nottingham Hip Fracture Score) British Journal of Anaesthesia (2008) [25], The Bone & Joint Journal (2015) [26], Injury (2015) [27] Prospective Cohort 4967 patients Age, Sex AMTS, Hb on admission, Comorbidities, Active malignancy history Residence Fracture prediction: NHFS Score Mortality prediction Forward Univariate and Multivariate Logistic Regression 5 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Cont. Table 4. Cont. 3. Results Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development ACS NSQIP Surgical Risk Calculator Journal of Neurosurgery. Spine (2017) [28], The Journal of Arthroplasty (2018) [29], Spine (2020) [30], Clinical Orthopaedics and Related Research (2016) [31], The Journal of Arthroplasty (2015) [32], Journal of the American College of Surgeons (2013) [33] Retrospective Cohort 1,414,006 patients Age, Sex, Height, Weight Procedure, Functional Status, Emergency Case, ASA Class, Steroid use, Ascites, Systemic Sepsis, Ventilator Dependent, Disseminated Cancer, Diabetes, Hypertension, CHF, Dyspnea, History of Severe COPD, Dialysis, Acute Renal Failure Smoking status Mortality prediction Clinical events prediction: Adverse events Processes prediction: Length of stay Random Intercept and Fixed Slope Hierarchical Models E-PASS (Estimation of Physiologic Ability and Surgical Stress) Surgery Risk Calculator Journal of Bone and Mineral Research [34], Injury (2015) [27], Surgery Today (1999) [35], Surgery (2004) [36] Retrospective Cohort 3981 patients Age, Weight Cardiac arrhythmia, Pulmonary vital capacity, FEV1, Diabetes, Blood loss, OR time, Extent of skin incision at surgery, Heart failure, ECOG performance status, ASA class Clinical event prediction: Preoperative risk score, Surgical stress score, Comprehensive risk score Multiple Logistic Regression STTGMA (Score for Trauma Triage in Geriatric and Middle Aged Patients) The Journal of the American Academy of Orthopaedic Surgeons (2020) [37], Bulletin of the Hospital for Joint Disease (2016) [38] Retrospective Cohort 138,096 patients Age Injury mechanism, Glasgow Coma Scale (GCS), Abbreviated Injury Scale (AIS), Head/Neck, Chest, Extremity, Charlson Comorbidity Index (CCI) Mortality prediction: STIGMA Score Logistic Regression Analysis CCI (Charlson Comorbidity Index) Journal of Neurosurgery. 3. Results Spine (2017) [28], Clinical Orthopaedics and Related Research (2014) [39], Injury (2015) [27], Journal of Orthopaedic Research (2020) [40], Journal of Chronic Diseases (1987) [41] Prospective Cohort 1244 patients Age Myocardial infraction, CHF, Peripheral vascular disease, CVA or TIA, Dementia, COPD, Connective tissue disease, Peptic ulcer disease, Liver disease, Diabetes Mellitus, Hemiplegia, Moderate to severe CKD, Solid tumor, Leukemia, Lymphoma, AIDS Mortality prediction: CCI, 10-year survival Kaplan–Maier Method P-POSSUM The British Journal of Surgery (1991) [42], (1998) [43], Injury (2015) [27] Prospective Cohort 1440 patients Age Cardiac status, Respiratory status, ECG, Systolic BP, Pulse, Hemoglobin, WBC, Urea, Sodium, Potassium, GCS, Operation type, Number of procedures, Operative Blood Loss, Peritoneal Contamination, Malignancy Status, CEPOD Mortality prediction (for esophagogastric surgery) Clinical event prediction: Physiology score, Operative severity score, Morbidity Multiple Logistic Regression 6 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Cont. Table 4. Cont. Table 4. Cont. 3. Results Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development LACE+ Journal of Orthopaedic Research (2020) [40], Canadian Medical Association Journal (2010) [44], Open Medicine (2012) [45] Prospective Cohort 500,000 patients Length of stay, Acuity of admission, Comorbidities, Emergency department visits Mortality prediction: LACE+ score Clinical event prediction: Readmission risk Split-Sample Design, Fractional Polynomial Functions, Multiple Logistic Regression Chondrosarcoma Five-Year Survival Machine Learning Algorithm Clinical Orthopaedics and Related Research (2018) [46] Retrospective Cohort 1554 patients Age, Sex Histology, Size, Extension, Grade, Location Mortality prediction: 5-year survival Nonparametric Missforest Method, Boosted Decision Tree, Support Vector Machine, Bayes Point Machine, Neural Network Models, 10-Fold Crossvalidation Extremity Metastatic Disease Survival Prediction Machine Learning Algorithm Clinical Orthopaedics and Related Research (2020) [47] Retrospective Cohort 1090 patients Age Primary Tumor Histology, Visceral Metastasis, Brain Metastasis, Previous Systemic Therapy, Hemoglobin, Platelet, Absolute Lymphocyte, Absolute Neutrophil, Creatinine, White Blood Cell, Albumin, Alkaline Phosphatase, Sodium, Calcium Mortality prediction: 90-day and 1-year survival Missforest Methods, Random Forest Algorithms, Stochastic Gradient Boosting, Random Forest, Support Vector Machine, Neural Network, and Penalized Logistic Regression PathFX Clinical Orthopaedics and Related Research (2017) [48], BioMed Central (BMC) Cancer (2015) [49] Retrospective Cohort 1291 patients Age, Sex Oncologic Diagnosis, Pathologic Fracture, ECOG Performance Status, Hemoglobin concentration, Absolute lymphocyte count, Skeletal Metastases, Organ Metastases, Lymph Node Metastases, Physician’s Estimate of Survival, Skeletal Region Mortality prediction: Survival likelihood after treatment at multiple time intervals Clinical event prediction: Potential treatments Bayesian Belief Networks Demographic Input Length of stay, Acuity of admission, Comorbidities, Emergency department visits 7 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Cont. Table 4. Cont. Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development Incidental Durotomy Calculator Journal of Neurosurgery. Spine (2020) [50] Retrospective Review 1279 patients Age Revision Procedure, Procedure start after 4 pm, Surgery duration Clinical event prediction: Incidental durotomy likelihood Natural language processing (NLP), Multiple Logistic Regression, Bootstrapping Spinal RAT (Risk Assessment Tool) Journal of Neurosurgery. 3. Results Spine (2017) [28] Prospective and Retrospective Cohort 279,391 patients Age, Sex Spinal Area, Pre-op diagnosis, Use of BMP, Fusion, Surgery level, Instrumentation, Pulmonary dysfunction, Neurologic dysfunction, Hypercholesterolemia, Hypertension, Cardiac dysfunction, Diabetes mellitus, Systemic malignancy, Gastroesophageal dysfunction, Psychiatric disorder, Substance abuse Smoking status Clinical event prediction: Surgical complications risk (%), risk classiﬁcation (low, medium, high) Logistic Regression with Main Eﬀects, 2 and 3 Factor Interactions SpineSage The Spine Journal (2014) [51] Prospective Cohort 1532 patients Age, Gender, BMI Primary Diagnosis, Level of Surgery, Surgical Approach, Cerebrovascular Disease, Chronic Obstructive Pulmonary Disease, Asthma, Hypertension, Rheumatoid Arthritis, Renal Conditions, Pre-existing Neoplasm, Syncope or Seizure, Anemia, Bleeding disorder, Diabetes, CHF, Revision surgery, Previous spinal surgery, Previous cardiac complications Clinical event prediction: Complications risk Multivariate Analysis Back Treatment Outcomes Calculator Spine (2002) [52], (2018) [53] Prospective Cohort 289 patients Age, Sex, Height, Weight Condition, Symptoms, Episode, Hypertension, Physical therapy, Depression Smoking status, Activities, Work status, Worker’s compensation, Education, Expectation, Sleep, Sex life Clinical event prediction: Treatment risks, Outcomes with or without surgery (physical functioning, pain, sleep, sex life, satisfaction with symptoms) Multivariate Analysis Condition, Symptoms, Episode, Hypertension, Physical therapy, Depression 8 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Cont. Table 4. Cont. 3. Results Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development OaraScore (Outpatient Arthroplasty Risk Assessment Score) The Journal of Arthroplasty (2018) [54], (2017) [55] Retrospective Review 1120 patients BMI Chronic narcotic use, Chronic pain control diﬃculty, Chronic benzodiazepine use, Severe deconditioning; Additional inputs by topic Lack of home support Clinical event prediction: Outpatient surgery risk, Assessment score Multivariate Analysis Arthroplasty Size Predictor The Journal of Arthroplasty (2019) [56], (2017) [57] Retrospective Cohort 3491 primary TKAs Sex, Height, Weight Manufacturer, Model Clinical prediction: Predicted sizes Multivariate Linear Regression 90-Day Readmissions Risk Calculator The Journal of Bone and Joint Surgery (2019) [58] Retrospective Cohort 10,155 THAs and TKAs Age Joint, ASA, Duration of Surgery, Hemoglobin Level Postoperative, Cardiac Arrhythmia, CHF, Chronic Pulmonary Disease, Diabetes Complicated, Hypertension, Lymphoma, Neurologic Disease, Peripheral Vascular Disease, Pulmonary Circulation Disease, Renal Failure, Depression, Substance abuse Smoking Status, Alcohol use Clinical event prediction: 90-day readmission risk Multiple Logistic Regression QUALITOUCH Outcome Calculator Journal of Telemedicine and Telecare (2014) [59] Prospective Cohort 483 patients Age, Sex, Height, Weight Type of surgery, Hip replacement, Knee replacement, Spine surgery, Hypertension, Heart disease, Stroke, Depression, Diabetes, Cancer, Lung/Kidney/GI disease, Anemia, Substance abuse Pain during activities, Diﬃculty in activities/movements, Chronic pain, Back pain Clinical event prediction: Current pain level, 3-month post-op predicted pain level Multiple Regression STaRT Back Tool Arthritis Care & Research (2008) [60], The Journal of Arthroplasty (2019) [61], Spine (2002) [53] Prospective Cohort and Retrospective Review 1641 patients Symptoms Activity, Pain level, Mental state Clinical event prediction: Chronic pain risk level Forward Stepwise Binary Logistic Regression Analysis Estimated Blood Loss Calculator The Spine Journal (2020) [62] Retrospective Cohort 1281 patients BMI Pedicle Screws (T11-S1), Pelvic Screws, Laminectomy, Laminectomy Levels, Discectomies, ALIF Interbody Fusions, XLIF/OLIF Interbody Fusions, TLIF/PLIF Interbody Fusions, Schwab Osteotomies, TXA Use, Surgery duration Clinical event prediction: Blood loss (mL) Univariate Linear Regressions, Multivariate Analysis 9 of 20 J. Pers. Med. 2020, 10, 223 Table 4. Cont. 3. Results Tool Journal and (Year) of Publication [Ref] Tool Development Study Type and Size Demographic Input Clinical Input Patient Reported Input Tool Output Statistical Methods Used in Tool Development ShockNurd Clinical Orthopaedics and Related Research (2016) [63] Retrospective Review 382 patients Sex Tibial nail >4 weeks ago, Percentage Cortical Contact, Open Fracture, Compartment Syndrome, Soft Tissue Flap Required, Chronic Condition (HIV/HEP C/Diabetes), ASA Classiﬁcation, Low Energy Injury, Spiral Fracture Pattern Clinical event prediction: NURD Score, Non-Union Percentage, Conﬁdence Range Bivariate and Multivariate Regression, Stepwise Modeling Neuro Risk Opioid Use Calculator Spine (2018) [64] Retrospective Cohort 26,553 patients Age, Gender Cervical or Lumbar Spine, Operation type, Diabetes, Depression/Anxiety, Osteoporosis, Fibromyalgia, Morbid Obesity, Lower Back Pain, Motor Deﬁcits (plegia), Bowel/Bladder dysfunction, Substance abuse, Preoperative Opioid User (3 Months Prior to Surgery), Clinical event prediction: Narcotics use at 12-month postop Multiple Logistic Regression Opioid Calculator for Hand Surgery The Journal of Hand Surgery (2019) [65] Prospective Cohort 526 patients Age Can take Naproxen post-op, Can take Acetaminophen post-op, Currently taking Narcotics, Planned use of regional anesthesia, Procedure involves bone/ligament, Anticipated Surgical Time Clinical event prediction: Number of pills to prescribe Bivariate Analysis and Multiple Logistic Regressions Discharge to Rehabilitation and LOS Calculator The Spine Journal (2020) [66] Retrospective Cohort 257 patients Age, BMI, Insurance Diabetic, Type of spine surgery, Procedure time, Elective vs. Emergent Processes prediction: Risk of discharge to rehab, Length of stay Univariable And Multivariable Analyses RAPT (Risk Assessment and Prediction Tool) The Journal of Arthroplasty (2019) [67], (2020) [68], (2019) [61], Clinical Orthopaedics and Related Research (2015) [69] Prospective Cohort 3213 patients Age, Sex Functional Abilities, Social Support Processes prediction: Discharge requirements, Length of stay Binary Logistic Regression Abbreviations: ALIF—Anterior Lumbar Interbody Fusion, AMTS—Abbreviated Mental Test Score, BMD—Bone Mineral Density, BMP—Bone Morphogenetic Protein, CEPOD—Conﬁdential Enquiry into Peri-Operative Deaths, CHF—Congestive Heart Failure, CKD—Chronic Kidney Disease, COPD—Chronic Obstructive Pulmonary Disease, CVA—Cerebrovascular Accident, ECG—Echocardiogram, ECOG—Eastern Cooperative Oncology Group, FEV1—Forced Expiratory Volume, GCS—Glasgow Coma Scale, PLIF—Posterior Lumbar Interbody Fusion, SLE—Systemic Lupus Erythematosus, TIA—Transient Ischemic Attacks, TXA—Tranexamic Acid, XLIF—Extreme Lateral Interbody Fusion, OLIF—Oblique Lumbar Interbody Fusion, TLIF—Transforaminal Lumbar Interbody Fusion. 3. Results Abbreviations: ALIF—Anterior Lumbar Interbody Fusion, AMTS—Abbreviated Mental Test Score, BMD—Bone Mineral Density, BMP—Bone Morphogenetic Protein, CEPOD—Conﬁdential Enquiry into Peri-Operative Deaths, CHF—Congestive Heart Failure, CKD—Chronic Kidney Disease, COPD—Chronic Obstructive Pulmonary Disease, CVA—Cerebrovascular Accident, ECG—Echocardiogram, ECOG—Eastern Cooperative Oncology Group, FEV1—Forced Expiratory Volume, GCS—Glasgow Coma Scale, PLIF—Posterior Lumbar Interbody Fusion, SLE—Systemic Lupus Erythematosus, TIA—Transient Ischemic Attacks, TXA—Tranexamic Acid, XLIF—Extreme Lateral Interbody Fusion, OLIF—Oblique Lumbar Interbody Fusion, TLIF—Transforaminal Lumbar Interbody Fusion. J. Pers. Med. 2020, 10, 223 J Pers Med 2020 10 x FOR 10 of 20 11 f 21 11 of 21 We found that the frequency of orthopedic web-based tool publications has increased over time, with 7% of articles being published before the year 2000, 17% between 2000 and 2010, and the remaining 76% between 2010 and 2020 (Figure 1). Tool development was more often informed by exclusively retrospective studies (n = 17, 55%) than by exclusively prospective studies (n = 12, 39%), with only two tools using both retrospective and prospective data sources (n = 2, 6%). Additionally, the sizes of the studies used in tool development varied greatly, with a median study size of 2216 patients, an average study size of 241,644 patients, and a range of 257–4,726,046 patients (Figure 2). While nearly all tools were used clinical inputs (n = 30, 97%) and demographic inputs (n = 29, 94%), a minority used patient-reported inputs (n = 12, 39%). Age was the most commonly used demographic input (n = 25, 81%), with Sex/Gender as the second-most common (n = 16, 52%). Statistical methods used for the creation of tools varied, with most involving logistic regression or multivariate analysis (Table 4). Categorization of the tools by output yielded the following breakdown: fracture prediction (n = 7, 23%), mortality prediction (n = 9, 29%), clinical event prediction (n = 18, 58%), and processes prediction (n = 3, 10%) (Figure 3). Finally, we found that most tools could be further categorized into orthopedic categories, such as fractures (n = 8, 26%), spine (n = 8, 26%), total joint arthroplasty (n = 5, 16%), oncology (n = 3, 10%), general (n = 5, 16%), and miscellaneous (n = 3, 10%) (Figure 4). We found that the frequency of orthopedic web-based tool publications has increased over time, with 7% of articles being published before the year 2000, 17% between 2000 and 2010, and the remaining 76% between 2010 and 2020 (Figure 1). 3. Results Tool development was more often informed by exclusively retrospective studies (n = 17, 55%) than by exclusively prospective studies (n = 12, 39%), with only two tools using both retrospective and prospective data sources (n = 2, 6%). Additionally, the sizes of the studies used in tool development varied greatly, with a median study size of 2216 patients, an average study size of 241,644 patients, and a range of 257–4,726,046 patients (Figure 2). While nearly all tools were used clinical inputs (n = 30, 97%) and demographic inputs (n = 29, 94%), a minority used patient-reported inputs (n = 12, 39%). Age was the most commonly used demographic input (n = 25, 81%), with Sex/Gender as the second-most common (n = 16, 52%). Statistical methods used for the creation of tools varied, with most involving logistic regression or multivariate analysis (Table 4). Categorization of the tools by output yielded the following breakdown: fracture prediction (n = 7, 23%), mortality prediction (n = 9, 29%), clinical event prediction (n = 18, 58%), and processes prediction (n = 3, 10%) (Figure 3). Finally, we found that most tools could be further categorized into orthopedic categories, such as fractures (n = 8, 26%), spine (n = 8, 26%), total joint arthroplasty (n = 5, 16%), oncology (n = 3, 10%), general (n = 5, 16%), and miscellaneous (n = 3, 10%) (Figure 4). We found that the frequency of orthopedic web-based tool publications has increased over time, with 7% of articles being published before the year 2000, 17% between 2000 and 2010, and the remaining 76% between 2010 and 2020 (Figure 1). Tool development was more often informed by exclusively retrospective studies (n = 17, 55%) than by exclusively prospective studies (n = 12, 39%), with only two tools using both retrospective and prospective data sources (n = 2, 6%). Additionally, the sizes of the studies used in tool development varied greatly, with a median study size of 2216 patients, an average study size of 241,644 patients, and a range of 257–4,726,046 patients (Figure 2). While nearly all tools were used clinical inputs (n = 30, 97%) and demographic inputs (n = 29, 94%), a minority used patient-reported inputs (n = 12, 39%). Age was the most commonly used demographic input (n = 25, 81%), with Sex/Gender as the second-most common (n = 16, 52%). 3. Results Statistical methods used for the creation of tools varied, with most involving logistic regression or multivariate analysis (Table 4). Categorization of the tools by output yielded the following breakdown: fracture prediction (n = 7, 23%), mortality prediction (n = 9, 29%), clinical event prediction (n = 18, 58%), and processes prediction (n = 3, 10%) (Figure 3). Finally, we found that most tools could be further categorized into orthopedic categories, such as fractures (n = 8, 26%), spine (n = 8, 26%), total joint arthroplasty (n = 5, 16%), oncology (n = 3, 10%), general (n = 5, 16%), and miscellaneous (n = 3, 10%) (Figure 4). Figure 1. Article publication over time for web-based orthopedic predictive tools. 7% 5% 12% 19% 57% 0 10 20 30 40 Number of published articles Year of article publication Figure 1. Article publication over time for web-based orthopedic predictive tools. Figure 1. Article publication over time for web-based orthopedic predictive tools. 7% 5% 12% 19% 57% 0 10 20 30 40 Number of published articles Year of article publication Year of article publication Year of article publication Figure 1. Article publication over time for web-based orthopedic predictive tools. Figure 1. Article publication over time for web-based orthopedic predictive tools. Figure 1. Article publication over time for web-based orthopedic predictive tools. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. Figure 2. Study size used in tool creation. 11 of 20 J. Pers. Med. 2020, 10, 223 Figure 3. Tool breakdown by output. Figure 4. Tool breakdown by orthopedic category. 23% (N=7) 29% (N=9) 58% (N=18) 10% (N=3) Fracture Prediction Mortality Prediction Clinical Events Prediction Processes Prediction 8 8 5 3 5 3 0 2 4 6 8 Number of tools Tool topic Figure 3. Tool breakdown by output. Figure 3. Tool breakdown by output. Figure 4. Tool breakdown by orthopedic category. 23% (N=7) 29% (N=9) 58% (N=18) 10% (N=3) Fracture Prediction Mortality Prediction Clinical Events Prediction Processes Prediction 8 8 5 3 5 3 0 2 4 6 8 Number of tools Tool topic Figure 4. 3. Results Tool breakdown by orthopedic category. 23% (N=7) 29% (N=9) 58% (N=18) 10% (N=3) Fracture Prediction Mortality Prediction Clinical Events Prediction Processes Prediction 23% (N=7) 29% (N=9) 58% (N=18) 10% (N=3) Fracture Prediction Mortality Prediction Clinical Events Prediction Processes Prediction Figure 3. Tool breakdown by output. Figure 3. Tool breakdown by output. Figure 3. Tool breakdown by output. Figure 3. Tool breakdown by output. Figure 4. Tool breakdown by orthopedic category. 8 8 5 3 5 3 0 2 4 6 8 Number of tools Tool topic Figure 3. Tool breakdown by output. Figure 3. Tool breakdown by output. Figure 4. Tool breakdown by orthopedic category. 8 8 5 3 5 3 0 2 4 6 8 Number of tools Tool topic Figure 4. Tool breakdown by orthopedic category. Figure 4. Tool breakdown by orthopedic category. Figure 4. Tool breakdown by orthopedic category. Figure 4. Tool breakdown by orthopedic category. 4. Discussion 4. Discussion 4. Discussion In this scoping review, we systematically identified 31 web-based tools designed to provide personalized prediction in various of orthopedic settings. Overall, these tools provide orthopedic surgeons with information supporting the outcome prediction of fractures, mortality, other clinical events, such as surgical complications, and miscellaneous clinical processes, such as length of stay. This information applies to settings in different orthopedic subspecialties, in various points of the clinical pathway including pre- and post-operative surgical planning, and for shared decision- making with patients in discussions of elective surgeries. In this scoping review, we systematically identified 31 web-based tools designed to provide personalized prediction in various of orthopedic settings. Overall, these tools provide orthopedic surgeons with information supporting the outcome prediction of fractures, mortality, other clinical events, such as surgical complications, and miscellaneous clinical processes, such as length of stay. This information applies to settings in different orthopedic subspecialties, in various points of the clinical pathway including pre- and post-operative surgical planning, and for shared decision- making with patients in discussions of elective surgeries. In this scoping review, we systematically identiﬁed 31 web-based tools designed to provide personalized prediction in various of orthopedic settings. Overall, these tools provide orthopedic surgeons with information supporting the outcome prediction of fractures, mortality, other clinical events, such as surgical complications, and miscellaneous clinical processes, such as length of stay. This information applies to settings in diﬀerent orthopedic subspecialties, in various points of the clinical pathway including pre- and post-operative surgical planning, and for shared decision-making with patients in discussions of elective surgeries. 4.1. Limitations 4.1. Limitations 4.1. Limitations This work should be considered in light of some limitations. Firstly, many identified papers had tools that were inaccessible or had broken URLs. While this may indicate that a given tool was discontinued for technical reasons, it is challenging to ascertain whether there were modifications made for a newer version or alternative use. As tools continue to develop, iterative processes with well-documented updates and modifications will need to be utilized to better implement these tools in clinical practice. Secondly, papers presented varying levels of detail regarding the tool This work should be considered in light of some limitations. Firstly, many identified papers had tools that were inaccessible or had broken URLs. While this may indicate that a given tool was discontinued for technical reasons, it is challenging to ascertain whether there were modifications made for a newer version or alternative use. As tools continue to develop, iterative processes with well-documented updates and modifications will need to be utilized to better implement these tools in clinical practice. Secondly, papers presented varying levels of detail regarding the tool This work should be considered in light of some limitations. Firstly, many identiﬁed papers had tools that were inaccessible or had broken URLs. While this may indicate that a given tool was discontinued for technical reasons, it is challenging to ascertain whether there were modiﬁcations made for a newer version or alternative use. As tools continue to develop, iterative processes with well-documented updates and modiﬁcations will need to be utilized to better implement these tools in clinical practice. Secondly, papers presented varying levels of detail regarding the tool development J. Pers. Med. 2020, 10, 223 12 of 20 process which limited standardization of speciﬁc parameters. Future work should aim to increase transparency in the tool development and validation processes so that tool comparisons are uniform and systematic. Thirdly, only one database was used to search for these tools. While this raises the possibility of missed tools, the two searches conducted for this study were broad and anticipated the capture of the vast majority of these web-based orthopedic solutions. The selected electronic database (PubMed) is also the most relevant to clinical orthopedic practice. Finally, there may be more tools in development that are currently being validated at various institutions and remain unpublished at the time of this review. 4.1. Limitations 4.1. Limitations 4.1. Limitations While this is largely unavoidable in a fast-paced ﬁeld, further work may target the grey literature and other types of electronic databases or search engines to capture such tools. It is diﬃcult to quantify the current use of web-based orthopedic predictive tools in practice. This scoping review demonstrates a rapid increase in the frequency of tool-related publications over the last two decades, perhaps reﬂecting growing interest in web-based orthopedic predictive tools. Although specialized commercial software packages may overshadow the use of web-based tools in areas such as pre-operative planning, web-based tools oﬀer additional and unique functions, such as patient and surgical outcome predictions. In this way, web-based tools serve as potential complements to already established software packages. Identiﬁed tools consistently rely on more established statistical methods for their development, such as multivariate analysis and logistic regression. There were only two tools that demonstrate the use of machine-learning algorithms in their development, both of which were released within the past 5 years. Artiﬁcial intelligence and machine learning have the ability to process large amounts of data in diﬀerent forms, including actively and passively generated data from patients. It is likely that, as this technology evolves, future web-based medical tools will make increasing use of advanced predictive analytics and provide greater opportunities for more personalized patient care. 4.2. Future Work The proﬁle of tools identiﬁed in this study indicate two major areas for potential improvement. First, fewer than half of identiﬁed tools utilize patient-reported inputs. This may be related to the reliability of obtaining information from patients, as these data require active procurement in prospective cohorts or may not be consistently documented for retrospective cohorts. Despite these barriers, patient reported measures are important components of patient outcomes and ensure physician focus on subjective metrics that patients care about, such as perceived pain. As patient-reported outcome measures are increasingly used in both the ﬁeld of orthopedics and across medical specialties, greater incorporation of patient-reported data into these tools presents an area of potential improvement. Second, tools identiﬁed in this study fall into just a few orthopedic categories, such as fractures, total joint arthroplasty, spine, and oncology. These categories may reﬂect a lack of tools for other orthopedic subspecialties, such as orthopedic sports injuries. This lack may be related to the heterogeneity of injuries in such ﬁelds or insuﬃcient access to large existing patient databases for tool development. Continued expansion in the development of tools across orthopedic subspecialties would aﬀord a wider breadth of resources to orthopedic surgeons to better clinical outcomes for patients and should be a focus of improvement. 5. Conclusions The increasing number of web-based orthopedic tools is an opportunity for orthopedic surgeons to better predict outcomes and increase understanding of expectations with patients. The aim of this scoping review was to identify the current list of web-based orthopedic tools, as well as clearly outline their utility and validation. We provide orthopedic surgeons a repository of current and publicly available web-based tools which includes all the necessary information to determine what tools may apply to their practice. Areas for continued development of web-based tools are vast, with opportunities in both tool design and application. J. Pers. Med. 2020, 10, 223 13 of 20 13 of 20 Author Contributions: Conceptualization, E.S.; Methodology, E.S., P.C., L.M., and A.C.; Formal Analysis, E.S., P.C., A.C., L.M., E.L., and P.J.; Data Curation, L.M., P.C., and A.C.; Writing—Original Draft Preparation, P.C.; Writing—Review and Editing, E.S., P.C., A.C., L.M., E.L., and P.J.; Supervision, E.S. and P.J. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A Table A1. High Impact Orthopedic Journals Used in Secondary Search. The Journal of Bone and Joint Surgery, American Volume The Bone and Joint Journal Clinical Orthopaedics and Related Research The Journal of the American Academy of Orthopaedic Surgeons Journal of Orthopaedic Research: Oﬃcial Publication of the Orthopaedic Research Society Journal of Orthopaedics and Traumatology: Oﬃcial Journal of the Italian Society of Orthopaedics and Traumatology Injury The Journal of Arthroplasty The American Journal of Sports Medicine Arthroscopy: The Journal of Arthroscopy & Related Surgery: Oﬃcial Publication of the Arthroscopy Association of North America and the International Arthroscopy Association The Journal of Hand Surgery Journal of Shoulder and Elbow Surgery Foot & Ankle Orthopaedics Spine European Spine Journal: Oﬃcial Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society The Spine Journal: Oﬃcial Journal of the North American Spine Society Journal of Neurosurgery. Spine Joint Bone Spine Journal of Pediatric Orthopedics Table A1. High Impact Orthopedic Journals Used in Secondary Search. The Journal of Bone and Joint Surgery, American Volume The Bone and Joint Journal Clinical Orthopaedics and Related Research The Journal of the American Academy of Orthopaedic Surgeons Journal of Orthopaedic Research: Oﬃcial Publication of the Orthopaedic Research Society Journal of Orthopaedics and Traumatology: Oﬃcial Journal of the Italian Society of Orthopaedics and Traumatology Injury The Journal of Arthroplasty The American Journal of Sports Medicine Arthroscopy: The Journal of Arthroscopy & Related Surgery: Oﬃcial Publication of the Arthroscopy Association of North America and the International Arthroscopy Association The Journal of Hand Surgery Journal of Shoulder and Elbow Surgery Foot & Ankle Orthopaedics Spine European Spine Journal: Oﬃcial Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society The Spine Journal: Oﬃcial Journal of the North American Spine Society Journal of Neurosurgery. Spine Joint Bone Spine Journal of Pediatric Orthopedics Table A1. High Impact Orthopedic Journals Used in Secondary Search. 14 of 20 J. Pers. Med. 2020, 10, 223 J. Pers. Med. 2020, 10, 223 Figure A1. Search Strategy and Identification of Sources. Figure A1. Search Strategy and Identiﬁcation of Sources. igure A1. Search Strategy and Identification of Sources. Figure A1. Search Strategy and Identiﬁcation of Sources. 15 of 20 J. Pers. Med. 2020, 10, 223 References 1. Christensen, D.L.; Dickens, J.F.; Freedman, B.; Mauntel, T.; Owens, B.D.; Potter, B.K.; Provencher, M.; Tokish, J.M.; Waterman, B.R.; Antosh, I.; et al. Patient-Reported Outcomes in Orthopaedics. J. Bone Jt. Surg. 2018, 100, 436–442. [CrossRef] 1. Christensen, D.L.; Dickens, J.F.; Freedman, B.; Mauntel, T.; Owens, B.D.; Potter, B.K.; Provencher, M.; Tokish, J.M.; Waterman, B.R.; Antosh, I.; et al. Patient-Reported Outcomes in Orthopaedics. J. Bone Jt. Surg. 2018, 100, 436–442. [CrossRef] 2. Gagnier, J.J. Patient reported outcomes in orthopaedics. J. Orthop. Res. Oﬀ. Publ. Orthop. Res. Soc. 2017, 35, 2098–2108. [CrossRef] 3. Sepucha, K.R.; Atlas, S.J.; Chang, Y.; Freiberg, A.; Malchau, H.; Mangla, M.; Rubash, H.; Simmons, L.H.; Cha, T. Informed, Patient-Centered Decisions Associated with Better Health Outcomes in Orthopedics: Prospective Cohort Study. Med. Decis. Mak. Int. J. Soc. Med. Decis. Mak. 2018, 38, 1018–1026. [CrossRef] 4. Bernstein, D.N.; Fear, K.; Mesﬁn, A.; Hammert, W.C.; Mitten, D.J.; Rubery, P.T.; Baumhauer, J.F. Patient-reported outcomes use during orthopaedic surgery clinic visits improves the patient experience. Musculoskelet. Care 2019, 17, 120–125. [CrossRef] 5. Waheeb, A.; Zywiel, M.G.; Palaganas, M.; Venkataramanan, V.; Davis, A.M. The inﬂuence of patient factors on patient-reported outcomes of orthopedic surgery involving implantable devices: A systematic review. Semin. Arthritis Rheum. 2015, 44, 461–471. [CrossRef] 6. 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Appendix C Tool URL Additional Notes LACE+ https://www.besler.com/lace-risk-score/ (accessed on 26 October 2020) Neuro Risk Opioid Use Calculator http://neuro-risk.com/opiod-use/ (accessed on 26 October 2020) NHFS (Nottingham Hip Fracture Score) http://www.riskprediction.org.uk/index-nhfs.php (accessed on 26 October 2020) OaraScore (Outpatient Arthroplasty Risk Assessment Score) https://www.djoglobal.com/our-brands/djo-surgical/oara-score (accessed on 26 October 2020) Paid subscription required; option to print results Opioid Calculator for Hand Surgery https://jscalc.io/calc/9hH05AdFRt4iV6YD (accessed on 26 October 2020) PathFX https://www.pathfx.org/ (accessed on 26 October 2020) Free registration required P-POSSUM http://www.riskprediction.org.uk/index-op.php (accessed on 26 October 2020) QFracture https://qfracture.org/ (accessed on 26 October 2020) QUALITOUCH Outcome Calculator https://outcomecalculator.org/en/ (accessed on 26 October 2020) RAPT (Risk Assessment and Prediction Tool) https://www.lifespan.org/centers-services/total-joint-center-lifespan-orthopedics-institute/take- rapt-assessment (accessed on 26 October 2020) ShockNurd http://shocknurd.org/ (accessed on 26 October 2020) Link currently inactive Spinal RAT (Risk Assessment Tool) https://apps.apple.com/us/app/risk-assessment-tool-for-spine-surgery-procedures/id1087663216 (accessed on 26 October 2020) iOS application SpineSage https://depts.washington.edu/spinersk/ (accessed on 26 October 2020) STaRT Back Tool https://startback.hfac.keele.ac.uk/training/resources/startback-online/ (accessed on 26 October 2020) STTGMA (Score for Trauma Triage in Geriatric and Middle Aged Patients) https://sttgma.wordpress.com/ (accessed on 26 October 2020) Option to download Excel formula sheets URL 17 of 20 17 of 20 J. Pers. Med. 2020, 10, 223 Appendix C Table A2. Tool URLs. Tool URL Additional Notes 90-Day Readmissions Risk Calculator https://dukeriskcalculators.shinyapps.io/Readmissions/ (accessed on 26 October 2020) ACS NSQIP Surgical Risk Calculator https://riskcalculator.facs.org/RiskCalculator/ (accessed on 26 October 2020) Option to email or download report as PDF Arthroplasty Size Predictor https://apps.apple.com/us/app/arthroplasty-size-predictor/id1234761373 (accessed on 26 October 2020) iOS application Back Treatment Outcomes Calculator http://spinesurgerycalc.dartmouth.edu/calc/ (accessed on 26 October 2020) CAROC (Canadian Risk for Osteoporosis Calculator) https://osteoporosis.ca/health-care-professionals/tools/caroc/ (accessed on 26 October 2020) CCI (Charlson Comorbidity Index) https://www.mdcalc.com/charlson-comorbidity-index-cci (accessed on 26 October 2020) Free registration required; option to copy results to clipboard as text Chondrosarcoma Five-Year Survival Machine Learning Algorithm https://sorg-apps.shinyapps.io/chondrosarcoma/ (accessed on 26 October 2020) Discharge to Rehabilitation and LOS Calculator https://jhuspine1.shinyapps.io/RehabLOS/ (accessed on 26 October 2020) Edinburgh Wrist Calculator https://www.trauma.co.uk/wristcalc (accessed on 26 October 2020) E-PASS (Estimation of Physiologic Ability and Surgical Stress) Surgery Risk Calculator https://www.medicalalgorithms.com/surgery-risk-calculator-estimation-of-physiologic-ability- surgical-stress (accessed on 26 October 2020) Estimated Blood Loss Calculator https://jhuspine2.shinyapps.io/Estimated_Blood_Loss/ (accessed on 26 October 2020) Extremity Metastatic Disease Survival Prediction Machine Learning Algorithm https://sorg-apps.shinyapps.io/extremitymetssurvival/ (accessed on 26 October 2020) FRAX https://www.sheﬃeld.ac.uk/FRAX/ (accessed on 26 October 2020) Desktop application; payment required FRS (Fracture Risk Scale) https://www.fco.ngo/blog/fracture-risk-scale-frs-tool-assessing-fracture-risk-long-term-care-ltc (accessed on 26 October 2020) Garvan https://www.garvan.org.au/bone-fracture-risk (accessed on 26 October 2020) Option to print results Incidental Durotomy Calculator https://jhuspine3.shinyapps.io/Incidental_Durotomy_Calculator/ (accessed on 26 October 2020) J. 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https://openalex.org/W4392110499	https://sanscientific.com/journal/index.php/rbm/article/download/185/345	English	null	The Effect of In-store Logistics, Store Image, Sales Promotion, and Service Quality on Customer Satisfaction	Research of Business and Management	2,024	cc-by-sa	7,292	p-ISSN/e-ISSN: 2986-3732/2986-2825 Homepage: https://sanscientific.com/journal/index.php/rbm 2(1) 15-28 (2024) https://doi.org/10.58777/rbm.v2i1.185 https://doi.org/10.58777/rbm.v2i1.185 Received: 05-02-2024; Accepted: 23-02-2024 Received: 05-02-2024; Accepted: 23-02-2024 Research Article Yulia Kartika Sari1*, Ariel Nian Gani2 1,2 Faculty of Economics and Business, YARSI University, Jakarta This is an open-access article under the CC-BY-SA international license. This is an open-access article under the CC-BY-SA international license. Abstract This research aims to determine the effect of In-Store Logistics Performance, Store Image, Sales Promotion, and Service Quality on Satisfaction (Case Study of Matahari Department Store Consumers at Artha Gading Mall). The population in this study were consumers of the Matahari Department Store at Artha Gading Mall. The sampling technique used was purposive sampling. The number of respondents in this study was 100 respondents. Data was collected by distributing questionnaires. The data analysis methods used are multiple regression analysis. The results of this research show that: (1) partially In-Store Logistics Performance has a positive effect on consumer satisfaction. (2) Store Image partially has a positive effect on consumer satisfaction. (3) Sales Promotion has a positive effect on consumer satisfaction. (4) Service Quality has a partially positive effect on consumer satisfaction. (5) simultaneously, all independent variables have a positive influence on consumer satisfaction. The managerial implications of these findings are that improving in-store logistics performance, establishing a positive store image, implementing effective sales promotions, and improving service quality can increase customer satisfaction at Matahari Department Store. Keywords: In-Store Logistics Performance, Store Image, Sales Promotion and Service Quality JEL Classification: L22, M31, L15 How to cite: Sari, Y. K., Gani, A. N., (2024). The Effect of In-store Logistics, Store Image, Sales Promotion, and Service Quality on Customer Satisfaction, Research of Business and Management (RBM) 2(1), 15-28 JEL Classification: L22, M31, L15 How to cite: Sari, Y. K., Gani, A. N., (2024). The Effect of In-store Logistics, Store Image, Sales Promotion, and Service Quality on Customer Satisfaction, Research of Business and Management (RBM) 2(1), 15-28 Corresponding author: Yulia Kartika Sari (yuliakartika223@gmail.com) 1. Introduction Consumer satisfaction is something that is often observed in the world. Efforts to increase consumer satisfaction are one of the strategy formulations for forming customer loyalty through an earlier level of behavior, namely their good impression of the brand and company, as well as influencing consumer purchasing intentions (Munawaroh & Simon, 2023). To fulfill consumer satisfaction, companies are required to be astute in knowing shifts in customer needs and desires, which change almost all the time (Suwito, 2018). Currently, one of the main problems that the retail industry faces is the low level of consumer satisfaction (Gaol et al., 2016). This happens because consumers are used to shopping online or online retail (Suleman et al., 2020). 15 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 One company in Indonesia that is currently experiencing a decline in revenue is Matahari Department Store. Matahari Department Store Tbk is one of the largest retail companies in Indonesia, which was founded on 24 October, 1958, and continues to grow to this day and has 150 outlets in 80 cities throughout Indonesia. Initially, Matahari Department Store only sold textiles and clothing, but later developed into a department store that provides various lifestyle, beauty, fashion, and household equipment products. In 1998, Matahari Department Store joined the Lippo Group and began expanding its business throughout Indonesia. In 1998, Matahari Department Store conducted an initial public offering (IPO) and officially went public on the Jakarta Stock Exchange with the stock code "LPPF ."Matahari Department Store uses the term "word," which divides areas to organize its fashion products according to type and brand. The arrangements, such as men's, women's, and children's clothing, are grouped according to the brand; relatively large groups, such as cosmetics, shoes, and bags, are neatly grouped according to type. Source: Prepared by the author, May (2023) Figure 1. PT Matahari Department Store Tbk Revenue Data, 2018-2022 Rp0 Rp2.000.000.000 Rp4.000.000.000 Rp6.000.000.000 Rp8.000.000.000 Rp10.000.000.000 Rp12.000.000.000 2018 2019 2020 2021 2022 Source: Prepared by the author, May (2023) Figure 1. PT Matahari Department Store Tbk Revenue Data, 2018-2022 From the data above, there was a decline in income in 2018-2022. In 2018, the income was IDR 10,000,245,173; in 2019, it was IDR 10,000,276,431; in 2020, it was IDR 4,000,839,058; in 2021, it was IDR 5,000,585,975; and in 2022 it was IDR 4,000,964,474. 1. Introduction In 2018-2019, revenues at Matahari Department Store were still quite stable because Matahari was still the largest shopping center in Indonesia. Meanwhile, at the beginning of 2020, Matahari experienced a significant decline due to the impact of the outbreak (Covid-19) which occurred in various countries, including Indonesia which caused people to be restricted by the government from reducing various activities outside their homes had an impact on the sales income of various department stores including Matahari Department Store And took place the following year. Richard L. Oliver is a marketing professor from the United States who introduced Zhang's Expectancy Disconfirmation Model (EDM) (2021). EDM draws attention to the determinants of satisfaction that are internal to the individual, and the main idea of this model, which was originally developed in consumer behavior research, is that satisfaction or dissatisfaction is a function of and reference (standards used to compare) and perceived performance (Oliver, 2020). Usually, consumers who use retail services want to navigate store services as comfortably and conveniently as possible which can influence consumer satisfaction to support the store or not (Bitner, 2016). Although there is much research in the retail sector, only a few researchers discuss in-store logistics performance and store image in the retail industry (Sari et al., 2023) (Ltifi and Gharbi, 2015). 16 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 In-store logistics performance refers to convenience, including entering and exiting the store quickly and finding merchandise easily to identify and access products (Bouzaabia et al., 2016). Some stores focus on providing convenient infrastructure (for example, by using signage, dedicated recreation areas, or specially adapted shopping carts) or various services that facilitate the shopping process, such as information services, sales advice, and self-service technology (Sari et al., 2023) (Baker et al., 2022). According to Peter & Olson in Yudatama et al. (2021), Store Image is what consumers think about a store, including perceptions and attitudes that are based on sensations from stimuli related to the store received through the five senses. Store image is the personality of a store that describes what consumers see and feel about a particular store (Purwati, 2019). According to Kotler & Keller in Sarie (2018), to increase consumer satisfaction, sales promotions must be able to attract stronger and faster responses from buyers. 1. Introduction Sales promotions can use short-term effects such as highlighting product offers to get attention and direct people to the product. Sales promotion includes a wide variety of promotional tools designed to engineer a faster or stronger market response (Natalia and Mulyana, 2015). Consumer satisfaction also depends on the quality of services offered by the company (Maria & Anshori, 2020). Good service quality in a company will provide a sense of satisfaction to consumers. If there are demands from consumers, consumers hope that the company can provide services in the form of answers that are given in a friendly, fast, and precise manner (Mulyono, 2021). In the business world, shops have become important centers of economic activity. To run their operations successfully, stores need to manage logistics well, create a positive store image, implement effective sales promotions, and provide quality service to consumers (Maghfur et al., 2023). All these aspects influence consumer satisfaction. From an Islamic perspective, it is important to ensure that logistics, store image, sales promotions, and service quality are carried out with high ethics and morality and in accordance with Islamic teachings, which encourage honesty, transparency, fairness, and good service to others, in Islamic Business Principles and Ethics. In-store logistics performance must be carried out with Islamic ethics and morality, including in the procurement of goods, storage, and distribution in the store. The principle of honesty in business must be applied in all logistics processes. If logistics are carried out well and correctly, it will improve the store's image in this retail business. From an Islamic perspective, the image of this shop is very important. A good shop that has a positive image in the community will attract consumers to come back and shop there. Islam emphasizes the importance of maintaining a good name and good reputation. Presenting the shop as a safe, quality, and reliable place will attract and retain customers (Hidayat and Rifa'i, 2018) in Management Ethics from an Islamic Perspective. In-store logistics performance g p In-store logistics performance plays a very important role in influencing consumer satisfaction. In contrast, in the case of retail stores, convenience includes entering and leaving the store quickly and finding merchandise easily. According to the explanation (Bouzaabia et al., 2017), in-store logistics performance is how the company performs in creating an effective and efficient network in the store so that the store is smooth and under-stocked. In-store logistics performanceNowadays it is very important for retail stores to optimize sales space, not only displaying product samples on the front but also the inventory of goods stored in the store warehouse, and this will make the store more attractive to consumers followed by good in-store logistics performance and appropriately because if it is not done correctly, there will be excess or shortage of goods which will have an impact on consumer satisfaction (Sari et al., 2023) (Bienstock et al, 2020). In-store logistics performance is influenced by demands on consumer desires, where retail stores must be sensitive to consumer desires for retail stores, and the existence of this system will control retail store inventory. Store Image Store image is an impression that is interpreted because of the completeness felt by consumers in connection with the store and is interdependent on consumer impressions based on both current and previous exposure (Maharta et al., 2018). Store image is an overall picture that is more than just the sum of the parts, where each part interacts with each other in the consumer's mind. According to Agustin et al. (2019), store image is a combination of various attitudes that consumers have about a store or company, which are connected to what consumers expect when consumers choose a store and consumer expectations about the store, which are attitudes consumers towards the store itself (Noviana & Oktavia, 2023). Service Quality According to Cravens (2023), service quality is efforts to fulfill requests to specify products such as performance data, requests for details, processing purchase orders, order status inquiries, and warranty services. When a service is described as something that a corporation offers, it usually means that a party has performed an act for another party. Service quality is a main or complementary activity that is not directly involved in the product manufacturing process but places more emphasis on transaction services between buyers and sellers. Service quality is the expected level of excellence and control over this level of excellence will fulfill customer desires. Sales Promotion Sales promotion is a form of direct persuasion using various incentives that can be arranged to stimulate immediate product purchases and/or increase the number of goods purchased by customers. Promotional marketing means efforts to increase sales of products offered by providing information to the public about the existence of a product that has certain uses to meet consumer needs (Sentono, 2019). Meanwhile, sales promotion is a company's activity to sell the products it markets in such a way that consumers will find it easy to see them, and even with certain placement and arrangement, the product will attract consumers' attention. From the definition above, sales promotion is a company's effort to increase product sales using incentives and information to consumers to encourage quick purchases or increase the number of goods purchased. This promotion also involves placing and arranging products in such a way as to attract consumer attention (Assidiki & Budiman, 2023). 2. Literature Review and Hypothesis Customer Satisfaction Consumer happiness is the general disposition that customers display toward products or services following their acquisition and utilization. Kotler (2018) defines contentment as the emotion that occurs from contrasting the performance (results) of the product under consideration with the anticipated performance (results). After obtaining, evaluating, and utilizing a product in the form of commodities or services, a person's feelings of satisfaction-whether positive or negative-can be deduced from several meanings of the term. Their happy demeanor suggests that they are content with the food they are eating. Conversely, if consumers show feelings of disappointment with the product they have purchased, it means they are dissatisfied. Peter & Olson in Setiyaningrum (2021) stated that if consumers are satisfied with a product or brand, consumers will continue to buy it, use it, and tell other people about their pleasant experience. On the other hand, if consumers are not satisfied, they will tend to switch brands and file a complaint with the company. Retailers and influence others to avoid buying the brand. 17 Sari, Gani RBM 2(1) 2024 15-28 The Influence of Store Image on Consumer Satisfaction When consumers feel satisfied that the shop has a high store image, they also assume that the goods sold in the store are of high quality, so the more positive the store image of a store, the higher the consumer's intention to buy (Prakoso, 2020). Powered by a positive store image, a retailer will be able to create satisfaction after consumers experience shopping at the store. According to Arief and Widyatmoko (2018), shop image is an understanding of the shop in the minds of customers according to the characteristics/attributes of the shop so that customers can differentiate between one shop and other shops. According to research conducted by Sinatrya and Efendi (2020), the results were that store image had a positive effect on consumer satisfaction at the RJ Junior electronics store in Trenggalek. The population count was carried out by representing the number of transactions in one month, namely February, amounting to 7000 transactions. Furthermore, the results of research conducted by Joseph et al. (2018) found that store image is important for the satisfaction of consumers who visit a retail store for the first time. The results of this research show that the store image has a positive influence on supermarket consumer satisfaction. The population in this study were Supermarket consumers in Uyo Metropolitan City, with a sample of 244 consumers. Based on description theory and is also supported by previous research, the following hypothesis can be formulated: H2: Store Image has a positive effect on Consumer Satisfaction Hypothesis The Influence of In Store Logistics Performance on Consumer Satisfaction In-store logistics operations (in-store logistics performance) play a very important role in influencing customer experience and satisfaction. In the case of retail stores, customer comfort and satisfaction include entering and leaving the store quickly and finding merchandise easily. Layout is an example of a design cue that can influence expectations regarding their ability to move efficiently through a store. According to research by Bouzaabia et al. (2020) stated that in-store logistics performance has a positive effect on consumer satisfaction directly. In this study, the 18 Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 population used was supermarket consumers in Belgium. The number of samples used was 200 respondents. The results of this research are that consumers are satisfied with in-store logistics performance in the retail industry. Furthermore, research conducted by Moussaoui et al. (2022) found that in-store logistics performance had a positive effect on consumer satisfaction. The data collection instrument was carried out in the city of Nador, Morocco (consumers of the supermarket "Marjane") using a sample of 201 consumers. Based on the theoretical explanation and supported by previous research, the following hypothesis can be formulated: H1: In-store logistics Performance has a positive effect on Customer Satisfaction The Influence of Sales Promotion on Consumer Satisfaction Any type of temporary offer or incentive meant for consumers, retailers, or wholesalers with the goal of eliciting a response from the public is referred to as a sales promotion. Promotion encourages consumers to try new products so that it can increase sales. It is also useful for promoting greater consumer awareness of prices and providing customer satisfaction with these promotions. If a company's sales promotion is interesting and good and attracts buyers, then when their post-purchase expectations are met, consumers will feel satisfied (Sutrisno and Darmawan, 2022). According to research conducted by Sarie (2018) obtained results namely that the influence of sales promotions on consumer satisfaction had a positive effect, with the population in this study being all consumers who shopped at Hypermarket Pakuwon Supermall Surabaya with a sample size of 50 consumers. The results of other research conducted by Nurjuman et al. (2023) obtained results namely that the influence of sales promotions on consumer satisfaction had a positive effect on the population taken from customers who had purchased Flash Coffee products at Bintaro outlets from September 2022 to 10 November 2022 and the sample taken was 97 respondents. Based on description theory and is also supported by previous research, the following hypothesis can be formulated: H3: Sales promotions have a positive effect on consumer satisfaction The Influence of Service Quality on Consumer Satisfaction The Influence of Service Quality on Consumer Satisfaction Q y Service quality is the expected level of excellence and control over this excellence to fulfill customer desires. Kotler (2021) states that customers will feel satisfied if they receive good service or if they meet expectations. In the long term, a bond like this allows the company to thoroughly understand customer expectations and their needs and the company can increase customer satisfaction where the company maximizes pleasant customer experiences and minimizes unpleasant customer experiences. According to research conducted by Nugraha (2016), the results were that service 19 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 quality had a positive effect on consumer satisfaction. The population in this study was Circle KG- Walk Pakuwon Mall Surabaya. The sample taken in this research amounted to 110 respondents. Other research conducted by Paul et al. (2015) found that service quality has a positive effect on consumer satisfaction. In this study, the population used was private-sector bank customers and public-sector bank customers in India. The number of samples used was 250 private sector bank customers and 250 public sector bank customers. Based on description theory and is also supported by previous research, the following hypothesis can be formulated: y p g yp H4: Service Quality has a positive effect on Consumer Satisfaction Population Population is a generalization area that consists of objects or subjects that have certain quantities and characteristics and are determined by researchers to be studied and then concluded. Population is the subject of research. In this study, the population is all consumers who have made purchases from the Matahari Department Store at Artha Gading Mall, who may come from various regions, so the exact total of this research population is unknown. Data Types and Data Sources yp Internal data typeThis research is primary data. Primary data sources are individual respondents and focus groups; the internet can also be a primary data source if the questionnaire is distributed via the internet (Wahyuddin et al., 2023). Primary data in this research was obtained directly from respondents, namely consumers of the Matahari Department Store at Artha Gading Mall, with the help of a Google form. 3. Data and Method Explanatory study employs quantitative methodology in research. Research aimed at explaining the positions of the variables under study and the connections between them is known as explanatory research. Quantitative research is a research method used to examine a certain population or sample, collecting data using research instruments and quantitative or statistical data analysis with the aim of testing the hypothesis that has been established (Santosa & Hidayat, 2014; Sugiyono, 2014). Y = a + b1+ b2+ b3+ b4+ 0 (2) Sample The sample is part of the number and characteristics possessed by the population (Sugiyono, 2013). The sample used must be representative and reflect the existing population. Data collection in this research used a purposive sampling method. Purposive sampling is a sampling method that limits the special characteristics of a person who provides information and is in accordance with what the researcher wants (Sugiyono, 2013). The Lemeshow Formula was utilized by the author to calculate this sample size. Because the population is either unknown or infinite, the Lemeshow formula is applied. The following is the Lemeshow formula: 𝒏= 𝒁𝟐 𝑷(𝟏−𝑷) 𝒅𝟐 𝒏= 𝒁𝟐 𝑷(𝟏−𝑷) 𝒅𝟐 (1) (1) Information: n = number of samples z = z score at 95% confidence = 1.96 p = maximum error = 10% Using the formula above, the number of samples to be used can be calculated as follows: p Using the formula above, the number of samples to be used can be calculated as follows: p Using the formula above, the number of samples to be used can be calculated as follows: 20 Sari, Gani RBM 2(1) 2024 15-28 𝑛= 𝑍2 𝑃(1 −𝑃) 𝑑2 𝑛= 1,962 . 0,5(1 −0,5) 𝑑2 𝑛= 3,8416.0.25 0,01 𝑛= 96.04 = 100 𝑛= 𝑍2 𝑃(1 −𝑃) 𝑑2 𝑛= 1,962 . 0,5(1 −0,5) 𝑑2 𝑛= 3,8416.0.25 0,01 The sample value (n) produced by applying the Lemeshow formula is 96.04, which is rounded to 100 persons. The sample value (n) produced by applying the Lemeshow formula is 96.04, which is rounded to 100 persons. Test of Multilinear Regression Analysis Test of Multilinear Regression Analysis Test of Multilinear Regression Analysis the following formulation, multiple linear regression analysis is a statistical technique for examining the impact of the independent variable on the dependent variable: Y = a + b1+ b2+ b3+ b4+ 0 (2) Validity Test Results y Validation tests are used to measure whether a questionnaire is valid or not. An instrument or questionnaire is said to be valid if the questions on the instrument or questionnaire can reveal something that the questionnaire will measure. We use a validity test; it is necessary to determine the r table value. The way to do this is to look for the r table value through the r table (Pearson's Product Moment) with the formula: df (Degree of freedom) = N – 2 = 100 -2 = 98 (3) (3) With df = 100 and alpha = 0.05, we get r table = 0.195 (by looking at the r table at df = 98 with a two-tailed test). If the calculated r-value > r-table is positive, then the question item or indicator is declared valid. With df = 100 and alpha = 0.05, we get r table = 0.195 (by looking at the r table at df = 98 with a two-tailed test). If the calculated r-value > r-table is positive, then the question item or indicator is declared valid. Table 1. Validity Test Results Table 1. Validity Test Results Table 1. Validity Test Results 21 Table 1. Validity Test Results Validity Test Results Variable Question Items r Count r Table Information In-Store Logistics Performance INL1 0.684 0.195 Valid INL2 0.735 0.195 Valid INL3 0.794 0.195 Valid INL4 0.729 0.195 Valid INL5 0.741 0.195 Valid INL6 0.751 0.195 Valid INL7 0.869 0.195 Valid INL8 0.792 0.195 Valid INL9 0.856 0.195 Valid INL10 0.858 0.195 Valid INL11 0.879 0.195 Valid INL12 0.840 0.195 Valid INL13 0.768 0.195 Valid SI1 0.699 0.195 Valid SI2 0.707 0.195 Valid SI3 0.765 0.195 Valid SI4 0.669 0.195 Valid SI5 0.645 0.195 Valid 21 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 Store Image SI6 0.715 0.195 Valid SI7 0.732 0.195 Valid SI8 0.676 0.195 Valid SI9 0.751 0.195 Valid SI10 0.806 0.195 Valid SI11 0.813 0.195 Valid Sales promotion PP1 0.731 0.195 Valid PP2 0.699 0.195 Valid PP3 0.760 0.195 Valid Service Quality Consumer Satisfaction KL1 0.677 0.195 Valid KL2 0.724 0.195 Valid KL3 0.825 0.195 Valid KL4 0.744 0.195 Valid KL5 0.742 0.195 Valid KL6 0.735 0.195 Valid KL7 0.863 0.195 Valid KL8 0.783 0.195 Valid KL9 0.856 0.195 Valid KL10 0.854 0.195 Valid KL11 0.872 0.195 Valid KL12 0.836 0.195 Valid KL13 0.757 0.195 Valid KK14 0.835 0.195 Valid KK2 0.731 0.195 Valid KK3 0.699 0.195 Valid KK3 0.760 0.195 Valid Source: Processed data (2023) Based on the table above, all r counts > r table so that it can be concluded that all question items on the variables in-store logistics performance, store image, sales promotion, service quality, and customer satisfaction are declared valid. In this way, each statement from all the variables in this research has harmony to be used as a primary data collection tool that can describe the concept being researched. Normality test Normality test To know whether a variable can be said to be normal or not according to the residuals being studied is the aim of carrying out a normality test. To know whether a variable can be said to be normal or not according to the residuals being studied is the aim of carrying out a normality test. Table 3. Normality Test Results Unstandardized Residuals N 100 Normal Parameters, b Mean 0.0000000 Std. Deviation 0.65641553 Most Extreme Differences Absolute 0.086 Positive 0.086 Negative -0.086 Statistical Tests 0.086 Asymp. Sig. (2-tailed) c 0.065 Source: Processed data (2023) Table 3. Normality Test Results Theed on the table above shows that the significance is 0.065 > 0.05, so it can be concluded that the data tested is normally distributed. Reliability Test Results The reliability test is carried out after obtaining valid items from the validity test so that a reliability test can be carried out using SPSS ver.29 on a computer using the Cronbach alpha formula. Table 2. Reliability Test Results Table 2. Reliability Test Results Table 2. Reliability Test Results Variable Cronbach'Alpha r Table Information In-Store Logistics Performance 0.942 0.60 Reliable Store Image 0.934 0.60 Reliable Sales promotion 0.785 0.60 Reliable Service Quality 0.941 0.60 Reliable Consumer Satisfaction 0.706 0.60 Reliable Source: Processed data (2023) All of the variables have a Cronbach Alpha value more than 0.60, which indicates that the data processed above is credible for all of the variables. A study found that the following variables have significant Cronbach Alpha values: 0.942 for the in-store logistics performance variable, 0.934 for the Store Image variable, 0.785 for the Sales Promotion variable, 0.941 for Service Quality, and 0.706 for the Satisfaction variable among consumers. All of the variables have a Cronbach Alpha value more than 0.60, which indicates that the data processed above is credible for all of the variables. A study found that the following variables have significant Cronbach Alpha values: 0.942 for the in-store logistics performance variable, 0.934 for the Store Image variable, 0.785 for the Sales Promotion variable, 0.941 for Service Quality, and 0.706 for the Satisfaction variable among consumers. 22 Sari, Gani RBM 2(1) 2024 15-28 Sari, Gani RBM 2(1) 2024 15-28 Table 4. Multicollinearity Test Results Table 4. Multicollinearity Test Results Coefficients Model Collinearity Statistics Tolerance VIF 1 In-Store Logistics Performance 0.301 3,321 Store Image 0.499 2,004 Sales promotion 0.232 4,309 Service Quality 0.138 7,265 Source: Processed data (2023) Based on the table above, the tolerance value obtained for the In-Store Logistics Performance variable is 0.301, the Store Image variable is 0.499, and the Sales Promotion variable is 0.232. Multicollinearity Test Results y It can be inferred that multicollinearity does not exist in the study if the tolerance value is > 0.10 and the VIF is less than 10; on the other hand, multicollinearity does occur if the tolerance value is less than 0.10 and the VIF is greater than 10. Multiple Linear Regression Test Results Multiple Linear Regression Test Results Multiple Linear Regression Test Results A multiple linear regression test was carried out to determine the influence of In-Store Logistics Performance, Store Image, Sales Promotion, and Service Quality on Consumer Satisfaction. A multiple linear regression test was carried out to determine the influence of In-Store Logistics Performance, Store Image, Sales Promotion, and Service Quality on Consumer Satisfaction. Table 8. Multiple Linear Regression Test Results Model Unstandardized Coefficients Standardized Coefficients Beta t Sig. B Std. Error 1 (Constant) 3,549 1,075 3,302 0.001 In-Store Logistics Performance 0.132 0.007 0.011 2,192 0.048 Store Image 0.809 0.050 0.867 5,235 0.002 Sales promotion 0.103 0.054 0.026 2,691 0.031 Service Quality 0.081 0.066 0.066 2,029 0.022 Source: Processed data (2023) Table 8. Multiple Linear Regression Test Results 5. Discussion 5. Discussion In-Store Logistics Performance on Consumer Satisfaction g In summary, the research findings highlight that among the five indicators in the In-Store Logistics Performance variable, shopping aids and convenience at Matahari Department Store in Artha Gading Mall received the highest. This indicates that the store has effectively aided and facilities to enhance the consumer shopping experience. The findings support the fourth hypothesis about how logistics effectiveness affects customer happiness. Every aspect of logistics performance has an impact on customer satisfaction. The factor that most contributes to explaining satisfaction, however, is "product availability"; when customers can obtain the quantities they desire, their level of contentment increases. Though not as much as the prior element, checkout-level logistics performance plays a role in explaining satisfaction. Shelf-level logistics performance has the least impact on customer satisfaction. This result is consistent with the findings of studies conducted. Specifically, clear store maps, signage, directions, and comfortable changing rooms contribute to this positive perception. However, the returns indicator scored the lowest, suggesting potential areas for improvement in the product return process. Possible reasons for this lower score include vague return policies, complicated return procedures, and delays in the return process. Heteroscedasticity Test Results y If the variance of the residuals from one observation to another is constant, it is called homoscedasticity, and if the variance is different, it is called heteroscedasticity. Table 5. Heteroscedasticity Test Results Table 5. Heteroscedasticity Test Results Table 5. Heteroscedasticity Test Results Table 5. Heteroscedasticity Test Results Model Unstandardized Coefficients Standardized Coefficients Beta t Sig. B Std. Error 1 (Constant) 3,527 0.675 5,228 4,177 In-Store Logistics Performance -0.092 0.079 -0.110 -1,173 0.244 Store Image 0.038 0.062 -0.045 0.610 0.543 Sales promotion -0.008 0.088 0.009 -0.085 0.932 Service Quality -0.797 0.122 0.914 0.1765 0.080 Source: Processed data (2023) 23 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 It can be concluded that the data used in this research does not exhibit symptoms of heteroscedasticity based on the table above, which shows that the significance value for the in-store logistics performance variable is 0.244; the store image variable is 0.543; the sales promotion variable is 0.932; and the service quality variable is 0.080. It can be concluded that the data used in this research does not exhibit symptoms of heteroscedasticity based on the table above, which shows that the significance value for the in-store logistics performance variable is 0.244; the store image variable is 0.543; the sales promotion variable is 0.932; and the service quality variable is 0.080. In-Store Service Quality on Consumer Satisfaction S o e Se v ce Qu y o Co su e S s c o The research findings reveal a positive perception among respondents regarding the reliability and empathy of the Matahari Department Store at Mal Artha Gading. The highest average values for reliability and empathy indicators suggest that customers feel confident in the store consistently delivering reliable services. That staff effectively understands and responds to their needs. However, the assurance indicator received the lowest average score, indicating potential areas for improvement in customer confidence related to staff competence, information clarity, and professionalism. There are numerous measures and definitions of service quality without a single consensus. Several previous studies have stated that there is a service provided by an organization. Customer satisfaction is one of the factors that influences customer loyalty to organizations. Service quality is a complex construct that incorporates multiple attributes that may change rapidly and dramatically, which will then facilitate precise measuremen. Studies have shown that the quality of service is a critical success point for commercial and retail banks to win over one. As they daily perform face-to-face interaction with customers, banks must always maintain a professional environment. Possible reasons for the lower assurance score include uncertainty about staff competency, vague information, and perceived lack of professionalism. In-Store Image on Consumer Satisfaction In Store Image on Consumer Satisfaction The research findings indicate that respondents generally have a positive perception of the Matahari Department Store in Artha Gading Mall, particularly regarding the store image and layout. The high suggests that respondents find the visual presentation and organization of the store appealing. This positive perception can be attributed to factors such as attractive displays, convenient navigation, logical product placement, and a pleasant store atmosphere. This is in line with the understanding that store image is a combination of real or functional factors and intangible or psychological factors that consumers feel. Also defines store image as a complex of consumer perceptions of the different attributes of a store. Store descriptions explain a multidimensional structure, so many studies have been found that discuss attributes in building a store image. Companies can build an image positively in the minds of consumers using indicators that create an image and function in the right way. The 6 dimensions of store image mentioned in their research are personnel, price, product, convenience, atmosphere, and promotion. Proposed five elements of store image: assortment, price, styling, product, and location. Doyle and Fenwick’s work was modified, who suggested three more attributes-parking facilities, friendly personnel, and atmosphere. 24 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 In contrast, argued that store image is a complex of consumers' store perceptions of different attributes. Store image has been found to be influenced significantly by store attributes such as decor, lighting, layout, salespersons (Baker et al., 2020), visual communication, noise, and colors, music, crowding. These store attributes also help in determining store patronage by consumers. Meanwhile, in research regarding store image using 6 different attributes, namely Physical facilities/characteristics, Pricing Policy, Product Range, Customer Service, Store Character, and Store Reputation. However, the personnel indicator received a lower, suggesting that there are areas for improvement in respondents' perceptions of the store's staff. Possible reasons for this lower score include less friendly service and staff unavailability. In-Store Sales Promotion on Consumer Satisfaction The research findings highlight that the first statement regarding Matahari Department Store's price offers at Artha Gading Mall receiving the highest indicates respondents' agreement with the statement. This implies that regular price offers and sales promotions can instill hope among customers, fostering the belief that they can find products at more affordable prices. Additionally, attractive discounts and offers, coupled with appealing product choices, can enhance consumer interest and perceived value. Conversely, the third statement in the sales promotion variable has the lowest, suggesting a comparatively lower response from respondents. Possible reasons for this include past negative experiences with similar promotions or a lack of effective promotional communication. The results of this hypothesis test also reject the results of research conducted, showing that promotion is a factor that can influence Customer Satisfaction researched that promotion has a positive and significant effect on Customer Satisfaction and in line with research conducted shows that promotion does not have a completely significant effect on Customer Satisfaction. To address this, Matahari Department Store can take steps such as providing clear and comprehensive information through various channels, ensuring transparency in communication, and offering genuinely attractive promotions to enhance customer satisfaction and perception. In summary, the research underscores the importance of consistent and attractive promotions in attracting customers and increasing sales. It also emphasizes the need for clear communication and genuine value in promotional strategies to enhance customer satisfaction and perception. Recommendation Based on the research results, discussion, and conclusions obtained, the suggestions that can be given are as follows: Suggestions for future researchers. This research can be carried out again with a different research object, for example, regarding the restaurant business. This research can be carried out again by conducting different tests on competing companies that operate in the same way, namely retail businesses that offer daily and household necessities. For other researchers who are interested in conducting similar research, it is hoped that they can add other variables that are thought to influence retail consumer satisfaction, such as the Word of Mouth because it refers to communication between individuals regarding their experiences with a product, brand, or service. This can take the form of recommendations, reviews, or informative conversations, and the Shopping Experience variable, namely the overall experience when shopping at a store, can have a big impact on consumer satisfaction. 6. Conclusion Based on the results of the analysis and discussion that has been carried out previously, the following conclusions can be drawn: In-store logistics performance has a positive effect on consumer satisfaction, Store image has a positive effect on consumer satisfaction, Sales promotions have a positive effect on consumer satisfaction, Service quality has a positive effect on consumer satisfaction, In-store logistics performance, store image, sales promotion, and service quality together simultaneously influence consumer satisfaction at Matahari Department Store at Mal Artha Gading. 25 Sari, Gani Sari, Gani RBM 2(1) 2024 15-28 RBM 2(1) 2024 15-28 Managerial implications provide a roadmap for Matahari Department Store to improve its overall performance and enhance customer satisfaction by strategically addressing key components identified in the study. Regular assessments and adaptations based on customer feedback and market trends will be crucial for sustained success. 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https://openalex.org/W2464176087	https://hal.archives-ouvertes.fr/hal-01355643/document	English	null	Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV	Nature communications	2,016	cc-by	22,165	To cite this version: Abdellah Akil, Juan Peng, Mohyeddine Omrane, Claire Gondeau, Christophe Desterke, et al.. Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mech- anism hijacked by HCV. Nature Communications, 2016, 7, 10.1038/ncomms12203. hal-01355643 Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV Abdellah Akil, Juan Peng, Mohyeddine Omrane, Claire Gondeau, Christophe Desterke, Mickaël Marin, Hélène Tronchère, Cyntia Taveneau, Sokhavuth Sar, Philippe Briolotti, et al. Distributed under a Creative Commons Attribution 4.0 International License 1 INSERM, Unite´ 1193, F-94800 Villejuif, France. 2 University of Paris-Sud, UMR-S 1193, F-94800 Villejuif, France. 3 Laboratoire des He´patites Virales, De´partement de Virologie. Institut Pasteur du Maroc, BP 20360 Casablanca, Maroc. 4 Faculte´ des Sciences, Laboratoire de Biochimie-Immunologie, Univ. Mohammed V, Rabat, Maroc. 5 DHU Hepatinov, Villejuif F-94800, France. 6 INSERM U1183, Institute of Regenerative Medicine and Biotherapy, University of Montpellier, 34295 Montpellier, France. 7 Department of Hepato-Gastroenterology A, Hospital Saint Eloi, CHRU, 34295 Montpellier, France. 8 University of Paris-Sud, -UFR medecine- INSERM UMS33, Villejuif, France. 9 INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France. 10 Virologie Mole´culaire et Structurale CNRS UPR 3296 - INRA UsC 1358, 91198 Gif-sur-Yvette, France. 11 Univ. Internationale de Rabat, Sala Al Jadida, Maroc. 12 Institut Pasteur, 75724 Paris, France. 13 AP-HP Hoˆpital Paul-Brousse, Centre He´pato-Biliaire, Villejuif F-94800, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to A.G.-D. (email: ama.gassama@inserm.fr). ARTICLE Received 31 Aug 2015 \| Accepted 7 Jun 2016 \| Published 15 Jul 2016 HAL Id: hal-01355643 https://hal.science/hal-01355643v1 Submitted on 18 Nov 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Results T Transcriptomic analysis of septins in human cirrhosis. To determine the expression proﬁle of septin 9 in cirrhosis, we used the GSE14323 data set31, including transcriptomes of normal liver (n ¼ 19) and HCV-induced cirrhosis samples (n ¼ 49). Data revealed a signiﬁcant upregulation (two-sided Student’s t-test, P ¼ 0.012) of septin 9 transcriptional expression in cirrhosis samples compared to that in normal liver samples (Fig. 1a). p p p ( g ) Septin 9 occupies a terminal position in an octameric septin complex involved in the formation of higher order structures such as ﬁlaments and rings (Fig. 1b)32. According to the importance of hetero-oligomerisation in the assemblies and functions of septins23, we assessed the differential expression of septins in HCV-induced cirrhosis using the GSE14323 data set by unsupervised principal component analysis (Fig. 1c–f). Sample projection on the ﬁrst factorial map showed a signiﬁcant separation between the normal liver and HCV-induced cirrhosis samples according to the ﬁrst axis (P ¼ 2.67E 15) (Fig. 1c). Learning machine algorithm of Random Forest conﬁrmed a good separation of the normal liver and cirrhosis samples. The classiﬁcation error by class was low and especially null for the cirrhosis class after building 500 classiﬁcation trees and multidimensional Scaling plot (MDSplot), conﬁrming the good separation of the samples after learning machine analysis (Fig. 1d; Supplementary Fig. 1). Random Forest analysis predicted that most septins are affected in HCV-induced cirrhosis when compared to normal liver, while septin 10 was less affected. This was conﬁrmed by individual boxplot performed on each septin (Fig. 1e, P value were calculated by two-sided Student t-test). These analyses showed that the expression of septin 7 and 2 is massively affected in these samples (Fig. 1d,e). A weak error on misclassiﬁcation by class (confusion of 1.67%) was detected when using the learning machine algorithm (Supplementary Fig. 1c). Thus, we performed an unsupervised classiﬁcation on these samples with previously used septin expression (classiﬁcation tree with Euclidean distance and complete linkage (Fig. 1f)). This unsupervised algorithm performed on 58 clinical samples allowed p p p g p HCV is an enveloped positive single-stranded RNA virus from the Flaviviridae family. HCV genomic RNA encodes for a polyprotein precursor of B3,100 amino acids post-translationally cleaved by cellular and viral proteases into the structural (core, E1 and E2), non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) and p7 protein8. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 L ipid droplets (LDs) are highly dynamic organelles generated from the endoplasmic reticulum (ER)1, which can remain attached to the ER or generate contacts with other organelles, including the mitochondria, lysosomes, peroxisomes and Golgi2. LDs consist of a hydrophobic core of neutral lipids, which are primarily composed of triacylglycerols (TAGs) and cholesterol esters, surrounded by a monolayer of phospholipids and a large group of proteins that facilitate cellular signalling interactions, control the access of metabolic enzymes, and inﬂuence LD movement within the cell1,3. These proteins include predominantly the perilipin family (Plins) and proteins of the rab family of GTPases1,4. There are ﬁve known perilipins (PLINs 1–5) expressed in hepatocytes, which play distinct roles. PLIN2 also called ADRP (adipose differentiation-related protein) is a prominent LD protein, expression level of which is tied to LD accumulation. PLIN3 initially described as a tail-interacting protein of 47 kDa (TIP47) is also ubiquitously expressed5. LD growth and degradation are highly regulated processes and excessive accumulation of LDs in the liver causes steatosis, which may contributes to cirrhosis, the ﬁnal stage for several chronic liver diseases and often evolves to hepatocellular carcinoma. L size and composition between organisms19,20. In drosophila and mammalian septins, complexes contain in general three to four different septins present in two copies. The core hetero- oligomeric units of six to eight subunits serve to build higher order structures, including ﬁlaments and rings structures, which are important for septin in vivo biological function20. Septins are implicated in multiple cellular functions, including cytokinesis, ciliogenesis, cell migration, vesicle trafﬁcking, and cell polarity21–23. Septins associate with the cytoskeleton and are known as a fourth type of cytoskeletal structure23. Septins also bind to membranes, speciﬁcally to PIs, providing membrane stability and serving as diffusion barriers for membrane proteins20,21,23. Nevertheless, these septin/PI interactions have been scarcely investigated24–26 and their link with HCV life cycle and their potential role in LD biogenesis and growth remain unclear. Septins are also involved in infection by pathogens, including bacteria, fungi23,27,28 and HCV virus29. Alteration in expression proﬁle of septins are reported in different cancers and we determined that septin 9 is speciﬁcally upregulated in liver cancer30. Despite being involved in carcinogenesis, septin 9 expression and function have not been investigated in precancerous diseases such as cirrhosis. ARTICLE In this study, we investigate the expression of septin 9 and the differential expression of the other septins in HCV-induced cirrhosis. We also demonstrate that septin 9 regulates LD growth through binding to PtdIns5P in HCV- infected cells. Of interest, we show that this new mechanism is involved in lipid homoeostasis independently of HCV infection. p Chronic HCV infection is a major public health problem and a main risk factor for cirrhosis, and hepatocellular carcinoma, thus remaining the major cause of liver resection and transplantation6. There is no vaccine to protect against HCV, although major advances have been recently achieved regarding HCV infection treatment, and the combination of drugs, including protease inhibitors, represents a real breakthrough that cure almost 90% of infected patients. However, these treatments have remained very costly. Additionally, understanding the key mechanisms driving HCV replication and persistence will have a profound impact, beyond HCV, for the appraisal of the pathogenesis of this family of viruses. Each step of the replication cycle of HCV is tightly dependent on the host lipid metabolism, and liver steatosis associated with the accumulation of LDs is a hallmark of chronic HCV infection7,8. HCV and LD interactions are crucial for persistent viral propagation and virion production9. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV Abdellah Akil1,2,3,4,, Juan Peng1,2,5,, Mohyeddine Omrane1,2,5,, Claire Gondeau6,7, Christophe Desterke8, Mickae¨l Marin1,2, He´le`ne Tronche`re9, Cyntia Taveneau10, Sokhavuth Sar1,2, Philippe Briolotti6,7, Soumaya Benjelloun3, Abdelaziz Benjouad4,11, Patrick Maurel6,7, Vale´rie Thiers12, Ste´phane Bressanelli10, Didier Samuel1,2,5,13, Christian Bre´chot1,2,12 & Ama Gassama-Diagne1,2,5 The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a signiﬁcant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into ﬁlaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 ﬁlaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway. 1 NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Results T The core protein plays a major role in the virion assembly through interaction with LDs10. The proteins covering the LD surface and their link with HCV life cycle have been characterized, whereas the potential role of phospholipids in these events remains unclear. The composition of the phospholipid monolayer of LDs depends on the cell type, but is mostly composed of phosphatidylcholine, phosphatidylethanolamine, and, to a lesser extent, phosphatidylinositol (PtdIns) and lysophospholipids1,11,12. The phosphorylated derivatives of PtdIns (PIs) are important signalling molecules that are essential for a variety of cellular functions, including membrane remodelling and trafﬁcking13. Although recent studies revealed the role of PtdIns 4-kinases (PI4K-IIIa and PI4K-IIIb) and their lipid products, PtdIns4P, as critical regulators of the HCV life cycle8. The role of class II phosphoinositide 3-kinase (PI3K) was also demonstrated14. Furthermore we demonstrated that PIs control epithelial morphogenesis and polarity15,16 and represent HCV cellular targets17. g Septins form a family of GTP-binding proteins composed of 13 members in mammals and conserved from yeast to humans18. The complexity of this gene family is increased by the existence of alternative splicing, which markedly increases the number of potential isoforms. Results T Septins assemble into complexes variable in NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 2 P value=0.012 10.5 9.6 9.9 10.2 Septin_9 HCV-cirrhosis Control 9 7 6 2 2 2 6 7 GDP GTP GDP GDP GDP GDP GTP GDP 4 4 6 2 2 Dim 2 (11.70%) 0 0 –2 –2 –4 –4 –6 control HCV-cirrhosis P value= 2.670578e–15 Dim 1 (57.60%) Mean decrease accuracy 20 15 10 5 0 Septin_10 Septin_11 Septin_9 Septin_8 Septin_4 Septin_6 Septin_7 Septin_2 11.0 10.5 10.0 9.5 Septin_2 P value= 6.2e–14 HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control Septin_4 8 7 6 8 7 6 Septin_6 P value= 8.3e–09 P value= 1.868e–12 11.0 10.5 10.0 9.5 9.0 Septin_7 P value= 2.32e–13 8.0 7.5 Septin_8 7.0 6.5 P value= 2.78e–09 9.5 9.0 8.5 8.0 Septin_10 P value=0.158 8.8 8.4 8.0 7.6 P value= 0.00022 Septin_11 Color key and histogram 1 2 3 4 5 6 Count 6 7 8 9 10 11 Value Septin_2 Septin_7 Septin_9 Septin_10 Septin_11 Septin_4 Septin_6 Septin_8 Normal liver HCV-cirrhosis Contol13 Contol19 Contol9 Contol15 Contol7 Contol6 Contol3 Contol1 Contol2 Contol16 Contol18 Contol14 Contol11 Contol12 Contol8 Contol17 Contol10 Contol5 Contol4 Cirrhosis9 Cirrhosis10 Cirrhosis3 Cirrhosis13 Cirrhosis14 Cirrhosis39 Cirrhosis7 Cirrhosis12 Cirrhosis11 Cirrhosis28 Cirrhosis27 Cirrhosis24 Cirrhosis16 Cirrhosis30 Cirrhosis15 Cirrhosis17 Cirrhosis2 Cirrhosis40 Cirrhosis19 Cirrhosis25 Cirrhosis1 Cirrhosis33 Cirrhosis21 Cirrhosis22 Cirrhosis31 Cirrhosis29 Cirrhosis5 Cirrhosis4 Cirrhosis32 Cirrhosis41 Cirrhosis26 Cirrhosis18 Cirrhosis4 Cirrhosis35 Cirrhosis36 Cirrhosis6 Cirrhosis37 Cirrhosis20 Cirrhosis8 Cirrhosis38 Cirrhosis23 c b a d e f gure 1 \| Transcriptomic analysis of septin 9 in human cirrhosis. (a) R software was used to generate boxplot presents septin 9 expression in d in normal liver samples of GSE14323 data set and to calculate mentioned P value of two-sided Student’s t-test. (b) The octameric complex o ntains septin 2 group and septin 6 group members, septin 7 and septin 9 which caps the two extremities of the rod shaped complex. (c) R softw ctoMineR package was used to obtain unsupervised principal component analysis performed on GSE14323 with septin molecules: P value obt st principal axis allowed discriminating normal liver from cirrhosis in GSE14323. (d) Mean decrease accuracy of predict variables during the ‘ ndom Forest learning machine. (e) R software was used to generate boxplot of septins allows to discriminate cirrhosis from normal liver with G d to calculate mentioned P value of two-sided Student’s t-test. Results T (f) R software was used to perform heatmap by unsupervised classiﬁcation wit olecules in GSE14323 (classiﬁcation tree with Euclidean distance and complete linkage). ATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 ART ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 P value=0.012 10.5 9.6 9.9 10.2 Septin_9 HCV-cirrhosis Control 9 7 6 2 2 2 6 7 GDP GTP GDP GDP GDP GDP GTP GDP 4 4 6 2 2 Dim 2 (11.70%) 0 0 –2 –2 –4 –4 –6 control HCV-cirrhosis P value= 2.670578e–15 Dim 1 (57.60%) Mean decrease accuracy 20 15 10 5 0 Septin_10 Septin_11 Septin_9 Septin_8 Septin_4 Septin_6 Septin_7 Septin_2 c b a d P value=0.012 10.5 9.6 9.9 10.2 Septin_9 HCV-cirrhosis Control a a Mean decrease accuracy 20 15 10 5 0 Septin_10 Septin_11 Septin_9 Septin_8 Septin_4 Septin_6 Septin_7 Septin_2 d d 11.0 10.5 10.0 9.5 Septin_2 P value= 6.2e–14 HCV-cirrhosis Control HCV-cirrhosis Control Septin_4 8 7 6 P value= 8.3e–09 e e HCV-cirrhosis Control HCV-cirrhosis Control 8 7 6 Septin_6 P value= 1.868e–12 11.0 10.5 10.0 9.5 9.0 Septin_7 P value= 2.32e–13 HCV-cirrhosis Control 11.0 10.5 10.0 9.5 9.0 Septin_7 P value= 2.32e–13 HCV-cirrhosis Control 8 7 6 Septin_6 P value= 1.868e–12 HCV-cirrhosis Control HCV-cirrhosis Control HCV-cirrhosis Control 8.0 7.5 Septin_8 7.0 6.5 P value= 2.78e–09 9.5 9.0 8.5 8.0 Septin_10 P value=0.158 Septin 11 ol HCV-cirrhosis Control HCV-cirrhosis Control e= 09 9.5 9.0 8.5 8.0 Septin_10 P value=0.158 8.8 8.4 8.0 7.6 P value= 0.00022 Septin_11 HCV-cirrhosis Control 8.0 7.5 Septin_8 7.0 6.5 P value= 2.78e–09 Color key and histogram 1 2 3 4 5 6 Count 6 7 8 9 10 11 Value Septin_2 Septin_7 Septin_9 Septin_10 Septin_11 Septin_4 Septin_6 Septin_8 Normal liver HCV-cirrhosis Contol13 Contol19 Contol9 Contol15 Contol7 Contol6 Contol3 Contol1 Contol2 Contol16 Contol18 Contol14 Contol11 Contol12 Contol8 Contol17 Contol10 Contol5 Contol4 Cirrhosis9 Cirrhosis10 Cirrhosis3 Cirrhosis13 Cirrhosis14 Cirrhosis39 Cirrhosis7 Cirrhosis12 Cirrhosis11 Cirrhosis28 Cirrhosis27 Cirrhosis24 Cirrhosis16 Cirrhosis30 Cirrhosis15 Cirrhosis17 Cirrhosis2 Cirrhosis40 Cirrhosis19 Cirrhosis25 Cirrhosis1 Cirrhosis33 Cirrhosis21 Cirrhosis22 Cirrhosis31 Cirrhosis29 Cirrhosis5 Cirrhosis4 Cirrhosis32 Cirrhosis41 Cirrhosis26 Cirrhosis18 Cirrhosis4 Cirrhosis35 Cirrhosis36 Cirrhosis6 Cirrhosis37 Cirrhosis20 Cirrhosis8 Cirrhosis38 Cirrhosis23 f f Figure 1 \| Transcriptomic analysis of septin 9 in human cirrhosis. (a) R software was used to generate boxplot presents septin 9 expression in cirrhosis and in normal liver samples of GSE14323 data set and to calculate mentioned P value of two-sided Student’s t-test. ARTICLE Following the above described transcriptomic study, we hypothesized that HCV could be responsible of the upregulation of septin 9 in cirrhosis. To explore this possibility, Huh7.5 cells were inoculated for 24, 48 and 72 h with HCV JFH-1 (Japanese fulminate hepatitis 1), a genotype 2a strain, which produces infectious particles in culture and recapitulates all steps of the HCV life cycle33. Immunoblot data revealed a non-signiﬁcant, time-dependent increase of septin 9 expression in non-infected cells, while septin 9 expression was signiﬁcantly enhanced in JFH-1-infected cells (Fig. 2a), with the highest expression at 72 h of infection (Fig. 2a) when septin 9 formed bundles of ﬁlaments surrounding the clusters of HCV core (Fig. 2b). Treatment of Huh7.5 cells with two different septin 9 siRNAs (si1 and si3) before their infection strongly reduced core expression (Fig. 2b,c). These data suggested that HCV-regulated septin 9 assembly and expression was required for core protein expression. p q p p Thus, to further characterize the septin 9 ﬁlamentous structure, we determined the presence of other septins. We focussed on septin 2, the major ubiquitously expressed septin and the most deregulated in HCV-induced cirrhosis (Fig. 1). In non-infected cells, septin 2 formed short ﬁlaments that co-localized with septin 9 (Fig. 2d,e). These ﬁlamentous structures containing septin 2 and their co-localization with septin 9 were signiﬁcantly increased upon JFH-1 infection (Fig. 2d–f). Depletion of septin 9 using si3 disrupted septin 2 ﬁlaments and reduced its staining when compared with that in control cells in both non-infected and JFH-1-infected cells (Fig. 2d,e). These data were conﬁrmed by immunoblot analysis (Fig. 2g) and suggested the role of septin 9 in the formation of septin ﬁlamentous structure, consistent with previous reports32. Although the ﬁrst evidence for HCV interaction with LDs was obtained from a study on the core protein, recent studies indicated that the interaction of the HCV non-structural protein NS5A with PLIN3 is essential for the recruitment of NS5A on LDs to promote the release of HCV particles36,37. Interestingly, increases in PLIN3 and NS5A expression were observed in Huh7R cells expressing septin 9_i1 (Supplementary Fig. 6a). These results were conﬁrmed by immunoblot (Supplementary Fig. 6b) indicating that septin 9_i1 regulated LDs growth and HCV protein expression. Next, we assessed the effects of septin 9 expression on HCV-modulated microtubule (MT) network. Tubulin staining indicated a strong MT network in HCV-infected cells when compared with that of non-infected cells (Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 treatment (Supplementary Fig. 3) and emphasized the role of septin 9 in HCV-induced LD accumulation. Finally, we revealed a strong decrease of HCV genomic RNA after septin 9 knockdown (Fig. 3i), suggesting a critical role of septin 9 in HCV replication. We also analysed the transcript level of the ﬁve well-characterized variants of septin 9 (i1 to 5) after JFH-1 infection. Isoform 1 mRNAs were the most abundant, while isoform 3 mRNAs were almost undetectable (Supplementary Fig. 2b). Accordingly, the following studies focused on septin 9 isoform 1 (Septin 9_i1) and we used Huh7 cells stably expressing HCV full-length genomic replicon from genotype 1b (Huh7R)35. This system permitted the efﬁcient expression of all viral proteins and is a convenient and valuable tool for molecular studies. A decrease in both the core and envelope E2 proteins was observed in Huh7R cells transfected with si3 (Supplementary Fig. 4a). By opposite both core and E2 expression increased in cells expressing septin 9_i1 compared with control cells (Supplementary Fig. 4b). Later, septin 9_i1 and the LD characteristics were assessed by immunoﬂuorescence analysis. Septin 9_i1 assembled in ﬁlaments surrounding the large clusters of LD and transfection of si3 inhibited LD accumulation (Fig. 4a). Overall, proﬁles of LD distribution and changes in LD morphology (Fig. 4b,c) were similar to the results obtained using the JFH-1 strain (Fig. 3). Septin 9_i1 ﬁlaments co-localized with endogenous septin 2 (Supplementary Fig. 5a), indicating that septin 9_i1 is integrated in a hero-oligomeric structure composed of other septins consistent with data in Fig. 2. Moreover, the ratio of the intensity of exogenous septin 9_i1 revealed with an anti-V5 tag antibody to the intensity of endogenous septin 9 detected with septin 9 antibody was 1.1 (Supplementary Fig. 5b). Thus, we concluded that septin 9_i1 expression was similar to that of endogenous septin 9. the discrimination of two major groups of experiments with no error of misclassiﬁcation between normal liver and HCV-induced cirrhosis samples. This result suggests that the expression of septin protein family is deregulated in HCV-induced cirrhosis as compared with that in the normal liver. the discrimination of two major groups of experiments with no error of misclassiﬁcation between normal liver and HCV-induced cirrhosis samples. This result suggests that the expression of septin protein family is deregulated in HCV-induced cirrhosis as compared with that in the normal liver. HCV assembles septin 9 which stabilizes microtubules. ARTICLE 2h,i; Supplementary Fig. 2a). Importantly, MTs co-localized with septin 9 ﬁlaments and si3 severely destroyed MT ﬁlaments (Fig. 2h–j), indicating a regulatory role of septin 9 in HCV-dependent MT organization. Septin 9 regulates neutral lipid metabolism. LD growth can be mediated by several mechanisms, but, typically, its size expansion involved an increase in the neutral lipid content, which essentially consists of TAG and cholesterol ester5. Therefore, we analysed the neutral lipid content of septin 9_i1-expressing cells. Transfection of septin 9_i1 in Huh7R cells induced a threefold increase of TAG and correlated with a decrease of diacylglycerol (DAGs) (Fig. 4d). As a consequence, the ratio of TAG to DAG was greatly increased (Fig. 4e). The fatty acid composition of TAG and DAG was also analysed. The composition of these two lipids was very similar and consisted mostly of C16 and C18 long chains (Fig. 4f). Similar data were obtained using septin 9-depleted Huh7R cells (Fig. 4g-i). A signiﬁcant TAG decrease and an increase in DAG were observed (Fig. 4g), suggesting a conversion of the TAG pool into DAGs. Septin 9 regulates LD behaviour and HCV replication. The accumulation of LDs is an important feature of HCV infection4. Thus, to understate the importance of septin 9 in HCV life cycle, we investigated LDs. JFH-1 infection sharply increased LDs that formed clusters co-localized with the core protein around the nucleus (Fig. 3a), while si3 treatment markedly decreased LDs (Fig. 3b) and their co-localization with core (Fig. 3c,d). A typical proﬁle of LD distribution was presented (Fig. 3e,f). In non- infected cells, about 70% of LDs were present in the perinuclear area and si3 treatment decreased the percentage to 55%. JFH-1 infection increased the perinuclear LDs up to 92% and septin 9 depletion decreased this level to 62% (Fig. 3g). These changes in the level and distribution of LDs were accompanied by a 2.5-fold increase of LD size in JFH-1-infected cells, while si3 decreased the LD size by half (Fig. 3h). Similar changes were observed for LD number (Fig. 3h). PLIN2 is among the best studied protein associated with LDs and is considered as a marker of LDs34. The strong increase in PLIN2 expression and its co-localization with core observed in JFH-1-infected cells were abolished by si3 Diacylglycerol acyltransferase-1 (DGAT1) is one of the two known DGAT enzymes that catalyse the ﬁnal step in triglyceride biosynthesis38. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Results T (b) The octameric complex of septins contains septin 2 group and septin 6 group members, septin 7 and septin 9 which caps the two extremities of the rod shaped complex. (c) R software with FactoMineR package was used to obtain unsupervised principal component analysis performed on GSE14323 with septin molecules: P value obtained on ﬁrst principal axis allowed discriminating normal liver from cirrhosis in GSE14323. (d) Mean decrease accuracy of predict variables during the ‘Septin’ Random Forest learning machine. (e) R software was used to generate boxplot of septins allows to discriminate cirrhosis from normal liver with GSE14323 and to calculate mentioned P value of two-sided Student’s t-test. (f) R software was used to perform heatmap by unsupervised classiﬁcation with septin molecules in GSE14323 (classiﬁcation tree with Euclidean distance and complete linkage). NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 3 ARTICLE The bar graph prese Septin 9 Septin 9 Septin 9 Septin 9 Non infected Non infected JFH-1 Merge Merge Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 MTs MTs Septin 9 Septin 9 Control si3septin9 Control si3septin9 si3septin9 Control e i 1 0.8 0.6 0.4 0.2 0 40 70 37 20 Densitometry septin 2/actin JFH-1 JFH-1 Non infected Non infected Septin 2 Core Septin 9 Actin Septin 2 Ctrl Ctrl Ctrl Ctrl si3 si3 si3 si3 g 40 70 37 20 JFH-1 Non infected Septin 2 Core Septin 9 Actin Ctrl Ctrl si3 si3 0.5 1 0 Colocalisation index (Rr) No infected JFH-1 *** f f 1 0.8 0.6 0.4 0.2 0 Densitometry septin 2/actin JFH-1 Non infected Septin 2 Ctrl Ctrl si3 si3 * Control si3 Septin 9 JFH-1 Non infected Septin 9 / microtubule (MTs) h h i JFH-1 Non infected Merge Merge Merge Merge MTs MTs MTs MTs Septin 9 Septin 9 Septin 9 Septin 9 si3septin9 si3septin9 Control Control i 0.5 1 0 Colocalisation index (Rr) Non infected JFH-1 *** j j j Figure 2 \| Septin 9 increases in JFH-1-infected cells and regulates septin 2 and microtubules ﬁlaments. (a) Immunoblot of septin 9 and core in Huh7.5 cells infected or not with JFH-1 for 24, 48 and 72 h. Actin was used as a loading control. The bar graph presents the densitometry analysis of the immunoblots from three independent experiments. (b) Huh7.5 cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 24 h then infected or not with HCV JFH-1 for 72 h then stained for septin 9 (green) and core (red). (c) Immunoblot of septin 9 and core in Huh7.5 cells transfected with non-targeting (control) or septin 9 siRNA (si1 or si3) then infected with HCV JFH-1 for 72 h. (d) Huh7.5 treated as in b and stained for septin 9 (green) and septin 2 (red). (e) Dot rectangles in d are presented in higher magniﬁcation. (f) Bar graph shows Pearson’s correlation coefﬁcient (Rr) of septin 9 and septin 2 calculated in 30 cells from 2 independent experiments. (g) Immunoblot of septin 2, septin 9 and core in Huh7.5 cells treated as described in b. Actin was used as a loading control. Bar graph presents septin 2 expression from three independent experiments. ARTICLE (d) Huh7.5 treated as in b and stained for septin 9 (green septin 2 (red). (e) Dot rectangles in d are presented in higher magniﬁcation. (f) Bar graph shows Pearson’s correlation coefﬁcient (Rr) of septin 9 septin 2 calculated in 30 cells from 2 independent experiments. (g) Immunoblot of septin 2, septin 9 and core in Huh7.5 cells treated as described Actin was used as a loading control. Bar graph presents septin 2 expression from three independent experiments. (h) Huh7.5 cells were treated as described in b and stained for microtubules (MTs) with b tubulin (red) and septin 9 (green). (i) Dot squares in h present in higher magniﬁcation. (j graph shows Pearson’s correlation coefﬁcient (Rr) analysis of septin 9 and MTs calculated in 30 cells from two independent experiments. Values a means±s.e.m. Student’s t-test was used. Po0.05, *Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications c 50 20 75 JFH-1 Septin 9 Control si septin 9 si1 si3 Core Actin c g Control si3 Septin 9 JFH-1 Non infected Septin 9 / septin 2 d d g e e Septin 9 Septin 9 Septin 9 Septin 9 Non infected Non infected JFH-1 Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 Control si3septin9 Control si3septin9 e i Septin 9 Septin 9 Septin 9 Septin 9 0 0 0 0 Densitometry septin 2/actin Non infected Non infected JFH-1 N Merge Merge Merge Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 MTs MTs MTs Septin 9 Septin 9 Septin 9 Septin 2 Core Septin 9 Actin Control si3septin9 Control si3septin9 si3septin9 Control Control e i g regulates septin 2 and microtubules ﬁlaments. (a) n was used as a loading control. ARTICLE (c) Immunoblot of septin 9 and core in Huh7.5 cells transfec Septin 9 Septin 9 Septin 9 Septin 9 1 0.8 0.6 0.4 0.2 0 40 70 37 20 50 20 75 Densitometry septin 2/actin Control si3 Septin 9 JFH-1 JFH-1 JFH-1 Non infected Non infected JFH-1 JFH-1 JFH-1 Non infected Non infected Non infected Septin 9 Septin 9 Septin 9 Septin 9 Core Core Merge Merge Merge Merge Merge Merge Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 MTs MTs MTs MTs Septin 9 Septin 9 Septin 9 Septin 9 Septin 9 Septin 2 Core Septin 9 Actin Septin 2 Control si septin 9 Control si3septin9 Control si3septin9 si3septin9 si3septin9 Control Control si1 Ctrl Ctrl Ctrl Ctrl si3 si3 si3 si3 si3 Core Actin c e i g b 7 d regulates septin 2 and microtubules ﬁlaments. (a) Immunoblot of septin 9 and co tin was used as a loading control. The bar graph presents the densitometry analysis Huh7.5 cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 24 h 9 (green) and core (red). (c) Immunoblot of septin 9 and core in Huh7.5 cells tran n infected with HCV JFH-1 for 72 h. (d) Huh7.5 treated as in b and stained for septin 9 higher magniﬁcation. (f) Bar graph shows Pearson’s correlation coefﬁcient (Rr) of s riments. (g) Immunoblot of septin 2, septin 9 and core in Huh7.5 cells treated as de s septin 2 expression from three independent experiments. (h) Huh7.5 cells were tr b tubulin (red) and septin 9 (green). (i) Dot squares in h present in higher magniﬁc ysis of septin 9 and MTs calculated in 30 cells from two independent experiments. ARTICLE a 20 37 50 24 72 48 24 72 48 JFH-1 Non infected Septin 9 Core Actin Time (h) a 0 8 6 4 2 20 37 50 Densitometry septin 9/actin 24 72 48 24 (h) 72 48 24 72 48 JFH-1 Non infected No infected JFH-1 Septin 9 Core Septin 9 * * Actin Time (h) Control si3 Septin 9 JFH-1 Non infected Septin 9 Septin 9 Septin 9 Septin 9 Core Core Merge Merge c b b a 0 8 6 4 2 Densitometry septin 9/actin 24 (h) 72 48 No infected JFH-1 Septin 9 * * Septin 9 Septin 9 Septin 9 Septin 9 1 0.8 0.6 0.4 0.2 0 40 70 37 20 50 20 75 Densitometry septin 2/actin si3 S JFH-1 JFH-1 Non infected Non infected JFH-1 JFH-1 JFH-1 Non infected Non infected Merge Merge Merge Merge Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 MTs MTs MTs MTs Septin 9 Septin 9 Septin 9 Septin 9 Septin 9 Septin 2 Core Septin 9 Actin Septin 2 Control si septin 9 Control si3septin9 Control si3septin9 si3septin9 si3septin9 Control Control si1 Ctrl Ctrl Ctrl Ctrl si3 si3 si3 si3 si3 * Core Actin c e i g 0 8 6 4 2 0.5 0.5 1 0 1 0 Densitometry septin 9/actin Colocalisation index (Rr) Colocalisation index (Rr) Control si3 Septin 9 Control si3 Septin 9 24 (h) 72 48 JFH-1 Non infected JFH-1 Non infected No infected Non infected JFH-1 JFH-1 Septin 9 / septin 2 Septin 9 / microtubule (MTs) Septin 9 * * * * d f h j Figure 2 \| Septin 9 increases in JFH-1-infected cells and regulates septin 2 and microtubules ﬁlaments. (a) Immunoblot of septin 9 and core in Hu cells infected or not with JFH-1 for 24, 48 and 72 h. Actin was used as a loading control. The bar graph presents the densitometry analysis of the immunoblots from three independent experiments. (b) Huh7.5 cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 24 h then infe or not with HCV JFH-1 for 72 h then stained for septin 9 (green) and core (red). (c) Immunoblot of septin 9 and core in Huh7.5 cells transfected non-targeting (control) or septin 9 siRNA (si1 or si3) then infected with HCV JFH-1 for 72 h. ARTICLE The role of DGAT1 in the trafﬁcking of the HCV core protein to LDs and its involvement in the production of infectious HCV particles were reported39,40. Thus, we analysed DGAT expression and we observed a signiﬁcant increase in the total intensity of the DGAT1 signal in septin 9_i1-expressing NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 Septin 9 Septin 9 Septin 9 Septin 9 1 0.8 0.6 0.4 0.2 0 40 70 37 20 50 20 75 Densitometry septin 2/actin Control si3 Septin 9 JFH-1 JFH-1 JFH-1 Non infected Non infected JFH-1 JFH-1 JFH-1 Non infected Non infected Non infected Septin 9 Septin 9 Septin 9 Septin 9 Core Core Merge Merge Merge Merge Merge Merge Merge Merge Merge Merge Septin 2 Septin 2 Septin 2 Septin 2 MTs MTs MTs MTs Septin 9 Septin 9 Septin 9 Septin 9 Septin 9 Septin 2 Core Septin 9 Actin Septin 2 Control si septin 9 Control si3septin9 Control si3septin9 si3septin9 si3septin9 Control Control si1 Ctrl Ctrl Ctrl Ctrl si3 si3 si3 si3 si3 * Core Actin c e i g b a 0 8 6 4 2 0.5 0.5 1 0 1 0 20 37 50 Densitometry septin 9/actin Colocalisation index (Rr) Colocalisation index (Rr) Control si3 Septin 9 Control si3 Septin 9 24 72 48 24 (h) 72 48 24 72 48 JFH-1 JFH-1 Non infected JFH-1 Non infected No infected Non infected JFH-1 JFH-1 Septin 9 / septin 2 Septin 9 / microtubule (MTs) Non infected No infected JFH-1 Septin 9 Core Septin 9 * * * * Actin d f h j Time (h) e 2 \| Septin 9 increases in JFH-1-infected cells and regulates septin 2 and microtubules ﬁlaments. (a) Immunoblot of septin 9 and core nfected or not with JFH-1 for 24, 48 and 72 h. Actin was used as a loading control. The bar graph presents the densitometry analysis of noblots from three independent experiments. (b) Huh7.5 cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 24 h the t with HCV JFH-1 for 72 h then stained for septin 9 (green) and core (red). NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications ARTICLE (a) Huh7.5 cells were transfected non-targeting (control or Ctrl) or septin 9 siRNA (si3) for 24 h then infected with JFH-1 for further 72 h and stained for core (green) and L (b) LDs ﬂuorescence intensity in 30 cells from two experiments performed as described in a. (c) Dot squares in a are presented in higher m The right panels present green, red, line proﬁle blots of the pink lines. (d) Bar graphs show Pearson’s correlation coefﬁcient (Rr) for co-localiza core and LDs of 30 cells from two experiments. (e) Representation of the peripheral (black) and perinuclear (red) regions of the cell. (f) Radia showing LD distribution between the peripheral (black) and the perinuclear regions (red) of cells indicated with the white arrows in a. (g) Qua LDs in perinuclear and peripheral regions in 30 cells from two independent experiments performed as described in a. (h) LD size (left) and (right) in 30 cells from two independent experiments done as described in a. (i) Huh7.5 cells were transfected with non-targeting (control siRNA (si3) for 24 h and infected with JFH-1 as in a for 72 h. Cells were analysed for septin 9 mRNA and HCV RNA level by qRT–PCR. Bar esults from 3 independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, *Po0.0001. ARTICLE (h) Huh7.5 cells were treated as described in b and stained for microtubules (MTs) with b tubulin (red) and septin 9 (green). (i) Dot squares in h present in higher magniﬁcation. (j) Bar graph shows Pearson’s correlation coefﬁcient (Rr) analysis of septin 9 and MTs calculated in 30 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, *Po0.0001. Scale bar, 10 mm. 5 NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 ARTICLE MTs are required for septin 9-induced LD behaviour. Different reports indicated that MTs play a role in the dynamics and growth of LDs41,42. Furthermore, septins interact with MTs and are essential for MT-dependent cell processes12,23,43. Therefore, cells. Moreover, DGAT1 co-localized with septin 9_i1 ﬁlaments, as shown at the highest magniﬁcation (Fig. 4j). Together, these data indicated that septin 9 could also participate in LD growth by regulating lipid biosynthesis. MTs are required for septin 9-induced LD behaviour. Different reports indicated that MTs play a role in the dynamics and growth of LDs41,42. Furthermore, septins interact with MTs and are essential for MT-dependent cell processes12,23,43. Therefore, er, DGAT1 co localized with septin 9_i1 ﬁlaments, the highest magniﬁcation (Fig. 4j). Together, these d that septin 9 could also participate in LD growth lipid biosynthesis. MTs are required for septin 9 induced LD beha reports indicated that MTs play a role in the growth of LDs41,42. ARTICLE Furthermore, septins interact are essential for MT-dependent cell processes12,2 * *** * ** * ** LDs LDs LDs LDs Core Core Merge Merge Control Control si3 Septin 9 si3 Septin 9 Non infected JFH-1 Fluorescence intensity of LD per cell 100 80 60 40 20 0 Ctrl si3 Ctrl si3 Non infected JFH-1 LDs Core Merge LDs Core Merge 0 1 2 3 4 0 1 2 3 4 60 Intensity (a.u.) 40 20 0 60 40 20 0 Distance (µm) Ctrl si3 JFH-1 1 0.8 0.6 0.4 0.2 0 * Colocalisation index Periphery Perinuclear Fluorescence intensity of LDs (a.u.) 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 20 10 0 20 10 0 20 10 0 20 10 0 si3 Septin 9 si3 Septin 9 Control Control Distance (µm) Non infected JFH-1 e f g h si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected % of LDs intensities 0 50 100 % of LDs intensities 0 50 100 *** Perinuclear Periphery LD size (µm2) LD size LD number 2 1 0 0 LD number per cell 50 100 150 200 Ctrl si3 Ctrl si3 Septin 9 mRNA (%) 120 100 80 60 40 20 0 120 100 80 60 40 20 0 i HCV RNA (%) a b c d in 9 regulates core and LDs accumulation in JFH-1 infected cells and virus replication. (a) Huh7.5 cells were transfe control or Ctrl) or septin 9 siRNA (si3) for 24 h then infected with JFH-1 for further 72 h and stained for core (green) cence intensity in 30 cells from two experiments performed as described in a. (c) Dot squares in a are presented in hig present green, red, line proﬁle blots of the pink lines. (d) Bar graphs show Pearson’s correlation coefﬁcient (Rr) for co-loc 30 cells from two experiments. (e) Representation of the peripheral (black) and perinuclear (red) regions of the cell. (f) R ribution between the peripheral (black) and the perinuclear regions (red) of cells indicated with the white arrows in a. (g ear and peripheral regions in 30 cells from two independent experiments performed as described in a. (h) LD size (left lls from two independent experiments done as described in a. ARTICLE (i) Huh7.5 cells were transfected with non-targeting (co 24 h and infected with JFH-1 as in a for 72 h. Cells were analysed for septin 9 mRNA and HCV RNA level by qRT–PCR ndependent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, Po0.0001. Sc * Fluorescence intensity of LD per cell 100 80 60 40 20 0 Ctrl si3 Ctrl si3 Non infected JFH-1 b LDs LDs LDs LDs Core Core Merge Merge Control si3 Septin 9 Non infected JFH-1 Fluorescence intensity of LD per cell 100 80 60 40 20 0 C Non a b * LDs LDs LDs LDs Core Core Merge Merge Control si3 Septin 9 Non infected JFH-1 Fluorescence intensity of LD per cell 100 80 60 40 20 0 Ctrl si3 Ctrl si3 Non infected JFH-1 a b b a *** * ** * ** Control si3 si3 Septin 9 LDs Core Merge LDs Core Merge 0 1 2 3 4 0 1 2 3 4 60 Intensity (a.u.) 40 20 0 60 40 20 0 Distance (µm) Ctrl si3 JFH-1 1 0.8 0.6 0.4 0.2 0 * Colocalisation index Periphery Perinuclear Fluorescence intensity of LDs (a.u.) 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 20 10 0 20 10 0 20 10 0 20 10 0 si3 Septin 9 si3 Septin 9 Control Control Distance (µm) Non infected JFH-1 e f g h si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected % of LDs intensities 0 50 100 % of LDs intensities 0 50 100 *** Perinuclear Periphery LD size (µm2) LD size LD number 2 1 0 0 LD number per cell 50 100 150 200 Ctrl si3 Ctrl si3 Septin 9 mRNA (%) 120 100 80 60 40 20 0 120 100 80 60 40 20 0 i HCV RNA (%) c d Figure 3 \| Septin 9 regulates core and LDs accumulation in JFH-1 infected cells and virus replication. ARTICLE Scale b Ctrl si3 JFH-1 1 0.8 0.6 0.4 0.2 0 * Colocalisation index d 0 1 2 3 4 0 1 2 3 4 60 Intensity (a.u.) 40 20 0 60 40 20 0 Distance (µm) Ctrl si3 JFH-1 1 0.8 0.6 0.4 0.2 0 * Colocalisation index d 0 1 2 3 4 0 1 2 3 4 60 Intensity (a.u.) 40 20 0 60 40 20 0 Distance (µm) Colocalisation index d Control si3 Septin 9 LDs Core Merge LDs Core Merge 0 1 2 3 4 0 1 2 3 4 60 Intensity (a.u.) 40 20 0 60 40 20 0 Distance (µm) Ctrl si3 JFH-1 1 0.8 0.6 0.4 0.2 0 * Colocalisation index c d d Control si3 Septin 9 LDs Core Merge LDs Core Merge c c Periphery Perinuclear e f Fluorescence intensity of LDs (a.u.) 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 80 60 40 20 0 20 10 0 20 10 0 20 10 0 20 10 0 si3 Septin 9 si3 Septin 9 Control Control Distance (µm) Non infected JFH-1 f e * * ** * g h si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected si3 Ctrl si3 Ctrl JFH-1 Non infected % of LDs intensities 0 50 100 % of LDs intensities 0 50 100 * Perinuclear Periphery LD size (µm2) LD size LD number 2 1 0 0 LD number per cell 50 100 150 200 Ctrl si3 Ctrl si3 Septin 9 mRNA (%) 120 100 80 60 40 20 0 120 100 80 60 40 20 0 i HCV RNA (%) g h Figure 3 \| Septin 9 regulates core and LDs accumulation in JFH-1 infected cells and virus replication. (a) Huh7.5 cells were transfected with non-targeting (control or Ctrl) or septin 9 siRNA (si3) for 24 h then infected with JFH-1 for further 72 h and stained for core (green) and LDs (red). (b) LDs ﬂuorescence intensity in 30 cells from two experiments performed as described in a. (c) Dot squares in a are presented in higher magniﬁcation. The right panels present green, red, line proﬁle blots of the pink lines. (d) Bar graphs show Pearson’s correlation coefﬁcient (Rr) for co-localization between core and LDs of 30 cells from two experiments. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications ARTICLE (a) Huh7R cells transfected with empty vector (EV) or septin 9_i1 fo ed) and V5 tag (green). Cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 48 h were stained for LD b) Schematic representation shows the perinuclear and peripheral regions of the cell. Bar graphs represent LDs intensity in region of 36 cells from ﬁve independent experiments. (c) LD size and LD number in 36 cells from 5 independent experiment fected with either EV or septin 9_i1 cDNAs then analysed for triacylglycerol (TAG) and diacylglycerol (DAG). (d) Data rep Ratio of TAG to DAG. (f) Distribution of fatty acid species in TAG and DAG according to carbon number in the acyl group. R ependents experiments. (g–i) Huh7R cells transfected with non-targeting or septin 9 siRNA for 72 h were analysed for TAG cells transfected with EV or septin 9_i1 were stained for V5 tag (red) and diacylglycerol acyltransferase-1 (DGAT1) (green e area shown in higher magniﬁcation. Bar graph represents DGAT1 ﬂuorescence intensity in 50 cells from two independe means±s.e.m. Student’s t-test was used. Po0.05, Po0.0001. Scale bar, 10 mm. ARTICLE Perinuclear Periphery Perinuclear Periphery * * * * I1 EV si3 Ctrl 100 80 60 40 20 0 % of LDs intensities I1 EV si3 Ctrl 100 80 60 40 20 0 % of LDs intensities b Empty vector (EV) Septin 9_i1 (I1) V5 tag LDs Merge Septin 9 LDs Merge Control (Ctrl) si3 Septin 9 (si3) Perinuclear Periphery Perinuclear Periphery * * * * * * * *** I1 EV si3 Ctrl 100 80 60 40 20 0 % of LDs intensities I1 EV si3 Ctrl I1 EV si3 Ctrl I1 EV i3 Ct l 100 80 60 40 20 0 % of LDs intensities 300 200 100 0 0 1 2 LDs size (µm2) LDs number per cell b a c b a TAG (nmol per 106 cells) DAG (nmol per 106 cells) 60 40 20 0 0 0 1 2 3 4 * EV Septin 9_i1 EV Septin 9_i1 EV Septin 9_i1 60 40 20 Ratio of TAG/DAG d e f EV Septin 9_i1 EV Septin 9_i1 18-18-16 18-18-18 TAG 16-16-14 16-16-16 18-16-16 16-18 16-16 18-18 DAG 18-18 d * TAG (nmol per 106 cells) DAG (nmol per 106 cells) Ratio of TAG/DAG 20 10 0 0 5 10 15 10 5 0 Septin 9 siRNA Control Septin 9 siRNA Control Septin 9 siRNA Control g h h i i TAG DAG 18-16-16 16-16-16 18-18-18 18-18-16 Septin 9_siRNA Control Septin 9_siRNA Control 16-18 16-16 18-18 g j p _ DGAT1 / V5 tag DGAT1 EV Septin 9_i1 Fluorescence intensity of DGAT1 (a.u.) EV Septin 9_i1 *** 100 80 60 40 20 0 j Figure 4 \| Septin 9 regulates TAG and DAG levels in Huh7R cells. (a) Huh7R cells transfected with empty vector (EV) or septin 9_i1 for 48 h were stained for LDs (red) and V5 tag (green). Cells transfected with non-targeting (control) or septin 9 siRNA (si3) for 48 h were stained for LDs (red) and septin 9 (green). (b) Schematic representation shows the perinuclear and peripheral regions of the cell. Bar graphs represent LDs intensity in the perinuclear and peripheral region of 36 cells from ﬁve independent experiments. (c) LD size and LD number in 36 cells from 5 independent experiments. (d–f) Huh7R cells were transfected with either EV or septin 9_i1 cDNAs then analysed for triacylglycerol (TAG) and diacylglycerol (DAG). (d) Data represent nmol per 106 cells. (e) Ratio of TAG to DAG. ARTICLE (e) Representation of the peripheral (black) and perinuclear (red) regions of the cell. (f) Radial proﬁle plots showing LD distribution between the peripheral (black) and the perinuclear regions (red) of cells indicated with the white arrows in a. (g) Quantiﬁcation of LDs in perinuclear and peripheral regions in 30 cells from two independent experiments performed as described in a. (h) LD size (left) and LD number (right) in 30 cells from two independent experiments done as described in a. (i) Huh7.5 cells were transfected with non-targeting (control) or septin 9 siRNA (si3) for 24 h and infected with JFH-1 as in a for 72 h. Cells were analysed for septin 9 mRNA and HCV RNA level by qRT–PCR. Bar graphs show results from 3 independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, *Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 Empty vector (EV) Septin 9_i1 (I1) V5 tag LDs Merge Septin 9 LDs Merge Control (Ctrl) si3 Septin 9 (si3) Perinuclear Periphery Perinuclear Periphery * * * * * * * * I1 EV si3 Ctrl 100 80 60 40 20 0 % of LDs intensities I1 EV si3 Ctrl I1 EV si3 Ctrl I1 EV si3 Ctrl 100 80 60 40 20 0 % of LDs intensities 300 200 100 0 0 1 2 LDs size (µm2) LDs number per cell b a c TAG (nmol per 106 cells) DAG (nmol per 106 cells) 60 40 20 0 0 0 1 2 3 4 * EV Septin 9_i1 EV Septin 9_i1 EV Septin 9_i1 60 40 20 Ratio of TAG/DAG TAG (nmol per 106 cells) DAG (nmol per 106 cells) Ratio of TAG/DAG 20 10 0 0 5 10 15 10 5 0 Septin 9 siRNA Control Septin 9 siRNA Control Septin 9 siRNA Control d e g h i f EV Septin 9_i1 EV Septin 9_i1 18-18-16 18-18-18 TAG 16-16-14 16-16-16 18-16-16 16-18 16-16 18-18 DAG TAG DAG 18-18 18-16-16 16-16-16 18-18-18 18-18-16 Septin 9_siRNA Control Septin 9_siRNA Control 16-18 16-16 18-18 DGAT1 / V5 tag DGAT1 EV Septin 9_i1 Fluorescence intensity of DGAT1 (a.u.) EV Septin 9_i1 *** 100 80 60 40 20 0 j Septin 9 regulates TAG and DAG levels in Huh7R cells. ARTICLE (f) Distribution of fatty acid species in TAG and DAG according to carbon number in the acyl group. Results are obtained from 3 independents experiments. (g–i) Huh7R cells transfected with non-targeting or septin 9 siRNA for 72 h were analysed for TAG and DAG as in d–f. (j) Huh7R cells transfected with EV or septin 9_i1 were stained for V5 tag (red) and diacylglycerol acyltransferase-1 (DGAT1) (green). White squares indicate the area shown in higher magniﬁcation. Bar graph represents DGAT1 ﬂuorescence intensity in 50 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 we postulated that MTs could play a role in septin 9-induced LD growth. Consequently, we performed a nocodazole washout (Fig. 5). Treatment of Huh7R cells expressing septin 9_i1 with nocodazole resulted in the dispersion of LDs throughout the cell and decreased their size. One hour after nocodazole removal, LDs started to gather around the nucleus and increased in size. These changes were accompanied by the partial recovery of the microtubule ﬁlamentous structure (Fig. 5a). Finally, LDs were restored to their initial size and relocated around the nucleus 3 h after nocodazole removal (Fig. 5b–d). Taken together, these results indicate that MT dynamics are required for septin 9 to induce LD growth and perinuclear clustering. production of PtdIns5P (ref. 48). First, the effect of YM201636 was veriﬁed by PtdIns5P immunostaining in Huh7R cells (Fig. 7a). Subsequently, the cells were transfected either with an empty vector or a septin 9_i1 construct and treated with YM201636. Moreover LD size and level increased in cells expressing septin 9_i1 and were surrounded by septin 9_i1 ﬁlaments as described in Fig. 4. YM201636 treatment profoundly reduced the LD size (Fig. 7b,c and Supplementary Fig. 8a) and decreased TAGs concomitantly with an increase of DAGs (Supplementary Fig. 8b–d). Interestingly, YM201636 treatment disrupted septin 9_i1 (Fig. 7b; Supplementary Fig. 8a) and MT ﬁlaments and prevented both co-localization (Fig. 7c). Furthermore, PtdIns5P addition to Huh7R cells transfected with septin 9 siRNA partly rescued the disruption of MT ﬁlaments and LD clustering caused by septin 9 depletion (Supplementary Fig. 9). Thus, these data demonstrated that PtdIns5P is a crucial regulator of septin 9 structural and functional features. PtdIns5P binds to septin 9 and promotes LD growth. To further identify the mechanism underlying septin 9 assembly and its role on LD, we assessed the importance of phosphoinositides (PI). The phospholipid monolayer that surrounds LDs plays a role in the regulation of LDs morphology. Phosphatidylcholine stabilizes the tension and reduces LD growth, while phosphatidic acid contributes to LD expansion44. PtdIns was also detected on LD surface11, however, the presence and the role of its phosphorylated derivatives, PIs, on LD growth have not been investigated. Therefore, we identiﬁed the PIs that bind to septin 9 by performing a protein–lipid overlay assay using a glutathione- S-transferase (GST)-tagged recombinant protein (Fig. 6a). NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication Data from the assay revealed that septin 9 bound speciﬁcally to mono- phosphorylated PtdIns (PtdIns3P, PtdIns4P and PtdIns5P) and the strongest interaction was observed with PtdIns5P while no signal was detected using GST protein alone as a negative control (Fig. 6a), supporting the speciﬁcity of the observed binding signals. g p Interestingly our data revealed that both septin 9 and HCV-regulated PtdIns5P levels. Indeed infection of Huh7.5 cells using JFH-1 particles increased PtdIns5P (Supplementary Fig. 10a). Moreover PtdIns5P increased in Huh7R cells compared with naive Huh7 cells and transfection of the later cells with septin 9_i1 cDNA signiﬁcantly enhanced the PtdIns5P signal (Supplementary Fig. 10b,c). Septin 9 PBR is required for its assembly and LD growth. Septins bind PIs in vitro via the polybasic region (PBR) near their N terminus26,49. The conserved sequence encompassing residues aa289 to aa294 is boxed in Fig. 8a. Thus, as another approach to assess the importance of PI for septin 9 structural and functional features, we generated a PBR-deleted mutant (septin 9_del1) from the cDNA of V5-tagged septin 9_i1. The recombinant proteins from the entire septin 9_i1 and the deleted construct were produced in Escherichia coli, puriﬁed to near homogeneity, and analysed by SDS gel electrophoresis (Fig. 8b). Coomassie blue staining revealed a major band of the expected molecular weight (70 kDa) for both septin 9_i1 and the deleted mutant, while some lower molecular weight species co-puriﬁed with septin 9_i1 (Fig. 8b). Immunoblot (Fig. 8c) conﬁrmed that all detected protein bands were recognized by a V5-tag antibody. Puriﬁed recombinant proteins were used to perform a lipid overlay assay. Septin 9_i1 bound PtdIns3P, PtdIns4P, and PtdIns5P as expected, although a signal was observed with phosphatidic acid and PtdIns(3, 5)P2. The lipid-binding signal was strongly reduced for septin 9_del1 (Fig. 8d). Subsequently Huh7R cells35 were transfected with the septin 9_i1, septin 9_del1 cDNAs, or empty vector (EV). Septin 9_i1 formed ﬁlaments whereas septin 9_del1 could not form ﬁlaments (Fig. 8e). Expression of septin 9_i1 increased LD in the perinuclear region compared to EV-transfected cells (Fig. 8e,f). In contrast, septin 9_del1 expression signiﬁcantly reduced the total level of LDs and their perinuclear distribution (Fig. 8e,f). These changes in the LD distribution were accompanied by signiﬁcant decrease of the LD size and number (Fig. 8g). Furthermore septin 9_i1 ﬁlament structures co-localized with MTs. By contrast, the mutant septin 9_del1 lost the ﬁlamentous structure and co-localization with MTs. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication Time after nocodazole washout (WO) 1 h 0 Periphery Perinuclear Perinuclear Periphery % of LDs intensities % of LDs intensities 1 0.5 0 0 0 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 W Septi 1 2 LD size (µm2) * * * * 0.6 0.4 0.2 LD s b c d Periphery Perinuclear Perinuclear Periphery % of LDs intensities % of LDs intensities 1 0.5 0 0 0 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 1 2 LD size (µm2) ** * * * * 0.6 0.4 0.2 LD size b c d Figure 5 \| Septin 9 regulates LDs in microtubules-dependent manner. (a) Huh7R cells transfected with septin 9_i1 were treated with nocodazole (33 mM) for 2 h at 37 C and placed on ice for 1 h. Then cells were washed ﬁve times with ice-cold culture medium to remove the nocodazole and moved at 37 C for indicated time in the ﬁgure before staining for V5 tag (green), LDs (red) and microtubules (grey) with b tubulin. (b) Schematic representation shows the perinuclear and peripheral regions. (c) Percentage of LDs intensity in the perinuclear and peripheral regions of 20 cells from two independent experiments. (d) LD size analysed in 20 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, Po0 0001 Scale bar 10 mm b Periphery Perinuclear Perinuclear Periphery % of LDs intensities % of LDs intensities 1 0.5 0 0 0 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 1 2 LD size (µm2) * * * * 0.6 0.4 0.2 LD size b c d 0 Ctrl 0 1 2 WO (h) Septin 9_i1 1 2 LD size (µm2) ** * * LD size d d c Figure 5 \| Septin 9 regulates LDs in microtubules-dependent manner. (a) Huh7R cells transfected with septin 9_i1 were treated with nocodazole (33 mM) for 2 h at 37 C and placed on ice for 1 h. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication The Pearson co-localization coefﬁcient (Rr) between septin 9 and MTs dropped from 0.56±0.01 in cells expressing septin 9_i1 to 0.17±0.03 in cells expressing septin 9_del1 and the RGB line proﬁle showed a similarity between MTs and septin 9_i1 cellular distribution (Fig. 8h). These results indicate that PBR is necessary for the formation of septin 9 ﬁlamentous structures and LD growth. Moreover, PBR is involved in MTs organization and highlighted the critical role of septin 9 in cytoskeleton organization. g Furthermore, we showed that PtdIns3P, PtdIns4P and PtdIns5P promoted a signiﬁcant increase in LD size compared with non-treated cells, whereas PtdIns(4,5)P2 evaluated as a non-binding septin PI, had no signiﬁcant effect (Fig. 6b,c). PtdIns5P was the most efﬁcient (Fig. 6b,c), consistent with the results in Fig. 6a. Moreover, septin 9 assembled surrounding the LDs that became more visible upon the addition of PtdIns5P (Fig. 6b). Overall, these data revealed the crucial role of mono- phosphorylated PIs in the expansion of LDs and the organization of septin 9 high-order structures. Thus, we focused on PtdIns5P, which was the most effective on LDs. PtdIns5P speciﬁcally binds to the plant homeodomain (PHD) ﬁnger found in many chromatin-remodelling proteins, and the PHD domain of the tumour suppressor inhibitor of growth (ING2) was proposed to function as a receptor of PtdIns5P (ref. 45). We used the biotinylated GST-tagged PHD domain of ING2 as a probe to visualize cellular PtdIns5P, as previously described46. As expected, compared with untreated cells, PtdIns5P addition strongly increased both the PHD signal and the LD size and conﬁrmed that addition of exogenous PtdIns5P increased its intracellular level (Supplementary Fig. 7a–c). To validate the role of PtdIns5P on LDs, we used another approach based on IpgD, the virulence factor of Shigella ﬂexneri, which is a PtdIns(4,5)P2-4-phosphatase responsible for the profound increase of PtdIns5P in S. ﬂexneri-infected cells47. Transfection of Huh7R cells with GFP-tagged-IpgD cDNA (GFP-IpgD) increased PtdIns5P signal (Supplementary Fig. 7d) and LD size and accumulation (Fig. 6d). PIKfyve regulates septin 9 ﬁlaments and LD accumulation. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication To further study the interaction between septin 9 and PtdIns5P and its importance in LD accumulation, we used YM201636, a selective inhibitor of FYVE ﬁnger containing phosphoinositide kinase (PIKfyve), a major contributor to the intracellular NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 a Septin 9_i1 Time after nocodazole washout (WO) 0 1 h 3 h Control MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge a Septin 9_i1 Time after nocodazole washout (WO) 0 1 h 3 h Control MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge MTs LDs MTs/LDs V5 tag Merge Periphery Perinuclear Perinuclear Periphery % of LDs intensities % of LDs intensities 1 0.5 0 0 0 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 Ctrl 0 1 2 WO (h) Septin 9_i1 1 2 LD size (µm2) ** * * * * 0.6 0.4 0.2 LD size b c d Figure 5 \| Septin 9 regulates LDs in microtubules-dependent manner. (a) Huh7R cells transfected with septin 9_i1 were treated with nocodazole (33 mM) for 2 h at 37 C and placed on ice for 1 h. Then cells were washed ﬁve times with ice-cold culture medium to remove the nocodazole and moved at 37 C for indicated time in the ﬁgure before staining for V5 tag (green), LDs (red) and microtubules (grey) with b tubulin. (b) Schematic representation shows the perinuclear and peripheral regions. (c) Percentage of LDs intensity in the perinuclear and peripheral regions of 20 cells from two independent experiments. (d) LD size analysed in 20 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, *Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication Then cells were washed ﬁve times with ice-cold culture medium to remove the nocodazole and moved at 37 C for indicated time in the ﬁgure before staining for V5 tag (green), LDs (red) and microtubules (grey) with b tubulin. (b) Schematic representation shows the perinuclear and peripheral regions. (c) Percentage of LDs intensity in the perinuclear and peripheral regions of 20 cells from two independent experiments. (d) LD size analysed in 20 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, Po0.0001. Scale bar, 10 mm. Septin 9 regulates LD growth in naive cells. Unravelling this new role of septin 9 in LD behaviour led us to examine its relevance independently of HCV infection. We performed a set of experiments using naive Huh7 cells. Septin 9_i1 expression signiﬁcantly increases LD size comparing to EV-transfected cells (Supplementary Fig. 11a,b). Additionally, neutral lipids analysis revealed an increase TAG in septin 9_i1-transfected cells (Supplementary Fig. 11c,d). Expression of GFP-IpgD Septin 9 regulates LD growth in naive cells. Unravelling this new role of septin 9 in LD behaviour led us to examine its relevance independently of HCV infection. We performed a set of experiments using naive Huh7 cells. Septin 9_i1 expression 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 0 0.4 0.8 1.2 1.6 2 GST Septin 9 LPA LPC PtdIns PtdIns (3)P PtdIns (4)P PtdIns (5)P PtdIns (3,4)P2 PtdIns (3,5)P2 PtdIns (4,5)P2 PtdIns (3,4,5)P3 PA PS Blank Non treatment Non treatment PtdIns3P PtdIns4P PtdIns5P PtdIns(4,5)P2 Control IpgD-GFP 0.5 LD size (µm2) LD size (µm2) 0 1 1.5 2 2.5 3 3.5 4 IpgD-GFP Control GFP / LDs Septin 9 Septin 9 Septin 9 Septin 9 Septin 9 LDs LDs LDs LDs LDs Merge Merge Merge Merge Merge LDs 3D reconstruction GFP / LDs * LD size LD size + PtdIns3P + PtdIns5P + PtdIns(4,5)P2 + PtdIns4P PE PC S1P c d a b Figure 6 \| Mono-phosphate phosphoinositides regulate LDs size. (a) PIP strip overlay assay: PIP strips were incubated either with glutathion-S- transferase (GST) used as a negative control or with puriﬁed septin 9_i3 GST tagged at 0.5 mg ml 1 and analysed with anti GST antibody. LPA, lysophosphatidic acid, LPC, lysophosphocholine, PtdIns, phosphatidylinositol, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, PA, phosphatidic acid, PS, phosphatidylserine, PE, phosphatidylethanolamine, PC, phosphatidylcholine, S1P, sphingosine 1-phosphate. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication (b) Huh7R cells were treated with cell-permeant PtdIns3P, PtdIns4P, PtdIns5P or PtdIns (4, 5) P2 at 30 mM for 15 min be ﬁxing and staining for septin 9 (green) and LDs (red). The dot squares indicate the zoom area. (c) Bar graph shows the LD siz in Huh7R cells treat described in b. The results were obtained from at least 20 cells for each treatment from three independent experiments. (d) Huh7R cells were transf with IpgD-GFP construct or not (control) for 48 h and stained for LDs (red). Bar graph represents LDs size measured in 15 cells from two indepen experiments. Values are means±s.e.m. Student’s t-test was used. Po0.001, Po0.0001. Scale bar, 10 mm. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms1 GST Septin 9 LPA LPC PtdIns PtdIns (3)P PtdIns (4)P PtdIns (5)P PtdIns (3,4)P2 PtdIns (3,5)P2 PtdIns (4,5)P2 PtdIns (3,4,5)P3 PA PS Blank Non treatment Septin 9 Septin 9 Septin 9 Septin 9 Septin 9 LDs LDs LDs LDs LDs Merge Merge Merge Merge Merge + PtdIns3P + PtdIns5P + PtdIns(4,5)P2 + PtdIns4P PE PC S1P a b b a Septin 9 LDs Merge + PtdIns(4,5)P2 0 0.4 0.8 1.2 1.6 2 Non treatment PtdIns3P PtdIns4P PtdIns5P PtdIns(4,5)P2 LD size (µm2) LD size c IpgD-GFP Control GFP / LDs LDs 3D reconstruction GFP / LDs d d d Control IpgD-GFP 0.5 LD size (µm2) 0 1 1.5 2 2.5 3 3.5 4 * LD size c c Control Figure 6 \| Mono-phosphate phosphoinositides regulate LDs size. (a) PIP strip overlay assay: PIP strips were incubated either with glutathion-S- transferase (GST) used as a negative control or with puriﬁed septin 9_i3 GST tagged at 0.5 mg ml 1 and analysed with anti GST antibody. LPA, lysophosphatidic acid, LPC, lysophosphocholine, PtdIns, phosphatidylinositol, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, PA, phosphatidic acid, PS, phosphatidylserine, PE, phosphatidylethanolamine, PC, phosphatidylcholine, S1P, sphingosine 1-phosphate. (b) Huh7R cells were treated with cell-permeant PtdIns3P, PtdIns4P, PtdIns5P or PtdIns (4, 5) P2 at 30 mM for 15 min befor ﬁxing and staining for septin 9 (green) and LDs (red). The dot squares indicate the zoom area. (c) Bar graph shows the LD siz in Huh7R cells treated a described in b. The results were obtained from at least 20 cells for each treatment from three independent experiments. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication Arrows indicated the area present below at a higher magniﬁcation. Bar graph shows LD size in 30 cells from th experiments. (c) Huh7R cells transfected with septin 9_i1 and treated with YM201636 were stained for V5 tag (green) and microtubules (red) tubulin. The dot squares indicate the zoomed areas shown as a 2D images with a black background, and to the right is a 3D reconstruction imag grey background and longitudinal section (right down). Pearson’s Correlation coefﬁcient (Rr) for septin and microtubules was calculated from 20 c three independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, *Po0.0001. Scale bar, 10 mm. LD size (µm2) 0 0.5 1.5 1 2 Septin 9_i1 + YM 201636 Septin 9_i1 EV EV + YM 201636 ** LDs / V5 tag Empty vector Septin 9_i1 Septin 9_i1 + YM 201636 Empty vector + YM 201636 LDs size b b MTs / V5 tag Septin 9_i1 Rr = 0.53±0.02 Rr = 0.25±0.017 c Septin 9_i1 + YM 201636 c Figure 7 \| Huh7R treatment with YM201636 causes decrease of LDs size and disrupts septin 9 and microtubules ﬁlaments. (a) Huh7R cells treated or not with YM201636 were stained for PtdIns5P (PHD) (green). Dot yellow squares indicate the zoomed area shown in 2D image with a black background and in 3D reconstruction images with white background. Bar graph shows PtdIns5P ﬂuorescence intensity analysis of at least in 60 cells from three independent experiments. (b) Huh7R cells were transfected with either empty vector (EV) or septin 9_i1 then treated with YM201636 and stained for V5 tag (green) and LDs (red). The dot squares indicate the zoomed area shown in a 2D image with a black background, and to the right is a 3D reconstruction image with a grey background. Arrows indicated the area present below at a higher magniﬁcation. Bar graph shows LD size in 30 cells from three experiments. (c) Huh7R cells transfected with septin 9_i1 and treated with YM201636 were stained for V5 tag (green) and microtubules (red) with b tubulin. The dot squares indicate the zoomed areas shown as a 2D images with a black background, and to the right is a 3D reconstruction image with a grey background and longitudinal section (right down). Pearson’s Correlation coefﬁcient (Rr) for septin and microtubules was calculated from 20 cells from three independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, Po0.0001. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication (d) Huh7R cells were transfecte with IpgD-GFP construct or not (control) for 48 h and stained for LDs (red). Bar graph represents LDs size measured in 15 cells from two independen experiments. Values are means±s.e.m. Student’s t-test was used. Po0.001, *Po0.0001. Scale bar, 10 mm. septin 9_del1 expressing Huh7 cells (Supplementary Fig. 13d). Taken together, these results indicate the fundamental role of septin 9 and PtdIns5P in LD accumulation and cytoskeleton dynamic independently of HCV infection. (Supplementary Fig. 11e,f) or the addition of exogenous mono- phosphorylated PIs to huh7 cells increase LD size (Supplementary Fig. 12a). In contrast, septin 9_del1 transfection or treatment with YM201636 in septin 9_i1-transfected cells signiﬁcantly decrease LD size (Supplementary Fig. 12b), disrupted septin 9 ﬁlamentous structure and its co-localization with MTs (Supplementary Fig. 13a–c). Similar effects were observed on actin ﬁlaments in Septin 9/PtdIns5P regulates sodium oleate-induced LD growth. To emphasize the function of septin 9 and PtdIns5P in lipid 10 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 PHD PHD No treatment Fluorescence intensity of PHD (a.u.)/µm2 + YM 201636 Non treatment + YM 201636 * 0 50 100 a PHD PHD No treatment Fluorescence intensity of PHD (a.u.)/µm2 + YM 201636 Non treatment + YM 201636 *** 0 50 100 a a LD size (µm2) 0 0.5 1.5 1 2 * Septin 9_i1 + YM 201636 Septin 9_i1 EV EV + YM 201636 LDs / V5 tag MTs / V5 tag Empty vector Septin 9_i1 Septin 9_i1 Rr = 0.53±0.02 Rr = 0.25±0.017 Septin 9_i1 + YM 201636 Empty vector + YM 201636 LDs size + 201 treatment 201636 c b Septin 9_i1 + YM 201636 Figure 7 \| Huh7R treatment with YM201636 causes decrease of LDs size and disrupts septin 9 and microtubules ﬁlaments. (a) Huh7R cells t not with YM201636 were stained for PtdIns5P (PHD) (green). Dot yellow squares indicate the zoomed area shown in 2D image with a black bac and in 3D reconstruction images with white background. Bar graph shows PtdIns5P ﬂuorescence intensity analysis of at least in 60 cells from independent experiments. (b) Huh7R cells were transfected with either empty vector (EV) or septin 9_i1 then treated with YM201636 and staine tag (green) and LDs (red). The dot squares indicate the zoomed area shown in a 2D image with a black background, and to the right is a 3D recon image with a grey background. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 0 LD size (µm2) LD number LD size LD number / cell * * * * * * * * 0 1 2 3 4 5 0 1 2 3 4 5 Intensity (a.u.) * EV I1 Del1 EV I1 Del1 EV I1 Del1 EV I1 Del1 % of LDs intensities % of LDs intensities 50 0 100 0 100 200 300 0 10 20 30 0.5 1 1.5 2 NH2 COOH Proline rich domain GTP-binding domain Septin unique element 281 289 294 300 Septin 9_i1 Septin 9_del1 37 50 75 100 37 50 75 100 I1 Del1 Del1 I1 Del1 I1 Setpin 9_ Empty vector Septin 9_i1 V5 tag Septin 9_del1 Septin 9_i1 Septin 9_del1 V5 tag LDs Perinuclear Periphery LD distribution Distance (µm) Distance (µm) Perinuclear Periphery 100 100 200 200 Fluorescence (a.u.) 0 MTs Merge 10 20 30 5 10 15 20 25 50 100 150 200 50 100 150 200 50 100 150 200 0 10 20 30 0 Merge V5 tag LDs Merge V5 tag LDs Merge Setpin 9_ Setpin 9_ LPA S1P PtdIns(3,4)P2 PtdIns(3,5)P2 PtdIns(4,5)P2 PtdIns(3,4,5)P3 LPC PE PS PA Blank V5 tag PC PtdIns PtdIns(3)P PtdIns(4)P PtdIns(5)P Polybasic domain G1 G3 G4 a b c d e g h f letion of septin 9/PIs interaction domain disturbs localization of septin 9 with microtubules and affects LDs accumulation. domain organization: the polybasic domain is located at N-terminal of GTP-binding domain, which is recognized by three motifs G305QSGLGK311), G3 (DXXG ¼ D362TPG365), G4 (XKXD ¼A444KAD447). Down: septin 9_i1 and septin 9_del1 sequence in the . The boxed sequence (289RRKAMK294) has been deleted in septin 9_del1. (b) Coomassie blue stained SDS–PAGE gel of puriﬁed del1. (c) Immunoblot analysis of puriﬁed septin 9_i1 and septin 9_del1. (d) PIP strip overlay assay for V5 tag, puriﬁed septin 9_i1 med as in Fig. 6a. Lipid-bound V5 fusion proteins were detected with anti-V5 antibody. (e) Left: Huh7R cells were transfected eithe septin 9_del1 and stained for V5 tag (green) and LDs (red). Right: radial proﬁle plots of the presented cells show LDs intensity dis and perinuclear regions. (f) LDs intensity in the perinuclear and peripheral regions of 36 cells from 5 independent experiments pe e. (g) LD size and LD number in 36 cells from 5 independent experiments done as described in e. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunication Scale bar, 10 mm. 11 NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (h) Staining of V5 tag (gree (MTs) with b tubulin (red) in Huh7R cells transfected with septin 9_i1 or septin 9 del1. The Pearson’s correlation coefﬁcient (Rr) m two independent experiments is presented on the merge panels. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (a) Septin 9 domain organization: the polybasic domain is located at N-terminal of GTP-binding domain, which is recognized by three motifs G1 (GXXXXGK ¼G305QSGLGK311), G3 (DXXG ¼ D362TPG365), G4 (XKXD ¼A444KAD447). Down: septin 9_i1 and septin 9_del1 sequence in the polybasi domain region. The boxed sequence (289RRKAMK294) has been deleted in septin 9_del1. (b) Coomassie blue stained SDS–PAGE gel of puriﬁed septin 9_ and septin 9_del1. (c) Immunoblot analysis of puriﬁed septin 9_i1 and septin 9_del1. (d) PIP strip overlay assay for V5 tag, puriﬁed septin 9_i1 and sept 9_del1 performed as in Fig. 6a. Lipid-bound V5 fusion proteins were detected with anti-V5 antibody. (e) Left: Huh7R cells were transfected either with EV septin 9 i1 or septin 9 del1 and stained for V5 tag (green) and LDs (red) Right: radial proﬁle plots of the presented cells show LDs intensity distribution in 9/PIs interaction domain disturbs localization of septin 9 with microtubules and affects LDs accumulation. Figure 8 \| Deletion of septin 9/PIs interaction domain disturbs localization of septin 9 with microtubules and aff Figure 8 \| Deletion of septin 9/PIs interaction domain disturbs localization of septin 9 with microtubules and affects LDs accumulation. (a) Septin 9 domain organization: the polybasic domain is located at N-terminal of GTP-binding domain, which is recognized by three motifs G1 (GXXXXGK ¼G305QSGLGK311), G3 (DXXG ¼ D362TPG365), G4 (XKXD ¼A444KAD447). Down: septin 9_i1 and septin 9_del1 sequence in the polybasic domain region. The boxed sequence (289RRKAMK294) has been deleted in septin 9_del1. (b) Coomassie blue stained SDS–PAGE gel of puriﬁed septin 9_i1 and septin 9_del1. (c) Immunoblot analysis of puriﬁed septin 9_i1 and septin 9_del1. (d) PIP strip overlay assay for V5 tag, puriﬁed septin 9_i1 and septin 9_del1 performed as in Fig. 6a. Lipid-bound V5 fusion proteins were detected with anti-V5 antibody. (e) Left: Huh7R cells were transfected either with EV or septin 9_i1 or septin 9_del1 and stained for V5 tag (green) and LDs (red). Right: radial proﬁle plots of the presented cells show LDs intensity distribution in the peripheral and perinuclear regions. (f) LDs intensity in the perinuclear and peripheral regions of 36 cells from 5 independent experiments performed as described in e. (g) LD size and LD number in 36 cells from 5 independent experiments done as described in e. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 The right panels are green, red, line proﬁle blots of the pink lines Student’s t-test was used Po0 05 Po0 001 Po0 0001 Scale bar 10 mm NH2 COOH Proline rich domain GTP-binding domain Septin unique element 281 289 294 300 Septin 9_i1 Septin 9_del1 37 50 75 100 37 50 75 100 I1 Del1 Del1 I1 Del1 I1 Setpin 9_ Empty vector Septin 9_i1 Septin 9_del1 V5 tag LDs Perinuclear Periphery LD distribution Distance (µm) Fluorescence (a.u.) 0 10 20 30 5 10 15 20 25 50 100 150 200 50 100 150 200 50 100 150 200 0 10 20 30 0 Merge V5 tag LDs Merge V5 tag LDs Merge Setpin 9_ Setpin 9_ LPA S1P PtdIns(3,4)P2 PtdIns(3,5)P2 PtdIns(4,5)P2 PtdIns(3,4,5)P3 LPC PE PS PA Blank V5 tag PC PtdIns PtdIns(3)P PtdIns(4)P PtdIns(5)P Polybasic domain G1 G3 G4 a b c d e NH2 COOH Proline rich domain GTP-binding domain Septin unique element 281 289 294 300 Septin 9_i1 Septin 9_del1 37 50 75 100 37 50 75 100 I1 Del1 Del1 I1 Del1 I1 Setpin 9_ Setpin 9_ Setpin 9_ LPA S1P PtdIns(3,4)P2 PtdIns(3,5)P2 PtdIns(4,5)P2 PtdIns(3,4,5)P3 LPC PE PS PA Blank V5 tag PC PtdIns PtdIns(3)P PtdIns(4)P PtdIns(5)P Polybasic domain G1 G3 G4 a b c d a a 37 50 75 100 Del1 I1 Setpin 9_ b 37 50 75 100 37 50 75 100 Del1 I1 Del1 I1 Setpin 9_ Setpin 9_ b c b d I1 Del1 Setpin 9_ V5 tag c Empty vector Septin 9_i1 Septin 9_del1 V5 tag LDs Fl ( ) Merge V5 tag LDs Merge V5 tag LDs Merge e Perinuclear Periphery LD distribution Distance (µm) Fluorescence (a.u.) 0 10 20 30 5 10 15 20 25 50 100 150 200 50 100 150 200 50 100 150 200 0 10 20 30 0 e Fluorescence (a.u.) * * * * * * EV I1 Del1 EV I1 Del1 % of LDs intensities % of LDs intensities 50 0 100 0 10 20 30 Perinuclear Periphery f 0 1 2 0 1 2 Intensity (a.u.) V5 tag Septin 9_i1 Septin 9_del1 Distan 100 100 200 200 MTs Merge h h h 0 1 2 3 4 5 0 1 2 3 4 5 Intensity (a.u.) Distance (µm) 100 100 200 200 0 LD size (µm2) LD number LD size LD number / cell * * * EV I1 Del1 EV I1 Del1 0 100 200 300 0.5 1 1.5 2 g g Figure 8 \| Deletion of septin 9/PIs interaction domain disturbs localization of septin 9 with microtubules and affects LDs accumulation. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 Listeria monocytogenes23, Candida albicans50 and Chlamydia trachomatis27. Moreover, septin 6 controls HCV replication via a direct interaction with a RNA binding protein, acting as a scaffold protein to facilitate the positioning of the HCV replication complex29. However, the roles of the LDs were not investigated in these studies related to septin/pathogen interactions. Here we demonstrated that IpgD, the virulence effector of S. ﬂexneri increased the cellular content of PtdIns5P and induced LD growth. Even though this result conﬁrmed the role of PtdIns5P in LD growth and accumulation, its enabled speculation on the potential role of LDs in infection by S. ﬂexneri. Therefore, the ﬁndings of this study can be extended to other types of pathogens beyond HCV. homoeostasis, Huh7 cells were treated with sodium oleate (0–200 mM), commonly used to induce LD formation. Neutral lipid analysis revealed a concentration-dependent increase of TAG and a decrease of DAG due to the addition of sodium oleate, validating the effect of sodium oleate (Supplementary Fig. 14). The immunoﬂuorescence data indicated a dose-dependent enlargement of the LDs and an increase of endogenous septin 9. Remarkably, in cells supplemented with 200 mM sodium oleate, septin 9 ﬁlaments were visible around the very large clusters of LDs (Fig. 9a). Immunoblot data conﬁrmed the gradual increase of septin 9 expression with the sodium oleate concentration and a concomitant increase of PLIN2 expression (Fig. 9b). p g To further investigate the association of septin 9 with LD formation, we performed membrane ﬂotation assays37 (Fig. 9c,d) using Huh7 cells and the same cells treated with 100 mM sodium oleate as described in methods. Twenty-ﬁve fractions were collected and assessed by western blotting for septin 9, septin 2. PLIN2 and calnexin were analysed as markers of LDs and ER, respectively. The lowest-density membrane fractions (fractions 1–8) are called ‘LD-rich’ and the fractions (9–15) represented the ‘ER/endosome-rich’fractions, while the fractions (16–25) contained soluble and aggregated proteins. In non-treated Huh7 cells, septin 9 was mainly found in the ER/endosome-rich and the soluble/aggregated protein fractions, but not within low-density LD-rich fractions (Fig. 9c, upper panels). Similar distribution proﬁles were observed for septin 2 and calnexin, while PLIN2 was detectable in ‘LD-rich’ fractions. These data suggested that, in non-treated cells, septins are mainly associated with LDs in the ER. ARTICLE This distribution changed in the presence of sodium oleate, septin 9 and septin 2 increased in low-density LD-rich fractions with high expression of PLIN2 and decreased from the ER/endosome-rich fractions totally depleted of PLIN2. There was no visible change in the distribution proﬁle of the different proteins among the soluble/aggregated protein fractions. Overall, these results suggested that septin 9 and septin 2 are associated with the ER site of LD biogenesis and sodium oleate promoted LD biogenesis and enhanced septins association with LDs. Subsequently, we explored the role of septin 9 and PtdIns5P on oleate-induced LD accumulation. Transfection with septin 9_i1 cDNA of Huh7 cells treated with 100 mM sodium oleate increased the LD size, while transfection with the septin 9_del1 construct or with si3 septin 9 (Fig. 10a,b) and YM201636 treatment (Fig. 10c), produced the opposite effect, indicating that septin 9 and PtdIns5P regulated oleate-induced LD biogenesis. y Despite the numerous publications about LD biology, several questions related to their biogenesis, growth and dynamics remain unanswered1,4,44. LD formation likely occurs at the ER, and different mechanisms are involved in LD growth, including growth from nascent LDs in the ER or in the vicinity of the ER via the incorporation of TAGs and phospholipids locally synthesized into LDs. These proposed mechanisms arose from the observations that key enzymes of phospholipid and neutral lipid synthesis are present on the LD surface44. LDs might also expand via fusion of existing LDs. These fusion processes include the absorption of small LDs by large LDs mediated by Fsp27 (a 27 kDa fat-speciﬁc protein) enriched at the contact site between LDs51,52. Fusion mechanisms can also be mediated via phospholipid metabolizing enzymes that produce fusogenic compounds at the LD surface53,54.This fusion between LDs involves the SNARE proteins55. In this study, we demonstrated that septin 9 signiﬁcantly regulates the cellular content of TAG, a neutral lipid component essential for LD biogenesis. Furthermore, septin 9 promotes perinuclear accumulation of neutral lipid synthesis enzymes such as DGAT1 and regulates the expression of PLIN2 and PLIN3, two PLINfamily proteins crucial for LD formation. These data support a role of septin 9 in LD growth and biogenesis. Nonetheless, we cannot rule out a role for septin 9 in LD fusion mechanisms. LD fusions are MT-dependent processes42. In this study, we demonstrated the requirement for dynamic MTs for septin 9-induced LD growth. ARTICLE Furthermore, LD fusion and size is related to the composition of the surrounding monolayer of phospholipids, including PtdIns. Interestingly, we demonstrated that septin 9 binds to mono-phosphorylated PtdIns, which is consistent with previously published data for Saccharomyces cerevisiae49. These PIs promote LD growth and are reminiscent of the role of PtdIns4P in the homoeostasis of LDs in yeasts56,57. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (h) Staining of V5 tag (green) and microtubules (MTs) with b tubulin (red) in Huh7R cells transfected with septin 9_i1 or septin 9 del1. The Pearson’s correlation coefﬁcient (Rr) measured in 30 cells from two independent experiments is presented on the merge panels. The right panels are green, red, line proﬁle blots of the pink lines. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, *Po0.0001. Scale bar, 10 mm. Figure 8 \| Deletion of septin 9/PIs interaction domain disturbs localization of septin 9 with microtubules and affects LDs accumulation. (a) Septin 9 domain organization: the polybasic domain is located at N-terminal of GTP-binding domain, which is recognized by three motifs G1 (GXXXXGK ¼G305QSGLGK311), G3 (DXXG ¼ D362TPG365), G4 (XKXD ¼A444KAD447). Down: septin 9_i1 and septin 9_del1 sequence in the polybasic domain region. The boxed sequence (289RRKAMK294) has been deleted in septin 9_del1. (b) Coomassie blue stained SDS–PAGE gel of puriﬁed septin 9_i1 and septin 9_del1. (c) Immunoblot analysis of puriﬁed septin 9_i1 and septin 9_del1. (d) PIP strip overlay assay for V5 tag, puriﬁed septin 9_i1 and septin 9_del1 performed as in Fig. 6a. Lipid-bound V5 fusion proteins were detected with anti-V5 antibody. (e) Left: Huh7R cells were transfected either with EV or septin 9_i1 or septin 9_del1 and stained for V5 tag (green) and LDs (red). Right: radial proﬁle plots of the presented cells show LDs intensity distribution in the peripheral and perinuclear regions. (f) LDs intensity in the perinuclear and peripheral regions of 36 cells from 5 independent experiments performed as described in e. (g) LD size and LD number in 36 cells from 5 independent experiments done as described in e. (h) Staining of V5 tag (green) and microtubules (MTs) with b tubulin (red) in Huh7R cells transfected with septin 9_i1 or septin 9 del1. The Pearson’s correlation coefﬁcient (Rr) measured in 30 cells from two independent experiments is presented on the merge panels. The right panels are green, red, line proﬁle blots of the pink lines. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001, Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 different components are present on the LD addition, LDs interact with different cellular l di th ER G l i d it h d i 63 speciﬁc PI and Rab GTPase composition could b might help deﬁne LD identity as for endosomal Th t d f th t ib ti f PI i lli Septin 9 Septin 2 PLIN2 Calnexin Septin 9 Septin 2 PLIN2 c d Calnexin LD-rich LD-rich LD-rich ER/endosome-rich ER/endosome -rich ER/endosome -rich a b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 75 37 50 100 75 37 50 100 Non treatment Sodium oleate Septin 9 Septin 2 PLIN2 Calnexin Densitometry Densitometry Non treatment Sodium oleate 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 7 7 7 7 7 7 7 7 9 9 9 9 9 9 9 9 11 11 11 11 11 11 11 11 15 15 15 15 15 15 15 15 13 13 13 13 13 13 13 13 Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction 8,000 4,000 0 2,000 1,000 0 0 2,000 1,000 0 3,000 2,500 0 5,000 2,000 2,000 0 0 4,000 4,000 6,000 6,000 0 10,000 15,000 5,000 4,000 2,000 Soluble/ aggregated proteins Sodium oleate (µM) 0 50 100 200 0 0 0.5 0.5 1 1 1.5 Densitometry Septin 9/actin Sodium oleate (µM) Densitometry PLIN2/actin Septin 9 PLIN2 * * * * Sodium oleate (µM) 0 0 50 50 100 100 200 200 Septin 9 / LDs 3D reconstruction Sodium oleate (µM) 0 50 100 200 Septin 9 PLIN2 Actin 75 50 50 oleate treatment increases septin 9 and LDs in the perinuclear region and in LD-rich fractions. (a) Huh7 cells w m was supplemented with 0, 50, 100 or 200 mM sodium oleate complex for 24 h then stained for septin 9 (green indicate the zoomed area shown as a 3D reconstruction image with a white background. (b) Immunoblot analysis of n a. Bar graphs below show the analysis from three independent experiments. (c) Huh7 cells were treated or not w mitted to membrane ﬂotation assay and analysed by western blot for septin 9, septin 2, PLIN2 and Calnexin. Discussion In this study, we showed a deregulation of septin expression in HCV-induced cirrhosis compared with that in the normal liver. Subsequent studies using HCV-infected cells revealed an unexpected role for septin 9 in the control of cellular lipid homoeostasis through its binding to PtdIns5P required for microtubules stabilization and LD perinuclear accumulation. As depicted in our model in Fig. 10d this new role of septin 9 was also observed independently of the presence of HCV in naı¨ve cells and in sodium oleate-induced LD accumulation. Additionally, septin 9 regulates HCV replication and assembles around the perinuclear clusters of the core in HCV-infected cells. Septin 9 promotes the perinuclear recruitment of proteins coating the LD surface such as PLIN2 and PLIN3 as well as the HCV core and the non-structural NS5A. Furthermore, PLIN3 playing an essential role in the HCV replication process37.Thus, one could speculate that HCV recruits septin 9 to control LD growth and creates a lipid-enriched environment, facilitating HCV replication. Septins are crucial cytoskeletal components that are exploited by many pathogens such as S. ﬂexneri, y PtdIns5P is mainly present in the plasma and intracellular membranes, including the smooth ER and Golgi58, and remains the least characterized among the eight different PIs59. Notably, PtdIns5P regulates septin 9 assemblies in high-order structures. PtdIns5P partly rescued the loss of microtubule ﬁlaments induced by septin 9 knockdown. These results are consistent with the reported role for PIKfyve in microtubule-dependent vesicular trafﬁcking60 and strongly indicate a new role for PtdIns5P as a partner of septin 9 in the regulation of MT and LD dynamics, providing new insights on PtdIns5P biological role. Consequently, we proposed that septin 9 could act as a scaffold that establishes contacts between MTs and LDs via binding to PtdIns5P that coats the surface of LDs and promotes LD growth and intracellular movement. These results revealed an unprecedented role for PIs in LD biology in mammals, although a detailed mechanism awaits further investigations. PIs are important regulators of membrane trafﬁcking in coordination with small GTPase proteins such as ARF GTPase, Rab GTPases, and the subunits of the COPI 13 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (d) Dens proﬁle from western blot in c is shown. Values are means±s.e.m. Student’s t-test was used. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 Po0.05, Po0.00 \| b 0 0.5 1 1.5 Densitometry Septin 9/actin Sodium oleate (µM) Septin 9 * 0 50 100 200 Sodium oleate (µM) 0 50 100 200 Septin 9 PLIN2 Actin 75 50 50 a Sodium oleate (µM) 0 50 100 200 Septin 9 / LDs 3D reconstruction b a 0 3D reconstruction 0 0.5 1 µ Densitometry PLIN2/actin PLIN2 ** * Sodium oleate (µM) 0 50 100 200 Septin 9 Septin 2 PLIN2 Calnexin Septin 9 Septin 2 PLIN2 c Calnexin LD-rich ER/endosome-rich 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 75 37 50 100 75 37 50 100 Non treatment Sodium oleate Soluble/ aggregated proteins 0 0.5 1 µ Densitometry PLIN2/actin PLIN2 ** * Sodium oleate (µM) 0 50 100 200 nstruction Septin 9 Septin 2 PLIN2 Calnexin Septin 9 Septin 2 PLIN2 c Calnexin LD-rich ER/endosome-rich 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 75 37 50 100 75 37 50 100 Non treatment Sodium oleate Soluble/ aggregated proteins c d LD-rich LD-rich ER/endosome -rich ER/endosome -rich Septin 9 Septin 2 PLIN2 Calnexin Densitometry Densitometry Non treatment Sodium oleate 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 7 7 7 7 7 7 7 7 9 9 9 9 9 9 9 9 11 11 11 11 11 11 11 11 15 15 15 15 15 15 15 15 13 13 13 13 13 13 13 13 Fraction Fraction Fraction Fraction Fraction Fraction Fraction Fraction 8,000 4,000 0 2,000 1,000 0 0 2,000 1,000 0 3,000 2,500 0 5,000 2,000 2,000 0 0 4,000 4,000 6,000 6,000 0 10,000 15,000 5,000 4,000 2,000 d LD-rich ER/endosome -rich Septin 9 Septin 2 PLIN2 Calnexin Densitometry Densitometry Non treatment 1 1 1 1 3 3 3 3 5 5 5 5 7 7 7 7 9 9 9 9 11 11 11 11 15 15 15 15 13 13 13 13 Fraction Fraction Fraction Fraction 8,000 4,000 0 2,000 1,000 0 0 2,000 0 4,000 6,000 4,000 2,000 d LD-rich ER/endosome -rich Densitometry Sodium oleate 1 1 1 1 3 3 3 3 5 5 5 5 7 7 7 7 9 9 9 9 11 11 11 11 15 15 15 15 13 13 13 13 Fraction Fraction Fraction Fraction 2,000 1,000 0 3,000 2,500 0 5,000 2,000 0 4,000 6,000 0 10,000 15,000 5,000 Densitometry Figure 9 \| Sodium oleate treatment increases septin 9 and LDs in the perinuclear region and in LD-rich fractions. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (d) Proposed model: HCV infection, oleic acid treatment and IpgD, the virulence fac neri may increase the cellular level of both septin 9 and PtdIns5P promoting their interaction and the subsequent organization of MTs wth and accumulation. TURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 0 2 4 6 0 0.5 1 1.5 2 2.5 Control si3 septin 9 Control si3 septin 9 LD size (µm2) LD size (µm2) * * HCV, oleic acid, IpgD (Shigella) Septin 9 PtdIns5P LD LD LD LD LD Microtubules Septin 9 PtdIns5P LD size LD size Control si3 Septin 9 Control YM 201636 LDs LDs LDs LDs c b Endoplasmic reticulum d Figure 10 \| Septin 9 and PtdIns(5)P are required for LDs accumulation in Huh7 sodium oleate-treated cells. (a) Huh7 cells transfected with EV, septin 9_i1, or septin 9_del1 and treated with 100 mM sodium oleate complex were strained for LDs and V5 tag. Dot squares indicates the zoom area shown below in 3D reconstruction images. Bar graph shows LD size analysis of 33 cells from two independent experiments. (b) Huh7 cells transfected with non-targeting (control) or septin 9 siRNA (si3) were treated with 100 mM sodium oleate complex then strained for LDs and V5 tag. Bar graph shows LD size analysis of 30 cells from two independent experiments. (c) Huh7 cells treated with 100 mM sodium oleate then with 160 mm YM201636 for 1 h before staining for LDs (red). Bar graphs present mean LD size of 60 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.001, Po0.0001. Scale bar, 10 mm. (d) Proposed model: HCV infection, oleic acid treatment and IpgD, the virulence factor of Shigella ﬂexneri may increase the cellular level of both septin 9 and PtdIns5P promoting their interaction and the subsequent organization of MTs to control LD growth and accumulation. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 (a) Huh7 cells were grown for 24 h. The culture medium was supplemented with 0, 50, 100 or 200 mM sodium oleate complex for 24 h then stained for septin 9 (green) and LDs (red). The white squares indicate the zoomed area shown as a 3D reconstruction image with a white background. (b) Immunoblot analysis of septin 9 and PLIN2 in cells treated as in a. Bar graphs below show the analysis from three independent experiments. (c) Huh7 cells were treated or not with sodium oleate at 100 mM were submitted to membrane ﬂotation assay and analysed by western blot for septin 9, septin 2, PLIN2 and Calnexin. (d) Densitometry analysis of protein expression proﬁle from western blot in c is shown. Values are means±s.e.m. Student’s t-test was used. Po0.05, *Po0.001. Scale bar, 10 mm. Figure 9 \| Sodium oleate treatment increases septin 9 and LDs in the perinuclear region and in LD-rich fractions. (a) Huh7 cells were grown for 24 h. The culture medium was supplemented with 0, 50, 100 or 200 mM sodium oleate complex for 24 h then stained for septin 9 (green) and LDs (red). The white squares indicate the zoomed area shown as a 3D reconstruction image with a white background. (b) Immunoblot analysis of septin 9 and PLIN2 in cells treated as in a. Bar graphs below show the analysis from three independent experiments. (c) Huh7 cells were treated or not with sodium oleate at 100 mM were submitted to membrane ﬂotation assay and analysed by western blot for septin 9, septin 2, PLIN2 and Calnexin. (d) Densitometry analysis of protein expression proﬁle from western blot in c is shown. Values are means±s.e.m. Student’s t-test was used. Po0.05, Po0.001. Scale bar, 10 mm. complex. These different components are present on the LD surface61,62. In addition, LDs interact with different cellular organelles, including the ER, Golgi and mitochondria63. Therefore, the existence of a different pool of LDs with a speciﬁc PI and Rab GTPase composition could be postulated and might help deﬁne LD identity as for endosomal compartments64. Thus, a study of the contribution of PI signalling to LD biogenesis and growth is of great interest in LD biology. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 14 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 0 2 4 6 0 0.5 1 1.5 2 2.5 Control si3 septin 9 Control si3 septin 9 LD size (µm2) LD size (µm2) LD size (µm2) * * * HCV, oleic acid, IpgD (Shigella) Septin 9 PtdIns5P LD LD LD LD LD Microtubules Septin 9 PtdIns5P 0 0.5 1 1.5 2 2.5 3 EV Septin 9_i1 Septin 9_del1 LD size LD size LD size EV Septin 9_i1 Septin 9_del1 V5 tag/LDs V5 tag/LDs V5 tag/LDs Control si3 Septin 9 Control YM 201636 LDs LDs LDs LDs a c b Endoplasmic reticulum d ptin 9 and PtdIns(5)P are required for LDs accumulation in Huh7 sodium oleate-treated cells. (a) Huh7 cells transfected with EV, LD size (µm2) * 0 0.5 1 1.5 2 2.5 3 EV Septin 9_i1 Septin 9_del1 LD size EV Septin 9_i1 Septin 9_del1 V5 tag/LDs V5 tag/LDs V5 tag/LDs a Septin 9_i1 0 2 4 6 0 0.5 1 1.5 2 2.5 Control si3 septin 9 Control si3 septin 9 LD size (µm2) LD size (µm2) LD size (µm2) * * * HCV, oleic acid, IpgD (Shigella) Septin 9 PtdIns5P LD LD LD LD LD Microtubules Septin 9 PtdIns5P 0 0.5 1 1.5 2 2.5 3 EV Septin 9_i1 Septin 9_del1 LD size LD size LD size Control si3 Septin 9 Control YM 201636 LDs LDs LDs LDs c b Endoplasmic reticulum d ure 10 \| Septin 9 and PtdIns(5)P are required for LDs accumulation in Huh7 sodium oleate-treated cells. (a) Huh7 cells transfected tin 9_i1, or septin 9_del1 and treated with 100 mM sodium oleate complex were strained for LDs and V5 tag. Dot squares indicates the wn below in 3D reconstruction images. Bar graph shows LD size analysis of 33 cells from two independent experiments. (b) Huh7 cel h non-targeting (control) or septin 9 siRNA (si3) were treated with 100 mM sodium oleate complex then strained for LDs and V5 tag. Bar size analysis of 30 cells from two independent experiments. (c) Huh7 cells treated with 100 mM sodium oleate then with 160 mm YM2 before staining for LDs (red). Bar graphs present mean LD size of 60 cells from two independent experiments. Values are means±s.e.m. St s used. Po0.001, *Po0.0001. Scale bar, 10 mm. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Methods The cDNA of GFP-IpgD was a gift from He´le`ne Tronche`re (INSERM UMR-S 1048, I2MC and Universite´ Paul Sabatier). The cDNA of GFP-IpgD was a gift from He´le`ne Tronche`re (INSERM UMR-S 1048, I2MC and Universite´ Paul Sabatier). Chemicals. Phosphoinositides (PtdIns4P diC8 Cat#P4008, PtdIns5P diC4 Cat#P5004, PtdIns3P diC8 Cat#P3008, PtdIns(4,5)P2 diC8 Cat#P4508) and PIP strip Cat#P-6001 were purchased from Echelon. Nocodazole Cat#487928 from Calbiochem, YM201636 Cat#sc-204193 from Santa Cruz. Sodium oleate Cat#o-7501 and Isopropyl b-D-1-thiogalactopyranoside (Cat# I6758) were purchased from Sigma-Aldrich. Albumin BSA FFA Cat#126575 was from Calbiochem. Puriﬁed septin 9-GST isoform 3 Cat# H00010801-P01 was purchased from Clinisciences. Puriﬁed GST was from GenScript and V5 peptide control for V5 antibody was from Novus Biologicals. The transfection of cDNA and siRNA were performed using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics) and Lipofectamine RNAiMAX reagent (Invitrogen) respectively following the manufacturer’s protocol. The transfection was performed for 48 h unless indicated otherwise in the text. The transfection of cDNA and siRNA were performed using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics) and Lipofectamine RNAiMAX reagent (Invitrogen) respectively following the manufacturer’s protocol. The transfection was performed for 48 h unless indicated otherwise in the text. Protein production and puriﬁcation. E. coli BL21(DE3) Rosetta cells (#70953-3 Novagen) were transformed with pET21d septin 9_i1 V5/His and PET21d septin 9_del1 V5/His plasmids and all culture media contained 100 mg ml 1 ampicillin, 34 mg ml 1 chloramphenicol. Cells from a single colony were used to seed an overnight 20 ml preculture of LB medium. One litre LB medium cultures from these precultures were grown at 37 C with shaking to an optical density (OD600 nm) of 0.9. The temperature was brought down to 28 C and protein expression was then induced with 1 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) from Sigma (Cat# I6758) for 4 h. The cells were harvested by centrifugation and the pellet was stored at 80 C until used. The cell pellet was suspended in 15 ml of 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 10% glycerol, 20 mM imidazole, 0.1% Triton X-100, and one protease inhibitor cocktail EDTA free tablet (Roche). Cell lysis was performed by sonication on ice and the cell lysate was clariﬁed by centrifugation for 30 min at 4 C to 40,000g. Methods Anti septin 9 Cat#ab38314 (WB:1/500, IF:1/25), anti HCV core Cat#ab2740 (WB:1/1,000, IF:1/100), anti atubulin Cat#ab15246 (WB:1/1,000, IF:1/100), anti septin 2 Cat#ab88657(WB:1/500, IF:1/50), mouse and rabbit anti-V5 tag Cat#ab27671 (WB:1/1,000, IF:1/400), Cat#ab9116 (WB:1/1,000, IF:1/400) respectively and anti ADFP/PLIN2 Cat#ab52355 (WB:1/2,000, IF:1/100) were from abcam; anti btubulin Cat# T4026 (WB:1/1,000, IF:1/100) and anti TIP47/PLIN3 Cat#HPA006427 (WB:1/1,000, IF:1/100) from Sigma-Aldrich; anti-Actin Cat#sc-1616 (WB:1/1,000) and anti-PLIN2 Cat#sc-32450 (WB:1/250, IF:1/25), anti DGAT1 Cat#sc-32861 (IF:1/100) from Santa Cruz Biotechnology, anti HCV NSA Cat#HCM-131-5 (WB:1/1,000, IF:1/100) from amsbio. The mouse antibody to HCV envelope protein E2 (WB:1/500) was a gift from Dr Jean Dubuisson (CIIL, Lille, France)66. JFH-1/HCVcc production and inoculation. Huh7.5 were infected with cell culture-grown HCV JFH-1 at a multiplicity of infection (MOI) of 0.5 TCID50 cell 1 (50% tissue culture infective doses). After incubation at 37 C for 16 h, the inoculum was removed then cells were washed three times with DMEM and were further maintained in culture for the indicated time in the legend and text. Reverse transcription and real-time PCR analysis. Total RNA was isolated using RNAble, solution (Eurobio) and HCV RNA copy number was quantiﬁed using the SuperScript III Platinium One-Step quantitative RT–PCR system (Invitrogen) and a Ligth Cycler Fast Start DNA MasterPlus SYBR Green I mix (Roche). PCR was carried on a Light Cycler 480 Real-Time PCR System (Roche). The sequence of the Primer used in real-time RT–PCR analysis was presented in Supplementary Tables 1 and 6-FAM-CCTTGTGGTACTGCCTGA-MGB (Applied Biosystems, Foster City, CA, USA) was used as internal probe. To analyse the septin 9 transcript variants, total RNA was isolated using RNeasy Mini Kit 50 (Cat# 74104 QIAGEN) and applied to reverse transcription using RevertAid First Strand cDNA Synthesis Kit (Cat#K1622 Fermentas). The cDNA was analysed by qPCR using QuantiTect SYBR Green PCR Kit (Cat#204143 QIAGEN) and a 7500 Fast Real-Time PCR System (Applied Biosystems). Reaction parameters were 30 min at 50 C, 15 min at 95 C, followed by 45 cycles of 15 s at 94 C, 30 s at 55 C and 30 s at 72 C. The triplicate mean values were calculated using GAPDH gene transcription as reference for normalization. Used qRT–PCR primers sequences are presented in the Supplementary Table 1. Secondary antibodies and dyes. Anti mouse IgG-HRP and anti rabbit IgG-HRP form GE healthcare (WB:1/1,000). Anti-goat IgG-HRP Cat#sc-2020 (WB:1/1,000) from Santa Cruz. Methods After ﬁltering the supernatant the protein was isolated on a 1 ml metal afﬁnity column (HisTrap HP, GE Healthcare) pre equilibrated in 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 10% glycerol, 20 mM imidazole and eluted with 50 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 300 mM imidazole. The fractions containing the protein of interest were further puriﬁed by cation exchange chromatography. The pooled fractions were diluted with a 1:5 ratio to a ﬁnal solution of 30 mM tris pH8, 100 mM NaCl, 1 mM EDTA and then incubated with 1 ml of a strong cation exchange resin (Macro-Prep 25S, BioRad). The resin was packed in a column and eluted with a 100–600 mM NaCl linear gradient. Fractions containing the majority of protein were obtained B350 mM NaCl. Septins were then ﬂash-frozen and stored at 80 C. Cell lines and culture conditions. Human hepatocarcinoma cells Huh7.5, Huh7 and stable Huh7 cells expressing HCV genomic replicon (Huh7R) were used35. Cells were maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Invitrogen) containing 4.5 g l 1 glucose and supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids (GibcoBRL) and 1% penicillin/streptomycin (GibcoBRL). For Huh7R the described medium was supplemented with G418 at 400 mg ml 1. Cells were tested for mycoplasma contamination weekly. Cell treatments. Cells treatment with sodium oleate: complexes of sodium oleate with BSA were prepared as previously reported65. Brieﬂy, the solution containing 20 mM sodium oleate and 2.4 mM bovine serum albumin-fatty acid free (BSA FFA) was heated to 55 C. The obtained complex was diluted in a pre-warmed culture medium at indicated concentration before addition to the cells. The treated cells were maintained in culture for 24 h and collected either for immunoblot or immunoﬂuorescence experiments. p Cells treatment with YM201636: cells were incubated for 1 h at 37 C in the pre-warmed culture medium containing 160 mM of YM201636 and collected for analyses. Cells treatment with PIs: PIs as a lyophilized powder were solubilized at 500 mM in water by vortex. The solution was then diluted in Dulbecco’s phosphate-buffered saline (DPBS) to the concentration indicated in the legends of the experiment. Cells were washed with DPBS and the PIs solution was added to the cell for indicated time. Primary antibodies. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 from their pcDNA3.1/V5-His-TOPO vector. The resulting DNA fragments have been inserted in a pET21d vector using In-Fusion HD Cloning System Cat#639648 (Clontech) pET21d in-frame with the C-terminal 6-histidine coding sequence of the vector and according to manufacturer recommendations to obtain pET21d septin 9_i1 V5/His and PET21d septin 9_del1 V5/His. All the primers sequences were presented in the Supplementary Table 1. In conclusion, this study provides new insights for research on the increasing pathologies associated with lipid metabolism disorders and cancer. Methods Alexa Fluor 633 Cat#A21136, A21070 and A21082 (IF:1/100), Alexa Fluor 568 Cat#A11004, A11011 and A11057 (IF:1/100), Alexa Fluor 488 Cat#A11001, A21206 and A21202 (IF:1/100), streptavidin Alexa-488 Cat#S32354 (IF:1/100), streptavidin Alexa-568 Cat#S11226 (IF:1/100). Actin was stained with Alexa Fluor 594 phalloidin Cat#a12381 (IF:1/100) and nuclei with Hoechst Cat#H21486 (IF:1/5,000), both are from Invitrogen. Lipophilic ﬂuorescence dye LD 540 (IF:1/100) from Christoph Thiele (Bonn University, Germany), was used to stain lipid droplets67. GST-2XPHD preparation. The GST-2XPHD probe was produced in BL21 RIL þ bacteria strain (Stratagene) and puriﬁed with glutathione Sepharose 4B (Amersham). After dialysis in PBS overnight and concentration with Vivaspin 500, MCO 10000, the recombinant protein was biotinylated using the BiotinTag Micro biotinylation kit (Sigma-aldrich) according to the manufacturer. siRNA, plasmids and transfection reagents. siRNAs from septin 9 were a Stealth RNAi siRNA (si1: Cat#SEPT9HSS173895, si3: Cat# SEPT9HSS173897) from Invitrogen and non-targeting siRNA (Cat#sc-37007) was from Santa Cruz. The sequences targeting septin 9 (si1 and si3) are presented in the Supplementary Table 2. siRNA, plasmids and transfection reagents. siRNAs from septin 9 were a Stealth RNAi siRNA (si1: Cat#SEPT9HSS173895, si3: Cat# SEPT9HSS173897) from Invitrogen and non-targeting siRNA (Cat#sc-37007) was from Santa Cruz. The sequences targeting septin 9 (si1 and si3) are presented in the Supplementary Table 2. Immunoﬂuorescence. Cells were grown on coverslips, ﬁxed with paraformalde- hyde 4% for 20 min and permeabilized for 20 min at 37 C using the permeabilizing buffer (PFS): DPBS containing saponin (Cat#10294440 Fisher scientiﬁc) 0.025% m.v 1, gelatin from cold water ﬁsh skin (Cat#G7041 Sigma 0.7% m.v 1). Then cells were incubated with primary antibody for 2 h washed three times for 5 min with PFS and incubated with the appropriate secondary antibodies or with the dye for 90 min. The coverslips were mounted using Prolong Gold (Cat#P36934 Invitrogen). For PtdIns5P staining the cells on coverslips were ﬁxed and permeabilized as described above and then incubated with recombinant The cDNA of septin 9 isoform 1 transcript within pcDNA3.1/V5-His-TOPO vector (cDNA septin 9_i1) was a gift from Dr Russell (CCRCB, Queens University Belfast). The pET21d vector was a gift from Dr Pierre Nioche (INSERM UMR-S 1124, University of Paris Descartes). The cDNA of septin 9 isoform 1 transcript within pcDNA3.1/V5-His-TOPO vector (cDNA septin 9_i1) was a gift from Dr Russell (CCRCB, Queens University Belfast). The pET21d vector was a gift from Dr Pierre Nioche (INSERM UMR-S 1124, University of Paris Descartes). NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 0 2 4 6 0 0.5 1 1.5 2 2.5 Control si3 septin 9 Control si3 septin 9 LD size (µm2) LD size (µm2) * * LD size LD size Control si3 Septin 9 Control YM 201636 LDs LDs LDs LDs c b d 0 2 4 6 0 0.5 1 1.5 2 2.5 Control si3 septin 9 LD size (µm2) LD size (µm2) * LD size Control si3 Septin 9 Contro LDs LDs c b d b c d LD LD LD LD LD Microtubules Septin 9 PtdIns5P Endoplasmic reticulum Figure 10 \| Septin 9 and PtdIns(5)P are required for LDs accumulation in Huh7 sodium oleate-treated cells. (a) Huh7 cells transfected with EV, septin 9_i1, or septin 9_del1 and treated with 100 mM sodium oleate complex were strained for LDs and V5 tag. Dot squares indicates the zoom area shown below in 3D reconstruction images. Bar graph shows LD size analysis of 33 cells from two independent experiments. (b) Huh7 cells transfected with non-targeting (control) or septin 9 siRNA (si3) were treated with 100 mM sodium oleate complex then strained for LDs and V5 tag. Bar graph shows LD size analysis of 30 cells from two independent experiments. (c) Huh7 cells treated with 100 mM sodium oleate then with 160 mm YM201636 for 1 h before staining for LDs (red). Bar graphs present mean LD size of 60 cells from two independent experiments. Values are means±s.e.m. Student’s t-test was used. Po0.001, Po0.0001. Scale bar, 10 mm. (d) Proposed model: HCV infection, oleic acid treatment and IpgD, the virulence factor of Shigella ﬂexneri may increase the cellular level of both septin 9 and PtdIns5P promoting their interaction and the subsequent organization of MTs to control LD growth and accumulation. NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications 15 ARTICLE References Microbiol. Immunol. 369, 263–288 (2013). q g g Blot Scans are presented in the ﬁgures and the uncropped scan are supplied in Supplementary Fig. 15. Blot Scans are presented in the ﬁgures and the uncropped scan are supplied in Supplementary Fig. 15. Supplementary Fig. 15. 7. Barba, G. et al. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc. Natl Acad. Sci. USA 94, 1200–1205 (1997). PIP strip overlay assay. Membranes PIP Strips (Echelon Biosciences) were blocked with 3% BSA FFA dissolved in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (3% BSA FFA PBS-T) at room temperature for 60 min, then incubated overnight at 4 C with the same buffer containing the puriﬁed protein of interest at a concentration of 0.5 mg ml 1 or the puriﬁed corresponding tag (V5 or GST) at equivalent molar concentration used as controls. The membranes were washed and bounded proteins were detected with suitable antibody. 8. Paul, D., Madan, V. & Bartenschlager, R. Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host Microbe 16, 569–579 (2014). 9. Miyanari, Y. et al. The lipid droplet is an important organelle for hepatitis C virus production. Nat. Cell Biol. 9, 1089–1097 (2007). 10. Boulant, S. et al. Structural determinants that target the hepatitis C virus core protein to lipid droplets. J. Biol. Chem. 281, 22236–22247 (2006). 11. Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R. & Fujimoto, T. The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition. J. Biol. Chem. 277, 44507–44512 (2002). Neutral lipids analysis. This analysis was performed using the facility service of Metatoul in Toulouse (http://www.metatoul.fr). One million of cells were used for lipid extraction according to Bligh and Dyer68 using dichloromethane/methanol/ water (2.5:2.5:2.1, v/v/v) mix, in the presence of the internal standards: 4 mg of stigmasterol, 4 mg of glycerid dimyristoleate, 4 mg of cholesteryl heptadecanoate, 8 mg of glyceryl trinonadecanoate. Organic phases were collected and evaporated to dryness and neutral lipids were separated through a SPE cartridge. Brieﬂy SiOH cartridge (200 mg, Macherey Nagel) was equilibrated with 2 ml of dichloromethane and the lipid extract was put down in 20 ml of a solution of dichloromethane containing 10% omethanol and loaded. Neutral lipids were eluted with 2 ml of the same mixture. References The ﬁnal extract were concentrated and dissolved in 20 ml of ethyl acetate. One microlitre of the lipid extract was analysed by gas-liquid chromatography on a FOCUS Thermo Electron system using an Zebron-1 Phenomenex fused silica capillary columns (5 m 0,32 mm i.d., 0.50 mm ﬁlm thickness)69. The temperature of the Oven was programmed from 200 to 350 C at a rate of 5 C per min and the carrier gas was hydrogen (0.5 bar). The injector and the detector were at 315 and 345 C, respectively. p 12. Olofsson, S.-O. et al. Lipid droplets as dynamic organelles connecting storage and efﬂux of lipids. Biochim. Biophys. Acta 1791, 448–458 (2009). and efﬂux of lipids. Biochim. Biophys. Acta 1791, 448–458 (20 13. Balla, T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137 (2013). 14. Maehama, T., Fukasawa, M., Date, T., Wakita, T. & Hanada, K. A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication. Biochem. Biophys. Res. Commun. 440, 150–156 (2013). 15. Gassama-Diagne, A. et al. Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells. Nat. Cell Biol. 8, 963–970 (2006). 16. Martin-Belmonte, F. et al. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128, 383–397 (2007). 17. Awad, A. et al. SHIP2 regulates epithelial cell polarity through its lipid product, which binds to Dlg1, a pathway subverted by hepatitis C virus core protein. Mol. Biol. Cell 24, 2171–2185 (2013). Membrane ﬂotation assay. The assay was performed as recently reported37. Huh7 cells treated with 100 mM sodium oleate for 24 h were trypsinized, washed and counted. Cells (15 106) total for each sample were resuspended in 1.75 ml PBS-containing 0.25 M sucrose (PBS/sucrose) supplemented with protease inhibitor cocktail (Sigma). The cells were then lysed with 200 passages in a tight ﬁtting dounce homogenizer to ensure B90% lysis. The cell lysate was then spun at 2,500g for 10 min at 4 C to pellet cellular debris and nuclei. Equal amounts of protein (2.5 mg) for each cell type was adjusted in volume to 1 ml with PBS/sucrose and mixed with 1 ml of 60% iodixanol (Sigma) resulting in a 30% iodixanol concentration. A discontinuous iodixanol gradient (10%, 20%) was layered on top 18. Nishihama, R., Onishi, M. & Pringle, J. R. New insights into the phylogenetic distribution and evolutionary origins of the septins. Biol. References Immunoblot. Cells were washed with ice-cold DPBS and lysed on ice using the following buffer: 20 mM Tris, HCl, 100 mM NaCl, 1% Triton X-100 at PH 7.4 containing protease inhibitors (cOmplete ULTRA Cat#05892970001 Roche). The proteins were separated on SDS–PAGE and electrotransferred onto nitrocellulose membrane. After transfer, the membrane was saturated in DPBS containing 0.1% Tween 20 and 5% milk. Primary antibodies were added overnight at 4 C or for 2 h at room temperature depending on the antibody. The membranes were washed with DPBS and incubated for 1 h at room temperature with appropriate secondary antibody coupled with peroxidase. ECL plus kit (Cat#32132 Thermo Scientiﬁc) was used for protein detection. Chemiluminescent signal was detected by G:BOX Chemi Fluorescent & Chemiluminescent Imaging System from SYNGENE. Blot quantiﬁcation was done using ImageJ software. Immunoblot. Cells were washed with ice-cold DPBS and lysed on ice using the following buffer: 20 mM Tris, HCl, 100 mM NaCl, 1% Triton X-100 at PH 7.4 containing protease inhibitors (cOmplete ULTRA Cat#05892970001 Roche). The proteins were separated on SDS–PAGE and electrotransferred onto nitrocellulose membrane. After transfer, the membrane was saturated in DPBS containing 0.1% Tween 20 and 5% milk. Primary antibodies were added overnight at 4 C or for 2 h at room temperature depending on the antibody. The membranes were washed with DPBS and incubated for 1 h at room temperature with appropriate secondary antibody coupled with peroxidase. ECL plus kit (Cat#32132 Thermo Scientiﬁc) was used for protein detection. Chemiluminescent signal was detected by G:BOX Chemi Fluorescent & Chemiluminescent Imaging System from SYNGENE. Blot quantiﬁcation was done using ImageJ software. 1. Pol, A., Gross, S. P. & Parton, R. G. Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites. J. Cell Biol. 204, 635–646 (2014). 2. Welte, M. A. Expanding roles for lipid droplets. Curr. Biol. 25, R470–R481 (2015). 3. Walther, T. C. & Farese, R. V. Lipid droplets and cellular lipid metabolism. Annu. Rev. Biochem. 81, 687–714 (2012). 4. Ohsaki, Y., Suzuki, M. & Fujimoto, T. Open questions in lipid droplet biology. Chem. Biol. 21, 86–96 (2014). 5. Bickel, P. E., Tansey, J. T. & Welte, M. A. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim. Biophys. Acta 1791, 419–440 (2009). 6. Yamane, D., McGivern, D. R., Masaki, T. & Lemon, S. M. Liver injury and disease pathogenesis in chronic hepatitis C. Curr. Top. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 of the lysate/iodixanol mixture, and the gradient was spun at 200,000g for 16 h at 4 C in a MLS 50 Rotor. A total of 25 fractions were collected from top to bottom. The fractions were mixed 1:1 with Laemmli buffer for immunoblot. Bio-GST-2XPHDING2 (prepared as describe above) in 1% BSA-PBS for 1 h. Following incubation, cells were washed and incubated for another hour with streptavidin Alexa-488 (1/300) (Invitrogen) and Hoechst for nuclei staining, mounted and analysed for immunoﬂuorescence. Transcriptome analysis. Transcriptome data set GSE14323 (ref. 31) was downloaded from NCBI website (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE14323): normalized matrix by users (RMA method in the R affy package) was employed to realize analysis. This data set is composed of 19 control samples of normal liver and 49 samples of cirrhosis. R software version 3.2.2 was employed to perform, principal component analysis, boxplots, heatmap, two-sided Student t-test, also algorithm of random forest (RF). The learning machine application RF was performed by initializing the function tuneRF to determine the mtry parameter with the optimal error of bag. The algorithm was implemented with a learning based on 500 classiﬁcation trees with the optimal mtry parameter previously determined70. Images acquisition and analysis. Images acquired with a Leica TCS SP5 AOBS tandem confocal microscope were analysed by Icy bioimage analysis software for 3D reconstruction. For colocalization analysis, images were treated by ImageJ software, the plugin ‘Intensity Correlation Analysis’ was used to generate the Pearson’s correlation coefﬁcient (Rr) which range from 1 (perfect exclusion) to þ 1 (perfect correlation). To determine LD distribution in cells, the plugin ‘Radial proﬁl’ was used. For analysis, a circle was deﬁned at the periphery of each cell and the plugin produces a proﬁle plot of normalized integrated intensities around concentric circles as a function of distance from a point in the image, considered here as the centre of the cell. The concentric circles were assembled in three circle bands, the ﬁrst corresponding to the area of the nuclei and the rest corresponding to the cytoplasm was divided in two equal bands (the band near the nuclei is considered as the ‘perinuclear’ and the other the ‘periphery’). The intensity in each band was calculated from the total integrated intensities around concentric circles present in the band. Statistical analyses. Unpaired Student’s t-tests was used and statistical signiﬁcance was determined at Po0.05; Po0.001, *Po0.0001. Data availability. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 The data that support the ﬁndings of this study are available from the corresponding author upon request. Transcriptome data set GSE14323 (ref. 31) was downloaded from NCBI website (http://www.ncbi.nlm.nih.gov/geo/ query/acc.cgi?acc=GSE14323). To calculate the size and number of LDs, images obtained by confocal microscopy were processed by background subtraction and standardized thresholding (default), cell ROI was made by free hand selection tool, then LDs size and number was obtained by ImageJ analyse particles function (particle area less than 0.01 mm2 were excluded). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 NATURE COMMUNICATIONS \| 7:12203 \| DOI: 10.1038/ncomms12203 \| www.nature.com/naturecommunications Methods cDNA septin 9_i1 vector was used as a template to generate pcDNA V5 septin 9_del1 using the QuikChange II XL Site-Direced Mutagenesis Kit (Cat#200521) from agilent following the manufacturer’s recommendations. Therefore the septin 9_i1 V5/His tag and septin 9_del1 V5/His sequences have been ampliﬁed by PCR 16 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12203 22. Fung, K. Y. Y., Dai, L. & Trimble, W. S. Cell and molecular biology of septins. Int. Rev. Cell Mol. Biol. 310, 289–339 (2014). 53. Guijas, C., Rodrı´guez, J. P., Rubio, J. M., Balboa, M. A. & Balsinde, J. Phospholipase A2 regulation of lipid droplet formation. Biochim. Biophys. Acta 1841, 1661–1671 (2014). Phospholipase A2 regulation of lipid droplet formation. Biochim. Biophys. Acta 1841, 1661–1671 (2014). 23. Mostowy, S. & Cossart, P. Septins: the fourth component of the cytoskeleton. Nat. Rev. Mol. Cell Biol. 13, 183–194 (2012). 54. Andersson, L. et al. PLD1 and ERK2 regulate cytosolic lipid droplet formation. J. Cell Sci. 119, 2246–2257 (2006). 24. Bertin, A. et al. Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin ﬁlament assembly and organization. J. Mol. Biol. 404, 711–731 (2010). 55. Bostro¨m, P. et al. SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity. Nat. Cell Biol. 9, 1286–1293 (2007). 25. Tanaka-Takiguchi, Y., Kinoshita, M. & Takiguchi, K. Septin-mediated uniform bracing of phospholipid membranes. Curr. Biol. 19, 140–145 (2009). 56. Foti, M., Audhya, A. & Emr, S. D. Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology. Mol. Biol. Cell 12, 2396–2411 (2001). g p p p 26. Zhang, J. et al. Phosphatidylinositol polyphosphate binding to the mammalian septin H5 is modulated by GTP. Curr. Biol. 9, 1458–1467 (1999). 27. Volceanov, L. et al. Septins arrange F-actin-containing ﬁbers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion. mBio 5, e01802–e01814 (2014). morphology. Mol. Biol. Cell 12, 2396–2411 (2001). 57. Ren, J. et al. A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis. Mol. Biol. Cell 25, 712–727 (2014). 28. Bridges, A. A. & Gladfelter, A. S. Fungal pathogens are platforms for discovering novel and conserved septin properties. Curr. Opin. Microbiol. 20, 42–48 (2014). 58. Sarkes, D. & Rameh, L. E. A novel HPLC-based approach makes possible the spatial characterization of cellular PtdIns5P and other phosphoinositides. Biochem. J. 428, 375–384 (2010). 29. Kim, C. S., Seol, S. K., Song, O.-K., Park, J. H. & Jang, S. K. An RNA-binding protein, hnRNP A1, and a scaffold protein, septin 6, facilitate hepatitis C virus replication. J. Virol. 81, 3852–3865 (2007). 59. Acknowledgements 42. Bostro¨m, P. et al. Cytosolic lipid droplets increase in size by microtubule- dependent complex formation. Arterioscler. Thromb. Vasc. Biol. 25, 1945–1951 (2005). A.A. was supported by a fellowship of RII Pasteur, University of Paris XI and CHB association. C.G. was supported by a grant from Agence nationale de recherches sur le sida et les he´patites virales (ANRS). This work was supported by a grant from Association pour la Recherche sur le Cancer (ARC/SUBV/CKLQ6) to AGD. We thank T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan) for providing the pJFH1 plasmid, C. Rice (Rockefeller University, New York, NY) for the Huh7.5 cell line and H.Russell (Queen University, Belfast, UK.) for providing Septin 9_i1 construct. We also thank C. Comera (INRA, UMR1331, Toulouse) for advices on lipid staining and Christopher Thiele (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden) for providing the LD450 Dye and all the members of the INSERM 1193 for helpful discussions. We thank A. Jalil and E. Perret from the imagery services at IGR (Villejuif) and Institut Pasteur (Paris) We thank Justine Bertrand-Michel (Metatoul platform, Toulouse, France) for lipids analysis. 43. Sellin, M. E., Stenmark, S. & Gullberg, M. Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers. Mol. Biol. Cell 23, 4242–4255 (2012). 44. Yang, H., Galea, A., Sytnyk, V. & Crossley, M. Controlling the size of lipid droplets: lipid and protein factors. Curr. Opin. Cell Biol. 24, 509–516 (2012). 45. Gozani, O. et al. The PHD ﬁnger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 114, 99–111 (2003). 46. Coronas, S. et al. Elevated levels of PtdIns5P in NPM-ALK transformed cells: implication of PIKfyve. Biochem. Biophys. Res. Commun. 372, 351–355 (2008). 47. Niebuhr, K. et al. Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.ﬂexneri effector IpgD reorganizes host cell morphology. EMBO J. 21, 5069–5078 (2002). ARTICLE p y p Interaction with a Putative Cellular Receptor, CD81. J. Virol. 73, 6235–6244 (1999). 37. Vogt, D. A. et al. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein. PLoS Pathog. 9, e1003302 (2013). 67. Spandl, J., White, D. J., Peychl, J. & Thiele, C. Live cell multicolor imaging of lipid droplets with a new dye, LD540. Trafﬁc 10, 1579–1584 (2009). g 38. Yen, C.-L. E., Stone, S. J., Koliwad, S., Harris, C. & Farese, R. V. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J. Lipid Res. 49, 2283–2301 (2008). 68. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and puriﬁcation. Can. J. Biochem. Physiol. 37, 911–917 (1959). 8. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid ext puriﬁcation. Can. J. Biochem. Physiol. 37, 911–917 (1959). 69. Barrans, A. et al. Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases. J. Biol. Chem. 269, 11572–11577 (1994). p 39. Herker, E. et al. Efﬁcient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. Nat. Med. 16, 1295–1298 (2010). 40. Camus, G. et al. Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core. J. Biol. Chem. 288, 9915–9923 (2013). 70. Breiman, L. Random forests. Mach. Learn. 45, 5–32 (2001). p 41. Thiam, A. R., Farese, R. V. & Walther, T. C. The biophysics and cell biology of lipid droplets. Nat. Rev. Mol. Cell Biol. 14, 775–786 (2013). Author contributions 48. Sbrissa, D., Ikonomov, O. C., Filios, C., Delvecchio, K. & Shisheva, A. Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636. Am. J. Physiol. Cell Physiol. 303, C436–C446 (2012). A.A., J.P. and M.O. contributed equally to design and performed most of the experiments, analysed the data and prepared the ﬁgures. C.G. and P.B. performed HCV infection. M.M., V.T. and S.S. performed experiments. C.D. performed transcriptomic analyses. H.T. provided reagents. C.T and S.Br prepared recombinant proteins and analysed the data. S.Be., A.B., P.M., D.S. and C.B. discussed the data and edited the manuscript. A.G.-D. conceived and supervised the project, designed experiments and wrote the manuscript. 49. Casamayor, A. & Snyder, M. Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function. Mol. Cell. Biol. 23, 2762–2777 (2003). 50. Phan, Q. T. et al. Role of endothelial cell septin 7 in the endocytosis of Candida albicans. mBio 4, e00542–513 (2013). 51. Gong, J., Sun, Z. & Li, P. CIDE proteins and metabolic disorders. Curr. Opin. Lipidol. 20, 121–126 (2009). References Chem. 392, 681–687 (2011). rich, C. S., Erzberger, J. P. & Barral, Y. The septin family of GTP 19. Weirich, C. S., Erzberger, J. P. & Barral, Y. The septin family of GTPases architecture and dynamics. Nat. Rev. Mol. Cell Biol. 9, 478–489 (2008). g p y architecture and dynamics. Nat. Rev. Mol. Cell Biol. 9, 478–489 (2008). 20. Barral, Y. & Kinoshita, M. Structural insights shed light onto septin assemblies and function. Curr. Opin. Cell Biol. 20, 12–18 (2008). 20. Barral, Y. & Kinoshita, M. Structural insights shed light on and function. Curr. Opin. Cell Biol. 20, 12–18 (2008). 21. Dolat, L., Hu, Q. & Spiliotis, E. T. Septin functions in organ system physiology and pathology. Biol. Chem. 395, 123–141 (2014). 17 ARTICLE Viaud, J., Boal, F., Tronche`re, H., Gaits-Iacovoni, F. & Payrastre, B. Phosphatidylinositol 5-phosphate: a nuclear stress lipid and a tuner of membranes and cytoskeleton dynamics. BioEssays News Rev. Mol. Cell. Dev. Biol. 36, 260–272 (2014). p 30. Dos Santos, A. et al. Identiﬁcation of cellular targets in human intrahepatic cholangiocarcinoma using laser microdissection and accurate mass and time tag proteomics. Mol. Cell. Proteomics 9, 1991–2004 (2010). 60. Ikonomov, O. C. et al. Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi network trafﬁc of furin. J. Biol. Chem. 284, 3750–3761 (2009). g p 31. Mas, V. R. et al. Genes involved in viral carcinogenesis and tumor initiation in hepatitis C virus–induced hepatocellular carcinoma. Mol. Med. 15, 85–94 (2009). 32. Kim, M. S., Froese, C. D., Estey, M. P. & Trimble, W. S. 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https://openalex.org/W2519325934	https://kclpure.kcl.ac.uk/ws/files/57571796/The_antidepressant_roles_ZHOU_Accepted_9May2016_GOLD_VoR.pdf	English	null	The antidepressant roles of Wnt2 and Wnt3 in stress-induced depression-like behaviors	Translational psychiatry	2,016	cc-by	12,621	Citation for published version (APA): Zhou, W.-J., Xu, N., Kong, L., Sun, S.-C., Xu, X.-F., Jia, M.-Z., Wang, Y., & Chen, Z.-Y. (2016). The antidepressant roles of Wnt2 and Wnt3 in stress-induced depression-like behaviors. Translational psychiatry, 6(9), Article e892. https://doi.org/10.1038/tp.2016.122 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Zhou, W.-J., Xu, N., Kong, L., Sun, S.-C., Xu, X.-F., Jia, M.-Z., Wang, Y., & Chen, Z.-Y. (2016). The antidepressant roles of Wnt2 and Wnt3 in stress-induced depression-like behaviors. Translational psychiatry, 6(9), Article e892. https://doi.org/10.1038/tp.2016.122 1Department of Neurobiology, Shandong Provincial Key Laboratory of Mental Disorders, CAS Center for Excellence in Brain Science and Intelligence Technology, School of Medicine, Shandong University, Jinan, Shandong, China and 2Department of Clinical Laboratory, Second Hospital of Shandong University, Jinan, Shandong, China. Correspondence: Professor Y Wang or Professor Z-Y Chen, Department of Neurobiology, Shandong Provincial Key Laboratory of Mental Disorders, CAS Center for Excellence in Brain Science and Intelligence Technology, School of Medicine, Shandong University, No.44 Wenhua Xi Road, Jinan, Shandong 250012, China. E-mail: wangyue@sdu.edu.cn or zheyuchen@sdu.edu.cn 3These authors contributed equally to this work. 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Download date: 24. Oct. 2024 OPEN Citation: Transl Psychiatry (2016) 6, e892; doi:10.1038/tp.2016.122 www.nature.com/tp ORIGINAL ARTICLE The antidepressant roles of Wnt2 and Wnt3 in stress-induced depression-like behaviors W-J Zhou1,3, N Xu1,2,3, L Kong1, S-C Sun1, X-F Xu1, M-Z Jia1, Y Wang1 and Z-Y Chen1 Wnts-related signaling pathways have been reported to play roles in the pathogenesis of stress-induced depression-like behaviors. However, there is relatively few direct evidence to indicate the effect of Wnt ligands on this process. Here, we investigated the role of Wnts in mediating chronic restraint stress (CRS)-induced depression-like behaviors. We found that CRS induced a signiﬁcant decrease in the expression of Wnt2 and Wnt3 in the ventral hippocampus (VH) but not in the dorsal hippocampus. Knocking down Wnt2 or Wnt3 in the VH led to impaired Wnt/β-catenin signaling, neurogenesis deﬁcits and depression-like behaviors. In contrast, overexpression of Wnt2 or Wnt3 reversed CRS-induced depression-like behaviors. Moreover, Wnt2 and Wnt3 activated cAMP response element-binding protein (CREB) and there was CREB-dependent positive feedback between Wnt2 and Wnt3. Finally, ﬂuoxetine treatment increased Wnt2 and Wnt3 levels in the VH and knocking down Wnt2 or Wnt3 abolished the antidepressant effect of ﬂuoxetine. Taken together, our study indicates essential roles for Wnt2 and Wnt3 in CRS-induced depression-like behaviors and antidepressant. Translational Psychiatry (2016) 6, e892; doi:10.1038/tp.2016.122; published online 13 September 2016 Animals Adult male C57BL/6 mice (8 weeks old) were kept in cages in a 12 h light/ dark cycle with food and water available unless otherwise noted (Vital River Laboratories, Beijing, China). Three to four mice were housed in one cage. The room temperature was maintained at 20 ± 2 °C. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of Shandong University. Chronic stress is a risk factor for developing psychiatric diseases, such as major depression and anxiety disorders.19 Increasing evidence shows that some Wnt-related signaling molecules are altered under stress condition. For example, chronic unpredictable stress decreases the expression of Frizzled 6 (Fzd6), a Wnt receptor.20 The expression of dishevelled-2 (DVL2), a key molecule in the Wnt signaling cascade, is also downregulated in the nucleus accumbens (NAc) subjected to chronic social defeat stress.21 Acute and chronic stress increases the expression of Dickkopf-1 (Dkk-1), an endogenous inhibitor of Wnt/β-catenin signaling, in the INTRODUCTION hippocampus.22 However, there is few evidence to indicate the effect of Wnt ligands on the process of stress. Although previous microarray studies of the hippocampus have identiﬁed decreases in the Wnt2 transcript after chronic restraint stress (CRS),23 no direct results could verify the relationships between Wnts and stress-induced depression behaviors. In the present study, we have used CRS in mice as a useful animal model of depression and aimed to investigate whether selective Wnts participated in stress- induced depression-like behaviors. Major depression is a widespread psychiatric disorder with very high prevalence rate up to 15% in the world.1 Over the past decade, a number of studies have found mechanisms underlying depression and available antidepressants according. For example, glycogen synthase kinase 3 (GSK3) has been shown to participate in the pathogenesis and treatment of major depression disorders.2–5 Lithium, a well-used mood stabilizer for both bipolar and depression, can inhibit the activity of GSK3β.6 Wnt pathway plays essential roles during embryogenesis, nervous system development and adult hippocampal neuro- genesis (AHN).7–9 Wnts are secreted glycoproteins that activate different signaling pathways, including canonical Wnt/β-catenin pathway, non-canonical planar cell polarity (PCP) pathway and Wnt/Ca2+ pathway. GSK3 is primarily regulated by Wnt,10 and interestingly, it has been shown that Wnt/β-catenin signaling participates in depression disorders.11–13 The activation of Wnt/ β-catenin signaling promotes antidepressant effect, whereas the blockade of Wnt/β-catenin signaling increases depression behaviors.3,14–18 Forced swim test The forced swim test (FST) was performed in a glass cylinder (25 cm in height and 18 cm in diameter, containing18 cm of water at 22 °C) as previously described, with some modiﬁcations.30,31 Mice were forced to swim for 6 min, and the sessions were videotaped for later analysis by a blind observer. Immobility time during the last 4 min of the testing period was recorded. Immobility was deﬁned as being stationary and ﬂoating in the water with minimal movements to keep the head above water. After the swim test, mice were dried and returned to their home cages. Quantiﬁcation and imaging Images were acquired using a confocal ﬂuorescence LSM-780 microscope (Carl Zeiss, Jena, Germany) ﬁtted with standard ﬁlter sets and a standard (1 Airy disk) pinhole (Microstructural platform of Shandong University). BrdU+ and BrdU+NeuN+ cells were counted using the MetaMorph software package (Molecular Devices, Sunnyvale, CA, USA) with an unbiased stereological protocol as previously described.33 The observer was blind to the experimental design to prevent any inadvertent bias in cell counting. Every sixth section was counted for positive cells in the ventral dental gyrus (DG), which ensured that the same cell was not counted repeatedly. Quantitative real-time PCR Mice were killed immediately after CRS. Brains were removed after decapitation and different brain regions were acquired using a mouse brain slicer. Total RNA was extracted using TRNzol-A+ RNA isolation reagent (Tiangen, Beijing, China) according to the manufacturer’s instructions. Puriﬁed total RNA (500 ng) was then reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). Quantitative Real-time PCR was performed in a Cycler (Bio-Rad, Hercules, CA, USA) using SYBR Green (Roche Diagnostics, Mannheim, Germany). Primer sequences used in the paper were listed in Supplementary Table S1. Each sample was analysed in duplicate and the relative levels of mRNA were normalized for each well of the β-actin mRNA levels using the 2 −ΔΔCT method. Stereotaxic surgery and microinjection Mice were anesthetized by an i.p. injection of 5% chloral hydrate (400 mg kg −1) and then placed in a stereotaxic apparatus. Mice received VH micro-infusions. The injection coordinates, relative to bregma, were as follows: AP, −2.8 mm; lateral (L), ± 2.5 mm; and dorsoventral (V), −2.3 mm. Lentiviruses (1 μl per side) were infused into the VH, and the infusion micro-syringe was then left for diffusion for an additional 2 min. The Wnt2 or Wnt3 siRNA sequence used for lentiviruses was as followed: Wnt2 siRNA antisense, 5′-GAGGTCATTTTTCGTTGGC-3′; Wnt3 siRNA antisense, 5′ -TCGTAGATGCGAATACACT-3′. The siRNA plasmids and scramble plasmid used to package lentiviruses were purchased by Thermo Scientiﬁc Open Biosysterms (Waltham, MA, USA). The pUltra lentivirus vector was used to package Wnt2 or Wnt3-overexpression lentivirus with coexpressing a green-ﬂuorescent protein (GFP). The promoter used in the overexpression lentiviral vector is the ubiquitin promoter and the promoter used in the siRNA lentiviral vector is the U6 promoter. The lentiviruses had no cell speciﬁcity. p For evaluation of cell proliferation, mice were i.p. injected with 50 mg kg−1 5-bromo-2-deoxyuridine (BrdU, Sigma Aldrich) in 0.9% NaCl at 0800 hours and perfused 2 h later. BrdU-positive cells represented the newborn cells. For estimating the number of newly generated neurons, mice were given four injections of BrdU every 24 h at 0800 hours and killed 4 weeks after the last injection. BrdU+/NeuN+ double-labeled cells were the survived newborn neurons. For BrdU staining, brain sections were pretreated with 2N HCl for 30 min and then washed in 0.1 M borate solution (pH 8.5) for 10 min. After incubation in a blocking solution, brain sections were incubated with primary antibodies: sheep anti-BrdU (1:1000, Abcam, Cambridge, MA, USA) and mouse anti-NeuN (1:1000, Millipore, Billerica, MA, USA), followed by donkey anti-sheep Alexa 594 (1:1000, Invitrogen) and donkey anti-mouse Alexa 488 (1:1000, Life Technologies, Carlsbad, CA, USA) for 1 h. Statistical analysis For comparison of two groups, data were analyzed by Student’s t-test, followed by Bonferroni's correction. One-way analysis of variance (ANOVA) was performed for three or more groups, followed by the LSD post hoc test. When more than one factor was examined simultaneously, data were analyzed by two-way ANOVA, followed by the LSD post hoc test. For body weight, data were analyzed by repeated measures two-way ANOVA. A Dunnett’s T3 test was used to compare the differences between groups when equal variances were not assumed. P-valueso0.05 were considered statistically signiﬁcant and results were represented as means ± s.e.m. Data analyses were performed using SPSS statistical program, version 19.0 (IBM, Armonk, NY, USA). Sucrose preference test The sucrose preference test (SPT) was measured as previously described.29 Mice were accustomed to sucrose solution (1%) for 2 days after the last day of CRS to avoid neophobia. On 16th day, 12 h after water deprivation, mice were given free access to two bottles ﬁlled with either 1% sucrose solution or water for 1 h. Then the weights of the bottles were measured and ﬂuid consumption was determined. Sucrose preference was calculated as: sucrose preference (%) = sucrose intake/total ﬂuid intake (sucrose +water) × 100. BrdU administration and immunohistochemistry BrdU administration and immunohistochemistry Mice were anesthetized with 5% chloral hydrate, perfused with 4% paraformaldehyde and the brains were postﬁxed overnight. Brains were cut into 30-μm-thick sections on a freezing microtome and stored at −20 °C for immunohistochemical analysis. Brain sections were washed three times with PBS, and then incubated in a blocking solution (0.3% Trition X-100 and 10% normal donkey serum in TBS) for 1 h. After that brain sections were incubated overnight at 4 °C with primary antibodies: rabbit anti-GFP (1:1000, Invitrogen, Carlsbad, CA, USA), followed by incubation with donkey anti-rabbit Alexa 488 (1:1000, Invitrogen) for 1 h at room temperature. Behavioral procedures The behavioral tests were administered between 0800 and 1600 hours. All behavioral tests were performed 24 h after the last restraint treatment. Different cohorts of male mice were used for these behavioral experiments. The sample sizes were chosen based on common practice in animal behavior experiments (7–10 animals per group). Investigators were not blinded to groups. The animals were allowed to habituate to the context before test. After the end of the behavioral research, mice that received a lentivirus microinjection were killed, and the GFP was detected via ﬂuorescence microscopy to indicate the location of the virus. Animals were excluded from the analysis when the microinfusion sites were not correct. Translational Psychiatry (2016), 1 – 13 Brain area microdissection Hippocampal neuronal cultures and transfection Brains were removed and placed in a mouse brain slicer (Braintree Scientiﬁc, Braintree, MA, USA). The dorsal hippocampus (DH) was deﬁned as anteroposterior (AP): −0.94 to −2.30 mm and the ventral hippocampus (VH) as AP: −2.46 to −3.80 mm.24,25 Coronal sections (1-mm thick) were collected and the DH and VH were isolated under the dissecting microscope following delineations from the mouse brain atlas.24–26 Then the tissues were kept at −80 °C until use. The exact separation of DH and VH was further conﬁrmed by the expression of two speciﬁc genes lactose- phlorizinhydrolase (Lct) and Decorin (Dcn), which are primarily expressed in the DH and VH, respectively.27,28 Cultured hippocampal neurons were prepared from newborn (P0) mice. Cultured neurons (7 DIV) were infected with lentivirus for 6 h. The medium was then replaced with fresh medium. CBP–CREB (cAMP response element-binding protein) interaction inhibitor was applied 3 days after lentivirus infection. Neurons were lysed 10 h after the administration of the CREB inhibitor. Chronic restraint stress Mice were assigned randomly into the CRS group or the no CRS (No-CRS) group. The CRS group was restrained during the morning (0800-1000 hours) daily for 2 h for 14 days in well-ventilated polypropylene restrainers without food and water. After CRS treatment, mice were back to the home cage. Mice were weighed daily before the restraint stress treatment. In some series of experiments (Figure 5), stressed mice were once-daily administered with saline or ﬂuoxetine (10 mg kg−1, Sigma Aldrich, St Louis, MO, USA) i.p. Wnts and stress-induced depression-like behaviors W-J Zhou et al Wnts and stress-induced depression-like behaviors W-J Zhou et al 2 Brain area microdissection RESULTS Knockdown of Wnt2 or Wnt3 in the No-CRS group could mimic depression-like behaviors p Our results showed that CRS could selectively decrease the expression of Wnt2 and Wnt3, so we next investigated whether endogenous Wnt2 and Wnt3 are functionally related to depression-like behaviors. We generated lentiviruses expressing siRNA sequences against Wnt2 or Wnt3 (Lenti-siWnt2 and Lenti- siWnt3) and a control lentivirus with a scrambled sequence (Lenti- siSCR). To exclude the potential off-target effect of siWnt2 and siWnt3, an overexpressed Wnt2 or Wnt3 HEK293 cells system was introduced. Respective siRNAs were transfected into the over- expressed HEK293 cells, 48 h later the Wnt2 and Wnt3 expression levels were detected by western blot. We found that the expression of Wnt2 but not Wnt3 was attenuated by 60% after transfection of Lenti-siWnt2. Similarly, Wnt3 expression was decreased after infection of Lenti-siWnt3, while Wnt2 was not affected (Supplementary Figures S5A and B: Supplementary Figure S5A; F2,15 = 22.287, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2+Wnt2, Po0.001; Lenti-siWnt3+Wnt2, P = 0.359; Supplementary Figure S5B; F2,15 = 16.379, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2+Wnt3, P = 0.564; Lenti-siWnt3 +Wnt3, Po0.001 ). Then, mice were stereotactically injected with Lenti-siWnt2, Lenti-siWnt3 and Lenti-siSCR virus into the VH. The GFP gene was co-expressed with siRNA on the lentivirus vector. Therefore, the location of the microinjected lentivirus was showed by green ﬂuorescence on the slices, while the effect of the virus was evaluated by the levels of Wnt2/3 protein with western blot. We carried these experiments every week after microinjection and found that there were a maximum GFP expression (Supplementary Figure S5C) and the lowest levels of Wnt2/3 at 5 weeks post injection (Supplementary Figure S5D; Wnt2, t11 = 6.677, Po0.001; Wnt3, t11 = 6.349, Po0.001, two-tailed t-test), indicating that the effect of virus was optimal at this time point. Therefore, in the following studies, behavioral tests were performed 5 weeks after virus injection. y We initially examined whether the expression levels of various Wnts were altered after CRS. RESULTS We focused on Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a and Wnt7a, which have been reported to be highly expressed in the hippocampus.9,38–41 Previous studies have shown that the DH appears to be more involved in learning and memory functions, whereas the VH seems to participate in emotional modulation.42,43 Thus, we divided the hippocampus into the VH and DH according to the previous references,27,28 and proved the veracity of these regions by differential expression of the Dcn and Lct further (Supplementary Figure S2). Real-time PCR results showed that among the six different Wnts, only Wnt2 and Wnt3 mRNA levels were signiﬁcantly reduced in the VH (Figure 1a; Wnt2, t12 = 3.945, P = 0.002; Wnt3, t12 = 4.342, P = 0.001, two-tailed t-test). There were no signiﬁcant changes in any of the six Wnts examined in the DH after CRS (Figure 1b). Certainly, there is another explanation for this result that a single restraint operation is powerful enough to induce the decrease of Wnt2/3 in the VH. To exclude the possibility, mice were subjected to a single restraint stress for 2 h and the mRNA levels of Wnt2/3 in the VH were detected immediately. The results revealed that there were no signiﬁcant changes of Wnt2/3 mRNA (Supplementary Figure S3; Wnt2, t18 = 0.019, P = 0.985; Wnt3, t18 = −0.468, P = 0.645, two- tailed t-test), suggesting that the decreased Wnt2/3 gene expression was speciﬁc to CRS. We next investigated whether decreasing Wnt2 or Wnt3 could mimic depression-like behaviors (Figure 2a). Mice in the Lenti- siWnt2 and Lenti-siWnt3 groups showed decreased sucrose consumption compared with mice in the Lenti-siSCR group (Figure 2b; F2,27 = 19.394, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, Po0.001; Lenti-siWnt3, Po0.001). There was a signiﬁcant increase in immobility time in both FST (Figure 2c; F2,27 = 21.964, Po0.001, one-way ANOVA; post hoc test, Lenti- siWnt2, Po0.001; Lenti-siWnt3, Po0.001) and TST (Figure 2d; F2,27 = 15.686, Po0.001, one-way ANOVA; post hoc test, Lenti- siWnt2, Po0.001; Lenti-siWnt3, Po0.001) in the Lenti-siWnt2 and Lenti-siWnt3 groups compared with the Lenti-siSCR group. RESULTS Selective reduction of Wnt2 and Wnt3 in the VH after CRS CRS, a well-established animal model used to induce depression- and anxiety-like behaviors, was applied in our experiments.19,34–37 Mice were subjected to restraint treatment for 2 h per day for 14 days. Mice in the CRS group showed more anhedonic (that is, decreased sucrose consumption in SPT, t14 = 6.551, Po0.001, two- tailed t-test) and despair behaviors (that is, increased immobility in FST, (t14= −4.378, P = 0.001, two-tailed t-test) and TST, (t14 = −4.224, P = 0.001, two-tailed t-test)) compared with the No-CRS group (Supplementary Figure S1A–C). In the open ﬁeld test (OFT), there was no difference between the CRS group and the No-CRS group in the total distance traveled, indicating that locomotor activity was unaffected (Supplementary Figure S1D; t14 = 0.381, P = 0.709, two- tailed t-test). Compared with the No-CRS group, mice in the CRS group spent less time in the center compartment (Supplementary Figure S1E; t14= 2.544, P = 0.034, two-tailed t-test). In elevated plus maze (EPM), the CRS group of mice also displayed a signiﬁcant decrease in the percentage of time spent in the open arms (Supplementary Figure S1F; t14= 5.987, Po0.001, two-tailed t-test) and in the percentage of entries into the open arms (Supplementary Figure S1G; t14= 5.910, Po0.001, two-tailed t-test), which suggested that mice in the CRS group displayed anxiety-like behaviors. We also detected the effects of daily restraint stress on body weight. Prior to CRS exposure, the body weights of mice in the No-CRS and CRS groups were similar (Supplementary Figure S1H; t14 = 0.553, P = 0.589). A two-way repeated ANOVA revealed that CRS had a signiﬁcant effect on body weight, and that the stress × day interaction was signiﬁcant (two-way repeated ANOVA: interaction: F13,182= 46.298, Po0.001; stress: F1,182 = 7.808, P = 0.027; day: F13,182= 16.662, Po0.001). An LSD post hoc test revealed that the CRS group showed a signiﬁcant decrease in body weight compared with the No-CRS group starting from the sixth day and lasting throughout the remainder of stress exposure days (Po0.05). These results validate the use of CRS as an animal depression and anxiety model in our experiments. p g The selective reduction of Wnt2 and Wnt3 expression in the VH following CRS suggested that Wnt2 and Wnt3 may play roles in CRS-induced depression-like behaviors. Tail suspension test Tail suspension tests (TSTs) were performed using a procedure described previously, with slight modiﬁcations.32 The tail was wrapped with tape, leaving ~ 1 cm of the tail protruding. The long length of the tape was used to reduce tail-climbing. Mice were suspended by the tail for 6 min, and the test procedures were videotaped for analysis by a blind observer. Mice that climbed their tails were removed from the experimental analysis. Mobility was deﬁned as movement of the hind legs. Wnts and stress-induced depression-like behaviors Wnts and stress-induced depression-like behaviors W-J Zhou et al 3 alternative control, we did not detect a signiﬁcant alteration in the Wnt3a protein level in either the VH or the DH (Figures 1c and d). RESULTS In the OFT, the Lenti-siWnt2 and Lenti-siWnt3 groups exhibited no signiﬁcant difference in locomotor activity or in the percentage of time spent in the central areas compared with the Lenti-siSCR group (Supplementary Figure S5E; F2,27 = 0.476, P = 0.627, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.383; Lenti-siWnt3, P = 0.928; Supplementary Figure S5F; F2,27 = 0.430, P = 0.655, one- way ANOVA; post hoc test, Lenti-siWnt2, P = 0.362; Lenti-siWnt3, P = 0.625). In EPM, neither the percentage of time spent in the open arms (Supplementary Figure S5G; F2,27 = 0.480, P = 0.624, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.445; Lenti-siWnt3, P = 0.895) nor the percentage of entries into the open arms (Supplementary Figure S5H; F2,27 = 1.153, P = 0.331, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.826; Lenti- siWnt3, P = 0.244) was signiﬁcantly different among Lenti-siWnt2, We next determined whether the decrease observed in Wnt2 and Wnt3 mRNA levels would lead to reduction in protein levels after CRS. Western blots analysis showed that the protein levels of Wnt2 and Wnt3 were signiﬁcantly decreased after CRS in the VH but not in the DH (Figure 1c; Wnt2, t16 = 5.175, Po0.001; Wnt3, t16 = 3.334, P = 0.004, two-tailed t-test; Figure 1d; Wnt2, t16 = 0.766, P = 0.455; Wnt3, t16 = −0.213, P = 0.834, two-tailed t-test). As an Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors W-J Zhou et al 0 50 100 150 Wnt2 Relative levels of mRNA (%) Relative levels of mRNA (%) VH VH No-CRS CRS Wnts protein / actin Normalized to control CRS CRS 0 50 100 150 No-CRS No-CRS No-CRS DH DH Wnt2 Wnt3a VH Wnt2 Wnt3 Wnt3a CRS Actin DH CRS No-CRS 0 50 100 150 CRS No-CRS 0 50 100 150 Wnts protein / actin Normalized to control Wnt2 Wnt3 Wnt3a Actin Wnt3 Wnt3a Wnt4 Wnt5a Wnt7a Wnt2 Wnt3 Wnt3a Wnt4 Wnt5a Wnt7a Wnt3 Wnt2 Wnt3a Wnt3 Figure 1. Selective reduction of Wnt2 and Wnt3 in the VH after CRS. (a) Changes in Wnt (2, 3, 3a, 4, 5a and 7a) mRNA levels in the VH after CRS. n = 7 per group; Po0.01 versus the No-CRS group. (b) Changes in Wnt (2, 3, 3a, 4, 5a and 7a) mRNA levels in the DH after CRS. n = 7 per group. RESULTS As shown in Figures 2e and f, knockdown of either Wnt2 or Wnt3 resulted in a signiﬁcant increase in corticosterone and ACTH levels compared with Lenti- siSCR group (corticosterone; F2,14 = 6.773, P = 0.009, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.003; Lenti-siWnt3, P = 0.022; ACTH; F2,14 = 4.627, P = 0.029, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.029; Lenti-siWnt3, P = 0.013). Lenti-siWnt3 and Lenti-siSCR groups. Together, our results suggested that knockdown of Wnt2 or Wnt3 expression in the VH could mimic CRS-induced depression-like, but not anxiety-like behaviors. Previous studies have provided extensive evidence for the hypothalamic–pituitary–adrenal axis dysregulation in depression.44,45 We next detected changes in serum corticoster- one and adrenocorticotropin hormone (ACTH) levels following knockdown of Wnt2 or Wnt3. As shown in Figures 2e and f, knockdown of either Wnt2 or Wnt3 resulted in a signiﬁcant increase in corticosterone and ACTH levels compared with Lenti- siSCR group (corticosterone; F2,14 = 6.773, P = 0.009, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.003; Lenti-siWnt3, P = 0.022; ACTH; F2,14 = 4.627, P = 0.029, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.029; Lenti-siWnt3, P = 0.013). p g g We further investigated whether knockdown of Wnt2 or Wnt3 would lead to impaired neurogenesis. First, the number of surviving newborn neurons was analysed by cells that were double-labeled for BrdU and NeuN 4 weeks after the last BrdU injection. The results showed that CRS could signiﬁcantly decrease survived newborn neurons in the VH but not DH (Supplementary Figure S6; DH: t7 = 0.351, P = 0.736, two-tailed t-test; VH: t7 = 5.637, P = 0.006, two-tailed t-tes). Therefore, we focused on the alteration of adult neurogenesis in the VH in the following experiments. Similarly, knockdown of Wnt2 or Wnt3 could lead to a signiﬁcant reduction in BrdU+NeuN+ cells, compared with Lenti-siSCR group (Figure 2i; F2,12 = 14.071, P = 0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, Po0.001; Lenti-siWnt3, P = 0.001), suggesting that knockdown of Wnt2 or Wnt3 decreases the number of surviving hippocampal newborn neurons. Then, we evaluated whether the decreased number of surviving hippocampal newborn neurons was related with the decrease of cell proliferation. RESULTS (c, d) Changes in Wnt (2, 3 and 3a) protein levels in the VH (c) and DH (d) after exposure to CRS, as detected by western blot. n = 9 per group; Po0.01 versus the No-CRS group. All values are denoted as the mean ± s.e.m. CRS, chronic restraint stress; DH, dorsal hippocampus; VH, ventral hippocampus. 4 Relative levels of mRNA (%) CRS 0 50 100 150 No-CRS DH DH Wnt2 Wnt3 Wnt3a Wnt4 Wnt5a Wnt7a 0 50 100 150 Wnt2 Relative levels of mRNA (%) VH VH No-CRS CRS Wnt3 Wnt3a Wnt4 Wnt5a Wnt7a Wnts protein / actin Normalized to control CRS No-CRS No-CRS Wnt2 Wnt3a VH Wnt2 Wnt3 Wnt3a CRS Actin 0 50 100 150 Wnt3 DH CRS No-CRS CRS No-CRS 0 50 100 150 Wnts protein / actin Normalized to control Wnt2 Wnt3 Wnt3a Actin Wnt2 Wnt3a Wnt3 Wnt3a Wnt3 Wnt3 Figure 1. Selective reduction of Wnt2 and Wnt3 in the VH after CRS. (a) Changes in Wnt (2, 3, 3a, 4, 5a and 7a) mRNA levels in the VH after CRS. n = 7 per group; Po0.01 versus the No-CRS group. (b) Changes in Wnt (2, 3, 3a, 4, 5a and 7a) mRNA levels in the DH after CRS. n = 7 per group. (c, d) Changes in Wnt (2, 3 and 3a) protein levels in the VH (c) and DH (d) after exposure to CRS, as detected by western blot. n = 9 per group; Po0.01 versus the No-CRS group. All values are denoted as the mean ± s.e.m. CRS, chronic restraint stress; DH, dorsal hippocampus; VH, ventral hippocampus. (the cytosolic marker) and calnexin (the membrane marker), which excluded the possibility of the contamination by cytoplasmic and membrane proteins (Figure 2h and Figure 3h). Lenti-siWnt3 and Lenti-siSCR groups. Together, our results suggested that knockdown of Wnt2 or Wnt3 expression in the VH could mimic CRS-induced depression-like, but not anxiety-like behaviors. Previous studies have provided extensive evidence for the hypothalamic–pituitary–adrenal axis dysregulation in depression.44,45 We next detected changes in serum corticoster- one and adrenocorticotropin hormone (ACTH) levels following knockdown of Wnt2 or Wnt3. Translational Psychiatry (2016), 1 – 13 Wnt2 and Wnt3 could buffer CRS-induced depression-like behaviors Wnt2 and Wnt3 could buffer CRS-induced depression-like behaviors We next tested whether overexpressing Wnt2 or Wnt3 in the VH is sufﬁcient for alleviating CRS-induced depression-like behaviors. We injected lentiviruses expressing Wnt2 or Wnt3 into the VH of adult mice (Figure 3a). First, we evaluated the effect of the Lenti- Wnt2 and Lenti-Wnt3 viruses on the expression of Wnt2 and Wnt3 (Supplementary Figure S7A). Western blot results showed that Wnt2 and Wnt3 levels were signiﬁcantly increased 5 weeks after injection of Lenti-Wnt2 and Lenti-Wnt3, respectively, compared with injections of Lenti-GFP in the VH (Supplementary Figure S7A; Wnt2, t9 = −2.731, P = 0.023; Wnt3, t10 = −4.029, P = 0.002, two- tailed t-test). 1,32 2,32 Our results demonstrated that overexpressing Wnt2 or Wnt3 was sufﬁcient for alleviating CRS-induced depression-like, but not anxiety-like behaviors. Wnt2 and Wnt3 rescue CRS-induced Wnt/β-catenin signaling impairment and deﬁcits in neurogenesis Under basal condition, behavioral tests were performed 5 weeks after virus injection (Figure 3a). We found that sucrose consump- tion was signiﬁcantly increased in the Lenti-Wnt2 group (Figure 3b; P = 0.012). However, there was no signiﬁcant difference in the immobility time in FST and TST among the Lenti-Wnt2, Lenti-Wnt3 and Lenti-GFP groups of No-CRS mice (Figures 3c and d). As expected, under stress condition, the CRS-Lenti-GFP group showed depression-like behaviors compared with the Lenti-GFP group (Figures 3b–d; Po0.001). Interestingly, we found that sucrose consumption was signiﬁcantly increased in the CRS-Lenti- Wnt2 and CRS-Lenti-Wnt3 groups compared with the CRS-Lenti- GFP group (Figure 3b; Po0.001). Analysis by two-way ANOVA found a signiﬁcant stress × virus interaction effect and a signiﬁcant effect of stress and virus (two-way ANOVA: interaction: F2,54 = 7.946, P = 0.001; stress: F1,54 = 20.384, Po0.001; virus: F2,54 = 20.010, Po0.001). Mice in the CRS-Lenti-Wnt2 or CRS- Lenti-Wnt3 group displayed signiﬁcantly decreased immobility time in FST and TST compared with the CRS-Lenti-GFP group (Figures 3c and d; Po0.001). Two-way ANOVA revealed a signiﬁcant effect of stress and virus in FST and TST, where the effect of virus was dependent on CRS (two-way ANOVA: FST: Interaction: F2,54 = 12.627, Po0.001; stress: F1,54 = 13.166, P = 0.001; virus: F2,54 = 13.362, Po0.001; TST: Interaction: F2,54 = 5.599, P = 0.006; stress: F1,54 = 8.588, P = 0.005; virus: F2,54 = 8.638, P = 0.001). Wnt2 and Wnt3 could buffer CRS-induced depression-like behaviors Our results suggested that the effects of overexpressing Wnt2 and Wnt3 more speciﬁcally resulted in a reversal of stress- induced depression-like behaviors. Previous studies reported that chronic stress leads to Wnt/β- catenin signaling impairment and deﬁcits in neurogenesis.22,46 Our results, described above, showed that overexpressing Wnt2 and Wnt3 rescued CRS-induced depression-like behaviors. There- fore, we next examined whether overexpressing Wnt2 or Wnt3 could rescue the CRS-induced Wnt/β-catenin signaling impair- ment and deﬁcits in neurogenesis. We found that the levels of p-GSK3β (Ser9) and nuclear β-catenin were signiﬁcantly increased in the Lenti-Wnt2 or Lenti-Wnt3 group compared with the Lenti- GFP group under basal condition, which suggested that over- expressing Wnt2 or Wnt3 enhanced Wnt/β-catenin signaling (Figure 3g; p-GSK3β: Lenti-Wnt2, P = 0.001; Lenti-Wnt3, P = 0.016; Figure 3h; β-catenin: Lenti-Wnt2, Po0.001; Lenti-Wnt3, P = 0.009). Under stress condition, overexpression of Wnt2 or Wnt3 could rescue the CRS-induced decreases in p-GSK3β (Ser9) (CRS-Lenti- Wnt2, P = 0.013; CRS-Lenti-Wnt3, P = 0.005) and nuclear β-catenin (CRS-Lenti-Wnt2, Po0.001; CRS-Lenti-Wnt3, Po0.001), suggest- ing that Wnt2 and Wnt3 rescued the CRS-induced Wnt/β-catenin signaling impairment. g g p Previous studies reported that overexpression of Wnt3 was sufﬁcient for increasing neurogenesis in vitro and in vivo.9 However, whether Wnt2 is involved in adult neurogenesis remains unclear. Our results showed that the number of BrdU+NeuN+ cells was signiﬁcantly increased in the Lenti-Wnt2 or Lenti-Wnt3 group compared with the Lenti-GFP group under basal condition (Figure 3i; Lenti-Wnt2, P = 0.002; Lenti-Wnt3, P = 0.001), which suggests that Wnt2 and Wnt3 are involved in adult neurogenesis. Under stress condition, overexpression of Wnt2 or Wnt3 blocked the CRS-induced decrease in BrdU+NeuN+ cells compared with the CRS-Lenti-GFP group (Figure 3i; CRS-Lenti-Wnt2, P = 0.010; CRS- Lenti-Wnt3, P = 0.015), which suggested that overexpression of Wnt2 or Wnt3 rescued CRS-induced hippocampal neurogenesis deﬁcits. The above results indicated that Wnt/β-catenin signaling and hippocampal neurogenesis, coupled with Wnt2 and Wnt3, may mediate CRS-induced depression-like behaviors. p We also investigated the role of Wnt2 and Wnt3 in anxiety-like behaviors. In OFT, there was no signiﬁcant difference in total distance traveled among the Lenti-Wnt2, Lenti-Wnt3 and Lenti- GFP groups under basal or stress condition, indicating that locomotor activity was unaffected (Supplementary Figure S7B). RESULTS The result showed that there was a signiﬁcant decrease in BrdU+-labeled cells 2 h after BrdU injection in the Lenti-siWnt2 or Lenti-siWnt3 group compared with Lenti-siSCR group (Figure 2j; F2,15 = 26.248, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, Po0.001; Lenti-siWnt3, Po0.001), which suggested that knockdown of Wnt2 or Wnt3 impaired cell proliferation in the adult VH. p We next investigated the mechanisms underlying Wnt2- or Wnt3-mediated depression-like behaviors. Previous studies have reported that Wnt/β-catenin signaling regulates neurogenesis and is associated with depression disorders.9,11 We therefore ﬁrst examined whether the Wnt/β-catenin signaling pathway was altered at 5 weeks after the injection of the Lenti-siWnt2, Lenti- siWnt3 or Lenti-siSCR virus. Western blot analysis showed that the total GSK3β levels were not signiﬁcantly changed. However, Lenti-siWnt2 or Lenti-siWnt3 injection signiﬁcantly decreased the level of p-GSK3β (Ser9) and nuclear β-catenin compared with Lenti-siSCR injection (Figure 2g; p-GSK3β: F2,15 = 19.717, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, Po0.001; Lenti- siWnt3, P = 0.001; Figure 2h; nuclear β-catenin: F2,15 = 39.312, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, Po0.001; Lenti-siWnt3, Po0.001), which suggested that knock- down of Wnt2 or Wnt3 led to impaired Wnt/β-catenin signaling. The nuclear fraction we puriﬁed had no expression of α-tubulin Together, our results demonstrated that knockdown of Wnt2 or Wnt3 in the VH could lead to depression-like behaviors, impaired Wnt/β-catenin signaling and decreased neurogenesis, which suggested that the induced depression-like behaviors may be Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors Wnts and stress-induced depression-like behaviors W-J Zhou et al 5 the results of impaired Wnt/β-catenin signaling or decreased neurogenesis in the VH. condition, whereas overexpression could reverse the CRS- induced increase in serum levels of corticosterone and ACTH (Figure 3e; CRS-Lenti-Wnt2, Po0.001; CRS-Lenti-Wnt3, Po0.001; Figure 3f; CRS-Lenti-Wnt2, Po0.001; CRS-Lenti-Wnt3, P = 0.001). Analysis by two-way ANOVA indicated a signiﬁcant stress × virus interaction effect and a signiﬁcant effect of stress and virus (corticosterone; two-way ANOVA: interaction: F2,32 = 9.532, P = 0.001; stress: F1,32 = 33.674, Po0.001; virus: F2,32 = 14.243, Po0.001; ACTH; two-way ANOVA: interaction: F2,32 = 7.176, P = 0.003; stress: F1,32 = 4.589, P = 0.04; virus: F2,32 = 3.490, P = 0.04). Our results demonstrated that overexpressing Wnt2 or Wnt3 was sufﬁcient for alleviating CRS-induced depression-like, but not anxiety-like behaviors. Wnt2 and Wnt3 could buffer CRS-induced depression-like behaviors No signiﬁcant stress × virus interaction effect or virus effect was observed between the groups for the percentage of time spent in the center, but a signiﬁcant effect for stress treatment was noted (Supplementary Figure S7C; two-way ANOVA: interaction: F2,54 = 0.488, P = 0.617; stress: F1,54 = 69.602, Po0.001; virus: F2,54 = 0.134, P = 0.875). In EPM, no signiﬁcant stress × virus interaction effect or virus effect was observed between groups for percentage of time spent in or entries into open arms; however, a signiﬁcant effect of stress was found (Supplementary Figure S7D; two-way ANOVA: interaction: F2,54 = 0.112, P = 0.894; stress: F1,54 = 64.419, Po0.001; virus: F2,54 = 0.513, P = 0.602; Supplementary Figure S7E; two-way ANOVA: interaction: F2,54 = 0.415, P = 0.662; stress: F1,54 = 115.221, Po0.001; virus: F2,54 = 0.388, P = 0.680). Together, these data suggested that Wnt2 and Wnt3 had no effect on locomotor activity or anxiety- like behaviors. Wnt2 and Wnt3 positively regulate each other via CREB Wnt2 and Wnt3 positively regulate each other via CREB Wnt2 and Wnt3 positively regulate each other via CREB Our above experiments showed that CRS induces a synchronous reduction in Wnt2 and Wnt3, and that Wnt2 and Wnt3 participate in depression-like behaviors. We next sought to determine the relationship between Wnt2 and Wnt3. In our experiments, we found that knockdown of Wnt2 decreased, whereas overexpres- sion of Wnt2 increased Wnt3 levels compared with the Lenti-GFP group in vivo and vice versa (Figure 4a; F2,15 = 51.806, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2, P = 0.001; Lenti-Wnt2, Po0.001; Figure 4b; F2,15 = 27.506, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt3, P = 0.002; Lenti-Wnt3, P = 0.002). Moreover, the potential off-target effect of siWnt2 and siWnt3 could be excluded (Supplementary Figures S5A and B). These results suggested that the expression levels of Wnt2 and Wnt3 are associated with each other. In addition, we detected serum corticosterone and ACTH levels (Figure 3a). We found that overexpression of Wnt2 or Wnt3 had no effect on serum corticosterone or ACTH levels under basal Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors W-J Zhou et al 6 Previous studies have reported that transcription of both Wnt2 and Wnt3 is CREB dependent.47,48 Cultured neurons (7 DIV) were infected with Lenti-Wnt2 or Lenti-Wnt3 virus for 3 days, and we observed that although the total CREB levels exhibited no signiﬁcant alteration, the levels of p-CREB were signiﬁcantly increased after Lenti-Wnt2 or Lenti-Wnt3 virus infection, which suggested that both Wnt2 and Wnt3 activated CREB (Figure 4c; F2,12 = 36.713, Po0.001, one-way ANOVA; post hoc test, Lenti- Wnt2, Po0.001; Lenti-Wnt3, Po0.001). Furthermore, we found that administration of a CREB inhibitor (CBP–CREB interaction inhibitor) blocked the positive feedback effect between Wnt2 and Wnt3 (Figure 4d; P = 0.002; Figure 4e; P = 0.022). Two-way ANOVA analysis found a signiﬁcant virus × inhibitor interaction effect and a signiﬁcant effect of virus and inhibitor (two-way ANOVA: F2,12 = 36.713, Po0.001, one-way ANOVA; post hoc test, Lenti- Wnt2, Po0.001; Lenti-Wnt3, Po0.001). Furthermore, we found that administration of a CREB inhibitor (CBP–CREB interaction inhibitor) blocked the positive feedback effect between Wnt2 and Wnt3 (Figure 4d; P = 0.002; Figure 4e; P = 0.022). Wnt2 and Wnt3 positively regulate each other via CREB Two-way ANOVA analysis found a signiﬁcant virus × inhibitor interaction effect and a signiﬁcant effect of virus and inhibitor (two-way ANOVA: nd Wnt3 is CREB dependent.47,48 Cultured neurons (7 DIV) were fected with Lenti-Wnt2 or Lenti-Wnt3 virus for 3 days, and we bserved that although the total CREB levels exhibited no gniﬁcant alteration, the levels of p-CREB were signiﬁcantly creased after Lenti-Wnt2 or Lenti-Wnt3 virus infection, which uggested that both Wnt2 and Wnt3 activated CREB (Figure 4c; Wnt2, Po0.001; Lenti-Wnt3, Po0.001). Furthermore, we foun that administration of a CREB inhibitor (CBP–CREB interactio inhibitor) blocked the positive feedback effect between Wnt2 an Wnt3 (Figure 4d; P = 0.002; Figure 4e; P = 0.022). Two-way ANOV analysis found a signiﬁcant virus × inhibitor interaction effect an a signiﬁcant effect of virus and inhibitor (two-way ANOV Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors Wnts and stress-induced depression-like behaviors W-J Zhou et al 7 difference in sucrose consumption or immobility times in FST and TST compared with the Lenti-siWnt2+SAL+CRS group or the Lenti-siWnt3+SAL+CRS group. These ﬁndings suggested that knockdown of Wnt2 or Wnt3 could abolish the antidepressant effect of ﬂuoxetine. We also found that combined stress and knockdown of Wnt2/3 could not cause a more severe phenotype (Figures 5c–e), which may be resulted from the presence of a basement effect in the experiments. Figure 4d; Interaction: F1,13 = 4.922, P = 0.04; virus: F1,13 = 15.044, P = 0.002; inhibitor: F1,13 = 62.161, Po0.001; Figure 4e; Interaction: F1,18 = 8.475, P = 0.009; virus: F1,18 = 10.781, P = 0.004; inhibitor: F1,18 = 41.585, Po0.001). These results suggested that there is positive feedback between Wnt2 and Wnt3 that depends on the activation of CREB. nt2 and Wnt3 mediate the effect of antidepressant Wnt2 and Wnt3 mediate the effect of antidepressant Previous studies have reported that Wnt2 expression and Wnt/β-catenin signaling are increased by different classes of antidepressant treatments and by electroconvulsive seizures under basal condition.49,50 However, whether Wnt2 is necessary for the effect of antidepressants remains unclear. Previous studies have reported that chronic ﬂuoxetine treatment reverses chronic stress-induced depression-like behaviors.51,52 Here, we investi- gated whether Wnt2 or Wnt3 is necessary for the effects of ﬂuoxetine treatment under stress condition. To examine whether Wnt2 or Wnt3 expression is altered in response to ﬂuoxetine treatment under CRS condition, mice were randomly assigned into three groups: No-CRS+saline (No-CRS+SAL), CRS+saline (CRS+SAL) and CRS+ﬂuoxetine (CRS+FLX). Wnt2 and Wnt3 positively regulate each other via CREB After 2 weeks of CRS and ﬂuoxetine treatment, mice were killed, and the protein levels of Wnt2 and Wnt3 were detected. We found that two weeks of i.p. injections of ﬂuoxetine signiﬁcantly reversed the CRS-induced decrease in Wnt2 and Wnt3 levels compared with CRS+SAL group (Figure 5a; Wnt2: F6,45 = 29.602, Po0.001, one-way ANOVA; post hoc test, P = 0.002; Wnt3: F6,45 = 30.120, Po0.001, one-way ANOVA; post hoc test, Po0.001), suggesting that Wnt2 and Wnt3 may be involved in the ﬂuoxetine effect. We further examined the role of Wnt2 or Wnt3 in antidepressant effects by using lentivirus- mediated knockdown of Wnt2 or Wnt3 (Figure 5b). We found that the mice in the Lenti-siWnt2+SAL+CRS or Lenti-siWnt3+SAL+CRS group exhibited similar sucrose consumption in SPT (Figure 5c; F6,59 = 22.353, Po0.001, one-way ANOVA; post hoc test, Lenti- siWnt2+SAL+CRS, P = 0.715, Lenti-siWnt3+SAL+CRS, P = 0.689) and similar immobility times in FST (Figure 5d; F6,59 = 27.039, Po0.001, one-way ANOVA; post hoc test, Lenti-siWnt2+SAL+CRS, P = 0.723, Lenti-siWnt3+SAL+CRS, P = 0.879) and TST (Figure 5e; F6,59 = 16.472, Po0.001, one-way ANOVA; post hoc test, Lenti- siWnt2+SAL+CRS, P = 0.756, Lenti-siWnt3+SAL+CRS, P = 0.443) compared with the Lenti-GFP+SAL+CRS mice, which suggested that knockdown of Wnt2 or Wnt3 did not exacerbate CRS-induced depression-like behaviors. Treatment with ﬂuoxetine counteracted the depression-like behaviors induced by CRS (Figures 5c–e; Po0.001). However, mice in the Lenti-siWnt2+FLX+CRS or Lenti- siWnt3+FLX+CRS group showed signiﬁcantly decreased sucrose consumption in SPT and increased immobility times in FST and TST compared with the Lenti-GFP+FLX+CRS group (Figures 5c–e; Lenti-siWnt2+FLX+CRS, Po0.001, Lenti-siWnt3 +FLX+CRS, Po0.001). Moreover, the Lenti-siWnt2+FLX+CRS or the Lenti-siWnt3+FLX+CRS group displayed no signiﬁcant In addition, we found that there was a synchronous decrease of Wnt2 and Wnt3 in both the Lenti-siWnt2+CRS+SAL group and the Lenti-siWnt3+CRS+SAL group compared with the CRS+SAL group (Figure 5a; Wnt2: Lenti-siWnt2+CRS+SAL, P = 0.013, Lenti-siWnt3 +CRS+SAL, P = 0.021; Wnt3: Lenti-siWnt2+CRS+SAL, P = 0.019, Lenti-siWnt3+CRS+SAL, P=0.011). The ﬂuoxetine-induced increase in Wnt2 and Wnt3 was also synchronously blocked in both the Lenti-siWnt2+CRS+FLX group and the Lenti-siWnt3+CRS+FLX group compared with the CRS+FLX group (Figure 5a; Wnt2: Lenti-siWnt2+CRS+FLX, Po0.001, Lenti-siWnt3+CRS+FLX, Po0.001; Wnt3: Lenti-siWnt2+CRS+FLX, Po0.001, Lenti-siWnt3+CRS+FLX, Po0.001). These results further conﬁrmed that the expression levels of Wnt2 and Wnt3 are associated with each other. Figure 2. Knockdown of Wnt2 or Wnt3 in the VH could mimic depression-like behaviors. (a) Schematic of the experimental schedule used to investigate the effect of siWnt2 and siWnt3 virus injections into the VH on depression behaviors and hormone levels. The effects of Lenti- siWnt2 or Lenti-siWnt3 on depression-like behaviors in mice were examined, including (b) sucrose consumption in sucrose preference test (SPT), (c) immobility time in forced swim test (FST) and (d) immobility time in tail suspension test (TST). n = 10 per group; Po0.05, Po0.01 versus the Lenti-siSCR group. (e, f) Lenti-siWnt2 or Lenti-siWnt3 resulted in higher serum corticosterone (e) and ACTH (f) levels compared with Lenti-siSCR group. Lenti-siSCR group: n = 5; Lenti-siWnt2 group: n = 6; Lenti-siWnt3 group: n = 6. Po0.05, Po0.01 versus the Lenti-siSCR group. (g) Representative blots and densitometric analysis of p-GSK3β (Ser9) in the cytoplasmic fractions of VH after knockdown of Wnt2 or Wnt3. n = 6 per group; Po0.01 versus the Lenti-siSCR group. (h) Representative blots and densitometric analysis of β-catenin in the nuclear fractions of VH after knockdown of Wnt2 or Wnt3. n = 6 per group; Po0.01 versus the Lenti-siSCR group. (i) Knockdown of either Wnt2 or Wnt3 decreased the number of newborn neuron. Representative images showed BrdU+NeuN+ cells in the DG (scale bar, 20 μm) and quantiﬁcation of BrdU+NeuN+ cells. n = 5 per group; Po0.01 versus the Lenti-siSCR group. (j) Knockdown of either Wnt2 or Wnt3 impaired cell proliferation. Left, representative images showed BrdU+ cells in the DG (scale bar, 200 μm) and quantiﬁcation of BrdU+ cells. Right, a schematic diagram from ref. 26, which represents the VH. n = 6 per group; Po0.01 versus the Lenti-siSCR group. All values are denoted as the mean ± s.e.m. ACTH, adrenocorticotropin hormone; DG, dental gyrus; VH, ventral hippocampus. DISCUSSION Consistent with our results, knockdown of Fzd6 by injection of shRNA virus into the hippocampus resulted in depression-like behaviors in SPT.20 Increasing corticosterone levels could induce depression-like behaviors and decrease hippocampal neurogenesis.44,53,54 Our nslational Psychiatry (2016), 1 – 13 Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors W J Zh t l Wnts and stress-induced depression-like behaviors W-J Zhou et al 9 behaviors. Many studies have shown that the VH had glutama- tergic input to the paraventricular hypothalamic nucleus (PVN), while PVN is responsible for coordinating the regulation of results showed that knockdown of Wnt2 or Wnt3 increased serum corticosterone and ACTH levels, which suggested that corticoster- one and ACTH might be involved in Wnt-related depression-like one and ACTH might be involved in Wnt-related depression-like while PVN is responsible for coordinating the regulation of Figure 4. Wnt2 and Wnt3 positively regulate each other via CREB. (a) Levels of Wnt3 in the VH of mice injected with Lenti-GFP, Lenti-siWnt2 or Lenti-Wnt2, analyzed by western blot. n = 6 per group; Po0.01 versus the Lenti-GFP group. (b) Levels of Wnt2 in the VH of mice injected with Lenti-GFP, Lenti-siWnt3 or Lenti-Wnt3, analyzed by western blot. n = 6 per group; Po0.01 versus the Lenti-GFP group. (c) Representative blots and densitometric analysis showing p-CREB levels in cultured neurons that overexpressed Wnt2 or Wnt3. n = 5 per group; Po0.01 versus the Lenti-GFP group. (d) Representative blots and densitometric analysis showing Wnt3 levels in cultured neurons that overexpressed Wnt2 after administration of a CREB inhibitor. n = 4, 5, 4, 4, respectively; *Po0.01 versus the Lenti-GFP+Vehicle (Veh) group. (e) Representative blots and densitometric analysis showing Wnt2 levels in cultured neurons that overexpressed Wnt3 after administration of a CREB inhibitor. n = 6, 6, 5, 5, respectively; Po0.05, Po0.01 versus the Lenti-GFP+Veh group. All values are denoted as the mean ± s.e.m. CREB, cAMP response element-binding protein; GFP, green-ﬂuorescent protein; VH, ventral hippocampus. Figure 4. Wnt2 and Wnt3 positively regulate each other via CREB. (a) Levels of Wnt3 in the VH of mice injected with Lenti-GFP, Lenti-siWnt2 or Lenti-Wnt2, analyzed by western blot. n = 6 per group; Po0.01 versus the Lenti-GFP group. (b) Levels of Wnt2 in the VH of mice injected with Lenti-GFP, Lenti-siWnt3 or Lenti-Wnt3, analyzed by western blot. n = 6 per group; Po0.01 versus the Lenti-GFP group. DISCUSSION (c) Representative blots and densitometric analysis showing p-CREB levels in cultured neurons that overexpressed Wnt2 or Wnt3. n = 5 per group; Po0.01 versus the Lenti-GFP group. (d) Representative blots and densitometric analysis showing Wnt3 levels in cultured neurons that overexpressed Wnt2 after administration of a CREB inhibitor. n = 4, 5, 4, 4, respectively; *Po0.01 versus the Lenti-GFP+Vehicle (Veh) group. (e) Representative blots and densitometric analysis showing Wnt2 levels in cultured neurons that overexpressed Wnt3 after administration of a CREB inhibitor. n = 6, 6, 5, 5, respectively; Po0.05, *Po0.01 versus the Lenti-GFP+Veh group. All values are denoted as the mean ± s.e.m. CREB, cAMP response element-binding protein; GFP, green-ﬂuorescent protein; VH, ventral hippocampus. Figure 3. Wnt2 and Wnt3 buffer CRS-induced depression-like behaviors. (a) Schematic of the experimental schedule used to investigate the effect of Wnt2 or Wnt3 overexpression in the VH on depression-like behaviors and hormone levels under basal or stress condition. The effect of Lenti- Wnt2 and Lenti-Wnt3 viruses on (b) sucrose consumption in SPT, (c) immobility time in FST, and (d) immobility time in TST under basal or stress condition. n = 10 per group; Po0.05, Po0.01 versus the Lenti-GFP group; ##Po0.01 versus the CRS-Lenti-GFP. (e, f) The effect of overexpression of Wnt2 or Wnt3 on serum corticosterone (e) and ACTH (f) levels under basal or stress condition. n = 5, 6, 7, 6, 8, 6, respectively; Po0.01 versus the Lenti-GFP group; ##Po0.01 versus the CRS-Lenti-GFP group. (g) Representative blots and densitometric analysis of p-GSK3β (Ser9) in the cytoplasmic fractions of VH after overexpression of Wnt2 or Wnt3. under basal or stress condition. n = 7 per group; Po0.05, Po0.01 versus the Lenti-GFP; #Po0.05, ##Po0.01 versus the CRS-Lenti-GFP group. (h) Representative blots and densitometric analysis of β-catenin in the nuclear fractions of VH after overexpression of Wnt2 or Wnt3 under basal or stress condition. n = 7 per group; Po0.05, *Po0.01 versus the Lenti-GFP; #Po0.05, ##Po0.01 versus the CRS-Lenti-GFP group. (i) Overexpression of Wnt2 or Wnt3 rescued newborn neuron survival under basal or stress condition. Left, representative images showed BrdU+NeuN+ cells in the DG (scale bar, 20 μm). Right, quantiﬁcation of BrdU+NeuN+ cells. n = 5 per group; Po0.05, Po0.01 versus the Lenti-GFP group; #Po0.05, ##Po0.01 versus the CRS-Lenti- GFP group. All values are denoted as the mean ± s.e.m. DISCUSSION In the present study, we showed that CRS exposure selectively decreased Wnt2 and Wnt3 expression in the VH but not in the DH, and that knocking down Wnt2 or Wnt3 could mimic depression-like behaviors. Moreover, overexpression of Wnt2 or Wnt3 in the VH was sufﬁcient to buffer CRS-induced depression- like behaviors. Wnt2 and Wnt3 could activate CREB and we showed evidence for CREB-dependent positive feedback between Wnt2 and Wnt3. Finally, we determined that Wnt2 and Wnt3 in the VH could mediate the antidepressant effect of ﬂuoxetine. Previous studies have shown that Wnt/β-catenin signaling is involved in depression- but not anxiety-like behaviors by using β- catenin overexpressing transgenic mice or conditional β-catenin knockout mice.16,17 Our data provided several new insights into the roles of Wnt2 and Wnt3 in CRS-induced depression-like behaviors. First, we found that CRS exposure could selectively induce decreased Wnt2 and Wnt3 expression in the VH but not in the DH. A previous study reported that Dkk-1, an inhibitor of the canonical Wnt pathway, is increased after exposure to stress.22 Wilkinson et al.21 also found that one member of the Wnt-DVL- GSK3β pathway, DVL, was downregulated by social defeat stress. Chronic unpredictable stress decreased the expression of Fzd6, one of the receptors involved in Wnt signaling.20 These studies suggest that Wnt signaling pathway is involved in stress-induced depression-like behaviors. However, the studies about the effects of wnt ligands on the pathogenesis, development and treatment of depression are not available. A microarray studies of the hippocampus after CRS have identiﬁed decreases in the Wnt2 Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors W-J Zhou et al 8 transcript,23 and Okamato et al.49 have proved that Wnt2 is a common target of different classes of antidepressants, but there is no direct evidence to verify the functional correlation of Wnts and depression caused by stress. With the aid of positive and negative regulation of Wnt2/3, our study indicated a speciﬁc involvement of Wnt2 and Wnt3 in CRS for the ﬁrst time. Moreover, we found that knockdown of Wnt2 or Wnt3 could mimic the depression- but not anxiety-like behaviors. DISCUSSION ACTH, adrenocorticotropin hormone; CRS, chronic restraint stress; DG, dental gyrus; FST, forced swim test; GFP, green-ﬂuorescent protein; SPT, sucrose preference test; TST, tail suspension test; VH, ventral hippocampus. Translational Psychiatry (2016), 1 – 13 hypothalamic pituitary adrenal axis 55,56 Therefore the alteration knocking down Wnt2 or Wnt3 induced impaired Wnt/β Figure 5. Wnt2 and Wnt3 mediate the effect of antidepressants. (a) Representative blots and densitometric analysis showing Wnt2 an protein levels in the VH of mice within the No-CRS+SAL, CRS+SAL, CRS+FLX, Lenti-siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lent +CRS+SAL and Lenti-siWnt3+CRS+FLX treatment groups. n = 7, 6, 7, 8, 8, 8, respectively; Po0.01 versus the No-CRS+SAL group; #P ##Po0.01 versus the CRS+SAL group; $$Po0.01 versus the CRS+FLX group; &Po0.05, &&Po0.01 versus the Lenti-siWnt2+CRS+SAL o siWnt3+CRS+SAL group. (b) Schematic showing the experimental schedule used to investigate the effect of Wnt2 and W antidepressant responses to ﬂuoxetine. (c) Sucrose consumption in SPT, (d) immobility time in FST and (e) immobility time in TST for the Lenti-GFP+SAL, Lenti-GFP+CRS+SAL, Lenti-GFP+CRS+FLX, Lenti-siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lenti-siWnt3+CRS+S Lenti-siWnt3+CRS+FLX groups. Po0.01 versus the Lenti-GFP+SAL group; ##Po0.01 versus the CRS+Lenti-GFP+SAL group; $$Po0.0 the CRS+Lenti-GFP+FLX group. (f) In the OFT, all groups showed no difference in locomotor activity. n = 10, 10, 10, 9, 10, 7, 10, respect values are denoted as the mean ± s.e.m. CRS, chronic restraint stress; FLX, ﬂuoxetine; FST, forced swim test; GFP, green-ﬂuorescent OFT, open ﬁeld test; SAL, saline; SPT, sucrose preference test; VH, ventral hippocampus. Wnts and stress-induced depression-like behaviors W-J Zhou et al epresentative blots and densitometric analysis showing Wnt2 and Wn CRS+FLX, Lenti-siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lenti-siWn 8, 8, 8, respectively; Po0.01 versus the No-CRS+SAL group; #Po0. group; &Po0.05, &&Po0.01 versus the Lenti-siWnt2+CRS+SAL or Len al schedule used to investigate the effect of Wnt2 and Wnt3 PT, (d) immobility time in FST and (e) immobility time in TST for mice -siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lenti-siWnt3+CRS+SAL a group; ##Po0.01 versus the CRS+Lenti-GFP+SAL group; $$Po0.01 vers ifference in locomotor activity. n = 10, 10, 10, 9, 10, 7, 10, respectively. s; FLX, ﬂuoxetine; FST, forced swim test; GFP, green-ﬂuorescent prote ntral hippocampus. Wnts and stress-induced depression-like behaviors W-J Zhou et al 10 Figure 5. Wnt2 and Wnt3 mediate the effect of antidepressants. (a) Representative blots and densitometric analysis showing Wnt2 and Wnt3 protein levels in the VH of mice within the No-CRS+SAL, CRS+SAL, CRS+FLX, Lenti-siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lenti-siWnt3 +CRS+SAL and Lenti-siWnt3+CRS+FLX treatment groups. DISCUSSION Wnt2 regulates progenitor proliferation and increases the number of dopaminergic neurons in the developing ventral midbrain.38 However, it is unclear what the role of Wnt2 in AHN is. Our results showed that knocking down Wnt2 impaired cell proliferation and decreased the number of newly generated neurons, which suggested that Wnt2 was also involved in AHN. Second, we found that Wnt2 or Wnt3 was sufﬁcient for buffering CRS-induced depression-like behaviors. Overexpression of Wnt2 by infusion of AAV-Wnt2 had an antidepressant effect under basal condition.49 However, the effect of overexpressing Wnt2 or Wnt3 on CRS-induced depression-like behaviors remains unknown. To the best of our knowledge, our results provided the ﬁrst evidence showing that overexpressing Wnt2 or Wnt3 could produce an antidepressant effect under stress condition. Further- more, we found that overexpression of Wnt2 or Wnt3 could rescue CRS-induced Wnt/β-catenin signaling and hippocampal neuro- genesis deﬁcits. Recently, ketamine, a fast-acting antidepressant, could inhibit the activity of GSK3β in the brain. Moreover, the inhibition of GSK3β activity is necessary for rapid antidepressant effect of ketamine, which suggested that GSK3β activity plays an important role in antidepressant.57,59 To date, although the effects of reducing hippocampal neurogenesis on producing depressive behaviors are controversial, it is conﬁrmed that AHN is essential for the therapeutic effects of ﬂuoxetine in stressed mice.60–62 Therefore, the rescue of CRS-induced depression-like behaviors by overexpressing Wnt2 or Wnt3 might be mediated by regulating the GSK3β activity and/or hippocampal neurogenesis. Taken together, our results demonstrated that Wnt2 or Wnt3 in the VH is necessary and sufﬁcient for alleviating depression-like behaviors. Wnts are secreted glycoproteins that can not only act locally but may also have functions in other brain regions. Recent studies have reported that, besides the VH, Wnt2/3 are abundantly expressed in other brain regions, such as the striatum.68,69 This suggests that the functions of Wnt2/3 in other brain regions also need to be considered in the future. Moreover, we found that Wnt2 and Wnt3 could activate CREB and that there is CREB- dependent positive feedback between Wnt2 and Wnt3. Another novel and signiﬁcant ﬁnding of this study was that Wnt2 or Wnt3 is necessary for the actions of ﬂuoxetine under stress condition. Our study provided novel insight into the relationship between Wnts and stress-induced depression-like behaviors. Importantly, the screening of small molecule compounds to increase levels of Wnt2 or Wnt3 could be a method for identifying potential new targets for interventions for depression. The authors declare no conﬂict of interest. The authors declare no conﬂict of interest. DISCUSSION β y pp p g Third, we found that there is CREB-dependent positive feedback between Wnt2 and Wnt3. Our results showed that knockdown of Wnt2 could decrease, whereas overexpression of Wnt2 could increase Wnt3 levels and vice versa. These results suggested that the expression levels of Wnt2 and Wnt3 are associated with each other. Studies have reported that the Wnt signaling pathway could regulate the expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV).63 CaMKIV activates the CREB/CRE transcriptional pathway via the phosphorylation of CREB.64 The transcription of Wnt2 and Wnt3 are CREB dependent.47,48 However, there is no direct evidence showing that Wnt2 or Wnt3 could activate CREB. We found that overexpressing Wnt2 or Wnt3 in neurons could increase the level of p-CREB, and administration of CREB inhibitor could block the positive feedback effect between Wnt2 and Wnt3. The above data suggested that there is positive feedback between Wnt2 and Wnt3 that depends on the activation of CREB. For this reason, our current results are unable to clarify the role of wnt2 and wnt3 individually. Wnt3's effects have already been well-characterized in AHN,9 which is necessary for most antidepressants to have efﬁcacy. Given this ﬁnding, in the future, experiments modulating levels of Wnt2 on the background of a Wnt3 deletion (and vice versa) will be necessary to resolve the function of each. ACKNOWLEDGMENTS This work was supported by the National 973 Basic Research Program of China (No. 2012CB911000), the National Natural Science Foundation of China (No. 31371138, 91432306, 31571100), Shandong Province Natural Science Foundation (No. ZR2014CM036) and the Fundamental Research Funds of Shandong University. This work was supported by the National 973 Basic Research Program of China (No. 2012CB911000), the National Natural Science Foundation of China (No. 31371138, 91432306, 31571100), Shandong Province Natural Science Foundation (No. ZR2014CM036) and the Fundamental Research Funds of Shandong University. DISCUSSION n = 7, 6, 7, 8, 8, 8, respectively; Po0.01 versus the No-CRS+SAL group; #Po0.05, ##Po0.01 versus the CRS+SAL group; $$Po0.01 versus the CRS+FLX group; &Po0.05, &&Po0.01 versus the Lenti-siWnt2+CRS+SAL or Lenti- siWnt3+CRS+SAL group. (b) Schematic showing the experimental schedule used to investigate the effect of Wnt2 and Wnt3 on antidepressant responses to ﬂuoxetine. (c) Sucrose consumption in SPT, (d) immobility time in FST and (e) immobility time in TST for mice in the Lenti-GFP+SAL, Lenti-GFP+CRS+SAL, Lenti-GFP+CRS+FLX, Lenti-siWnt2+CRS+SAL, Lenti-siWnt2+CRS+FLX, Lenti-siWnt3+CRS+SAL and Lenti-siWnt3+CRS+FLX groups. Po0.01 versus the Lenti-GFP+SAL group; ##Po0.01 versus the CRS+Lenti-GFP+SAL group; $$Po0.01 versus the CRS+Lenti-GFP+FLX group. (f) In the OFT, all groups showed no difference in locomotor activity. n = 10, 10, 10, 9, 10, 7, 10, respectively. All values are denoted as the mean ± s.e.m. CRS, chronic restraint stress; FLX, ﬂuoxetine; FST, forced swim test; GFP, green-ﬂuorescent protein; OFT, open ﬁeld test; SAL, saline; SPT, sucrose preference test; VH, ventral hippocampus. hypothalamic–pituitary–adrenal axis.55,56 Therefore the alteration of CORT and ACTH levels may be related with the PVN, which is regulated by the VH directly. However, the detailed mechanism still needs to be studied in the future. Furthermore, we found that knocking down Wnt2 or Wnt3 induced impaired Wnt/β-catenin signaling and deﬁcits in neurogenesis. Previous studies found that GSK3β has been implicated in the regulation of mood-related behaviors and the treatment of antidepressants.57,58 Our result Translational Psychiatry (2016), 1 – 13 Wnts and stress-induced depression-like behaviors W-J Zhou et al Wnts and stress-induced depression-like behaviors 11 reported that Wnt2 and Wnt3 transcription are CREB dependent.47,48 Fluoxetine treatment could increase the transcrip- tional activity of CREB.67 These studies suggested that ﬂuoxetine might increase Wnt2 and Wnt3 levels by activating the transcrip- tional activity of CREB. Moreover, our study provided evidence that knockdown of Wnt2 or Wnt3 abolished the antidepressant effect of ﬂuoxetine, suggesting that Wnt2 and Wnt3 are essential for mediating the antidepressant effects of ﬂuoxetine under stress condition. In addition, we found that knockdown of Wnt2 could synchronously exacerbate the CRS-induced decrease in Wnt3 and block the ﬂuoxetine-induced increase in Wnt3 and vice versa. 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https://openalex.org/W4210244804	https://europepmc.org/articles/pmc5983151?pdf=render	English	null	Stress‐testing the brain to understand its breaking points	Journal of physiology	2,018	cc-by	1,477	TRANSLATIONAL PERSPECTIVES Aside from learning how to treat these refra- ctory cases, epilepsy retains many other mysteries: for most seizures, we have no idea what provokes them, what dictates how they spread through the brain, and why, just as suddenly, they stop, the vast majority terminating spontan- eously, without medical intervention. Many, probably even most, seizures arise from a focal site, and then spread into territories that just before may have been functioning perfectly normally. Given this interplay between focal pathology and surrounding functional tissue, and the chronic nature of the condition, one might wonder what could be learnt from an acute experimental preparation that is entirely bathed in a pro-epileptic medium. Such preparations, though, have proved to be a mainstay for epilepsy research for more than 30 years now, in particular, providing insights into thenatureofparoxysmaldepolarisingshifts, ictogenesis and spreading ictal activity (Traub & Miles, 1991). Epilepsy is a common and serious neuro- logicalcondition,characterizedbyrecurrent seizures. In fact it is one of the better managed brain disorders, with up to 70% of people acquiring good control on medi- cation, but that still leaves huge numbers of people with uncontrolled seizures. Aside from learning how to treat these refra- ctory cases, epilepsy retains many other mysteries: for most seizures, we have no idea what provokes them, what dictates how they spread through the brain, and why, just as suddenly, they stop, the vast majority terminating spontan- eously, without medical intervention. Many, probably even most, seizures arise from a focal site, and then spread into territories that just before may have been functioning perfectly normally. Given this interplay between focal pathology and surrounding functional tissue, and the chronic nature of the condition, one might wonder what could be learnt from an acute experimental preparation that is entirely bathed in a pro-epileptic medium. Such preparations, though, have proved to be a mainstay for epilepsy research for more than 30 years now, in particular, providing insights into thenatureofparoxysmaldepolarisingshifts, ictogenesis and spreading ictal activity (Traub & Miles, 1991). The Journal of Physiology The Journal of Physiology That isn’t the whole picture, though, because the same gradient in seizure sus- ceptibility was also seen in disinhibited tissue; this was attributed to differences in intrinsic cellular properties along the dorso-ventral axis of the mEC. C⃝2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Ridler T, Matthews P, Phillips K, Randall A & Brown J (2018). Initiation and slow propagation of epileptiform activity from ventral to dorsal medial entorhinal cortex is constrained by an inhibitory gradient. J Physiol 596, 2251–2266. TRANSLATIONAL PERSPECTIVES The more general point is that when considering how seizures spread, we must take into account both the axonal pathways, but also how the target brain areas respond (Trevelyan, 2016); a critical feature is the endogenous protective mechanisms resistingspreadingepileptiformactivitythat have been identiﬁed using similar in vitro preparations. Ridler et al. have provided a glimpse into the continued utility of in vitro models, to ‘stress-test’ the brain. This approach can help us explore how different parts of the brain may vary in their susceptibility to seizure activity, arising from differences in their inputs, the local microcircuitry and cellular properties. Interpretation of the recordings, though, must focus on both commonalities and differences between the models used, especially if we are to extrapolate these ﬁndings for clinical use. This study also has relevance to whether GABAergic activity promotes or restrains epileptic activity. The debate on this point has arisen in part because of model-speciﬁc differences in how epileptiform activity develops. The low Mg2+ model enhances excitation, and the earliest epileptiform discharges have a very large glutamatergic component, and yet the postsynaptic response is disproportionately small, or even absent, a fact that is explained by the parallel bombardment from interneurons. In this case, what requires explanation is the minimal response in the presence of the large glutamatergic drive, and GABA is clearly acting as a restraint. In contrast, 4-aminopyridine initially induces almost purely interneuronal bursts, which may give rise to full ictal events by chloride-loading pyramidal neurons, with a secondary surge TRANSLATIONAL PERSPECTIVES inextracellular[K+],aschlorideisremoved, coupled to K+, by the co-transporter KCC2 (Viitanen et al. 2010). Interestingly, Ridler et al. used 4-aminopyridine, so one might haveexpectedeventsthereforetohavearisen from the ‘inhibitory-dense’ dorsal territory, but instead their data add further evidence for the restraining role of interneurons in epilepsy. Regardless of that debate, the data fully support the idea that the local microcircuitry and cellular behaviour will inﬂuence how new territories are recruited to a seizure event. There are likely to be multiple ways in which seizures start, but during the generalization process, when the seizure propagates away from the ictal focus into functional cortical areas, GABA will surely act in its traditional sense, to inhibit activity. This, though, can be short-lived, with GABA quickly becoming excitatory (as has also been shown using the 0 Mg model; Ellender et al. 2014), because the combined glutamatergic and GABAergic bombardment is one of the fastest ways of chloride-loading cells. This merely re-emphasises the point that GABAergic inhibition is clearly a double-edged sword. Stress-testing the brain to understand its breaking points Stress-testing the brain to understand its breaking points rhinal cortex has become a paradigm model for understanding cognition. In the process, many groups have noted gradients of cellular properties across this area. Its relevance to epilepsy is that entorhinal cortex is likely to be an important conduit for seizures arising in the temporal lobe, the most common site of focal epilepsy. Ridler and colleagues now show that propagation through this brain area is shaped strongly by the pattern of inhibitory connectivity; dorsal mEC stellate neurons have more inhibitory inputs compared to ventral stellate cells, and previous work has shown that this may explain a dorso-ventral gradient in gamma oscillations (Beed et al. 2013). Similarly, Ridler et al. show that epileptiform activity arises predominantly from ventral mEC, the side with less inhibition, and spreads at a slow rate to the dorsal side. R. Ryley Parrish and Andrew J . Trevelyan Institute of Neuroscience, Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK Email: andrew.trevelyan@ncl.ac.uk Edited by: Ole Paulsen & Matthew Nolan Edited by: Ole Paulsen & Matthew Nolan Edited by: Ole Paulsen & Matthew Nolan Epilepsy is a common and serious neuro- logicalcondition,characterizedbyrecurrent seizures. In fact it is one of the better managed brain disorders, with up to 70% of people acquiring good control on medi- cation, but that still leaves huge numbers of people with uncontrolled seizures. Beed P, Gundlﬁnger A, Schneiderbauer S, Song J, Bohm C, Burgalossi A, Brecht M, Vida I & Schmitz D (2013). Inhibitory gradient along the dorsoventral axis in the medial entorhinal cortex. Neuron 79, 1197–1207. 2033 2033 J Physiol 596.11 (2018) pp 2033–2034 TRANSLATIONAL PERSPECTIVES Author contributions Viitanen T, Ruusuvuori E, Kaila K & Voipio J (2010). The K+–Cl−cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus. J Physiol 588, 1527–1540. Competing interests Trevelyan AJ (2016). Do cortical circuits need protecting from themselves? Trends Neurosci 39, 502–511. None declared. References Beed P, Gundlﬁnger A, Schneiderbauer S, Song J, Bohm C, Burgalossi A, Brecht M, Vida I & Schmitz D (2013). Inhibitory gradient along the dorsoventral axis in the medial entorhinal cortex. Neuron 79, 1197–1207. In this issue of The Journal of Physiology, Ridler et al. (2018) use brain slices from rodent medial entorhinal cortex (mEC) to ask how inhomogeneity in the tissue affects epileptiform discharges. In essence, by pharmacologically challenging the tissue, theywereseekingtoidentifyfaultlinesinthe tissue, the sites where a seizure could take hold. It is akin to how an engineer might ‘stress-test’ the parts of a ship, separate from the whole structure. Ellender TJ, Raimondo JV, Irkle A, Lamsa KP & Akerman CJ (2014). Excitatory effects of parvalbumin-expressing interneurons maintain hippocampal epileptiform activity via synchronous afterdischarges. J Neurosci 34, 15208–15222. On the back of the seminal work by O’Keefe, the Mosers, and others, the ento- DOI: 10.1113/JP276184 2034 Perspectives J Physiol 596.11 Traub RD & Miles R (1991). Neuronal Networks of the Hippocampus. Cambridge University Press, Cambridge, UK. ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. RRP and AJT are supported by grants from MRC (MR/R005427/1) and BBSRC (BB/P019854/1). Funding Both authors have read and approved the ﬁnal version of this manuscript and agree to be accountable for all aspects of the work in RRP and AJT are supported by grants from MRC (MR/R005427/1) and BBSRC (BB/P019854/1). C⃝2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society
https://openalex.org/W2094497039	https://bmcecolevol.biomedcentral.com/counter/pdf/10.1186/1471-2148-7-52	English	null	Mature habitats associated with genetic divergence despite strong dispersal ability in an arthropod	BMC evolutionary biology	2,007	cc-by	10,994	BioMed Central BioMed Central BMC Evolutionary Biology Open Access Received: 15 September 2006 Accepted: 2 April 2007 © 2007 Ishida and Taylor; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Acc Research article Mature habitats associated with genetic divergence despite strong dispersal ability in an arthropod Seiji Ishida* and Derek J Taylor Address: Department of Biological Sciences, The State University of New York at Buffalo, Buffalo NY 14260, USA Email: Seiji Ishida* - sishida@buffalo.edu; Derek J Taylor - djtaylor@buffalo.edu * Corresponding author Received: 15 September 2006 Accepted: 2 April 2007 Background clonal selection [5] and persistent priority effects [8]. There is now considerable evidence for the rapid evolu- tion of population-specific adaptations in cladocerans such as body size, defensive structures, phototactic behav- ior, resistance to algal toxins, and parasite resistance[7,10- 12]. There is also growing direct evidence that these local adaptations arise in species with strong dispersal of prop- agules by avian vectors [3,9,10] and wind [11,12]. Colo- nization studies indicate that cyclical parthenogens such as daphniids appear to have strong dispersal capacity [13]. Additional evidence for the strong natural dispersal capac- ity of cladocerans comes from paleogenetic analysis of cladoceran embryos in aged African lake sediments [14]. A single asexual hybrid clone of North American Daphnia pulex appears to have been introduced into a central Afri- can lake and spread naturally throughout the continent in less than 60 years. Likewise, clones in the Daphnia pulex group appear to have dispersed naturally across immense distances in the Arctic [15]. Although nearly all cladocer- ans lack the potential heterotic advantage and asexual propagules of the D. pulex group, the examples do speak to the strong passive dispersal capacity of daphniids. Thus, cladocerans appear to be biologically predisposed to the scenario envisioned by Ehrlich and Raven where local selection disrupts populations despite dispersal. g The Biological Species Concept assumes that gene flow limits population differentiation and acts as a cohesive process for species [1]. Related potential cohesive forces are selective sweeps of advantageous alleles, hybrid vigour involving immigrant alleles, and genetic rescue from fre- quent bouts of extinction and recolonization [2-4]. How- ever, Ehrlich and Raven [2] argued that gene flow itself is rarely sufficient to bind populations of a species together as a unit of concerted evolution even in the face of strong dispersal. Instead of dispersal, natural selection is the pri- mary force governing gene flow among populations. Although Ehrlich and Raven [2] recognized that selection may act as a cohesive force, they posited that in most cases selection keeps populations on independent evolutionary trajectories, and predicted that the increasing use of refined genetic tools would eventually reveal that gene flow is unimportant in unifying populations. Similarly, in their monopolization hypothesis, De Meester et al. [3] concluded that many aquatic populations possess weak ongoing gene flow – local adaptations, priority effects, and large egg/seed banks may restrict gene flow despite pronounced dispersal from nearby populations. Background Thus, the disruptive selection and monopolization schools view the population as the primary unit of evolution whereas the cohesion school views the species as the primary unit of evolution. Does the available indirect evidence then support low gene flow despite strong dispersal in cladocerans? There are many population genetic studies on cladocerans and most report moderate to strong population structure using relative estimates [3]. However, this result consti- tutes weak evidence for low gene flow because it is well known that founder effects and long-distance dispersal events (which both inflate FST values) have been common in cladocerans [9,19-21]. Additional evidence is necessary to tease apart ongoing gene flow from monopolization. Analysis of population history offers one possible solu- tion as the monopolization hypothesis and the ongoing gene flow hypothesis lead to different predictions about population genetic differentiation over time. Under ongo- ing gene flow, population differentiation should decrease with time since founding [8,16]. Moreover, gene flow and recombination should reduce allelic sequence divergence and the proportion of private alleles. But, with little or no ongoing gene flow, haplotype frequency differences, the proportion of private alleles, and allelic sequence diver- gences should increase among populations with time. Despite the predictions by Ehrlich and Raven [2], modern gene flow studies have strongly favoured the primacy of cohesive gene flow over disruptive local selection [4,5]. Indeed, Slatkin [6] declared that he knows of no cases where molecular estimates of gene flow are low despite a high level of observed dispersal (i.e., direct estimates). Moreover, nearly all of the low molecular estimates of gene flow in animals appear inflated by historical proc- esses (e.g. the inclusion of potentially vicariant phylo- groups) [4]. So, Ehrlich & Raven's key prediction of marked local population genetic differentiation in the face of dispersal appears unsupported. An additional blow against disruption/monopolization hypotheses is the recent theoretical and empirical evidence that the main role of selection may be as an enhancer (not a dis- ruptor) of gene flow via heterosis or universal selective sweeps [4,7]. Still, it is possible that the existing molecular estimates of gene flow, which are based largely on obligate sexual spe- cies, are unrepresentative of animals. De Meester et al. [3], for example, ranked cyclical parthenogens as the animal breeding system with the greatest potential for monopoli- zation. Cyclical parthenogens (e.g. http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 Abstract Background: Populations may be bound by contemporary gene flow, selective sweeps, and extinction-recolonization processes. Indeed, existing molecular estimates indicate that species with low levels of gene flow are rare. However, strong priority effects and local selective regimes may hinder gene flow (despite dispersal) sending populations on independent evolutionary trajectories. In this scenario (the monopolization hypothesis), population differentiation will increase with time and genealogical evidence should yield ample private haplotypes. Cyclical parthenogens (e.g. rotifers and cladocerans such as Daphnia) have an increased capacity for rapid local adaptation and priority effects because sexual reproduction is followed by multiple generations of clonal selection and massive egg bank formation. We aimed to better understand the history of population differentiation and ongoing gene flow in Daphnia rosea s.l., by comparing population and regional divergences in mature unglaciated areas and younger previously glaciated areas. We also examined the timing and paths of colonization of previously-glaciated areas to assess the dispersal limitations of D. rosea s.l. We used DNA sequence variation (84 populations and >400 individuals) at the mitochondrial ND2 and nuclear HSP90 loci from Holarctic populations for our genetic analyses. Results: The genetic evidence indicated pronounced historical structure. Holarctic mtDNA phylogenies of D. rosea s.l. revealed three geographically restricted and divergent clades: European, Siberian and Japanese/American. The Japanese/American clade showed marked population genetic structure (FST > 0.8) that was weakly associated with geographic distance, and a high proportion of private haplotypes. Populations from older unglaciated habitats (i.e., Japan) showed higher DNA sequence divergences than populations from presumed younger habitats (i.e. non-Beringian North America) with nDNA and with mtDNA. Mismatch analyses of mtDNA and nDNA were consistent with a single rapid post-glacial expansion of D. rosea that covered most of the New World. Conclusion: Our evidence agrees with negligible gene flow after founding, and the accumulation of genetic divergence with habitat age. Existing direct evidence and our mismatch analyses indicate that the pronounced population differentiation is unlikely to be due to dispersal limitation. Instead, priority effects and local selection regimes may play a role in limiting gene flow. The results challenge the notion that lacustrine populations of cladocerans are generally unified by contemporary gene flow. Page 1 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 Background The direct evidence for strong dispersal, potential regional genetic structuring, cyclical parthenogenetic breeding sys- tem, rapid local adaptations [26] and frequent occupancy of permanent habitats suggests that D. rosea s.l. is a good candidate for testing the historical predictions of the monopolization hypothesis. float forming a surface scum and shoreline deposit [25]. The direct evidence for strong dispersal, potential regional genetic structuring, cyclical parthenogenetic breeding sys- tem, rapid local adaptations [26] and frequent occupancy of permanent habitats suggests that D. rosea s.l. is a good candidate for testing the historical predictions of the monopolization hypothesis. Here, we tested the hypothesis that D. rosea s.l. conforms to the population structure of a sexual vagile species, where ongoing gene flow is sufficient to offset population genetic divergence. We aimed to compare populations from regions that are potentially of different ages by sam- pling in well-established glacial refugia (Japan and Ber- ingia) and in younger glaciated areas (temperate North America) [27]. We also assessed the timescale and routes of recolonization of glaciated areas to understand if evo- lutionary significant dispersal limitation exists in D. rosea s.l. We found evidence for marked historical structure and little evidence for ongoing gene flow. Populations from unglaciated regions exhibited significantly higher sequence divergences for nuclear and mitochondrial DNA compared to populations from glaciated North America. We found the genetic signature of a single rapid popula- tion expansion for most of the glaciated New World, sug- gesting that dispersal limitation is insignificant on an evolutionary timescale. The results are consistent with the monopolization hypothesis where priority effects and selection trump contemporary gene flow, rendering the population or local metapopulation as the units of evolu- tion. Studies of sexual Daphnia that use the absolute estimates of population divergences yielded by DNA sequences are still uncommon (see Paland et al[18] for an asexual exam- ple). This absolute measure of population divergence will be sensitive to sampling error, but should be less inflated by founder effect. De Gelas and De Meester [19] provided evidence from COI mtDNA sequences that the present day population structure in European Daphnia magna is largely the result of a mosaic formed by historical recolo- nization from four glacial refugia rather than from ongo- ing gene flow. They detected some divergent lineages in refugial areas but lacked samples for a detailed population analysis of refugial regions. Background In the present study we obtained both relative and absolute estimates of popula- tion genetic divergence; used both nuclear and mtDNA genomic sequences; sampled populations from through- out the Holarctic range of the study species (including at a local scale to understand structure from long distance dispersal); and studied a sexual species of Daphnia that commonly occupies permanent, weakly-disturbed waters with a large range of habitat ages. Background rotifers and cladocer- ans) have an increased capacity for rapid local adaptation over other breeding systems because of their frequent sex- ual reproduction followed by multiple generations of Existing population history studies of Daphnia do support the predictions of ongoing gene flow, but the generality of these results for the genus are uncertain. Potentially older populations from large lakes show less genetic differenti- ation (i.e. lower FST's) than populations from presumed younger habitats such as small ponds [22-28]. However, the higher turnover rates in ponds compared to large lakes Page 2 of 13 (page number not for citation purposes) Page 2 of 13 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:52 http://www.biomedcentral.com/1471-2148/7/52 http://www.biomedcentral.com/1471-2148/7/52 may lead to relatively high FST's in ponds [17]. A detailed 17-year longitudinal genetic study of two Daphnia species with rock-pool subpopulations of known age showed a clear erosion of allozyme-based FST's with time [11]. Still, the rock pools also appear to lack the large effective egg banks and strong spatial variation in biotic selection pres- sures (e.g. fish versus no fish) that are characteristic of cladoceran populations from larger water bodies. Finally, a Holarctic analysis of the Daphnia tenebrosa s. l. using mtDNA RFLP's showed significant genetic structure among younger glaciated regions but no genetic structure among older non-glaciated regions. D. tenebrosa is a mem- ber of the D. pulex group that normally produces strictly asexually at higher latitudes, making it less susceptible to the proposed mechanisms of monopolization in sexual cladocerans [15]. may lead to relatively high FST's in ponds [17]. A detailed 17-year longitudinal genetic study of two Daphnia species with rock-pool subpopulations of known age showed a clear erosion of allozyme-based FST's with time [11]. Still, the rock pools also appear to lack the large effective egg banks and strong spatial variation in biotic selection pres- sures (e.g. fish versus no fish) that are characteristic of cladoceran populations from larger water bodies. Finally, a Holarctic analysis of the Daphnia tenebrosa s. l. using mtDNA RFLP's showed significant genetic structure among younger glaciated regions but no genetic structure among older non-glaciated regions. D. tenebrosa is a mem- ber of the D. pulex group that normally produces strictly asexually at higher latitudes, making it less susceptible to the proposed mechanisms of monopolization in sexual cladocerans [15]. float forming a surface scum and shoreline deposit [25]. Results f We found marked regional structure of D. rosea s.l with three divergent allopatric mtDNA clades: European; Sibe- rian and Japanese-American and no indication of recent long-distance dispersal among regions (Fig. 1 and 2) [see Additional file 1]. The ND2 alignment was 937 bp with 122 unique ingroup haplotypes, no indels (insertions or deletions), and 421 variable sites. The best-fit ML model was TIM + I + G (transition model with invariable sites plus gamma distribution). The ML distance between the European and Siberian clades was 4.47%, while the ML distances from the American-Japanese clade to the Euro- pean clade and the Siberian clade were 7.61% and 8.13% respectively. We suspected the existence of a pseudogene in two Japanese populations (Kazefuki-o-ike and Hakuba- o-ike in Nagano) because of ambiguous sequences, and cloned PCR products to further examine the possibility. Cloning of the PCR products revealed that each individual of the populations had two sets of haplotypes (Fig. 1); one with a complete reading frame and one with stop codons at 127–129 bp. Ambiguous sites and stop codons were absent from the mtDNA of all individuals sampled out- side of these two populations. The study species, Daphnia rosea s.l. is a common compo- nent of oligotrophic permanent lakes and ponds in the boreal, alpine, and subarctic zones of the Holarctic [30- 34]. Although habitat-specific head shapes are common in D. rosea, the most recent morphological treatments have supported one Holarctic species [20,21]. Allozyme and mtDNA phylogenetic analyses of a few specimens from the extremes of the geographic range reported a western European clade (Daphnia rosea) and a North American clade (D. dentifera) [22,23], but provided no evidence for more than one species in each of these allo- patric clades. D. rosea s.l. hatch from resting eggs in the Spring and reproduce asexually for at least eight genera- tions [11]. In the fall, some individuals produce sexual propagules that sink to the sediment and function as egg banks [24], but vast numbers of resting propagules may Page 3 of 13 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:52 http://www.biomedcentral.com/1471-2148/7/52 l phylogroups in Holarctic Daphnia rosea s.l nal phylogroups in Holarctic Daphnia rosea s.l. Holarctic Neighbor-joining phylogeny based uences of D. rosea s.l. and its closely related species. Numbers on branches indicate bootstrap s eographical groups (Japanese/America, Siberia and Europe). Results f Each individual of two Japanese pop Hakuba-o-ike, Nagano) had two copies of sequences (connected with a line): one (cross) was a ad one stop codon; and stop codons and frameshifts where absent from the other (circle). 0.05 Daphnia cucullata Daphnia galeata Daphnia longispina North American and Japanese D. rosea s.l. Siberian D. rosea s.l. European D. rosea s.l. 100 99 100 100 97 96 99 62 Siberian D. rosea s.l. Daphnia longispina Three regional phylogroups in Holarctic Daphnia rosea s.l Figure 1 Three regional phylogroups in Holarctic Daphnia rosea s.l. Holarctic Neighbor-joining phylogeny based on mitochon- drial ND2 sequences of D. rosea s.l. and its closely related species. Numbers on branches indicate bootstrap support. D. rosea s.l. has three geographical groups (Japanese/America, Siberia and Europe). Each individual of two Japanese populations (Kaze- fuki-o-ike and Hakuba-o-ike, Nagano) had two copies of sequences (connected with a line): one (cross) was a putative pseudo- gene, which had one stop codon; and stop codons and frameshifts where absent from the other (circle). Page 4 of 13 (page number not for citation purposes) BMC Evolutionary Biology 2007, 7:52 http://www.biomedcentral.com/1471-2148/7/52 Holarctic map showing populations and regions studied for Daphnia rosea Figure 2 Holarctic map showing populations and regions studied for Daphnia rosea. Holarctic map of the 84 sampling loca- tions of D. rosea s.l. Japan (orange) and Beringia (green) represent possible refugial areas while North America (blue) indicates a potentially younger region. Siberian and European populations (gray) were not used for phylogenetic analysis only because of reduced samples and less information about putative refugia. Japan Beringia North America Siberia Europe p g p p g p g Holarctic map showing populations and regions studied for Daphnia rosea. Holarctic map of the 84 sampling loca- tions of D. rosea s.l. Japan (orange) and Beringia (green) represent possible refugial areas while North America (blue) indicates a potentially younger region. Siberian and European populations (gray) were not used for phylogenetic analysis only because of reduced samples and less information about putative refugia. mtDNA haplotype diversities were high for each region: 0.939 ± 0.017 (95% CI) for non-Beringian North Amer- ica, 0.869 ± 0.017 for Beringia, and 0.963 ± 0.006 for Japan. Private mtDNA haplotypes (which occur in only one population) were common: 94% of the 48 North American haplotypes and 91% of 34 Japanese haplotypes. Results f 3C) and FST = 0.915 in eastern Hokkaido, Japan (N = 2, Jp-1 in Fig. 3C). For Japan, AMOVA revealed that 55.84% of the variation in ND2 sequences occurred among regions, 34.62% of the varia- tion occurred among populations within a region and 9.54% of the variation occurred within populations. Although geographic distance among populations explained a significant amount of the variation in pair- wise FST's in Japan and in non-glaciated North America, the amount of variation explained was low (Fig. 4; 6– 20%). distance) was also extremely pronounced; FST = 1.000 in western Alaska, USA (N = 5); FST = 0.988 in Colorado, USA (N = 3); FST = 0.935 in Michigan, USA (N = 3); FST = 0.916 in Ontario, Canada (N = 3); FST = 0.833 in New York, USA (N = 3); FST = 0.645 in central Honshu, Japan (N = 8, Jp-7 in Fig. 3C); FST = 0.917 in western Hokkaido, Japan (N = 3, Jp-2 in Fig. 3C) and FST = 0.915 in eastern Hokkaido, Japan (N = 2, Jp-1 in Fig. 3C). For Japan, AMOVA revealed that 55.84% of the variation in ND2 sequences occurred among regions, 34.62% of the varia- tion occurred among populations within a region and 9.54% of the variation occurred within populations. Although geographic distance among populations explained a significant amount of the variation in pair- wise FST's in Japan and in non-glaciated North America, the amount of variation explained was low (Fig. 4; 6– 20%). Unlike the universally high relative measures, absolute measures of population divergences populations from presumed older habitats (i.e. Japan and Beringia) were generally greater than the divergences among populations from glaciated habitats (i.e. non-Beringian North Amer- ica) in mtDNA and in nDNA sequences (Fig. 3). The ND2 alignment of the Japanese-American clade contained 82 unique haplotypes (937 bp), no indels, and 199 variable sites. The best-fit ML model was TrN + G (Tamura – Nei with gamma distribution). Average pairwise ML distances (using the best fit model and parameters) among popula- tions were much higher in Japan (2.84% ± 0.14% in 95% CI) than in non-Beringian North America (1.01% ± 0.08%) and Beringian North America (0.88% ± 0.18%). mtDNA nucleotide diversities were also greater in Japan (0.0234 ± 0.0026) than in non-Beringia (0.0104 ± 0.0020) and in Beringia (0.0088 ± 0.0010). Results f Similar to the mtDNA, we detected a clade comprised of the non-Beringian North American populations and two Yukon populations with nDNA (Fig. 3B) [see Additional file 3]. The clade had significant negative values of neu- trality tests (p < 0.05 in Tajima's D and p < 0.001 in Fu's FS). Three nuclear haplotypes were detected as shared among populations in this North American clade. Pair- wise differences of all sequences (n = 43) in the clade were distributed in a unimodal pattern, which is similar to the expected distributions under models of spatial and sud- den expansions (Fig 5B). The parameter Tau in the spatial expansion model was 2.427. However, the least-square fitting algorithm in ARLEQUIN [28] did not converge, suggesting that the nuclear data poorly fit the available models. Thus, the P-value for the available models and the 95% CI of the expected distributions were not available. Similarly, pairwise differences of the non-Beringian nuclear sequences (n = 35) were distributed in a unimodal pattern, and the least-square fitting algorithm did not con- verge. The HSP90 tree consisted of a subset of specimens repre- sented on the ND2 tree of the Japanese-American clade. The total alignment was total 784 bp with 192 variable sites and 95 unique alleles. Two putative introns were detected: Intron I (69 bp) between 87 bp position and 155 bp position; and Intron II (69 bp) between 448 bp position and 516 bp position. No indels were found in exons. 2–5 bp and 3–7 bp indels were found in Intron I and Intron II, respectively. The best-fit ML model was GTR + I + G (General time-reversible model with invariable site plus gamma distribution). Average pairwise ML distances (using the best-fit model and parameters) among popula- tions (nDNA) were much higher in nonglaciated regions (Japan 5.04% ± 0.14%; Beringia 4.15% ± 0.37%) than in non-glaciated North America (1.07% ± 0.06%). Results f Population structure among local sites (within 100 km We found extremely high estimates of relative population structure within both glaciated and non-glaciated regions of the North American – Japanese mtDNA clade; for non- Beringian North America FST = 0.936 (Nm = 0.02) and Snn = 0.744 (P < 0.001); for Beringia FST = 0.869 (Nm = 0.04) and Snn = 0.751 (P < 0.001) and for Japan FST = 0.804 (Nm = 0.09) and Snn = 0.834 (P < 0.001). Likewise, Page 5 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 closely related haplotypes (Fig. 3A) [see Additional file 2]. The clade had significant negative values of neutrality tests (p < 0.001 in Tajima's D and Fu's FS). Still, just two mito- chondrial haplotypes were shared among populations in the star-like North American clade. One of these shared haplotypes formed the central haplotype in a haplotype network [see Additional file 2] and was widely distributed in North America: one population in Alaska (USA), one population in Yukon (Western Canada), one population in Wisconsin (USA), three populations in Michigan (USA) and two populations in Indiana (USA). The other shared haplotype was detected only within a 120 km diameter region near the Michigan – Indiana border. Pair- wise differences of all sequences (n = 199) in the clade formed a unimodal distribution pattern, which fit the expected distributions of spatial expansion (Fig 5A; P[simu- lated SSD > observed SSD] = 0.93) and pure demographic expan- sion (P = 0.38). The parameter Tau in the spatial expansion model was 3.228 (1.582–4.288 in 95 % CI). Pairwise differences of the non-Beringian sequences (n = 175) in the clade formed a unimodal pattern, which fit the expected distributions of spatial expansion (P = 0.45) but not of pure demographic expansion (P < 0.05). The parameter Tau in the spatial expansion model was 3.088 (1.544–4.080 in 95 % CI). distance) was also extremely pronounced; FST = 1.000 in western Alaska, USA (N = 5); FST = 0.988 in Colorado, USA (N = 3); FST = 0.935 in Michigan, USA (N = 3); FST = 0.916 in Ontario, Canada (N = 3); FST = 0.833 in New York, USA (N = 3); FST = 0.645 in central Honshu, Japan (N = 8, Jp-7 in Fig. 3C); FST = 0.917 in western Hokkaido, Japan (N = 3, Jp-2 in Fig. Page 6 of 13 (page number not for citation purposes) Discussion 0.01 Jp2 Jp4 Jp1 Jp7 Jp7 Jp1 Jp5 Jp8 Jp6 Jp3 Jp7 Jp7 Jp7 Jp6 Jp6 Jp7 Jp1 Jp3 Colorado Washington Vermont Jp1 Jp2 Jp8 Jp5 Jp7 Jp3 Jp4 Jp7 Jp7 Jp6 Washington 0.01 Jp6 Jp6 Jp7 A B 40ûN 30ûN 140ûE 130ûE C Jp4 Jp6 Jp8 Jp5 Jp3 Jp2 Jp1 Pacific Ocean Jp7 0 500 km North America North America J Colorado Washington Vermont Jp1 Jp2 Jp8 Jp5 Jp7 Jp3 Jp4 Jp7 Jp7 Jp6 Washington 0.01 Jp7 A North America B A Washington Colorado North America North America Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daph- nia rosea Figure 3 Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daphnia rosea. Unrooted Neighbor-joining phylograms of mitochondrial ND2 gene sequences (A) and nuclear HSP90 gene sequences (B) for Japanese and North American D. rosea s.l. The HSP90 sequences are from a subset of specimens represented on the ND2 tree. Colors represent geographic location: blue (North America except for Alaska, USA and Yukon, Canada); green (Beringian North America, i.e. Alaska and Yukon); and gold (Japan). Thick branches have more than 70% boot- strap support, and dot branches have less than 50% bootstrap support. The map (C) shows Japanese regions (Jp1-8) and popu- lations. Note the lack of sharing of nuclear and mitochondrial haplotypes among Japanese regions (Jp1-8). 40ûN 30ûN 140ûE 130ûE C Jp4 Jp6 Jp8 Jp5 Jp3 Jp2 Jp1 Pacific Ocean Jp7 0 500 km 40ûN 30ûN 140ûE 130ûE C Jp4 Jp6 Jp8 Jp5 Jp3 Jp2 Jp1 Pacific Ocean Jp7 0 500 km C Discussion The phylogeographic and population genetic results are consistent with the monopolization hypothesis where gene flow is insufficient to prevent population divergence [2,3]. Daphnia rosea s.l. may represent Slatkin's [6] missing gene flow category with strong estimated population structure despite strong dispersal abilities. Morjan & Rieseberg's [4] review provides an indication of how North American populations showed genetic signatures of a bottleneck and postglacial expansion. Most North Amer- ican haplotypes were restricted to a star-like clade of Page 6 of 13 (page number not for citation purposes) Page 6 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 0.01 Jp2 Jp4 Jp1 Jp7 Jp7 Jp1 Jp5 Jp8 Jp6 Jp3 Jp7 Jp7 Jp7 Jp6 Jp6 Jp7 Jp1 Jp3 Colorado Washington Vermont Jp1 Jp2 Jp8 Jp5 Jp7 Jp3 Jp4 Jp7 Jp7 Jp6 Washington 0.01 Jp6 Jp6 Jp7 A B North America North America Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daph- nia rosea Figure 3 Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daphnia rosea. Unrooted Neighbor-joining phylograms of mitochondrial ND2 gene sequences (A) and nuclear HSP90 gene sequences (B) for Japanese and North American D. rosea s.l. The HSP90 sequences are from a subset of specimens represented on the ND2 tree. Colors represent geographic location: blue (North America except for Alaska, USA and Yukon, Canada); green (Beringian North America, i.e. Alaska and Yukon); and gold (Japan). Thick branches have more than 70% boot- strap support, and dot branches have less than 50% bootstrap support. The map (C) shows Japanese regions (Jp1-8) and popu- lations. Note the lack of sharing of nuclear and mitochondrial haplotypes among Japanese regions (Jp1-8). Greater ab nia rosea Figure 3 geographic distance (km) 1 5 50 500 0.0 0.4 0.8 Japan (R^2=0.149*) 0.0 0.4 0.8 geographic distance (km) pairwise FST 1 5 50 500 0.00 0.02 0.04 0.06 Japan (R^2=0.057) geographic distance (km) pariwise ML distance (ND2) 0.00 0.02 0.04 0.06 1 5 50 500 0.00 0.04 0.08 Japan (R^2=0.154*) 0.00 0.04 0.08 geographic distance (km) pariwise ML distance (HSP90) pariwise ML distance (ND2) pariwise ML distance (HSP90) pariwise ML distance (ND2) pariwise ML distance (HSP90) 1 5 50 500 0.0 0.4 0.8 Beringia (R^2=0.062) geographic distance (km) 0.0 0.4 0.8 1 5 50 500 0.00 0.02 0.04 0.06 Beringia (R^2=0.009) geographic distance (km) 0.00 0.02 0.04 0.06 1 5 50 500 0.00 0.04 0.08 Beringia (R^2=0.072) 0.00 0.04 0.08 1 5 50 500 5000 0.0 0.4 0.8 North America (R^2=0.200) geographic distance (km) 0.0 0.4 0.8 1 5 50 500 5000 5000 0.00 0.02 0.04 0.06 North America (R^2=0.069) geographic distance (km) 0.00 0.02 0.04 0.06 1 5 50 500 0.00 0.04 0.08 North America (R^2=0.029) 0.00 0.04 0.08 geographic distance (km) (ND2) pairwise FST (ND2) pairwise FST (ND2) 1 5 50 500 0.0 0.4 0.8 Beringia (R^2=0.062) geographic distance (km) 0.0 0.4 0.8 1 5 50 500 5000 0.0 0.4 0.8 North America (R^2=0.200) geographic distance (km) 0.0 0.4 0.8 pairwise FST (ND2) pairwise FST (ND2) 1 5 50 500 0.0 0.4 0.8 Japan (R^2=0.149) 0.0 0.4 0.8 geographic distance (km) pairwise FST (ND2) 1 5 50 500 5000 0.0 0.4 0.8 North America (R^2=0.200) geographic distance (km) 0.0 0.4 0.8 pairwise FST (ND2) Beringia (R^2=0.062) pariwise ML distance (ND2) 1 5 50 500 0.00 0.02 0.04 0.06 Beringia (R^2=0.009) geographic distance (km) 0.00 0.02 0.04 0.06 pariwise ML distance (ND2) 1 5 50 500 0.00 0.02 0.04 0.06 Beringia (R^2=0.009) geographic distance (km) pariwise ML distance (ND2) 1 5 50 500 5000 0.00 0.02 0.04 0.06 North America (R^2=0.069) geographic distance (km) 0.00 0.02 0.04 0.06 1 5 50 500 0.00 0.02 0.04 0.06 Japan (R^2=0.057) geographic distance (km) pariwise ML distance (ND2) 0.00 0.02 0.04 0.06 Japan (R^2=0.057) geographic distance (km) pariwise ML distance (HSP90) 1 5 50 500 0.00 0.04 0.08 Beringia (R^2=0.072) 0.00 0.04 0.08 geographic distance (km) 0.00 0.04 0.08 pariwise ML distance (HSP90) pariwise ML distance (HSP90) 1 5 50 500 0.00 0.04 0.08 Beringia (R^2=0.072) 0.00 0.04 0.08 5000 1 5 50 500 0.00 0.04 0.08 North America (R^2=0.029) 0.00 0.04 0.08 geographic distance (km) 1 5 50 500 0.00 0.04 0.08 Japan (R^2=0.154) 0.00 0.04 0.08 geographic distance (km) pariwise ML distance (HSP90) pariwise ML distance (HSP90) 5000 1 5 50 500 0.00 0.04 0.08 North America (R^2=0.029) 0.00 0.04 0.08 geographic distance (km) geographic distance (km) Summaries nia rosea Figure 4 Summaries of relative and absolute pairwise population differentiation and their associations with geographic distance in Daph- nia rosea Figure 4 Summaries of relative and absolute pairwise population differentiation and their associations with geographic distance in Daphnia rosea. Greater ab nia rosea Figure 3 Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daph nia rosea Figure 3 Greater absolute genetic divergences and regional divergences in unglaciated regions compared to glaciated regions for Daphnia rosea. Unrooted Neighbor-joining phylograms of mitochondrial ND2 gene sequences (A) and nuclear HSP90 gene sequences (B) for Japanese and North American D. rosea s.l. The HSP90 sequences are from a subset of specimens represented on the ND2 tree. Colors represent geographic location: blue (North America except for Alaska, USA and Yukon, Canada); green (Beringian North America, i.e. Alaska and Yukon); and gold (Japan). Thick branches have more than 70% boot- strap support, and dot branches have less than 50% bootstrap support. The map (C) shows Japanese regions (Jp1-8) and popu- lations. Note the lack of sharing of nuclear and mitochondrial haplotypes among Japanese regions (Jp1-8). Page 7 of 13 (page number not for citation purposes) Page 7 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 Summaries of relative and absolute pairwise population differentiation and their associations with geographic distance in Daph- nia rosea Figure 4 Summaries of relative and absolute pairwise population differentiation and their associations with geographic distance in Daphnia rosea. Row one: associations between pairwise FST's (from ND2 sequences) and geographic distances (log); Row two: associations between pairwise ML distances (from ND2 sequences) and geographic distances (log); and Row three: the associations between pairwise ML distances (from HSP90 sequences) and geographic distances (log). Population- level information for HSP90 FST estimates was unavailable. Boxplot summaries of the pairwise genetic comparisons are shown to the right of each scatterplot. Regression coefficients are given above each scatterplot ( – P < 0.001; – P < 0.01). The first two columns are from presumed glacial refugial regions of Daphnia rosea and the last column is from glaciated North America. Page 8 of 13 (page number not for citation purposes) Greater ab nia rosea Figure 3 Row one: associations between pairwise FST's (from ND2 sequences) and geographic distances (log); Row two: associations between pairwise ML distances (from ND2 sequences) and geographic distances (log); and Row three: the associations between pairwise ML distances (from HSP90 sequences) and geographic distances (log). Population- level information for HSP90 FST estimates was unavailable. Boxplot summaries of the pairwise genetic comparisons are shown to the right of each scatterplot. Regression coefficients are given above each scatterplot (* – P < 0.001; – P < 0.01). The first two columns are from presumed glacial refugial regions of Daphnia rosea and the last column is from glaciated North America. habitat maturity that older populations possessed as much or more genetic structure than presumed younger populations. Our results indicate that populations of Daphnia from large lakes and large ponds can possess strong structure – stronger than has been reported from small pond species [17,29]. Part of the difference between our estimates and prior studies arises from the increased mutation rates of our markers. But this cannot be the only explanation as some studies of pond cladocerans [29,30] have used rapidly evolving markers (i.e. control region of mtDNA and microsatellites). Nor is the pattern restricted to the present study species as Ishida & Taylor [31] have recently found similarly strong population structure using the same markers in a large study of the lacustrine special- ist, Daphnia galeata. The absolute measures of genetic divergence from presumed older habitats or metapopula- extreme the population structuring is for D. rosea. They listed a mean FST in animals of 0.45 (150 species) for mtDNA and 0.2 (781 species) for nuclear markers. A small percentage of studies (24 animal species) had a pop- ulation subdivision estimate of > 0.8. However, in each of these cases, the estimate was calculated across divergent geographic clades, suggesting that historical relationships inflated the estimate. Our finding of FST > 0.8 subdivision among populations within a regional clade and geneti- cally divergent haplotypes among Japanese populations reveals that the vagile D. rosea s.l. also possesses among the highest estimates of population subdivision yet recorded for animals. Expansion Figure 5 p p p p p p g Expansion patterns in mismatch distributions for temperate North American populations of Daphnia rosea. The distribution of pairwise haplotype differences (number of differences) within the major mitochondrial clade (A) and the major nuclear clade (B) of North American populations. Black line indicates the observed distribution. Red line indicates the expected distribution based on the spatial expansion model. Light blue zone indicates the 95% CI range of expected distribu- tion based on the spatial expansion model. The nuclear distribution lacks the 95% CI range, because the least-square fitting algorithm in ARLEQUIN failed to converge. [11,13], the massive production of resting propagules, and the evidence for rapid postglacial colonization of vast areas of the northern hemisphere indicate that dispersal limitation may also be weak. The population genetic sig- nature of D. rosea s.l. in North America is consistent with postglacial expansion from a Beringian refugium. A severe genetic bottleneck in glaciated North America was clear from both the nuclear and the mitochondrial results. Diverse nuclear lineages in the Northern Pacific Coast (especially Alaska) and reduced genetic diversity in glaci- ated regions indicates population expansion from Ber- ingia. The mitochondrial North American clade consistently showed the spatial expansion patterns in mis- match distributions with or without Alaska and Yukon populations. The spatial expansion model assumes that subdivided populations expand the distribution range and increase the total number of the individuals, while a pure demographic expansion model assumes that popula- tions are not subdivided. The observed high value of FST and the high frequency of private haplotypes among the Am1 populations [see Additional file 1] indicate subdi- vided populations. The star-like haplotype network [see Additional file 1] and the significant negative values of neutrality tests were also consistent with the expansion patterns of this clade. Using Tau = 2ut and assuming a tions in unglaciated areas (Japan and Alaska) were gener- ally much greater than those population genetic divergences among younger habitats (glaciated areas). The contrast between glaciated and unglaciated areas was apparent in both the mitochondrial and the nuclear genomes. Only the Beringian – North American contrast for ND2 distances revealed similar levels of structure. Analysis of more populations from central Beringia is nec- essary to determine if the contrast is real. Greater ab nia rosea Figure 3 Unlike previous studies, and contrary to the predictions of ongoing gene flow, we found by two crude measures of Page 8 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 Expansion patterns in mismatch distributions for temperate North American populations of Daphnia rosea Figure 5 Expansion patterns in mismatch distributions for temperate North American populations of Daphnia rosea. The distribution of pairwise haplotype differences (number of differences) within the major mitochondrial clade (A) and the major nuclear clade (B) of North American populations. Black line indicates the observed distribution. Red line indicates the expected distribution based on the spatial expansion model. Light blue zone indicates the 95% CI range of expected distribu- tion based on the spatial expansion model. The nuclear distribution lacks the 95% CI range, because the least-square fitting algorithm in ARLEQUIN failed to converge. 0% 2% 4% 6% 8% 10% 12% 0% 5% 10% 15% 20% 25% 30% 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 10 12 14 16 18 20 22 A B Frequency (ND2) pairwise haplotype differences pairwise haplotype differences Frequency (HSP90) 0% 2% 4% 6% 8% 10% 12% 0 2 4 6 8 10 12 14 16 18 20 22 B pairwise haplotype differences Frequency (HSP90) B 0% 5% 10% 15% 20% 25% 30% 0 1 2 3 4 5 6 7 8 9 10 A Frequency (ND2) pairwise haplotype differences A Frequency (ND2) pairwise haplotype differences pairwise haplotype differences Page 9 of 13 (page number not for citation purposes) Expansion Figure 5 divergence rate of 2.0% per million years for arthropod mitochondrial protein-coding genes [32], the expansion time is estimated to be 86 000 years ago (42 000–113 000 years ago in 95% CI) for populations including Alaska and Yukon, and 82 000 years ago (41 000–109 000 years ago in 95% CI) for populations excluding Alaska and Yukon. However, the relationship between the age and the evolutionary rate can be described by a vertically trans- lated exponential decay curve, where the short-term (< 1– 2 Myr) mutation rate gets exponentially higher, while the long-term (> 10 Myr) substitution rate is constant. The estimated expansion times could be overestimated more than 10 times because of the recent events (< 0.1 Myr B.P.) [33]. Thus, the spatial expansion of D. rosea s.l. in North America might have occurred rapidly after the last Ice Age (< 15 000 y B.P.). As both Japan and Alaska contain ample migratory waterfowl vectors, and our results provide evi- dence that much of the North American continent was rapidly colonized after glaciation, there are no reasons to believe that dispersal differences or limitations account for the differing divergence patterns between Japan and North America. Also, the refugial areas represent relatively small geographic areas (e.g. Japan) compared to the vast glaciated regions (e.g., much of North America), so the divergence patterns are not attributable solely to isolation by distance. local regions continue to diverge over time (perhaps over hundreds to thousands of years) in vagile D. rosea, sug- gests that the evolutionary legacy of monopolization is perhaps the stabilization of countless innovations and adaptations in local metapopulations. Some of these local adaptations such as parasite resistance [26] and defensive structures on the head [20] have already been identified for D. rosea. local regions continue to diverge over time (perhaps over hundreds to thousands of years) in vagile D. rosea, sug- gests that the evolutionary legacy of monopolization is perhaps the stabilization of countless innovations and adaptations in local metapopulations. Some of these local adaptations such as parasite resistance [26] and defensive structures on the head [20] have already been identified for D. rosea. Although we present a case of marked population differ- entiation and sequence divergence in cladocerans, it is dif- ficult to estimate how taxonomically widespread monopolization is. Sample collection Specimens of D. rosea s.l. were collected from 84 Holarctic locations [see Additional file 4]. As previous studies showed that D. cucullata and D. longispina were closely related species to D. rosea s.l. in the mitochondrial ND2 phylogeny [35], we used these species as outgroups [see Additional file 4]. All samples were preserved in absolute ethanol at room temperature, or frozen at -80°C. These specimens were morphologically identified according to Ueno [36], Brooks [20], Glagolev [37], Taylor et al. [22] and Flössner [21]. If populations of Daphnia rosea are indeed exposed to neg- ligible gene flow despite dispersal, then what are the evo- lutionary consequences? Speciation by habitat isolation or vicariance may occur if populations are separated in distant glacial refugia. Most populations of D. rosea have likely gone extinct from the regular waves of catastrophic Pleistocene glaciation or experienced turnover as local selection regimes changed. Indeed, the shape of the mtDNA tree for D. rosea is consistent with the recoloniza- tion of the Holarctic from at least three geographically – distant bottlenecked glacial refugia. Our evidence that Expansion Figure 5 However, if a mixed breeding system and differing local selection regimes are important drivers of monopolization, then the patterns we found in D. rosea s.l. are perhaps more widespread among eukaryotes. More work is necessary to understand the causes of monopoli- zation. Future genetic analysis of preserved egg/embryo banks of cladocerans in lake sediments and permafrost may offer detailed window into the process. Expansion Figure 5 Both the increased divergence with age of habitat and the very high proportion of private haplotypes found in most popula- tions are inconsistent with ongoing gene flow acting to homogenize populations. However, they do satisfy a major prediction of the monopolization hypothesis: increased differentiation and divergence with population age. The roles of local selection, ongoing gene flow with drift, and dispersal limitation are difficult to tease apart as con- tributors to the observed population structuring of D. rosea. Ongoing gene flow with drift probably occurs, but it seems unlikely given the lack of allele sharing and rela- tively high sequence divergences among geographically close regions in Japan with both nDNA and mtDNA (see also De Gelas and De Meester [19]). Colonization studies Page 9 of 13 (page number not for citation purposes) Page 9 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 http://www.biomedcentral.com/1471-2148/7/52 http://www.biomedcentral.com/1471-2148/7/52 divergence rate of 2.0% per million years for arthropod mitochondrial protein-coding genes [32], the expansion time is estimated to be 86 000 years ago (42 000–113 000 years ago in 95% CI) for populations including Alaska and Yukon, and 82 000 years ago (41 000–109 000 years ago in 95% CI) for populations excluding Alaska and Yukon. However, the relationship between the age and the evolutionary rate can be described by a vertically trans- lated exponential decay curve, where the short-term (< 1– 2 Myr) mutation rate gets exponentially higher, while the long-term (> 10 Myr) substitution rate is constant. The estimated expansion times could be overestimated more than 10 times because of the recent events (< 0.1 Myr B.P.) [33]. Thus, the spatial expansion of D. rosea s.l. in North America might have occurred rapidly after the last Ice Age (< 15 000 y B.P.). As both Japan and Alaska contain ample migratory waterfowl vectors, and our results provide evi- dence that much of the North American continent was rapidly colonized after glaciation, there are no reasons to believe that dispersal differences or limitations account for the differing divergence patterns between Japan and North America. Also, the refugial areas represent relatively small geographic areas (e.g. Japan) compared to the vast glaciated regions (e.g., much of North America), so the divergence patterns are not attributable solely to isolation by distance. Conclusion O fi di Our findings are consistent with negligible gene flow after founding, and the accumulation of genetic divergence with regional age. Daphnia rosea s.l. showed increased lev- els of population divergence in mature unglaciated regions compared to presumed younger glaciated regions. Previous direct evidence of vagility in D. rosea s. l. and our evidence for a rapid population expansion at the conti- nental scale, suggests that most population differentiation is unlikely to be due to dispersal limitation. Instead, pri- ority effects and selection may play roles in limiting gene flow. The results challenge the notion that sexual popula- tions of lacustrine cladocerans are generally unified by contemporary gene flow. Of course, indirect estimates of gene flow apply only to the loci under study. Selective sweeps of globally favoura- ble alleles may occur at unsampled loci [4]. We note that the inclusion of mtDNA in our study makes our conclu- sion of strong population differentiation conservative – mtDNA is perhaps one of the most susceptible loci to homogenizing selective sweeps in animals [34]. We also note that during the multiple generations of clonal repro- duction in D. rosea, mtDNA and nuclear genomes are linked, increasing the potential for background selection against immigrant genes [3,29]. Our finding of generally concordant divergence patterns for nuclear and mito- chondrial loci and the frequent observation of stable, lake-specific body shapes in D. rosea s.l. [20] suggests that the strong population differentiation is not merely a locus-specific phenomenon. DNA extraction, PCR and sequencing Total genomic DNA was extracted by using QuickExtract (Epicentre). Samples were homogenized in 30–50 μL of the QuickExtract solution, incubated at 65°C for 2 h and 98°C for 10 min, and stored at -20°C. A c. 1000 bp frag- ment of the mitochondrial protein-coding NADH-2 Page 10 of 13 (page number not for citation purposes) Page 10 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 (ND2) gene was amplified using the primers MetF3 (5'- GTT CAT GCC CCA TTT ATA GGT TA-3') and TrpR (5'- GAA GGT TTT TAG TTT AGT TAA CTT AAA ATT CT-3') [35]. A c. 760 bp fragment of the nuclear protein-coding HSP90 gene was amplified using the primers (5'-TTA CGA GTC CAG ATG GGC TT-3') and (5'-ATC CGT TAT GAA TCC CTG ACT GA-3') [38]. Each 50 μL PCR reaction con- sisted of 5 μL of extracted DNA, 10× PCR buffer [50 mM KCl, 1.5 mg MgCl2, 10 mM Tris-HCl pH 8.3, 0.01% (w/v) gelatin], 2 mM of each dNTP, 1 μM of each primer and 1 unit of Taq DNA polymerase. The PCR temperature profile for the mitochondrial ND2 gene was as follows: 40 cycles of 94°C for 30 s, 48°C for 30 s and 72°C for 1 min, and final extension at 72°C for 5 min. The PCR temperature profile for the nuclear HSP90 gene was as follows: 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 1 min, and final extension at 72°C for 5 min. Because ND2 PCR products from two Japanese populations had multiple peaks in their sequences, cloning was performed for the products using the TOPO TA Cloning Kit (Invitrogen). Then, three cultured colonies were used for sequencing. For HSP90, all PCR products were cloned using the TOPO TA Cloning Kit, and then four or five cultured colonies were sequenced. Sequences of ND2 and HSP90 were obtained in both directions by Genaissance pharmaceuti- cals (Connecticut, USA) or Roswell Park Cancer Institute (New York, USA). Sequences were assembled, edited with Sequencher 4.2 (Gene Code Corporation), and aligned manually with Se-Al 2.0 [39]. number of sequence differences among different haplo- types sampled from the same subpopulation and Hb is the mean number of sequence differences among samples from different subpopulations [42]. DNA extraction, PCR and sequencing This estimator is equivalent to the NST estimator of Lynch & Crease [45] without a Jukes-Cantor correction for multiple substitu- tions. Snn is a measure of how often the most closely related sequences (nearest-neighbors) occur in the same locality (water bodies in the present case). The number of private haplotypes (which occur in only one population) was counted. Haplotype diversities and nucleotide diver- sities were calculated using DnaSP [44]. We further analyzed phylogeography of the North Ameri- can and Japanese populations using mitochondrial and nuclear data. For mitochondrial analysis, we used the same data of ND2 sequences that was used in construc- tion of the Holarctic D. rosea s.l. phylogeny. For nuclear analysis, we used 36 individuals of the D. rosea s.l: 13 indi- viduals from 13 non-Beringia North American popula- tions, six individuals from six Beringia North America, and 17 individuals from 17 Japanese populations. Intron boundaries of the HSP90 sequences were identified by comparing arthropod HSP90 mRNA sequences (e.g. AY528900, AY423488) and by examining the intron- splicing signature sequences. Two introns were identified and aligned using clustalW. For mitochondrial and nuclear data of North American and Japanese D. rosea s.l., the best-fit ML model was selected by hierarchical likeli- hood ratio tests using Modeltest. ML distance was calcu- lated using PAUP. NJ phylogeny was constructed with ML distance and 1000 bootstrap replicates using PAUP. The Mantel test [46] was used to determine the relationship between genetic distance (pairwise FST from Arlequin 3.01 [28], and pairwise ML divergence) and geographic dis- tance to test an isolation-by-distance model. The geo- graphic distance was calculated using the great-circle distance formula. The Mantel test was implemented in IBD 1.52 [47] using 1000 permutations. The computer program TCS 1.21 [48] was used to estimate the mito- chondrial haplotype network that illustrates all connec- tions that have 95% probability of being the most parsimonious. Analysis of molecular variance (AMOVA) was carried out for Japanese regional groups (Jp1-8 in Fig. 3C) using the TrN + G (Tamura-Nei distance with gamma distribution) by Arlequin with 1000 permutations. Authors' contributions SI and DJT conceived and designed the research. SI per- formed the experiments and analyzed the data. SI and DJT wrote the paper. All authors read and approved the final manuscript. q g g 4. Morjan CL, Rieseberg LH: How species evolve collectively: implications of gene flow and selection for the spread of advantageous alleles. Mol Ecol 2004, 13(6):1341-1356. g ( ) 5. Cousyn C, Meester LD, Colbourne JK, Brendonck L, Verschuren D, Volckaert F: Rapid, local adaptation of zooplankton behavior to changes in predation pressure in the absence of neutral genetic changes. Proc Nat Acad Sci USA 2001, 98:6256–6260. References 1. Mayr E: Animal species and evolution. Cambridge, MA , Harvard University Press; 1963. y 2. Ehrlich PR, Raven PH: Differentiation of populations. Science 1969, 165:1228-1232. 3. De Meester L, Gómez A, Okamura B, Schwenk K: The Monopoli- zation Hypothesis and the dispersal-gene flow paradox in aquatic organisms. Acta Oecologica 2002, 23:121-135. Additional file 1 Neighbor-joining phylogeny based on mitochondrial ND2 sequences of D. rosea s.l. and its closely related species. Numbers on branches indi- cate bootstrap support. Cli k h f fil p p 8. Boileau MG, Hebert PDN, Schwartz. SS: Non-equilibrium gene frequency divergence: persistent founder effects in natural populations. J Evol Biol 1992, 4:25-39. p p J 9. Figuerola J, Green AJ, Michot T: Invertebrate eggs can fly: evi- dence of waterfowl-mediated gene flow in aquatic inverte- brates. Amer Natur 2005, 165:274-280. [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S1.eps] 10. Charalambidou I, Santamaría L: Waterbirds as endozoochorous dispersers of aquatic organisms: a review of experimental evidence. Acta Oecologica 2002, 23:165-176. Genetic analyses For the mitochondrial phylogeny of Holarctic D. rosea s.l., we used a total of 403 individuals of D. rosea s.l from 84 Holarctic locations: 212 individuals from 30 non-Beringia North American populations (in North America except Alaska and Yukon), 43 individuals from nine Beringia North American populations (in Alaska, USA and Yukon, Canada), 97 individuals from 19 Japanese populations, 25 individuals from six Siberian populations, and 26 indi- viduals from 20 European populations [see Additional file 4]. The best-fit maximum likelihood (ML) model was selected by hierarchical likelihood ratio tests in the pro- gram Modeltest 3.7 [40]. The ML distance was based on the best fit model and calculated using PAUP v4.0b10 [41]. An Neighbor-joining (NJ) tree was constructed with the ML distance and 1000 bootstrap replicates using PAUP. To address the population expansion of major North American clades in mitochondrial and nuclear phyloge- nies, we performed the neutrality tests of Fu's FS [49] and Tajima's D [50] and revealed the frequency distribution of the number of pairwise sequence differences (i.e. the mis- match distribution). The values of Fu's FS and Tajima's D are expected to have significantly negative values for pop- ulation expansion (other explanations are background Population level information was available for the mtDNA of specimens from North America and Japan. We calculated FST with the estimator of Hudson et al. [42] and the nearest-neighbor statistic (Snn) of Hudson [43] as implemented in DnaSP 4.10 [44]. With Hudson et al.'s estimator of FST, each polymorphic site in an alignment is treated as a locus and FST = 1-Hw/Hb. Hw is the mean Page 11 of 13 (page number not for citation purposes) Page 11 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2148/7/52 BMC Evolutionary Biology 2007, 7:52 Additional file 4 Lists of Daphnia specimens used in this study. The lists provide the information of sampling locations, geographical position, IDs of ND2 and HSP90 sequences, number of individuals analyzed for ND2 sequences, and number of cloned colonies analyzed for HSP90 sequences. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S4.xls] selection and hitch-hiking associated with selective sweeps). The significance of Fu's FS and Tajima's D were tested by random permutation using 1000 replicates in ARLEQUIN 3.01 [28]. The mismatch distributions were calculated using ARLEQUIN. The observed distributions were compared with the simulated distributions under the models of pure demographic expansion and spatial expansion. Additional file 2 Mitochondrial haplotype network of North American and Japanese clades. Haplotypes are coloured according to geographical regions: blue (North America), green (Beringia), and red (Japan). White dots repre- sent inferred haplotypes. The dot size is proportional to the number of dif- ferent populations with the identical haplotype. Two divergent lineages in North America and several divergent lineages in Japan were excluded, because the computer program TCS failed to connect them. Cli k h f fil g 11. Haag CR, Riek M, Hottinger JW, Pajunen VI, Ebert D: Genetic Diversity and Genetic Differentiation in Daphnia Metapopu- lations With Subpopulations of Known Age. Genetics 2005, 170:1809-1820. 12. Cáceres CE, Soluk DA: Blowing in the wind: a field test of over- land dispersal and colonization by aquatic invertebrates. Oecologia 2002, 131:402-408. g 13. Louette G, De Meester L: High dispersal capacity of cladoceran zooplankton in newly founded communities. Ecology 2005, 86:353-359. [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S2.eps] 14. Mergeay J, Declerck S, Verschuren D, De Meester L: Daphnia com- munity analysis in shallow Kenyan lakes and ponds using dor- mant eggs in surface sediments. Fresh Biol 2006, 51:399-411. Additional file 4 Lists of Daphnia specimens used in this study. The lists provide the information of sampling locations, geographical position, IDs of ND2 and HSP90 sequences, number of individuals analyzed for ND2 sequences, and number of cloned colonies analyzed for HSP90 sequences. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S4.xls] Acknowledgements g Kotov AA, Sheveleva NG, Timoshkin O, Yoshida T, Urabe J, Tanaka S, Cerny M, Svensson J-E, Belyaeva M, Piel WH, Faustova M contributed to the collection of valuable Daphnia specimens. This research was supported by an NSF PEET grant. The sampling of Japanese specimens was partly sup- ported by a Grant-in-Aid for Scientific Research B to JU from the MECSST of Japan. Additional material g g 6. Slatkin M: Gene flow and population structure. In Ecological genetics Edited by: Real LA. Princeton, NJ , Princeton University Press; 1994:3–17. g g 6. Slatkin M: Gene flow and population structure. In Ecological genetics Edited by: Real LA. Princeton, NJ , Princeton University Press; 1994:3–17. Additional file 1 Neighbor-joining phylogeny based on mitochondrial ND2 sequences of D. rosea s.l. and its closely related species. Numbers on branches indi- cate bootstrap support. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S1.eps] Additional file 2 Mitochondrial haplotype network of North American and Japanese clades. Haplotypes are coloured according to geographical regions: blue (North America), green (Beringia), and red (Japan). White dots repre- sent inferred haplotypes. The dot size is proportional to the number of dif- ferent populations with the identical haplotype. Two divergent lineages in North America and several divergent lineages in Japan were excluded, because the computer program TCS failed to connect them. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S2.eps] Additional file 3 Neighbor-joining phylogeny based on nuclear HSP sequences of Japa- nese and North American D. rosea s.l. Numbers on branches indicate bootstrap support. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-7-52-S3.eps] 7. Ebert D, Haag C, Kirkpatrick M, Riek M, Hottinger JW, Pajunen VI: A Selective Advantage to Immigrant Genes in a Daphnia Meta- population. Science 2002, 295:485-488. 7. Ebert D, Haag C, Kirkpatrick M, Riek M, Hottinger JW, Pajunen VI: A Selective Advantage to Immigrant Genes in a Daphnia Meta- population. Science 2002, 295:485-488. Genetic analyses The model of pure demographic expansion assumes that non-subdivided populations suddenly expand in population size and increase the total number of individuals [51]. The model of spatial expansion assumes that subdivided populations expand the distribu- tion range and increase the total number of individuals [52,53]. Both models estimate Tau = 2ut where u = mTμ. t is the estimated time of expansion, mT is the number of nucleotide sequences under study, and μ is the mutation rate per time. A least square procedure and bootstrap approach (1000 replications) provided the probability that the sum of squared deviations is lower in the observed distribution than in the simulated distributions, and calculated the 95% confidence intervals of the expected distribution and Tau. selection and hitch-hiking associated with selective sweeps). The significance of Fu's FS and Tajima's D were tested by random permutation using 1000 replicates in ARLEQUIN 3.01 [28]. The mismatch distributions were calculated using ARLEQUIN. The observed distributions were compared with the simulated distributions under the models of pure demographic expansion and spatial expansion. The model of pure demographic expansion assumes that non-subdivided populations suddenly expand in population size and increase the total number of individuals [51]. The model of spatial expansion assumes that subdivided populations expand the distribu- tion range and increase the total number of individuals [52,53]. Both models estimate Tau = 2ut where u = mTμ. t is the estimated time of expansion, mT is the number of nucleotide sequences under study, and μ is the mutation rate per time. A least square procedure and bootstrap approach (1000 replications) provided the probability that the sum of squared deviations is lower in the observed distribution than in the simulated distributions, and calculated the 95% confidence intervals of the expected distribution and Tau. http://www.biomedcentral.com/1471-2148/7/52 http://www.biomedcentral.com/1471-2148/7/52 17. Crease T, Lee SK, Yu SL, Spitze K, Lehman N, Lynch M: Allozyme and mtDNA variation in populations of the Daphnia pulex complex from both sides of the Rocky Mountains. Heredity 1997, 79:242-251. 44. Rozas J, Sánchez-DelBarrio JC, Messegyer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other meth- ods. Bioinformatics 2003, 19:2496-2497. f 45. Lynch M, Crease T: The analysis of population survey data on DNA sequence variation. Mol Biol Evol 1990, 7:377-394. 18. Paland S, Colbourne JK, Lynch M: Evolutionary history of conta- gious asexuality in Daphnia pulex. Evolution 2005, 59:800-813. q 46. Mantel N: The detection of disease clustering and a general- ized regression approach. Cancer Res 1967, 27:209-220. g y p p 19. De Gelas K, De Meester L: Phylogeography of Daphnia magna in Europe. Mol Ecol 2005, 14:753-764. g pp 47. Bohonak AJ: IBD (isolation by distance): a program for analy- ses of isolation by distance. J Hered 2002, 93:153-154. 20. Brooks JL: The systematics of North American Daphnia. Mem Conn Acad Arts Sci 1957, 13:1-180. y J 48. Clement M, Posada D, Crandall KA: TCS: a computer program to estimate gene genealogies. Mol Ecol 2000, 9:1657-1660. 21. Flössner D: Die Haplopoda und Cladocera Mitteleuropas. Lei- den, The Netherlands. , Backhuys Publishers; 2000. 49. Fu YX: Statistical tests of neutrality of mutations against pop- ulation growth, hitchhiking and background selection. Genet- ics 1997, 147:915-925. 22. Taylor DJ, Hebert PDN, Colbourne JK: Phylogenetics and evolu- tion of the Daphnia longispina group (Crustacea) based on 12S rDNA sequence and allozyme variation. Mol Phy Evol 1996, 5:495-510. 50. Tajima F: Statistical methods for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 1989, 123:585-595. 23. Schwenk K, Posada D, Hebert PDN: Molecular systematics of European Hyalodaphnia: the role of contemporary hybridi- zation in ancient species. Proc R Soc B 2000, 267:1833-1842. yp y p y p 51. Rogers AR, Harpending H: Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol 1992, 9:552-569. 52. Ray N, Currat M, Excoffier L: Intra-deme molecular diversity in spatially expanding populations. Mol Biol Evol 2003, 20:76-86. p 24. Wolf HG, Carvalho GR: Resting eggs of lake-Daphania II. In situ observations on the hatching of eggs and their contribution to population and community structure. Fresh Biol 1989, 22:471-478. p y p g p p 53. http://www.biomedcentral.com/1471-2148/7/52 Excoffier L: Patterns of DNA sequence diversity and genetic structure after a range expansion: lessons from the infinite- island model. Mol Ecol 2004, 13:853-864. 25. Brooks JL: Re-descriptions of Daphnia pulex var. pulicaria Forbes, D. thorata F. and D. dentifera F. Amer Midl Natur 1953, 49:772-800. 26. Duffy MA, Sivars-Becker L: Rapid evolution and ecological host- parasite dynamics. Ecol Lett 2007, 10(1):44-53. p y ( ) 27. Hewitt GM: The structure of biodiversity – insights from molecular phylogeography. Front Zool 2004, 1:4. 28. Excoffier L, Laval G, Schneider S: Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evol Bioinf Online 2005, 1:47-50. f 29. Pálsson S: Microsatellite variation in Daphnia pulex from both sides of the Baltic Sea. Mol Ecol 2000, 9:1075-1088. 30. Pfrender ME, Spitze K, Lehman N: Multi-locus genetic evidence for rapid ecologically based speciation in Daphnia. Mol Ecol 2000, 9:1717-1735. 31. Ishida S, Taylor DJ: Quaternary diversification in a sexual Hol- arctic zooplankter, Daphnia galeata. Mol Ecol in press. 32. DeSalle R, Freedman T, Prager EM, Wilson AC: Tempo and mode of sequence evolution in mitochondrial-DNA of Hawaiian Drosophila. J Mol Evol 1987, 26:157-164. p J 33. Simon YWH, Phillips MJ, Cooper A, Drummond AJ: Time depend- ency of molecular rate estimates and systematic overesti- mation of recent divergent times. Mol Biol Evol 2005, 22:1561-1568. 34. Bazin E, Glemin S, Galtier N: Population Size Does Not Influence Mitochondrial Genetic Diversity in Animals. Science 2006, 312(5773):570-572. 35. Ishida S, Kotov AA, Taylor DJ: A new divergent lineage of Daph- nia (Cladocera: Anomopoda) and its morphological and genetical differentiation from Daphnia curvirostris Eylmann, 1887. Zool Jour Linnean Soc 2006, 146:385-405. J 36. Ueno M: Daphnia of Hokkaido and their habitat - lakes. Konan Woman's Col Res 1972, 8:65-102. 37. Glagolev SM: Genus Daphnia. In A key-book of the freshwater inver- tebrates of Russia and its vicinity 2 Crustacea Edited by: Alekseev VR. St Petersburg , Zoological Institute Press; 1995:48-58. g g 38. Kotov AA, Ishida S, Taylor DJ: An assessment of species diversity in the Daphnia curvirostris (Crustacea: Cladocera) complex with phylogenetic evidence for the independent origin of neckteeth. J Plank Res in press. Additional file 3 15. Weider LJ, Hobæk A, Colbourne JK, Crease TJ, Dufresne F, Hebert. PDN: Holarctic phylogeography of an asexual species com- plex: I. mtDNA variation in arctic Daphnia. Evolution 1999, 53:777-792. 16. Haag CR, Riek M, Hottinger JW, Pajunen VI, Ebert D: Founder events as determinants of within-island and among-island genetic structure of Daphnia metapopulations. Heredity 2006, 96:150-158. 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Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge J p 39. Rambaut A: Se-Al: Sequence Alignment Editor. [http:// evolve.zoo.ox.ac.uk/]. 1996. ] 40. Posada D, Crandall KA: Modeltest: testing the model of DNA substitution. Bioinformatics 1988, 14(9):817-818. ( ) 41. Swofford DL: PAUP. Phylogenetic Analysis Using Parsimony (and Other Methods). Version 4. Sunderland, Massachusetts , Sinauer Associates; 2000. ; 42. Hudson RR, Slatkin M, Maddison WP: Estimation of levels of gene flow from DNA sequence data. Genetics 1992, 132:583-589. 42. Hudson RR, Slatkin M, Maddison WP: Estimation of lev 43. 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https://openalex.org/W2121846392	https://jneuroengrehab.biomedcentral.com/counter/pdf/10.1186/1743-0003-11-21	English	null	Research highlights in neurorehabilitation	Journal of neuroengineering and rehabilitation	2,014	cc-by	1,578	Pajaro-Blázquez and Pons Journal of NeuroEngineering and Rehabilitation 2014, 11:21 http://www.jneuroengrehab.com/content/11/1/21 Pajaro-Blázquez and Pons Journal of NeuroEngineering and Rehabilitation 2014, 11:21 http://www.jneuroengrehab.com/content/11/1/21 Pajaro-Blázquez and Pons Journal of NeuroEngineering and Rehabilitation 2014, 11:21 http://www.jneuroengrehab.com/content/11/1/21 JNER JOURNAL OF NEUROENGINEERING AND REHABILITATION © 2014 Pajaro-Blázquez and Pons; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. EDITORIAL Open Access Abstract Recent studies of the mechanisms underlying plasticity and recovery following neurological injuries have originated innovative lines of research in neurorehabilitation. Additionally, the development of new technologies to facilitate the performance of evaluation and intervention procedures has stimulated research on novel rehabilitation paradigms and more effective rehabilitation strategies. However, translation of novel interventions into clinical practice remains a challenge. Further investigation to evaluate the effectiveness of novel rehabilitation approaches is needed. In this thematic series, six manuscripts summarize the results of current research with focus on evaluation and treatment strategies of relevance in neurorehabilitation. and clinicians. These technologies provide the means to deliver intensive therapies and to lessen the physical effort required by clinical personnel during manual therapy. However, there is no clear evidence that better outcomes can be achieved using technology-assisted intervention modalities (such as robotic therapy) than with conventional therapies. This might be partially due to the fact that active participation of patients throughout the rehabilitative process is a prerequisite to facilitate recovery. The first robotic devices that were made available for clinical studies and subsequently for clinical interventions were predominantly systems that completely guided the subjects’ movements while the user remained passive during the therapy. Robotic systems recently developed for application in rehabilitation provide different control strategies that are designed to promote subjects’ engage- ment and participation during therapy. To achieve this goal, in addition to carrying out clinical research studies with focus on the assessment of different robotic and non-robotic technologies, researchers need to gain a better understanding of the processes underlying motor recovery and motor adaptation, and leverage scientific discoveries in these areas to develop new control strategies for both robotic and sensor-based devices [2]. * Correspondence: marta.pajaro@csic.es Spanish National Research Council (CSIC), Bioengineering Group, Ctra. Campo Real km 0.200, La Poveda, 28500 Madrid, Arganda del Rey, Spain Research highlights in neurorehabilitation Marta Pajaro-Blázquez* and Jose Luis Pons Editorial Assessment and treatment strategies in NR were the focus of many contributions many presentations at the conference [3]. The authors of outstanding presentations were encouraged to submit a manuscript to be published in this thematic series of the Journal of Neuroengineering and Rehabilitation (JNER). As a result, this thematic series includes six manuscripts highlighting major areas of ongoing research in the field of NR. Three articles focused on the development of assessment strategies of relevance to the field of NR. Coscia et al. explored how changes in the amount of arm weight support affect muscle activation, kinematics and motor control during arm reaching movements in healthy volunteers. They analyzed the hand, elbow, shoulder and trunk trajectories, and the activation of fourteen muscles of the upper extremity to derive muscle synergies during arm reaching movements in six different conditions or arm weight support. They found that the same eight muscle synergies were consistently observed during the performance of arm reaching movements across different levels of arm weight support. The magnitude of the activation of individual muscles showed a decrease with an increase in the amount of arm weight support. The authors found variability in the kinematic of arm reaching movements across different levels of arm weight support, although no specific trend was associated with the amount of arm weight support. Fleerkotte et al. evaluated the effects of an eight-week training program with a prototype of a robotic gait trainer (LOPES) in patients with iSCI, using an impedance control strategy. Improvements in functional outcomes were observed after training, which were retained at an eight-week follow-up. Significant improvements were also observed in the hip joint kinematics. The authors reported that participants showing pre-training lower walking function showed the largest relative improvements. Finally, del-Ama et al. presented a cooperative control strategy for an hybrid exoskeleton (Kinesis) that leveraged robotic actuation and functional electrical stimulation of lower limb muscles. This control approach was tested in healthy subjects showing that the system is able to monitor muscle performance during gait to estimate and manage muscle fatigue while training with the Kinesis system. The authors argue that the system has potential for gait rehabilitation in patients with SCI. Bravo-Esteban et al. Editorial Many relevant areas in the field of neurorehabilitation (NR) have witnessed significant developments over the last two decades. Nevertheless, there are still major challenges that neuroscientists need to address to achieve clinically-relevant results in this field. The manuscripts included in this thematic series deal with two main problems in NR: 1) The development of accurate assessment tools for the evaluation of neurological deficits, such as spasticity, sensorimotor impairments, weakness and functional limitations. 2) Improvement in therapies aimed at both minimizing chronic deficits and maximizing function after central nervous system (CNS) injury. On one hand, relatively simple techniques that can be easily utilized in the clinic to assess the severity of injuries of the CNS that affect descending motor commands and thus movement control are still lacking. These tools should allow clinicians to accurately diagnose and characterize different deficits and detect changes over time. In addition, the evaluation procedures should be easy to implement and hence applicable in a clinical setting. The outcomes of such evaluation procedures would be used to guide therapies, predict prognosis and monitor changes [1]. On the other hand, robotic and sensor-based systems enabling novel therapies have emerged and quickly gained the attention of researchers The 2012 International Conference in Neurorehabilita- tion (ICNR2012) was recently held in Toledo (November 14–16, 2012) and was partially founded by the HYPER pro- ject (Spanish CONSOLIDER-INGENIO 2010 programme, CSD2009-00067). ICNR2012 aimed to bring together scientists from multiple disciplines with a common interest in Neural Engineering and Rehabilitation. The meeting provided a suitable scientific environment to Pajaro-Blázquez and Pons Journal of NeuroEngineering and Rehabilitation 2014, 11:21 http://www.jneuroengrehab.com/content/11/1/21 Page 2 of 2 The remaining three articles are focused on the assessment of different treatment strategies in NR. Marchal-Crespo et al. assessed different robotic training strategies to evaluate the impact of movement error amplification and reduction on motor learning and muscle activation during the performance of a simple task. This study was performed with the robotic system MARCOS, suitable to be used simultaneously with Magnetic Resonance Imaging (MRI). Healthy subjects were asked to train under four control strategies: haptic guidance, no guidance, error amplification and noise disturbance while measuring muscle activation in addition to neuroimaging data. The authors found that strategies adding disturbances and amplifying error enhanced muscle activation and boosted motor learning. discuss the latest advances in the field. Editorial investigated the potential of the electromyographic (EMG) coherence analysis of the tibialis anterior (TA) muscle as a measure of strength, quality of movement during gait and severity of spasticity in subjects with an incomplete spinal cord injury (iSCI). They explored the coherence value within specific frequency bands during isometric, isokinetic and isotonic controlled movements of the ankle. They found that intramuscular 15–30 Hz TA coherence value during isometric contractions generating maximal voluntary torque (MVT) was correlated to residual strength and gait function after iSCI, and that several spastic symptoms were negatively correlated with 10–16 Hz and 40-60Hz TA coherence value during isometric contractions at MVT. Altogether, this thematic series present new approaches to evaluate CNS motor control and innovative treatment strategies in NR. Future research in this field should focus on providing evidence of usability and efficacy in the clinical settings, including a detailed description of the ideal conditions for application of these new NR technologies. Received: 13 February 2014 Accepted: 14 February 2014 Published: 4 March 2014 Published: 4 March 2014 References 1. Pajaro-Blázquez M, Miangolarra-Page JC: “Clinical use of emerging technologies for neurorehabilitation. Am. J. Phys. Med. Rehabil. Acad. Physiatr 2013, 92(Suppl 1):e1. no. 10. 2. Krebs HI, Hogan N: Robotic therapy: the tipping point. Am. J. Phys. Med. Rehabil. 2012, 91(Suppl 3):S290–S297. no. 11. 3. Pons JL, Torricelli D, Pajaro M: Converging Clinical and Engineering Research on Neurorehabilitation. Springer; 2013. http://www.link.springer.com/book/ 10.1007%978-3-642-34546-3. doi:10.1186/1743-0003-11-21 Cite this article as: Pajaro-Blázquez and Pons: Research highlights in neurorehabilitation. Journal of NeuroEngineering and Rehabilitation 2014 11:21. Received: 13 February 2014 Accepted: 14 February 2014 Published: 4 March 2014 References Meyer et al. presented results suggesting that pre-trial brain electroencephalographic (EEG) data analysis can be used to predict performance in a reaching task in healthy subjects. In order to measure the learning process, the authors used the normalized time-to-target parameter, defined as the time required to reach the target from the verbal command to begin the movement, divided by the distance from starting to target position. The analysis of EEG data revealed the involvement of brain areas that also play a role in the motor learning process, and that the α/μ frequency band contains the most relevant information to predict intervention outcomes. pp 3. Pons JL, Torricelli D, Pajaro M: Converging Clinical and Engineering Research on Neurorehabilitation. Springer; 2013. http://www.link.springer.com/book/ 10.1007%978-3-642-34546-3. doi:10.1186/1743-0003-11-21 Cite this article as: Pajaro-Blázquez and Pons: Research highlights in neurorehabilitation. Journal of NeuroEngineering and Rehabilitation 2014 11:21.
https://openalex.org/W3204425217	https://e-journal.staima-alhikam.ac.id/jis/article/download/782/pdf	Indonesian	null	PENERAPAN MODEL BLENDED LEARNING PADA MATA PELAJARAN FIKIH KELAS IV DI ERA NEW NORMAL	Journal Islamic Studies	2,021	cc-by-sa	6,680	Kata kunci: Blended Learning, Fikih, New Normal Kata kunci: Blended Learning, Fikih, New Normal Abstrak Penelitian ini dilatar belakangi dengan munculnya wabah covid-19 yang mengakibatkan pembelajaran di sekolah ditiadakan untuk sementara dan dirubah menggunakan pembelajaran secara online. Pada mata pelajaran yang materinya tidak hanya membutuhkan teori saja, melainkan juga membutuhkan praktek. Sehingga dalam menggunakan pembelajaran online kurang efektif. Setelah adanya era new normal, Mendikbud mengeluarkan Surat Edaran mengenai kebijakan untuk melakukan pembelajaran tatap muka di sekolah dengan syarat tertentu. MI Masyhariyah Kebontemu menggunakan kebijakan tersebut sebagai acuan dalam menerapkan model blended learning. Penerapan blended learning digunakan untuk mengatasi permasalahan pada pembelajaran online terutama untuk pembelajaran Fikih. Penelitian ini bertujuan untuk mengetahui perencanaan dan penerapan dalam menggunakan model blended learning pada mata pelajaran Fikih di era new normal. Metode penelitian yaitu metode deskriptif kualitatif dengan bentuk studi kasus. Kemudian untuk teknik pengumpulan data menggunakan wawancara, observasi, dokumentasi. Hasil dari penelitian ini yaitu: 1) Perencanaan model blended learning dilakukan dengan sitematis dari merencanakan waktu, lokasi, materi, metode, media, langkah- langkah pembelajaran, dan pembuatan RPP (Rencana Pelaksanaan Pembelajaran); 2) Penerapan model blended learning di MI Masyhariyah Kebontemu dapat peneliti simpulkan berlangsug dengan baik. 1 1 1 PENERAPAN MODEL BLENDED LEARNING PADA MATA PELAJARAN FIKIH KELAS IV DI ERA NEW NORMAL DI MI MASYHARIYAH KEBONTEMU PETERONGAN JOMBANG Robiatin Alvin Nadiroh Sekolah Tinggi Agama Islam Ma’had Aly Al-Hikam Malang Sekolah Tinggi Agama Islam Ma’had Aly Al-Hikam Malang Robiatinalvin96@gmail.com 1 Yo Ceng Giap, dkk., Pembelajaran E-Learning Di Masa Pandemi COVID-19 (Yogyakarta: Deepublish, 2020), hlm. 44. 2 Surat Edaran Nomor 4 Tahun 2020 tentang Pelaksanaan Kebijakan Pendidikan dalam Masa Darurat Penyebaran Coronavirus Disease (COVID-19). 3 Munir, Pembelajaran Jarak Jauh Berbasis Teknologi Informasi Dan Komunikasi (Bandung: Alfabeta, 2009), hlm. 18 Pendahuluan 2 Di akhir tahun 2019 ini dunia lagi digemparkan dengan wabah yang menyerang seluruh dunia yang berasal dari Wuhan China yaitu Covid-19 (Coronavirus Disease 2019). Virus tersebut mulai menyebar di Indonesia pada awal tahun 2020. Dengan kondisi seperti ini, Kementerian Kesehatan Republik Indonesia mengeluarkan kebijakan lima protokol kesehatan utama mengenai Covid-19 yaitu protokol untuk bidang kesehatan, bidang komunikasi, bidang pengawasan perbatasan, bidang pendidikan, dan bidang transportasi1. Kebijakan dalam bidang pendidikan, berdampak pada dunia pendidikan sehingga membuat sekolah harus ditutup sementara dan para siswa disarankan untuk belajar dari rumah guna menghindari tertularnya Covid-19. Pernyataan tersebut diperkuat oleh Mendikbud RI Bapak Nadiem Anwar Makarim. Kebijakan tersebut dikeluarkan melalui Surat Edaran Nomer 4 Tahun 2020. Salah satu isi kebijakan tersebut yaitu belajar dilakukan secara online dirumah masing-masing.2 Terdapatnya Surat Edaran tersebut, sebagian sekolah melaksanakan proses Pembelajaran Jarak Jauh (PJJ). Pembelajaran jarak jauh yaitu proses pembelajaran yang dilakukan tanpa adanya kontak secara langsung antara siswa dan guru, namun proses pembelajaran disampaikan melalui media seperti Televisi, radio dan jejaring internet.3 Namun kebanyakan sekolah menggunakan pembelajaran jarak jauh secara daring atau online. Meski dalam kondisi ditengah pandemi, proses pembelajaran harus tetap diberlangsungkan meskipun secara online agar siswa tidak ketinggalan pengetahuan. Di era Covid-19 banyak dijumpai fenomena yang terjadi dalam pembelajaran secara online, salah satunya adalah kendala dalam pelaksanaanya. Salah satu faktor yang mempengaruhi adalah faktor ekonomi. Dikarenakan pembelajaran online ini harus terhubung dengan internet, maka siswa diwajibkan untuk memiliki handphone yang spesifikasinya sudah android dan juga kuota internet. Hal ini membuat para wali murid yang ekonominya menengah kebawah mengeluh dan protes karena pembelajaran ini memakan banyak biaya untuk membeli kuota internet dan dibeberapa daerah banyak dijumpai permasalahan dalam susah sinyal. 3 Pasca beberapa bulan wabah Covid-19 menyerang Indonesia dan mempengaruhi proses pembelajaran dalam dunia pendidikan Indonesia. Pemerintah mengeluarkan peraturan baru terkait dengan pembelajaran era Covid-19 yaitu pembelajaran era new normal. Era new normal adalah kehidupan baru setelah adanya pandemi yang mana masyarakat kembali melakukan aktivitas seperti biasa namun dengan tetap mematuhi protokol kesehatan4. Hal tersebut diperkuat dengan adanya kebijakan berupa diterbitkannya Surat Keputuasan Bersama Nomer 116266/A5/HK/20205 dan Surat Edaran oleh Kemendikbud Nomer 15 Tahun 2020 pada bab III6. Kedua Surat Edaran tersebut berisi panduan dalam melaksanakan sekolah dimasa pandemi. Mata pelajaran Fikih merupakan salah satu mata pelajaran wajib di MI Masyhariyah Kebontemu. Pada materi pembelajaran Fikih tidak hanya membutuhkan teori saja, melainkan juga membutuhkan praktek dalam pembelajaran. j 6 Surat Edaran Nomor 15 Tahun 2020 tentang Pedoman Penyelenggaraan Belajar dari Rumah dalam Masa Darurat Penyebaran Corona Virus Disease (COVID019). ( gy j y , ), 5 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020 tentang Panduan Penyelenggaraan Pembelajaran. 7 Husamah, Pembelajaran Bauran (Blended Learning) (Jakarta: Prestasi Pustakaraya, 2014), hal. 11. 4 Erwan Agus Purwanto, New Normal : Perubahan Sosial Ekonomi Dan Politik Akibat COVID-19 Erwan Agus Purwanto, New Normal : Perubahan Sosial Ekonomi Dan Politik Akibat COVID-19 (Yogyakarta: Gadjah Mada University Press, 2020), hlm. 7. 4 Erwan Agus Purwanto, New Normal : Perubahan Sosial Ekonomi Dan Politik Akibat COVID-19 (Y k t G dj h M d U i it P 2020) hl 7 g , (Yogyakarta: Gadjah Mada University Press, 2020), hlm. 7. 5 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020 tentang Panduan Penyelenggaraan Pembelajaran. 6 4 Erwan Agus Purwanto, New Normal : Perubahan Sosial Ekonomi Dan Politik Akibat COVID-19 (Yogyakarta: Gadjah Mada University Press, 2020), hlm. 7. 5 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020 tentang Panduan Penyelenggaraan Pembelajaran. 6 Surat Edaran Nomor 15 Tahun 2020 tentang Pedoman Penyelenggaraan Belajar dari Rumah dalam Masa Darurat Penyebaran Corona Virus Disease (COVID019). 7 Pendahuluan Penggunaan pembelajaran melalui online kurang maksimal dalam menerapkan pembelajaran praktek. Karena dengan pembelajaran online siswa tidak bisa melihat prakteknya secara langsung. Sehingga pada materi yang membutuhkan praktek banyak siswa yang kurang faham. Oleh karena itu pembelajaran Fikih ini harus dipraktekkan secara langsung agar mudah difahami oleh siswa dan juga bisa mempraktekkan secara langsung dengan bimbingan oleh guru. Di MI Masyhariyah yang beralamatkan di Dsn. Temulawak RT 013 / RW 003 Desa Kebontemu Kec. Peterongan Kab. Jombang. Dalam menyikapi adanya kendala pada saat proses pembelajaran online, MI Masyhariyah menerapkan model pembelajaran Blended Learning. Blended Learning merupakan model pembelajaran yang menggabungkan antara model pembelajaran secara online dengan pembelajaran secara tatap muka.7 Alasan dipilihnya model blended learning karena untuk mengatasi kendala yang ada pada pembelajaram online. Dengan begitu diharpakan agar proses pembelajaran berjalan dengan efektif. Pada awal sebelum menggunakan blended learning, proses pembelajaran di MI Masyhariyah dilakukan secara home visit dan online. Pembelajaran secara home visit 4 diterapkan pada kelas 1 – 3. Sedangkan untuk pembelajaran secara online diterapkan pada kelas 4 – 6. Dalam pembelajaran online, MI Masyhariyah menggunakan Whatsapp group. Namun pembelajaran ini hanya berlangsung 2 bulan saja, sebab ditemukan beberapa kendala seperti: 1. Ada beberapa mata pelajaran yang membutuhkan praktek, tidak hanya teori saja. Salah satunya pada mata pelajaran Fikih yang didalam materinya banyak menggunakan praktek. 1. Ada beberapa mata pelajaran yang membutuhkan praktek, tidak hanya teori saja. Salah satunya pada mata pelajaran Fikih yang didalam materinya banyak menggunakan praktek. 2. Kekurang fahaman siswa dalam menangkap materi. 3. Terdapatnya kejenuhan siswa dan wali murid. 4. Wali murid kendala finansial mengenai kuota internet, bahkan juga terdapat wali murid yang belum mempunyai Handphone Android. Dan kendala susah sinyal dikarenakan sekolah berada di daerah pedesaan. Dari adanya kendala-kendala tersebut akhirnya pihak sekolah sepakat untuk menggunakan model blended learning. Pembelajaran ini dilaksanakan dengan satu hari dalam seminggu untuk tatap muka dan lima hari untuk pembelajaran online dan mandiri. Pembelajaran tatap muka dilakukan secara shift yaitu hari Senin untuk kelas 1 dan 2, Selasa untuk kelas 3 dan 4, dan Rabu untuk kelas 5 dan kelas 6. Setelah dilakukan model pembelajaran ini, siswa mengalami perkembangan pemahaman materi dilihat dari segi nilai dan keaktifan siswa dalam menjawab soal. Dalam hal ini peneliti tertarik untuk meneliti mengenai penerapan model blended learning pada mata pelajaran Fikih di Era New Normal di MI Masyhariyah Kebontemu, Peterongan, Jombang. 8 Wasis D. Dwiyogo, Pembelajaran Berbasis Blended Learning (Depok: Rajawali Pers, 2108). A. Blended Learning A. Blended Learning 1. Pengertian Blended Learning j 11 Siti Istiningsih, Hasbullah, “Blended Learning, Trend Strategi Pembelajaran Masa Depan”, Jurna Elemen, Vol. 1, No. 1, Januari 2015, hlm. 53. , ) 10 Husamah, Pembelajaran Bauran...., hlm. 11-14. 9 Subhan Adi Santoso, Pembelajaran Blended Learning Masa Pandemi (Pasuruan: CV. Penerbit Qiara Media, 2020) hlm. 96. j 5 Ibrahim ,”Perpaduan Model Pembelajaran Aktif Konvensional (Ceramah) Dengan Cooperatif (Make- A Match) untuk Meningkatkan Hasil Belajar Pendidikan Kewarganegaraan”, Jurnal Ilmu Pendidikan Sosial, Sains, dan Humaniora, Vol. 3, No.2, Juni 2017, hlm. 202. g , , g , 17 Riza Anugrah P., dkk., “Penerapan Metode Pembelajaran Mandiri Dalam Meningkatkan Hasil Belaja Peserta Didik”, Jurnal Pendidikan Luar Sekolah, Vol. 1, No. 1, April 2017, hlm. 26. , , , , p 18 Siti Istiningsih, Hasbullah, “Blanded Learning, Trend......, hlm. 55. g , , g, 19 Husamah, Pembelajaran Bauran...., hlm. 27-30. g , , g, , 14 Husamah, Pembelajaran Bauran....., hlm. 37. , , , , , , 16 Siti Istiningsih, Hasbullah, “Blanded Learning, Trend......, hlm. 54. 12 Wahyu Aji Fatma Dewi, “Dampak Covid-19 Terhadap Implementasi Pembelajaran Daring di Sekolah Dasar”, Jurnal Ilmu Pendidikan, Vol. 2, No. 1, April 2020, hlm. 56. , , , , p , 13 Siti Istiningsih, Hasbullah, “Blanded Learning, Trend........., hlm. 52 Dasar”, Jurnal Ilmu Pendidikan, Vol. 2, No. 1, April 2020, hlm. 56. 13 Siti Istiningsih, Hasbullah, “Blanded Learning, Trend........., hlm. 52 , , , p , 13 Siti Istiningsih, Hasbullah, “Blanded Learning, Trend........., hlm. 52 14 H h P b l j B hl 37 1. Pengertian Blended Learning Blended Learning memiliki dua suku kata, yaitu blended dan learning. Dalam bahasa inggris blended berarti campuran dan learning berarti belajar. Maka arti blended Learning dalam bahasa inggris adalah campuran pembelajaran. Namun, blended learning memiliki arti yaitu kegiatan pembelajaran offline dengan kegiatan pembelajaran online yang dikombinasikan dalam strategi penyampaian pembelajaran.8 5 Definisi blended learning dapat digambarkan seperti gambar berikut: Definisi blended learning dapat digambarkan seperti gambar berikut: Definisi blended learning dapat digambarkan seperti gambar berikut Gambar 2.1 Definisi Blended Learning Classrooms Lessons Online Learning Blended Learning Gambar 2.1 Definisi Blended Learning Berdasarkan gambar 2.1, blended learning dibentuk dengan cara mencampurkan pembelajaran tatap muka dengan pembelajaran online.9 Mengenai definisi dari blended learning beberapa ahli berbeda-beda dalam mendifinisikan tentang blended learning, diantaranya sebagai berikut:10 a. Moebs dan Weibelzahl, menurut mereka blended learning yaitu pembelajaran yang mencampurkan antara pembelajaran secara online dengan pembelajaran secara tatap muka. b. Menurut McDonald, blended learning adalah menyelipkan media online pada proses pembelajaran tatap muka dengan waktu bersamaan. c. Menurut purtadi, blended learning, merupakan kolaborasi berbagai media pembelajaran yang berbeda dalam segi teknologi, aktivitas, dan barbagai jenis peristiwa yang bertujuan membuat proses pembelajaran yang terbaik untuk peserta didik. Dari definisi dari para ahli diatas memberikan simpulan bahwa blended learning adalah pencampuran pembelajaran online dengan offline dengan tujuan menjadikan pembelajaran yang terbaik. 2. 2. Komponen Blended Learning Blended learning mempunyai tiga komponen yang terdiri dari pembelajaran secara daring (dalam jaringan) atau online, pembelajaran secara tatap muka, dan pembelajaran mandiri.11 a. Pembelajaran daring atau Online 6 Pembelajaran online disebut juga dengan pembelajaran daring. Menurut Isman pembelajaran daring adalah pembelajaran yang memanfaatkan koneksi internet dalam proses pembelajaran.12 Dalam mengakses materi pembelajaran pembelajaran daring mempergunakan teknologi internet.13 , p ( g , ), 21 Abdul Hayy Abdul’al, Pengantar Ushul Fikih (Jakarta: Pustaka Al-Kautsar, 2014), hlm. 5-6. 20 Kasuwi Saiban, Metode Penetapan Hukum Islam (Malang: Setara Press, 2019), hlm. 102. yy , g ( ) 22 Abdul Hayy Abdul’al, Pengantar Ushul....., hlm. 6-7. b. Pembelajaran tatap muka b. Pembelajaran tatap muka Pembelajaran tatap muka merupakan proses pembelajaran yang dilakukan dengan berinteraksi secara langsung antara guru dengan siswa14. Pembelajaran tatap muka termasuk salah satu bentuk model pembelajaran konvensional yaitu pembelajaran yang menggunakan siswa sebagai objek belajar untuk menerima pengetahuan15. Pembelajaran tatap muka merupakan proses pembelajaran yang dilakukan dengan berinteraksi secara langsung antara guru dengan siswa14. Pembelajaran tatap muka termasuk salah satu bentuk model pembelajaran konvensional yaitu pembelajaran yang menggunakan siswa sebagai objek belajar untuk menerima pengetahuan15. Pembelajaran ini tergolong dalam komponen blended learning, karena sebagai tambahan dalam menjelaskan secara rinci pembelajaran yang kurang faham saat pembelajaran online.16 c. Belajar mandiri Belajar mandiri merupakan proses belajar siswa dengan cara membaca, menelaah serta memahami pengetahuan sesuai dengan materi pembelajaran yang dilaksanakan dalam lingkungan sekolah maupun diluar sekolah.17 Belajar mandiri merupakan proses belajar siswa dengan cara membaca, menelaah serta memahami pengetahuan sesuai dengan materi pembelajaran yang dilaksanakan dalam lingkungan sekolah maupun diluar sekolah.17 Belajar mandiri merupakan termasuk komponen blended learning, karena didalam pembelajaran daring siswa dituntut untuk belajar secara mandiri, dengan begitu siswa dapat lebih memahami.18 3. Tahapan Blended Learning Tahapan blended learning digunakan untuk perancangan dan penerapan proses pembelajaran blended learning agar berjalan efektif. Berikut ini tahapan blended learning yang disarankan oleh Soekartawi:19 3. Tahapan Blended Learning Tahapan blended learning digunakan untuk perancangan dan penerapan proses pembelajaran blended learning agar berjalan efektif. Berikut ini tahapan blended learning yang disarankan oleh Soekartawi:19 7 a. Materi bahan ajar ditentukan terlebih dahulu b. Membuat rancangan yang akan dipakai. b. Membuat rancangan yang akan dipakai. c. Menerapkan proses blended learning dengan efektif. d. Menyiapkan penilaian untuk membuat evaluasi pembelajaran. B. Pembelajaran Fikih yy g ( 22 Abdul Hayy Abdul’al, Pengantar Ushul....., hlm. 6-7. , q ( g g, ), 24 Siti Rahayu, dkk., COVID-19 The Nightmare or Rainbow (Jakarta: Mata Aksara, 2020), hlm. 1-5. 23 Abdul Wahab Khalaf, Ilmu Ushul Fiqih (Semarang: Dina Utama Semarang, 2014), hlm. 27. 1. Pengetian Pembelajaran Fikih 1. Pengetian Pembelajaran Fikih Fikih menurut pengertian etimologi adalah lafal mashdar dari fi’il madhi َفَقَه yang mempunyai arti mengerti secara mendalam. Sedangkan menurut pengertian terminologi, Fikih berarti sekumpulan hukum syari’at islam bersifat praktis yang diambil dari dalil-dalil terperinci baik dari al- Qur’an maupun hadis.20 Beberapa ulama berbeda pendapat mengenai pengertian Fikih secara etimologi, berikut ini adalah pengertian Fikih secara etimologi menurut para ulama:21 a. Imam al-Ghozali dan al-Amidi: Fikih secara etimologi berarti pemahaman secara mutlak. b. Syaikh Abu Ishaq Asy-Syirazi dan yang sependapat dengannya: Fikih adalah pemahaman terhadap hal-hal rumit saja. c. Abu Hasan Al-Bashri dan Imam Ar-Razi dan yang sependapat dengannya: Fikih adalah pemahaman terhadap tujuan ungkapan si pembicara saja. Adapun pendapat para ulama mengenai pengertian Fikih secara terminologi sebagai berikut:22 a. Al-Baidhawi: Fikih yaitu ilmu yang membahas beberapa hukum syara’ tentang perilaku yang diperoleh dari dalil terperinci. b. Asy-Syirazi dan imam Al-Haramain: Fikih adalah pengetahuan beberapa hukum syara’ yang diperoleh melalui ijtihad. c. Al-Qadhi Abu Bakar Al-Baqillani: Fikih adalah dugaan tentang hukum syara’ yang diperoleh melalui ijtihad. Dari definisi para ulama diatas, pengertian Fikih secara etimologi peneliti memilih pendapat dari Imam al-Ghozali dan al-Amidi yang 8 menyatakan bahwa Fikih secara etimologi berarti pemahaman secara mutlak. Sedangkan dalam pengertian Fikih secara terminologi peneliti memilih pendapat dari Asy-Syirazi dan imam Al-Haramain yang menyatakan Fikih adalah pengetahuan hukum-hukum syara’ yang diperoleh melalui ijtihad. 2. Tujuan Pembelajaran Fikih Tujuan Pembelajaran Fikih Proses pembelajaran pada mata pelajaran Fikih yang diberikan pada siswa bertujuan untuk memahamkan pokok-pokok hukum islam dan tata cara pelaksanaanya kepada siswa, agar dapat diterapkan dikehidupan. Abdul Wahab Khallaf berpendapat bahwa tujuan Fikih adalah setiap bertindak dan berkata kepada manusia selalu menerapkan hukum-hukum syari’at islam.23 1. Pengertian COVID-19 1. Pengertian COVID-19 Covid-19 merupakan singkatan dari Coronavirus Disease 2019. Covid-19 merupakan penyakit jenis baru yang disebabkan oleh virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-Cov-2) yang sebelumnya disebut Novel Coronavirus (2019-nCov).24 1. Pengertian COVID 19 Covid-19 merupakan singkatan dari Coronavirus Disease 2019. Covid-19 merupakan penyakit jenis baru yang disebabkan oleh virus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-Cov-2) yang sebelumnya disebut Novel Coronavirus (2019-nCov).24 Gambar 2.3 Ilustrasi Virus SARS-Cov-2 Gambar 2.3 Ilustrasi Virus SARS-Cov-2 p p y g 28 Febrianty, dkk., New Normal Edisi II (Yogyakarta: Zahir Publishing, 2020), hlm. 39. (t.t.:t.p.,2020), hlm. 1. 27 Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus......, hlm. 6-10. g p ( p ) 26 Kementrian Pendidikan dan Kebudayaan, Buku Saku Pedoman Edukasi Perubahan Perilaku (t.t.:t.p.,2020), hlm. 1. 25 Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus dilakukan masyaraka untuk cegah penularan COVID-19?, (t.t.: t.p.,2020), hlm. 4. 2. Penularan dan Gejala Covid-19 2. Penularan dan Gejala Covid-19 Covid-19 merupakan penyakit yang disebabkan oleh virus dengan sifat mudah menular dengan cepat, penularan terjadi melalui 2 jalan, yang pertama melalui kontak langsung dengan orang yang terinfeksi. Yang kedua melalui permukaan yang telah terkontaminasi dari orang terinfeksi, maksutnya dengan menyentuh benda atau permukaan yang terkena droplet Covid-19 merupakan penyakit yang disebabkan oleh virus dengan sifat mudah menular dengan cepat, penularan terjadi melalui 2 jalan, yang pertama melalui kontak langsung dengan orang yang terinfeksi. Yang kedua melalui permukaan yang telah terkontaminasi dari orang terinfeksi, maksutnya dengan menyentuh benda atau permukaan yang terkena droplet 9 (tetesan caira yang berasal dari batuk dan bersin) dari orang yang tertular, kemudian menyentuh mulut, hidung, atau mata sebelum mencuci tangan.25 Orang yang terserang Covid-19 memiliki gejala-gejala yang dialami, gejala tersebut muncul dengan bertahap. Gejala yang paling sering dirasakan yaitu suhu tubuh di atas 37 derajat celciu, adanya sakit tenggorokan, dan mudah merasakan lelah. Namun pada sebagian dari orang terinfeksi terdapat gejala yang lainnya.26 25 Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus dilakukan masyaraka untuk cegah penularan COVID-19?, (t.t.: t.p.,2020), hlm. 4. Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus......, hlm. 6-10. 28 Febrianty, dkk., New Normal Edisi II (Yogyakarta: Zahir Publishing, 2020), hlm. 39. 25 Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus dilakukan masyarakat untuk cegah penularan COVID-19?, (t.t.: t.p.,2020), hlm. 4. 26 Kementrian Pendidikan dan Kebudayaan, Buku Saku Pedoman Edukasi Perubahan Perilaku, (t.t.:t.p.,2020), hlm. 1. 27 Kementrian Kesehatan Republik Indonesia dan GERMAS, Apa yang harus......, hlm. 6-10. 28 F b i dkk N N l Edi i II (Y k Z hi P bli hi 2020) hl 39 3. Pencegahan COVID-19 3. Meskipun Covid-19 merupakan virus yang mudah menular, namun masih dapat dicegah agar tidak tertular. Berikut beberapa beberapa hal yang bisa mencegah tertularnya Covid-19:27 a. Rajin mencuci tangan pakai sabun dengan air atau membawa handsanitizer saat berpergian. b. Menjaga agar tubuh tetep sehat. c. Ketika bersin dan batuk menggunakan etika yang baik d. Social distancing dan physical distancing (menjaga jarak dan pembatasan interaksi fisik). D. Era New Normal g p 30 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020 tentang Panduan Penyelenggaraan Pembelajaran. D. Era New Normal 1. Pengertian Istilah new normal memang sedang banyak digunakan kembali setelah menyebarnya Covid-19. Tanpa terlihat kemungkinan ada obat ditemukan dalam waktu dekat, semua orang mulai membayangkan hidup dalam new normal ini.28 Istilah new normal memang sedang banyak digunakan kembali setelah menyebarnya Covid-19. Tanpa terlihat kemungkinan ada obat ditemukan dalam waktu dekat, semua orang mulai membayangkan hidup dalam new normal ini.28 New normal (kebiasaan baru) yang mempunyai arti sebagai tatanan kehidupan baru dimana sesuatu yang tidak biasa dilakukan sebelumnya menjadi hal normal untuk dilakukan. Dalam kaitannya dengan pandemi Covid-19 yang melanda dunia saat ini, new normal diartikan sebagai 10 perubahan perilaku masyarakat yang akan mempengaruhi kegiatan sehari- hari masyarakat selanjutnya.29 2. Pembelajaran di Era New Normal Berkenaan dengan telah ditetapkannya Surat Keputusan Bersama Nomor 04/KB/2020, Pada keputusan tersebut berisi tentang pemberian izin melakukan pembelajaran tatap muka pada satuan pendidikan.30 Dalam melaksanakan pembelajaran tatap muka di satuan pendidikan, terdapat prosedur yang telah ditetapkan melalui keputusan bersama menteri, antara lain sebagai berikut:31 a. Kondisi kelas, tempat duduk harus berjarak minimal 1 meter. b. Dilaksanakan dengan sistem shift saat masuk kelas. c. Di lingkungan sekolah wajib menyediakan tempat cuci tangan disetiap tempat yang banyak dikunjungi oleh orang. d. Seluruh warga sekolah wajib memakai masker. p , 2 Lexy J. Moleong, Metodelogi Penelitian Kualitatif (Bandung: PT Remaja Rosdakarya, 2017), hlm. 6. 29 Andika Chandra P., COVID-19 Dan NEW NORMAL Seri 3 (Bogor: Guepedia, 2020), hlm. 13. j 1 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020....., hlm. 5-7. Pembelajaran. 31 Keputusan Bersama Menteri Nomer 116266/ A5/ HK/ 2020....., hlm. 5-7. 3. Subyek Penelitian Subyek penelitian merupakan seseorang yang ingin diperoleh keterangannya.33 Data yang digunakan oleh peneliti berupa informasi yang berkaitan dengan penerapan blended learning pada mata pelajaran Fikih dalam memasuki era new normal di MI Masyhariah Kebontemu, Kecamatan Peterongan, Kabupaten Jombang. Adapun subyek sumber data yang diperlukan dalam hal ini adalah: a. Kepala Sekolah MI Masyhariyah Kebontemu. b. Guru Fikih MI Masyhariyah Kebontemu. c. Murid MI Masyhariyah Kebontemu. d. Wali murid MI Masyhariyah Kebontemu. 33 Zainal Arifin, Penelitian Pendidikan Metode Dan Paradigma Baru (Bandung: PT Remaja Rosdakarya, 2011), hlm. 300. 34 Sugiyono, Metode Penelitian Kuantitatif, Kualitatif Dan R & D (Yogyakarta: Alfabeta, 2011), hlm. 296. 296. 35 Margono, Metodologi Penelitian Pendidikan (Jakarta: PT. Rineka Cipta, 2000), hlm 165. 4. Teknik Pengumpulan Data Pengumpulan data merupakan langkah yang paling penting dalam penelitian, karena tujuan utamanya dalam pengumpulan data ini adalah mendapatkan data yang diperlukan.34 Adapun teknik yang digunakan oleh peneliti sebagai berikut: 33 Zainal Arifin, Penelitian Pendidikan Metode Dan Paradigma Baru (Bandung: PT Remaja Rosdakarya 2011), hlm. 300. 1. Pendekatan dan Jenis Penelitian 1. Pendekatan dan Jenis Penelitian Dalam penelitian ini peneliti menggunakan pendekatan penelitian kualitatif jenis deskriptif. Menurut Lexy J. Moleong penelitian kualitatif merupakan penelitian yang bertujuan untuk memahami suatu fenomena mengenai apa yang dialami oleh subyek penelitian misalnya perilaku, persepsi, motivasi, tindakan, dll., secara holistik dengan cara deskripsi dalam bentuk kata-kata dan bahasa, pada suatu konteks khusus yang alamiah dan dengan memanfaatkan berbagai metode alamiah.32 Dalam hal ini adalah Penerapan Model Blended Learning Pada Mata Pelajaran Fikih pada Era New Normal. Dalam penelitian ini peneliti menggunakan pendekatan penelitian kualitatif jenis deskriptif. Menurut Lexy J. Moleong penelitian kualitatif merupakan penelitian yang bertujuan untuk memahami suatu fenomena mengenai apa yang dialami oleh subyek penelitian misalnya perilaku, persepsi, motivasi, tindakan, dll., secara holistik dengan cara deskripsi dalam bentuk kata-kata dan bahasa, pada suatu konteks khusus yang alamiah dan dengan memanfaatkan berbagai metode alamiah.32 Dalam hal ini adalah Penerapan Model Blended Learning Pada Mata Pelajaran Fikih pada Era New Normal. 2. Instrumen Penelitian Instrumen penelitian yaitu alat yang digunakan oleh peneliti dalam mengumpulkan data yang digunakan untuk menjawab persoalan penelitian. Adapun instrumen yang digunakan dalam penelitian ini berupa pedoman 2. Instrumen Penelitian Instrumen penelitian yaitu alat yang digunakan oleh peneliti dalam mengumpulkan data yang digunakan untuk menjawab persoalan penelitian. Adapun instrumen yang digunakan dalam penelitian ini berupa pedoman 11 wawancara, chek-list, catatan dokumentasi, alat perekam berupa handphone untuk merekam suara saat melakukan wawancara. 3. Subyek Penelitian ), 34 Sugiyono, Metode Penelitian Kuantitatif, Kualitatif Dan R & D (Yogyakarta: Alfabeta, 201 296 36 J.R. Raco, Metode Penelitian Kualitatif: Jenis, Karakteristik, Dan Keunggulannya (Jakarta: PT Gramedia Widiasarana Indonesia, 2010), hlm. 112. g y 38 Husamah, Pembelajaran Bauran...., hlm. 11 Dokumentasi Penelitian ini menggunakan metode dokumentasi untuk mencari data otentik sebagai pelengkap seperti gambaran umum tentang MI Masyhariyah Kebontemu secara detail. , ), 37 Sugiyono, Metode Penelitian,......,hlm. 298. a. Wawancara a. Wawancara Menurut Margono, wawancara merupakan salah satu cara dalam pengumpulan data dengan perantara kontak langsung antara peneliti dengan narasumber dengan mengajukan pertanyaan-pertanyaan.35 Menurut Margono, wawancara merupakan salah satu cara dalam pengumpulan data dengan perantara kontak langsung antara peneliti dengan narasumber dengan mengajukan pertanyaan-pertanyaan.35 Dalam kesempatan wawancara peneliti akan mengambil data atau informasi dari beberapa sumber mengenai penerapan blended learning pada mata pelajaran Fikih di MI Masyhariyah Kebontemu, Peterongan, Jombang. b. Observasi Observasi merupakan bagian dari pengumpulan data. Obeservasi mempunyai arti pengumpulan data secara langsung dari tempat yang akan b. Observasi Observasi merupakan bagian dari pengumpulan data. Obeservasi mempunyai arti pengumpulan data secara langsung dari tempat yang akan Observasi merupakan bagian dari pengumpulan data. Obeservasi mempunyai arti pengumpulan data secara langsung dari tempat yang akan 12 diteliti oleh peneliti dengan melalui pengamatan dan pencatatan secara sistematis36. Dalam penelitian ini jenis observasi partisipasi digunakan oleh peneliti. Observasi partisipatif yaitu peneliti secara langsung dan mengikuti aktivitas obyek yang diteliti saat melakukan pengamatan.37 Dengan melakukan observasi partisipatif, maka gambaran data mengenai penerapan blended learning pada mata pelajaran Fikih akan lebih lengkap. g y , , , 9 8 Husamah, Pembelajaran Bauran...., hlm. 11 36 J.R. Raco, Metode Penelitian Kualitatif: Jenis, Karakteristik, Dan Keunggulannya (Jakarta: PT. Gramedia Widiasarana Indonesia, 2010), hlm. 112. 37 Sugiyono, Metode Penelitian,......,hlm. 298. 38 Husamah Pembelajaran Bauran hlm 11 A. Perencanaan Model Blended Learning Pada Mata Pelajaran Fikih di Era New Normal di MI Masyhariyah Kebontemu Peterongan Jombang MI Masyhariyah Kebontemu di era new normal menerapkan model blended learning. Berdasarkan pendapat Moebs dan Weibelzahl, menjelaskan bahwasanya model blended learning yaitu pencampuran antara pembelajaran secara online dengan pembelajaran secara tatap muka38. Pembelajaran Fikih di MI Masyhariyah Kebontemu dengan menggunakan model pembelajaran blended learning itu terbagi menjadi 2 kelas, yaitu kelas online dan kelas tatap muka. Dalam melaksanakan pembelajaran tatap muka yang terkandung dalam model blended learning di era pandemi Covid-19, MI Masyhariah melakukan perizinan kepihak berwajib seperti di kantor Kementrian Agama kota, Satgas tingkat desa, dan lain sebagainya sesuai Surat Keputusan Bersama Menteri Nomer 116266/A5/HK/2020. MI Masyhariyah Kebotemu mempersiapkan pembelajaran tatap muka yang terkandung dalam model blended learning di era pandemi Covid-19 sesuai Surat Keputusan Bersama Menteri Nomer 116266/A5/HK/2020 sebagai berikut: 13 1. Mewajibkan siswa, guru, dan semua warga sekolah untuk menggunakan masker. 1. Mewajibkan siswa, guru, dan semua warga sekolah untuk menggunakan masker. 1. Mewajibkan siswa, guru, dan semua warga sekolah untuk menggunakan masker. 1. Mewajibkan siswa, guru, dan semua warga sekolah untuk menggunakan masker. 2. Mengecek suhu tubuh siswa dan orang yang mengantarkan didepan gerbang sekolah, jika suhu tubuh siswa atau yang mengantarkan suhu tubuh dibawah 37 derajat maka murid diperkenankan untuk masuk kelas mengikuti kegiatan belajar mengajar, namun jika diatas 37 derajat atau mengalami flu, batuk, dan hilang rasa penciuman maka murid diwajibkan untuk istirahat di rumah atau langsung ke Puskesmas untuk penanganan. 2. Mengecek suhu tubuh siswa dan orang yang mengantarkan didepan gerbang sekolah, jika suhu tubuh siswa atau yang mengantarkan suhu tubuh dibawah 37 derajat maka murid diperkenankan untuk masuk kelas mengikuti kegiatan belajar mengajar, namun jika diatas 37 derajat atau mengalami flu, batuk, dan hilang rasa penciuman maka murid diwajibkan untuk istirahat di rumah atau langsung ke Puskesmas untuk penanganan. 3. Menyiapkan tempat cuci tangan disemua depan kelas dan semua tempat yang menjadi lewatan banyak orang, dan handsanitizer didalam ruang kelas. 3. Menyiapkan tempat cuci tangan disemua depan kelas dan semua tempat yang menjadi lewatan banyak orang, dan handsanitizer didalam ruang kelas. 4. Mengatur masuk siswa dengan sistem shift yaitu hari senin untuk tatap muka kelas 1 dan kelas 2, hari selasa untuk kelas 3 dan kelas 4, dan hari rabu untuk kelas 5 dan kelas 6. Kemudian membagi 1 kelas menjadi 2 ruangan dan mengatur tempat duduk siswa dengan jarak kurang lebih 1,5 meter. 4. A. Perencanaan Model Blended Learning Pada Mata Pelajaran Fikih di Era New Normal di MI Masyhariyah Kebontemu Peterongan Jombang Mengatur masuk siswa dengan sistem shift yaitu hari senin untuk tatap muka kelas 1 dan kelas 2, hari selasa untuk kelas 3 dan kelas 4, dan hari rabu untuk kelas 5 dan kelas 6. Kemudian membagi 1 kelas menjadi 2 ruangan dan mengatur tempat duduk siswa dengan jarak kurang lebih 1,5 meter. Sebelum melaksanakan penerapan model blended learning guru membuat perencanaan pembelajaran terlebih dahulu. Berdasarkan hasil wawancara dan observasi peneliti, ditemukan beberapa perencanaan dalam pembelajaran menggunakan model blended learning yang terbagi menjadi dua pembelajaran yaitu pembelajaran online dan pembelajaran tatap muka yang nantinya dua pembelajaran tersebut dikolaborasikan. Adapun perencanaan yang dilaksanakan oleh guru mata pelajaran Fikih di MI Masyhariyah Kebontemu pada pembelajaran online dan tatap muka sebagai berikut: 1. Menentukan tujuan pembelajaran 5. Guru menentukan media yang akan digunakan 1. Menentukan tujuan pembelajaran 1. Menentukan tujuan pembelajaran Adapun tujuan menerapkan model blended learning pada mata pelajaran Fikih di MI Masyhariyah Kebontemu yaitu agar proses pembelajaran siswa-siswi di masa pandemi dapat berjalan dengan baik, khususnya dalam mata pelajaran Fikih yang membutuhkan materi praktek dari pada teorinya. 2. Menentukan waktu dan lokasi pembelajaran 2. Menentukan waktu dan lokasi pembelajaran Pembelajaran online lokasinya dilaksanakan dirumah masing-masing sedangkan untuk waktunya mengikuti jadwal yang sudah ditentukan dari Pembelajaran online lokasinya dilaksanakan dirumah masing-masing sedangkan untuk waktunya mengikuti jadwal yang sudah ditentukan dari Pembelajaran online lokasinya dilaksanakan dirumah masing-masing sedangkan untuk waktunya mengikuti jadwal yang sudah ditentukan dari 14 sekolah. Untuk mata pelajaran Fikih menggunakan waktu 1,5 jam dalam pembelajaran online. Sedangkan untuk pembelajaran offline atau tatap muka lokasinya di sekolah dengan tetap mengikuti protokol kesehatan yang sudah ditentukan, untuk waktunya dilaksanakan dari jam 07.00 - 10.00 WIB dan tiap jam pelajaran menggunakan waktu 35 menit. 3. Menyiapkan materi pembelajaran Adapun persiapan guru mata pelajaran Fikih di MI Masyhariah Kebontemu dalam menyiapkan materi pembelajaran online dan tatap muka antara lain sebagai berikut: a. Pembelajaran online : Dengan cara meringkas materi yang akan dipelajari kemudian dibuat menjadi video atau PPT (power point) untuk diupload dimedia group whatsapp. b. Pembelajaran tatap muka : Dengan cara memilah-milah materi terlebih dahulu seperti materi yang lebih membutuhkan pemahaman, materi yang belum difahami oleh siswa saat pembelajaran online, dan materi yang membutuhkan praktek. 4. Guru menyiapkan metode pembelajaran. 4. Guru menyiapkan metode pembelajaran. Adapun metode yang dilaksanakan oleh guru mata pelajaran Fikih di MI Masyhariyah antara lain sebagai berikut: a. Pembelajaran online : Menggunakan metode tanya jawab, yaitu dengan cara setelah guru mengunggah materinya di whatsapp group dan setelah siswa membaca dan memahami materi yang diunggah di whatsapp group, guru bertanya pada siswa seperti “apakah ada yang perlu dipertanyakan mengenai pembahasan materi tersebut?”. Kemudian guru menjawab pertanyaan siswa. Setelah itu guru memberi soal kepada siswa mengenai materi yang dipelajari sebagai evaluasi apakah siswa sudah faham apa belum. b. Pembelajaran tatap muka : Menyesuaikan dengan materi yang akan dipelajari. Jika materi yang membutuhkan praktek maka guru menggunakan metode demonstrasi, selebihnya menggunakan metode diskusi, tanya jawab, dan ceramah. b. Pembelajaran tatap muka : Menyesuaikan dengan materi yang akan dipelajari. Jika materi yang membutuhkan praktek maka guru menggunakan metode demonstrasi, selebihnya menggunakan metode diskusi, tanya jawab, dan ceramah. 15 Adapun media yang digunakan oleh guru mata pelajaran Fikih di MI Masyhariyah Kebontemu antara lain sebagai berikut : a. 1. Menentukan tujuan pembelajaran Dalam model blended learning terdapat dua proses pembelajaran yang telah di kolaborasikan yaitu proses pembelajaran online dan pembelajaran offline. Di MI Masyhariyah Kebontemu Peterongan Jombang membuat dua RPP yang sudah dibedakan antara materi yang lebih membutuhkan pembelajaran tatap muka atau yang hanya butuh pembelajaran lewat online. 7. Guru menyusun RPP (Rencana Pelaksanaan Pembelajaran) Merencanakan proses kegiatan belajar mengajar, guru menyiapkan RPP terlebih dahulu. Dalam model blended learning terdapat dua proses pembelajaran yang telah di kolaborasikan yaitu proses pembelajaran online dan pembelajaran offline. Di MI Masyhariyah Kebontemu Peterongan Jombang membuat dua RPP yang sudah dibedakan antara materi yang lebih membutuhkan pembelajaran tatap muka atau yang hanya butuh pembelajaran lewat online. Dari penelitian tentang perencanaan model blended learning pada mata pelajaran Fikih di MI Masyahariyah Kebontemu di era new normal yang telah dilakukan oleh peneliti, peneliti menemukan bahwasannya sebelum melaksanakan penerapan model blended learning maka terlebih dahulu melakukan perencanaan pembelajaran agar proses pembelajaran bisa berlangsung secara sistematis dan bisa berlangsung secara terarah sehingga proses pembelajaran bisa maksimal meskipun dalam keadaan pandemi Covid-19. B. Penerapan Model Blended Learning pada Mata Pelajaran Fikih di Era New B. Penerapan Model Blended Learning pada Mata Pelajaran Fikih di Era New 1. Menentukan tujuan pembelajaran Pembelajaran online : Menggunakan media whatsapp group. Dalam proses pembuatan materi, guru terlebih dahulu meringkas materi yang kemudian dibuat video atau PPT (power point) yang kemudian diunggah guru di media whatsapp group. Alasan dipilihnya menggunakan media whatsapp group yaitu karena di aplikasi whatsapp tidak terlalu menggunakan kuota banyak sehingga mudah diakses dan juga dapat mengirim foto atau video sehingga bisa dibaca dan dilihat sama wali murid. a. Pembelajaran online : Menggunakan media whatsapp group. Dalam proses pembuatan materi, guru terlebih dahulu meringkas materi yang kemudian dibuat video atau PPT (power point) yang kemudian diunggah guru di media whatsapp group. Alasan dipilihnya menggunakan media whatsapp group yaitu karena di aplikasi whatsapp tidak terlalu menggunakan kuota banyak sehingga mudah diakses dan juga dapat mengirim foto atau video sehingga bisa dibaca dan dilihat sama wali murid. b. Pembelajaran tatap muka : Menggunakan buku paket, LKS (Lembar Kerja Siswa), papan tulis, gambar atau poster yang berisi tentang tata cara sholat, wudhu yang benar dan lain-lain sebagainya. Pada pembelajaran tatap muka di kelas guru menggunakan buku paket dan LKS (Lembar Kerja Siswa) sebagai rujukan materi yang digunakan dalam menyampaikan materi ke siswa. Kemudian papan tulis digunakan guru untuk mencatat materi yang perlu dijelaskan melalui papan tulis. Jika materinya mengenai tata cara sholat, wudhu, dan lain-lain sebagainya yang materinya terdapat pada gambar atau poster guru menggunakan gambar atau poster tersebut sebagai media untuk menyampaikan materi pembelajaran. b. 6. Guru menyiapkan langkah-langkah pembelajaran. a. Langkah-langkah yang dilakukan saat pembelajaran online yaitu dengan cara mengetik pesan melalui whatsapp group mengenai langkah-langkah saat tanya jawab. Adapun langkah-langkah pembelajaran online sebagai berikut: 1) Guru mengunggah materi di media whatsapp group 2) Siswa mendownload materi kemudian memahami materi 3) Setelah beberapa menit guru membuka pertanyaan jika ada perlu ditanyakan. 4) Guru menjawab pertanyaan dari siswa. 4) Guru menjawab pertanyaan dari siswa. 5) Guru memberikan pertanyaan kepada siswa mengenai materi yang dipelajari di media whatsapp group. 6) Siswa menjawab pertanyaan dari guru. 6) Siswa menjawab pertanyaan dari guru. 16 b. Langkah-langkah untuk pembelajaran tatap muka dilaksanakan secara berbeda-beda menyesuaikan dengan materi yang akan dipelajari dan metode yang akan digunakan. Jika materinya membutuhkan praktek, guru menggunakan langkah-langkah pembelajaran yang menggunakan metode demontrasi. Sedangkan jika meteri tersebut tidak membutuhkan praktek guru menggunakan langkah-langkah pembelajaran dengan menggunaka metode tanya jawab. 7. Guru menyusun RPP (Rencana Pelaksanaan Pembelajaran) Merencanakan proses kegiatan belajar mengajar, guru menyiapkan RPP terlebih dahulu. Normal di MI Masyhariyah Kebontemu Peterongan Jombang Setelah melakukan perencanaan model blended learning pada mata pelajaran Fikih di era new normal selanjutnya melaksanakan penerapan model blended learning. Berdasarkan hasil wawancara dan observasi peneliti, berikut ini penerapan dalam model blended learning pada mata pelajaran Fikih di MI Masyahariyah Kebontemu yang terbagi menjadi dua pembelajaran yaitu pembelajaran online dan pembelajaran tatap muka yang nantinya dua pembelajaran tersebut dikolaborasikan, antara lain sebagai berikut: 1. Kegiatan pendahuluan 17 Adapun kegiatan pendahuluan yang dilaksanakan pembelajaran online dan pembelajaran tatap muka pada mata pelajaran Fikih di MI Masyhariyah Kebontemu sebagai berikut : a. Pembelajaran online a. Pembelajaran online 1) Salam, doa, memberikan motivasi, menyampaikan tujuan pembelajaran terkait materi yang diajarkan. 2) Memeriksa kehadiran siswa melalui aplikasi whatsapp group. b l j k 2) Memeriksa kehadiran siswa melalui aplikasi whatsapp group. Pembelajaran tatap muka : b. Pembelajaran tatap muka : 1) Salam, doa, memberikan motivasi, menyampaikan tujuan pembelajaran terkait materi yang diajarkan. 2) Mengingatkan tentang prtotokol kesehatan. 3) Mengabsen kehadiran siswa. 3) Mengabsen kehadiran siswa. 2. Kegiatan inti 2. Kegiatan inti Adapun kegiatan inti yang dilaksanakan pembelajaran online dan pembelajaran tatap muka pada mata pelajaran Fikih di MI Masyhariyah Kebontemu sebagai berikut : a. Pembelajaran online 1) Guru menguploud video atau PPT (Power Point) mengenai materi yang akan dipelajari melalui via whatsapp group. 2) Siswa diarahkan untuk mengunduh video atau PPT (Power Point) yang telah diuploud oleh guru melalui whatsapp group kelas. 3) Siswa membaca dan memahami materi tersebut. 4) Guru melakukan tanya jawab dan memberikan kesempatan siswa untuk bertanya mengenai materi yang belum difahami. 5) Guru memberikan tugas siswa untuk mengerjakan soal yang ada dibuku paket. 6) Siswa mengirimkan hasil tugas lewat foto via whatsapp. 2) Guru memberikan kesempatan siswa untuk bertanya materi kemarin pada pembelajaran online. 3) Guru menjelaskan materi hari ini. 4) Guru mendemonstrasikan materi yang membutuhkan praktek. 18 5) Guru memberikan kesempatan siswa untuk bertanya mengenai materi yang hari ini dipelajari. 6) Guru melakukan quiz dan memberikan soal mengenai materi saat pembelajaran online dan hari ini. 7) Guru mengecek hasil tugas siswa dan memberikan koreksi jika masih ada kesalahan. 3. Kegiatan penutup 3. Kegiatan penutup 3. Kegiatan penutup Adapun kegiatan penutup yang dilaksanakan pada pembelajaran online dan pembelajaran tatap muka pada mata pelajaran Fikih di MI Masyhariyah Kebontemu sebagai berikut : a. Pembelajaran online 1) Guru mengecek hasil tugas siswa dan memberikan koreksi jika masih ada kesalahan. 2) Guru memberikan kesimpulan materi yang telah dipelajari. 3) Guru memberikan motivasi belajar dan mengingatkan tentang protokol kesehatan dan diakhiri dengan salam. b. Pembelajaran tatap muka 1) Guru memberikan kesimpulan materi yang telah dipelajari. 2) Guru memberikan motivasi belajar dan mengingatkan tentang protokol kesehatan dan diakhiri dengan salam 4. Penilaian 4. Penilaian 4. Penilaian Adapun penilaian pada pembelajaran online dan pembelajaran tatap muka pada mata pelajaran Fikih di MI Masyhariyah Kebontemu sebagai berikut : Adapun penilaian pada pembelajaran online dan pembelajaran tatap muka pada mata pelajaran Fikih di MI Masyhariyah Kebontemu sebagai berikut : a. Pembelajaran online : Dari kehadiran siswa, keaktivan siswa saat tanya jawab, hasil mengerjakan tugas, disiplin dalam pengumpulan tugas. Dari kehadiran siswa, keaktivan siswa saat tanya jawab, hasil mengerjakan tugas, disiplin dalam pengumpulan tugas. b. Pembelajaran tatap muka : b. Pembelajaran tatap muka : Dari sikap dan prilaku siswa saat pembelajaran, keaktivan siswa untuk bertanya atau menjawab selama di kelas, saat melakukan praktek, saat mengerjakan soal, dan saat menjawab soal quiz. Kesimpulan Kesimpulan Kesimpulan 19 Dari penelitian tentang penerapan model blended learning pada mata pelajaran Fikih di MI Masyahariyah Kebontemu di era new normal yang telah dilakukan oleh peneliti. 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Jurnal Pendidikan Luar Sekolah. Volume 1. Nomor 1. Peraturan Menteri Agama Republik Indonesia Nomor 2 Tahun 2008. Standar Kompetensi Lulusan dan Standar Isi Pendidikan Agama Islam dan Bahasa Arab di Madrasah. 6 Mei 2008. Jakarta. Perhimpunan Dokter Paru Indonesia (PDPI), Perhimpunan Dokter Spesialis Kardiovaskular Indonesia (PERKI), Perhimpunan Dokter Spesialis Penyakit Dalam Indonesia (PAPDI), Perhimpunan Dokter Anestesiologi dan Terapi 21 Intensif Indonesia (PERDATIN), Ikatan Dokter Anak Indonesia (IDAI). 2020. Pedoman Tatalaksana COVID-19 edisi 3. Jakarta: t.p. Intensif Indonesia (PERDATIN), Ikatan Dokter Anak Indonesia (IDAI). 2020. Pedoman Tatalaksana COVID-19 edisi 3. Jakarta: t.p. Purwanto, Erwan Agus. 2020. New Normal: Perubahan Sosial Ekonomi dan Politik Akibat Covid-19. Cetakan I. Referensi Yogyakarta: Gadjah Mada University Press. Raco, J.R. 2010. Metode Penelitian Kualitatif: Jenis, Karakteristik, dan Keunggulannya. Jakarta: PT. Gramedia Widiasarana Indonesia. Rahayu, Siti, dkk. 2020. COVID-19 The Nightmore or Rainbow. Cetakan I. Jakarta: Mata Aksara. Saiban, Kasuwi. 2019. Metode Penetapan Hukum Islam. Malang: Setara Press. Santoso, Subhan Adi. 2020. Pembelajaran Blended Learning Masa Pandemi. Pasuruan: CV. Penerbit Qiara Media. Sugiyono. 2011. Metode Penelitian kuantatif, kualitatif, dan R & D. Bandung: Alfabeta. Sugiyono. 2011. Metode Penelitian kuantatif, kualitatif, dan R & D. Bandung: Alfabeta. Surat Edaran Nomor 15 Tahun 2020 Pedoman Penyelenggaraan Belajar dari Rumah dalam Masa Darurat Penyebaran Corona Virus Disease (COVID019). 18 Mei 2020. Jakarta. Surat Edaran Nomor 4 Tahun 2020 Pelaksanaan Kebijakan Pendidikan dalam Masa Darurat Penyebaran Coronavirus Disease (COVID-19). 24 Maret 2020. Jakarta. Zain, Lukman. 2009. Pembelajaran Fikih. Jakarta: Direktorat Jendral Pendidikan Islam Departemen Agama RI.
https://openalex.org/W2201632998	http://pasca.unhas.ac.id/ojs/index.php/halrev/article/download/118/80	Indonesian	null	Urgensi Kejaksaan Diatur oleh Konstitusi	Hasanuddin Law Review	2,015	cc-by	7,825	HALREV Hasanud which p work is ALREV Hasanuddin Law Review is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. y , y, , Sulawesi, Indonesia. ISSN: 2442-9880 \| e-ISSN: 2442-9899. Open Access at: http://pasca.unhas.ac.id/ojs/index.php/halrev Hasanuddin Law Review is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Urgency of Attorney Governed by the Constitution Urgency of Attorney Governed by the Constitution Rommy Patra Fakultas Hukum Universitas Tanjungpura Jln. Ahmad Yani, Pontianak, Kalimantan Barat. Tel./Fax: +62-561-740187 E-mail: rommypatra@yahoo.co.id Submitted: Oct 23, 2015; Reviewed: Nov 28, 2015; Accepted: Dec 4, 2015 Rommy Patra Fakultas Hukum Universitas Tanjungpura Jln. Ahmad Yani, Pontianak, Kalimantan Barat. Tel./Fax: +62-561-740187 E-mail: rommypatra@yahoo.co.id Submitted: Oct 23, 2015; Reviewed: Nov 28, 2015; Accepted: Dec 4, 2015 Rommy Patra Fakultas Hukum Universitas Tanjungpura Jln. Ahmad Yani, Pontianak, Kalimantan Barat. Tel./Fax: +62-561-740187 E-mail: rommypatra@yahoo.co.id Submitted: Oct 23, 2015; Reviewed: Nov 28, 2015; Accepted: Dec 4, 2015 Abstract: Attorney existence in the Indonesian constitutional structure has a dilemma for this position. On one side is the Prosecutor’s law enforcement agencies to exercise power independently prosecution while on the other hand is part of a government institution under Law No. 16 of 2004 regarding the Attorney. The position of Attorney as an institution of government has been led to the independence of the Prosecutor is not optimal so that it appears stigma that the Prosecutor merely as a tool of the ruling power. In addition the terms of the arrangement just under the Act, the Attorney General has no legal standing as a constitutional organ that has the constitutional authority so that the current position does not reflect the urgency of its duties and functions. In an effort to organize the next Attorney institutions should be regulated directly by the Constitution. It is intended to make the Attorney as part of the main state organs have the same legal standing as other law enforcement agencies, the police and the courts (Supreme Court and Constitutional Court). As well as to strengthen and clarify the position as a state institution, prosecution authorities are focusing on the Attorney as central of authority, to fix the institutional relations between the members of law enforcement and related agencies and strengthen the independence of the Prosecutor in performing the function of prosecution in the constitutional structure of Indonesia. V Volume 1 Issue 3, December 2015: pp. 400-416. Copyright © 2015 HALREV. Faculty of Law, Hasanuddin University, Makassar, South Sulawesi, Indonesia. ISSN: 2442-9880 \| e-ISSN: 2442-9899. Open Access at: http://pasca.unhas.ac.id/ojs/index.php/halrev V Volume 1 Issue 3, December 2015: pp. 400-416. Copyright © 2015 HALREV. Faculty of Law, Hasanuddin University, Makassar, South Sulawesi, Indonesia. ISSN: 2442-9880 \| e-ISSN: 2442-9899. Open Access at: http://pasca.unhas.ac.id/ojs/index.php/halrev 1 Dio Ashar Wicaksana. (2013). “Kedudukan Kejak saan RI dalam Sistem Hukum Tata Negara Indone sia”. Jurnal Fiat Justitia, 1(1): 3. Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Hasanuddin Law Review Vol. 1 Issue 3, December (2015) yang mempunyai legal standing sama seperti lembaga penegak hukum lainya, yaitu Kepolisian dan Pengadilan (Mahkamah Agung dan Mahkamah Konstitusi). Selain itu, untuk memerkuat dan memerjelas kedudukan sebagai lembaga negara, memusatkan kewenangan penuntutan berada di kejaksaan sebagai central of authority, membenahi hubungan kelembagaan antar sesama penegak hukum maupun lembaga terkait dan memperkuat independensi kejaksaan dalam menjalankan fungsi penuntutan dalam struktur ketatanegaraan Indonesia. Keywords: Attorney; Constitution; Independence Abstrak: Eksistensi kejaksaan dalam struktur ketatanegaraan Indonesia memiliki posisi yang dilematis selama ini. Di satu sisi, kejaksaan adalah lembaga penegak hukum yang menjalankan kekuasaan penuntutan secara independen, sedangkan di sisi lain adalah bagian dari lembaga pemerintahan berdasarkan Undang-Undang Nomor Nomor 16 Tahun 2004 tentang Kejaksaan Republik Indonesia. Kedudukan kejaksaan sebagai lembaga pemerintahan selama ini dirasakan menyebabkan independensi kejaksaan tidak optimal sehingga muncul stigma bahwa kejaksaan hanyalah sebagai alat kekuasaan dari yang memerintah. Selain itu, ditinjau dari segi pengaturan yang hanya berdasarkan undang-undang, kejaksaan tidak mempunyai legal standing sebagai organ konstitusi yang mempunyai kewenangan konstitusional sehingga kedudukannya saat ini tidak merefleksikan urgensitas tugas dan fungsi yang dimilikinya. Dalam upaya menata institusi kejaksaan ke depan sebaiknya diatur langsung oleh konstitusi. Hal ini dimaksudkan untuk menjadikan kejaksaan sebagai bagian dari main state organ 400 PENDAHULUAN landasan konstitusional keberadaan masing- masing lembaga. Timbul pertanyaan, men gapa keberadaan kejaksaan tidak diatur, sedangkan kepolisian diatur langsung oleh UUD 1945. Kejaksaan sebagai institusi penegak hu kum di Indonesia memiliki tugas dan fungsi yang sangat penting dalam melaksanakan kekuasaan negara di bidang penuntutan dan kewenangan lainnya yang diberikan oleh undang-undang. Secara kelembagaan, kejaksaan merupakan penghubung antara masyarakat dengan negara dalam menjaga tegaknya hukum dan norma yang berlaku di masyarakat. Oleh karena itu, dalam melak sanakan fungsinya, kejaksaan haruslah be- kerja secara merdeka dan bebas dari inter vensi manapun, termasuk dari pemerintah.1 Kemudian, jika ditinjau dari segi ke- dudukan, berdasarkan konsideran UU No. 16 Tahun 2004, bahwa kejaksaan termasuk salah satu badan yang fungsinya berkaitan dengan kekuasaan kehakiman menurut UUD 1945. Sedangkan di dalam Pasal 2 ayat (1) menyebutkan, “Kejaksaan adalah lembaga pemerintahan yang melaksanakan kekuasaan negara di bidang penuntutan, serta kewe- nangan lain berdasarkan undang-undang.” Persoalannya apakah kejaksaan merupakan bagian dari badan yudikatif karena memiliki keterkaitan dengan kekuasaan kehakiman atau memang murni sebagai lembaga yang merupakan bagian dari eksekutif karena disebut sebagai lembaga pemerintahan. Sayangnya, keberadaan institusi ke jaksaan yang sangat penting ini belum di barengi dengan pengaturan kelembagaan yang ideal dilihat dari segi dasar hukum pembentukan, kedudukan dan independen sinya. Ditinjau dari dasar hukum pembentu kannya, keberadaan kejaksaan sebagai lem baga negara tidak diatur langsung oleh UUD 1945 melainkan dengan UU No. 16 Tahun 2004 tentang Kejaksaan Republik Indone sia. Jika dibandingkan, eksistensi kejaksaan dengan kepolisian sebagai sesama penegak hukum di Indonesia, mengalami perbedaan yang cukup signifikan, khususnya terkait Karena persoalan kedudukan berkaitan erat dengan independensi kejaksaan dalam melaksanakan kekuasaan negara di bidang penuntutan, maka dalam melaksanakan tu gas, fungsi dan wewenangnya, kejaksaan haruslah terlepas dari pengaruh kekuasaan pemerintah dan kekuasaan lainnya. Namun oleh UU No. 16 Tahun 2004 dinyatakan bah wa kejaksaan sebagai bagian dari lembaga pemerintahan dan Jaksa Agung diangkat ser 401 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) ta diberhentikan berdasarkan kewenangan Presiden sebagai kepala eksekutif. jaksa penuntut Kerajaan di Perancis meru pakan pejabat publik yang mewakili raja, untuk melindungi hak kepemilikannya dan sebagai penuntut apabila adanya tindak ke jahatan.2 Tentu saja, pengaturan ini tidak mere fleksikan kejaksaan sebagai lembaga inde penden. 2 Beneč Štefan. (2003). “Independence of Pro- secution”. Makalah disampaikan dalam Seminar “The prosecutor’s office in a democratic and constitutional state” organized by The General Prosecutor’s Office and the Slovak National Supporting Committee of Europe 2000, April 25- 27, 2003, hlm. 14. 3 Ibid. PENDAHULUAN Sebab, melihat karakteristik suatu lembaga negara independen biasanya pen gangkatan jabatan maupun pemberhentian sebagai personil atau pimpinannya juga mel ibatkan persetujuan dari Dewan Perwakilan Rakyat (DPR), seperti dalam pengangkatan Panglima Tentara Nasional Indonesia (TNI), pengangkatan Kepala Kepolisian Republik Indonesia (Kapolri), pemilihan pimpinan Komisi Pemberantasan Korupsi (KPK) dan lain-lain. Munculnya persoalan mendudukan sistem penuntutan ke dalam suatu bagian kekuasaan negara di Perancis, dimulai pada saat “ministẻre public”, yaitu semacam ci kal bakalnya lembaga penuntut, menjadi sebuah jabatan yang tidak hanya memiliki fungsi yang bersifat publik, tapi juga me miliki fungsi pengawasan atas pengadilan. Selain itu, pada saat “Napoleon’s Criminal Code” diterbitkan, “procereur d’etat” mem peroleh kedudukannya sebagai penegak hu kum, sehingga mulailah timbul permasalah- an dan perlunya pemikiran kembali apakah lembaga kejaksaan bagian dari kekuasaan eksekutif atau yudikatif.3 Berdasarkan persoalan di atas, penulis dalam fokus tulisan ini mendasarkan kepada kajian terhadap eksistensi kejaksaan sebagai lembaga negara dalam struktur ketatanega raan Indonesia. Hal ini tentu saja berka- itan dengan kedudukan, tugas, fungsi dan wewenang, independensi, serta hubungan kelembagaan antara kejaksaan dengan lem baga-lembaga negara lainnya. Adanya permasalahan tersebut mela hirkan dilema keberadaan sistem penun- tutan dalam kekuasaan negara, karena apakah penuntutan sebagai badan publik memenuhi tugas eksekutif atau kekuasaan kehakiman? Kepada siapa hal tersebut dipertanggung jawabkan? Kepada pemerintah? Kepada pengadilan? Atau kepada parlemen? Jika bertitik tolak dari trias politica, maka jelas permasalahan tersebut tidak dapat terungkap karena Montesquieu tidak memberikan pe mikiran di mana letak sistem penuntutan. Munculnya teori separation of powers pada saat itu, hanya untuk mencegah terjadinya 7 Yusril Ihza Mahendra. (2010), “Kedudukan Ke jaksaan dan Posisi Jaksa Agung Dalam Sistem Presidensial Di Bawah UUD 1945”, http://yusril. ihzamahendra.com/2010/08/20/kedudukan-kejak saan-dan-posisi-jaksa-agung-dalam-sistem-pres Kedudukan Kejaksaan dalam Struktur Ketatanegaraan Indonesia Upaya untuk memahami dan menempatkan kedudukan kejaksaan dalam struktur keta- tanegaraan jika ditinjau dari teori pemisahan kekuasaan (separation of powers) tidaklah mudah. Karena akan muncul persoalan ter kait dengan apakah Kejaksaan itu sebagai sebuah lembaga eksekutif atau yudikatif. Menurut pendapat Beneč Štefan bahwa teori separation of powers dari Montesquieu tidak menjelaskan dimana cabang sistem penuntu tan diletakkan. Walaupun di abad 14, posisi 402 402 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) kekuasaan raja yang mutlak. Oleh karena itu, persoalan meletakkan sistem penuntutan harus menjadi pemikiran yang serius dalam teori ilmu hukum dan konsep separation of powers Montesquieu menjadi hal yang perlu dipikirkan kembali dalam mengklasifikasi kan lembaga-lembaga negara.4 pendudukan Jepang untuk menggantikan is tilah ”officer van justitie” bagi petugas yang melakukan penuntutan perkara di pengadi lan militer Jepang. Istilah ini secara resmi digunakan oleh undang-undang pemerintah pendudukan tentara Jepang No. 1/1942 yang kemudian diganti oleh Osamu Seirei No. 3/1942, No. 2/1944 dan No. 49/1944.6 Dalam konteks Indonesia, eksistensi kejaksaan sebagai lembaga penegak hukum sudah ada sejak lama. Jika ditarik jauh sam pai ke era Majapahit, sudah adanya pelak sanaan tugas dan fungsi yang dilakukan mi rip dengan tugas jaksa saat ini. Pada waktu itu dikenal dengan istilah Adhyaksa, salah satunya adalah peran yang dijalankan oleh Gadjah Mada selain melakukan penegakan hukum, juga sebagai pelaksana segala per aturan raja dan melaporkan perkara-perkara ke pengadilan. Tugas Gadjah Mada ini apa bila dibandingkan dengan zaman sekarang sangatlah mirip dengan tugas jaksa, se hingga bisa disimpulkan bahwa kedudukan institusi Kejaksaan sejak zaman dahulu kala adalah sebagai alat negara dan pertanggung jawabannya kepada kepala negara yang saat itu adalah raja Hayam Wuruk.5 Kemudian pada masa awal kemer- dekaan dengan menggunakan Pasal II Aturan Peralihan UUD 1945 yang mengatakan bah wa, ”segala badan negara dan peraturan yang ada masih langsung berlaku, selama belum diadakan yang baru menurut Un dang-Undang Dasar ini”, maka pengaturan berkaitan dengan Kejaksaan juga masih mengacu kepada model pada era Hindia Be landa yang bersumber model pengaturan nya kepada Kejaksaan yang ada di Belanda. Menurut Yusril Ihza Mahendra dalam tradisi di Belanda yang menganut sistem pemerin tahan parlementer, semua hakim dan jaksa, adalah pegawai negeri. Secara struktur or ganisasi, personil dan keuangan baik jaksa maupun pengadilan berada di bawah Minist rie van Justititie (Kementerian Kehakiman). Namun secara fungsional dalam menjalan- kan tugas dan kewenangannya di bidang yu dikatif, jaksa dan hakim adalah independen. 6 Memaknai Independensi Kejaksaan di Indone sia (Kekuasaan Penuntutan), https://ilhamen dra.wordpress.com/2008/05/27/kekuasaan- penuntutan/#more-29, diakses pada tanggal 1 Juli 2015. 4 Ibid. 5 Dio Ashar Wicaksana. (2013). Op.Cit, hlm. 4. 4 Ibid. Kedudukan Kejaksaan dalam Struktur Ketatanegaraan Indonesia Jadi, memang terdapat kerancuan kedudu kan kejaksaan dalam sistem Belanda, yakni berada di antara dua sisi, antara eksekutif Pada perkembangan berikutnya, di era Hindia Belanda keberadaan jaksa dike nal dengan istilah officer van justitie, yang tugas pokoknya adalah menuntut seseorang ke pengadilan dalam suatu perkara tindak pidana. Pengaturan terhadap officer van jus titie ini berdasarkan kepada Indische Sta atsregeling yang substansinya mengacu ke pada model pengaturan kejaksaan yang ada di Belanda. Istilah ”kejaksaan dan jaksa” itu sendiri baru secara resmi digunakan di masa 403 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) sional, lembaga ini bekerja dalam penye lenggaraan badan-badan peradilan, sehingga Jaksa Agung disebut sebagai Jaksa Agung pada Mahkamah Agung. Keberadaan kejak saan yang rancu antara eksekutif dan yudika tif ini, baru berakhir pada tahun 1959, ketika UUD 1945 diberlakukan kembali melalui Dekrit Presiden 5 Juli 1959. Kemudian ber dasarkan Keputusan Presiden No. 204 Tahun 1960, secara tegas memisahkan Kejaksaan dari Kementerian Kehakiman dan Mahka mah Agung, serta menjadikannya sebagai suatu institusi yang berdiri sendiri dan meru pakan bagian langsung dari kabinet. Kepu tusan Presiden tersebut merupakan landasan hukum pertama yang menempatkan kejak saan sepenuhnya sebagai bagian dari ranah kekuasaan eksekutif. Kemudian dibentuklah UU No. 15 Tahun 1961 tentang Ketentuan- ketentuan Pokok Kejaksaan Republik In donesia yang menyatakan bahwa kejaksaan bukan saja ”alat negara penegak hukum”, tetapi dalam konteks penyelesaian revolusi, kejaksaan adalah ”alat revolusi”, yang tugas utamanya adalah sebagai ”penuntut umum”.9 Dalam sistem hukum tata negara Be landa, Jaksa Agung diangkat oleh Perdana Menteri atas usul Menteri Kehakiman. Calon Jaksa Agung diambil dari pejabat karir berdasarkan kecakapan, pengalaman dan kemampuan. Jabatan Jaksa Agung bu kanlah jabatan politik. Oleh karena tugas jaksa terkait langsung dengan pengadilan, maka dalam tradisi Belanda, Jaksa Agung disebut sebagai ”Jaksa Agung (Hoofd Of ficer van Justitie) pada Mahkamah Agung (Hooge Raad)”. Sedangkan perkembangan berikutnya, pada masa berlakunya UU Re publik Indonesia Serikat (RIS) No. 1 Tahun 1950 tentang Susunan, Kekuasaan dan Jalan Pengadilan Mahkamah Agung Indonesia, pola penempatan Jaksa Agung di Mahkamah Agung seperti yang ada di Belanda tetap di lanjutkan. Pasal 2 UU tersebut mengatakan ”Pada Mahkamah Agung adalah seorang Jaksa Agung dan dua orang Jaksa Agung Muda”. idensial-di-bawah-uud-1945-oleh-prof-dr-yusril- ihza-mahendra-pendahuluan-hampir-seluruh-neg ara-modern-di-du/#, diakses pada tanggal 30 Juni 2015. 2015. 8 Ibid Kedudukan Kejaksaan dalam Struktur Ketatanegaraan Indonesia Sedikit perubahan terjadi para pro- ses rekruitmen Jaksa Agung dan Jaksa Agung Muda, yang dalam tradisi Belanda diangkat oleh Perdana Menteri atas usul Menteri Ke hakiman, dalam Undang-Undang RIS diang kat oleh Presiden yang dalam praktiknya di lakukan atas usul Perdana Menteri.8 Pada perkembangan berikutnya, ke tika kekuasaan Presiden Soekarno runtuh di tahun 1967, pemerintahan baru di bawah Presiden Soeharto tetap menggunakan UU No. 15 Tahun 1961 yang terus berlaku tanpa perubahan. Namun demikian, dalam prak tiknya pada era Orde Baru, Kejaksaan Agung tidak lagi disebut sebagai Departemen Ke jaksaan Agung dan Jaksa Agung tidak dise but pula sebagai Menteri Jaksa Agung. In stitusi ini secara faktual disebut sebagai Ke jaksaan Agung yang dipimpin oleh seorang Jaksa Agung. Namun kewenangan peng- Kedudukan kejaksaan dengan model seperti di atas, terus digunakan Indonesia sampai era awal kemerdekaan hingga tahun 1959 di mana kedudukan kejaksaan adalah mendua. Secara organisasi, personil dan keuangan institusi ini berada di bawah Ke menterian Kehakiman, namun secara fung- 9 Ibid Ibid 9 Ibid 404 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) angkatan dan pemberhentian Jaksa Agung, sepenuhnya tetap berada di tangan Presiden. UU No. 15 Tahun 1961 tidak secara spesifik menyebutkan berapa lama Jaksa Agung akan memegang jabatannya. Akan tetapi setelah Pemilihan Umum 1971, Presiden Soeharto memulai sebuah konvensi ketatanegaraan, yakni Jaksa Agung selalu diangkat di awal kabinet dan berakhir masa jabatannya den gan berakhirnya masa bakti kabinet itu. Jaksa Agung yang disebut sebagai Menteri di dalam UU No 15 Tahun 1961 tidak lagi disebut demikian, namun sebagai bagian dari kabinet, Jaksa Agung diberi kedudukan setingkat Menteri Negara.10 siden, berdasarkan pertimbangan subyektif Presiden sendiri.11 Perkembangan berikutnya pada era reformasi, keberadaan UU No. 5 Tahun 1991 diganti lagi dengan UU No. 6 tahun 2004 di mana secara substansi terkait dengan keberadaan Jaksa Agung tetaplah pejabat negara yang diangkat dan diberhentikan oleh Presiden. Dilihat dari sejarah lembaga kejaksaan di Indonesia dari awal terbentuk hingga sekarang memang merupakan su- atu institusi yang berada di bawah ranah eksekutif dimana proses pengangkatan dan pemberhentian Jaksa Agung selalu berada di tangan Presiden, walaupun pernah melalui usul Menteri Kehakiman namun tetap saja secara pengangkatannya tetap ada di tangan Presiden.12 Setelah berlaku selama 30 tahun ak- hirnya UU No. 15 Tahun 1961 diganti ke beradaannya dengan UU No 5 Tahun 1991 tentang Kejaksaan Republik Indonesia. 13 Menurut Jimly asshiddiqie yang dapat dikategori kan sebagai badan-badan lain yang fungsinya berkaitan dengan kekuasaan kehakiman adalah 12 Dio Ashar Wicaksana. (2013). Kedudukan...Op.Cit, hlm. 6. 10 Ibid 11 Ibid. 14 Yusril Ihza Mahendra. (2010). Kedudukan Kejak- saan dan Posisi Jaksa Agung Dalam Sistem Presidensial Di Bawah UUD 1945, http://yusril. ihzamahendra.com/2010/08/20/kedudukan- kejaksaan-dan-posisi-jaksa-agung-dalam-sistem- presidensial-di-bawah-uud-1945-oleh-prof-dr- yusril-ihza-mahendra-pendahuluan-hampir-selur uh-negara-modern-di-du/#, diakses pada tanggal 30 Juni 2015. Kedudukan Kejaksaan dalam Struktur Ketatanegaraan Indonesia Ber dasarkan konsideran dalam UU tersebut ti dak lagi menyebut kejaksaan sebagai ”alat negara” tetapi menyebutnya sebagai ”lemba ga pemerintahan yang melaksanakan kekua saan negara di bidang penuntutan dalam tatanan susunan kekuasaan badan-badan penegak hukum dan keadilan”. Jaksa Agung sendiri ”diangkat dan diberhentikan oleh serta bertanggung jawab kepada Presiden”. Penegasan bahwa pengangkatan dan pem berhentian Jaksa Agung adalah kewenangan Presiden, serta pertanggungjawabannya ke pada Presiden, sekali lagi mempertegas bah wa kejaksaan sepenuhnya berada di bawah ranah kekuasaan eksekutif. Dalam UU No 5 Tahun 1991 juga tidak ada pembatasan apa- kah Jaksa Agung diangkat dari jaksa karier ataukah pengangkatan itu bersifat politik. Kedua-duanya dapat dilakukan oleh Pre- Melihat kedudukan Kejaksaan yang berada di ranah eksekutif menimbulkan banyak perdebatan, apakah Kejaksaan se- laku institusi penegak hukum ditempatkan di dalam ranah eksekutif sudah sesuai dengan perspektif hukum tata negara atau tidak. Berdasarkan Pasal 24 ayat (3) UUD 1945 disebutkan bahwa, “Badan-badan lain yang fungsinya berkaitan dengan kekuasaan kehakiman diatur dengan undang-undang”. Mengacu kepada pasal tersebut, banyak pihak yang berpendapat bahwa kejaksaan merupakan salah satu badan yang fungsinya berkaitan dengan kekuasaan kehakiman, sehingga kejaksaan seharusnya berada di ranah yudikatif dan lepas dari pengaruh eksekutif.13 10 Ibid 405 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Berbeda dengan pandangan di atas, menurut Yusril Ihza Mahendra, seluruh ke tentuan dalam BAB IX UUD 1945 sebenar- nya membicarakan Kekuasaan Kehakiman dalam konteks Peradilan. Sementara lemba ga pelaksana kekuasaan kehakiman disebut kan secara limitatif, yakni ”dilakukan oleh sebuah Mahkamah Agung dan badan-badan peradilan yang berada di bawahnya…dan oleh sebuah Mahkamah Konstitusi” (Pasal 24 ayat (1) dan (2) UUD 1945). mana menurut Yusril kalau hanya “berka- itan” tidaklah harus diartikan kejaksaan itu sebagai bagian dari kekuasaan kehakiman. Karena bisa juga dikatakan bahwa institusi Rumah Tahanan dan Lembaga Pemasyara katan juga terkait dengan kekuasaan keha kiman, dalam konteks teori criminal justice system. Namun dalam sejarahnya, Rumah Tahanan dan Lembaga Pemasyarakatan tetap berada di bawah Kementerian Hukum dan HAM yang merupakan ranah kekuasaan eksekutif.14 Sementara Pasal 24 ayat (3) UUD 1945 menyatakan, ”badan-badan lain yang fungsinya berkaitan dengan kekuasaan ke hakiman diatur dalam undang-undang”. Ada yang menafsirkan bahwa kejaksaan merupakan salah satu badan yang fungsinya berkaitan dengan kekuasaan kehakiman, di Selain itu, apabila melihat ketentuan dari Pasal 24 ayat (3) UUD 1945 tidak me nyebutkan bahwa “badan-badan lain” terse but haruslah dimasukkan ke dalam ranah yudikatif melainkan hanya menyebutkan bahwa ketentuan “badan-badan lain” terse but diatur di dalam undang-undang. Sedang- kan di dalam UU Kejaksaan ditegaskan bahwa Kejaksaan adalah lembaga pemerin tahan yang melaksanakan kekuasaan negara di bidang penuntutan- serta kewenangan lain berdasarkan undang-undang. Hal yang bisa ditegaskan dari pasal tersebut adalah kejak saan merupakan lembaga pemerintahan, se hingga kedudukan kejaksaan dalam sistem ketatanegaraan Indonesia merupakan ba gian dari pemerintahan. Pendapat demikian juga diperkuat oleh pernyataan Bagir Manan yang menyebutkan bahwa kejaksaan adalah badan pemerintahan. Dengan demikian, pimpinannya juga adalah pimpinan dari lembaga-lembaga atau badan-badan yang tugas nya berkaitan dengan peradilan dan penegakan hukum, yaitu berhubungan dengan fungsi-fungsi: (a) Penyelidikan, (b) penyidikan, (c) penuntutan, (d) pembelaan atau advokasi, (e) penyelesaian sen gketa dan mediasi atau pendamaian, (f) peradilan, penghakiman dan penghukuman, (g) pemasyara katan, (h) pelaksanaan putusan pengadilan selain pemasyarakatan, dan (i) pemulihan nama baik atau rehabilitasi, (j) pemberian grasi, (k) pemberian am nesti, (l) pemberian abolisi, (m) persaksian, dan (n) pemberian keterangan berdasarkan keahlian. Dari semua fungsi tersebut, yang terpenting adalah fungsi penyelidikan, penyidikan, dan penuntutan. 18 Memaknai Independensi Kejaksaan di Indone sia (Kekuasaan Penuntutan), https://ilhamen dra.wordpress.com/2008/05/27/kekuasaan- penuntutan/#more-29, diakses pada tanggal 1 Juli 2015. 17 Ahmad Andriadi. (2012), Kedudukan Kejaksaan Dalam Sistem Ketatanegaraan Republik Indonesia (Telaah Kritis Terhadap Undang-Undang Nomor 16 Tahun 2004 tentang Kejaksaan Republik Indonesia). Skripsi. Bagian Hukum Tata Negara Fakultas Hukum Universitas Hasanudin Makassar, Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Persoalan seputar kedudukan dan independensi kejaksaan dalam struktur ketatanegaraan suatu negara tidak hanya terjadi di Indonesia saja. Di negara semaju Amerika Serikat pun, persoalan semacam ini juga terjadi. Situasi Jaksa Agung sebagai pim-pinan institusi kejaksaan di Amerika Serikat digambarkan oleh Robert Palmer sebagai kondisi yang “schizophrenic”, ka- rena memerankan dua fungsi sekaligus, yaitu sebagai “petugas hukum” (law officer) dan juga sebagai “petugas eksekutif” (executive officer).18 Kondisi seperti ini tentu saja dapat mempengaruhi independensi kejaksaan da- lam penegakan hukum. suatu badan pemerintahan dan ditafsirkan bahwa yang dimaksud badan pemerintahan adalah kekuasaan eksekutif.”15 15 Dio Ashar Wicaksana. (2013). Kedudukan...Op.Cit, hlm. 7. 16 Ibid. Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Badan-badan yang dapat melakukan fungsi pe nyelidikan pelanggaran hukum ataupun hak asasi manusia adalah (a) Kepolisian Negara, (b) Tentara Nasional Indonesia (TNI) Angkatan Laut, (c) para Pejabat Penyidik Pegawai Negeri Sipil (PPNS), (d) Komisi Nasional Hak Asasi Manusia (Kom nasham), (e) Komisi Pemberantasan Tindak Pidana Korupsi (KPK), (f) Badan Pemeriksa Keuangan dan Pembangunan (BPKP), dan (g) Badan Pemer iksa Keuangan (BPK). Badan-badan yang dapat menjalankan fungsi penyidikan pro-justisia adalah (a) Kejaksaan, (b) Komisi Pemberantasan Tindak Pidana Korupsi, dan (c) Pejabat Penyidik Pegawai Negeri Sipil (PPNS). Sedangkan badan-badan yang melakukan penuntutan adalah (a) Kejaksaan, dan (b) Komisi Pemberantasan Tindak Pidana Korupsi. Lihat Jimly Asshiddiqie, Lembaga-lembaga Nega ra, Organ Konstitusional Menurut UUD 1945, hlm. 3 http://www.jimly.com/makalah/namafile/50/OR GAN-ORGAN_KONSTITUSI.doc, diakses pada tanggal 2 Juli 2015. lembaga-lembaga atau badan-badan yang tugas nya berkaitan dengan peradilan dan penegakan hukum, yaitu berhubungan dengan fungsi-fungsi: (a) Penyelidikan, (b) penyidikan, (c) penuntutan, (d) pembelaan atau advokasi, (e) penyelesaian sen gketa dan mediasi atau pendamaian, (f) peradilan, penghakiman dan penghukuman, (g) pemasyara katan, (h) pelaksanaan putusan pengadilan selain pemasyarakatan, dan (i) pemulihan nama baik atau rehabilitasi, (j) pemberian grasi, (k) pemberian am nesti, (l) pemberian abolisi, (m) persaksian, dan (n) pemberian keterangan berdasarkan keahlian. Dari semua fungsi tersebut, yang terpenting adalah fungsi penyelidikan, penyidikan, dan penuntutan. Badan-badan yang dapat melakukan fungsi pe nyelidikan pelanggaran hukum ataupun hak asasi manusia adalah (a) Kepolisian Negara, (b) Tentara Nasional Indonesia (TNI) Angkatan Laut, (c) para Pejabat Penyidik Pegawai Negeri Sipil (PPNS), (d) Komisi Nasional Hak Asasi Manusia (Kom nasham), (e) Komisi Pemberantasan Tindak Pidana Korupsi (KPK), (f) Badan Pemeriksa Keuangan dan Pembangunan (BPKP), dan (g) Badan Pemer iksa Keuangan (BPK). Badan-badan yang dapat menjalankan fungsi penyidikan pro-justisia adalah (a) Kejaksaan, (b) Komisi Pemberantasan Tindak Pidana Korupsi, dan (c) Pejabat Penyidik Pegawai Negeri Sipil (PPNS). Sedangkan badan-badan yang melakukan penuntutan adalah (a) Kejaksaan, dan (b) Komisi Pemberantasan Tindak Pidana Korupsi. Lihat Jimly Asshiddiqie, Lembaga-lembaga Nega ra, Organ Konstitusional Menurut UUD 1945, hlm. 3 http://www.jimly.com/makalah/namafile/50/OR GAN-ORGAN_KONSTITUSI.doc, diakses pada tanggal 2 Juli 2015. 406 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) hlm. 44-45. Independensi Kejaksaan Berbicara tentang independensi kejaksaan pasti tidak dapat dilepaskan dari pertanyaan tentang bagaimana kejaksaan dapat bekerja secara independen dalam fungsinya seba- gai penegak hukum, karena kedudukannya seba-gai bagian dari lembaga pemerintahan. Ini menimbulkan suatu kontradiksi di mana satu sisi kejaksaan adalah bagian ranah ekse kutif yang tidak lain berada di bawah Pre- siden selaku pemegang kekuasaan eksekutif, namun di sisi lain Kejaksaan harus melaku kan fungsi, tugas dan wewenangnya sebagai institusi penegak hukum.16 Perwujudan lembaga kejaksaan yang independen sebenarnya sangat dipenga ruhi oleh independensi pimpinan lembaga penuntutan tersebut. Pimpinan lembaga penuntutan yang sering disebut sebagai general prosecutor/attorney general (yang dalam bahasa Indonesianya Jaksa Agung) sebagai puncak pimpinan lembaga penun tutan memiliki tanggung jawab yang besar. Independensi Jaksa Agung ini sangat dipe- ngaruhi oleh pola pemilihan, pemberhentian dan pertanggung jawaban. Ditinjau dari as pek pemilihan, terdapat lima tipe pemilihan Jaksa Agung, yakni: (1) dipilih secara lang sung oleh masyarakat (direct election by citi zen voters), (2) dipilih oleh anggota legislatif (election by the legislature), (3) dipilih oleh anggota eksekutif (appointment by members of executive), (4) dipilih oleh anggota yudi katif (appointment by members of the Terkait independensi ini, sebenarnya mesti dibagi menjadi dua aspek, yakni: inde pendensi secara institusional (kelembagaan) dan independensi secara fungsional. Inde pendensi secara lembaga berarti bahwa ke jaksaan ditempatkan dalam posisi yang in dependen secara kelembagaan. Kejaksaan memang semestinya lebih baik ditempatkan mandiri secara kelembagaan dan lepas dari kekuasaan manapun. Namun yang terpen- ting dari persoalan independensi bukanlah independensi kelembagaan, melainkan in dependensi fungsional. Independensi fung sional adalah bahwa Jaksa itu bisa bebas dan merdeka dalam menjalankan tugasnya untuk menuntut ataukah tidak menuntut.17 15 Dio Ashar Wicaksana. (2013). Kedudukan...Op.Cit, hlm. 7. 16 Ibid. 407 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) judiciary), dan (5) dipilih oleh anggota ke jaksaan (appointment by members of the procuracy). Dari lima tipe pemilihan terse but, tipe pemilihan Jaksa Agung yang dipilih oleh parlemen dianggap lebih dapat mem berikan jaminan independensi. Selain mem berikan jaminan independensi pola pemi- lihan ini pun berakibat adanya masa jabatan Jaksa Agung yang lebih pasti “fix term”, bi asanya 5 (tahun).19 orang atau kelompok yang kritis terhadap nya dengan tuduhan subversif. Selain itu in tervensi juga dapat dilakukan oleh Presiden untuk memerintahkan kepada Jaksa Agung untuk tidak menyidik dan menuntut orang atau kelompok tertentu karena terkait de- ngan kepentingan politiknya. Kedua, mengenai pemberhentian Jaksa Agung. 21 Keberadaan Pasal 22 ayat (1) huruf d ini oleh MK dinyatakan konstitusional bersyarat (conditionally constitutional) sebelum dilakukannya legislative review yang berlaku prospektif ke depan. Artinya, masa jabatan Jaksa Agung dinyatakan konstitusional dengan tafsir berakhirnya masa jabatan Jaksa Agung berakhir bersamaan dengan masa jabatan Presiden yang mengangkatnya sesuai praktek ketatanegaraan di Indonesia. Lihat Putusan MK Nomor 49/PUU-VIII/2010. 19 Ibid 20 Ahmad Andriadi, (2012), Kedudukan....Op.Cit, hlm. 48-50. 24 Jimly Asshiddiqie. (2008). “Hubungan Antar Lembaga Negara Pasca Perubahan UUD 1945”, Bahan ceramah pada Pendidikan dan Latihan Kepemimpinan (Diklatpim) Tingkat I Angkatan XVII Lembaga Administrasi Negara. Jakarta, 30 Oktober 2008, hlm. 1-2. Independensi Kejaksaan Dalam Pasal 22 ayat (1) din yatakan bahwa Jaksa Agung diberhentikan dengan hormat dari jabatannya karena: a) meninggal dunia; b) permintaan sendiri; c) sakit jasmani atau rohani terus-menerus; d) berakhir masa jabatannya; e) tidak lagi memenuhi salah satu syarat sebagaimana di maksud dalam Pasal 21. Pada Pasal 22 ayat (1) huruf d dinyatakan bahwa Jaksa Agung berhenti apabila berakhir masa jabatan nya. Namun dalam pasal tersebut, tidak ada penjelasan yang rinci tentang berapa lama periode masa jabatan Jaksa Agung. Keadaan ini berpotensi menghilangkan independensi kekuasaan penuntutan karena Jaksa Agung dapat diberhentikan kapan pun tergantung pada keinginan Presiden.21 Dalam konteks Indonesia, berdasarkan UU No. 16 Tahun 2004 tentang Kejaksaan RI, pengaturan berkaitan dengan indepen densi institusi kejaksaan masih memiliki sejumlah persoalan, yaitu:20 Pertama, ber dasarkan Pasal 19 UU No. 16 Tahun 2004 dinyatakan bahwa Jaksa Agung adalah pe jabat negara yang diangkat dan diberhen tikan oleh Presiden. Dengan kedudukan nya yang diangkat dan diberhentikan oleh Presiden, maka Jaksa Agung menjadi tidak independen. Karena posisi Jaksa Agung adalah setingkat dengan menteri yang berarti merupakan bagian dari kabinet dan menjadi pembantu serta bertanggung jawab penuh kepada Presiden. Dikhawatirkan dengan kedudukan sebagai bawahan langsung atau pembantu Presiden, maka sewaktu-waktu dengan kekuasaan yang dimilikinya Pre- siden bisa saja mengintervensi dan mengen dalikan Jaksa Agung dengan jajaran yang dimilikinya untuk menggunakan kekuasaan penuntutan pidana menghantam lawan-la wan politiknya. Seperti pada era Orde Baru dimana instrumen dan aparat hukum terma suk kejaksaan digunakan oleh Presiden Soe harto untuk menghukum serta membungkam Oleh karena itu, berdasarkan Pasal 19 ayat (2) jo. Pasal 22 UU No. 16 Tahun 2004 dapat disimpulkan bahwa kedudukan Jaksa Agung tidak independen karena sangat ber gantung kepada kewenangan Presiden untuk diangkat dan diberhentikan. Posisi Jaksa Agung seperti itu dapat menimbulkan dua masalah yang dengan istilah “dual obliga 408 408 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Urgensi Kejaksaan Menjadi Organ Kon stitusi tion” dan “conflicting loyalties”.22 Karena di satu sisi sebagai penegak hukum yang harus bekerja mandiri dan obyektif, sedangkan di sisi lain merupakan lembaga pemerintahan yang tunduk kepada instruksi Presiden yang bisa saja mengintervensi tugas dan fungsi kejaksaan sebagai lembaga penegak hukum Materi muatan atau substansi konstitusi pada intinya menyangkut prinsip pengaturan dan pembatasan kekuasaan negara guna mewu judkan tujuan nasional. Karena itu, menurut William G. Andrews, “under constitutional ism, two types of limitations impinge on go- vernment. Power proscribe and procedures prescribed”. 22 Ahmad Andriadi, (2012), Kedudukan...Op.Cit, hlm. 51. 23 Memaknai Independensi Kejaksaan di Indone sia (Kekuasaan Penuntutan), https://ilhamen dra.wordpress.com/2008/05/27/kekuasaan- penuntutan/#more-29, diakses pada tanggal 1 Juli 2015. 22 Ahmad Andriadi, (2012), Kedudukan...Op.Cit, hlm. 51. 23 Memaknai Independensi Kejaksaan di Indone sia (Kekuasaan Penuntutan), https://ilhamen dra.wordpress.com/2008/05/27/kekuasaan- penuntutan/#more-29, diakses pada tanggal 1 Juli 2015. Independensi Kejaksaan Konstitusionalisme mengatur dua hubungan yang saling berkaitan satu sama lain, yaitu: pertama, hubungan antara pemerintahan dengan warga negara; dan kedua, hubungan antara lembaga pemerin tahan yang satu dengan lembaga pemerin tahan yang lain. Isi konstitusi dimaksudkan untuk mengatur mengenai tiga hal penting: (a) menentukan pembatasan kekuasaan or gan-organ negara, (b) mengatur hubungan antara lembaga-lembaga negara yang satu dengan yang lain, dan (c) mengatur hubu- ngan kekuasaan antara lembaga-lembaga negara dengan warga negara.24 Kedudukan kejaksaan yang kontra diktif tersebut, tentu akan memengaruhi independensinya, sehingga diperlukan ben tuk pengaturan yang tepat untuk menjaga independensinya itu melalui dua opsi: Per tama, jika kejaksaan merupakan bagian dari kekuasaan eksekutif, maka diperlukan suatu aturan hukum yang memberikan jami nan independensi dan membatasi intervensi kekuasaan lain; Kedua, membentuk kejak saan yang mandiri secara penuh yang tidak berada di bawah kekuasaan eksekutif tetapi membentuk mekanisme pertanggungjawa ban kepada parlemen. Olehnya itu, diper lukan adanya aturan hukum yang mengatur secara jelas keberadaan sistem penuntutan dan lembaga penuntutan dalam kekuasaan negara. Menurut Milan Hanzel, penentuan posisi lembaga penuntutan dalam aturan hu kum sangatlah penting, pengaturan hukum posisi lembaga ini tidak hanya cukup diatur dalam aturan hukum biasa (undang-undang) tetapi haruslah diatur dalam materi konsti tusi.23 Hal ini berarti, lembaga pelaksana kekuasaan penuntutan, yaitu Kejaksaan se baiknya merupakan lembaga konstitusional (constitutional body). Meski salah satu substansi dari kons- titusi adalah mengatur tentang lembaga negara, namun sayangnya, pada amendemen UUD 1945 berkaitan dengan pengaturan lembaga negara tidak terdapat ketentuan yang jelas mengenai apa yang menjadi definisi dari istilah lembaga negara dan apa yang menjadi ukuran bahwa suatu lembaga negara keberadaannya dapat dikategorikan sebagai lembaga yang diatur langsung de- ngan konstitusi. Tidak adanya pengaturan ini tentu menimbulkan banyak penafsiran 409 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Sebab menurut Hans Kelsen, pada dasarnya negara itu bertindak hanya melalui organ- organ atau lembaganya. Tercapai tidaknya tujuan bernegara berujung pada bagaimana lembaga-lembaga negara tersebut melak sanakan tugas dan wewenang konstitusio- nalnya, serta hubungannya antarlembaga negara. Pengaturan lembaga negara dan hubungan antarlembaga negara mereflek sikan pilihan dasar-dasar kenegaraan yang dianut oleh suatu negara.28 dalam mendefinisikan dan mengklasifikasi kan apa itu lembaga negara. Konsepsi tentang lembaga negara secara terminologis bukanlah istilah yang tunggal dan seragam. Di dalam kepusta kaan Inggris, untuk menyebut lembaga ne gara digunakan istilah political institution, sedangkan dalam terminologi Bahasa Be landa terdapat istilah staatorgan. Sementara itu, dalam Bahasa Indonesia menggunakan istilah lembaga negara, badan negara, atau organ negara25. 27 Firmansyah Arifin dkk, (2005), Lembaga Negara dan Sengketa Kewenangan Antarlembaga Negara, Jakarta, Konsorsium Reformasi Hukum Nasional bekerjasama dengan Mahkamah Konstitusi, hlm. 31. 25 Ni’matul Huda, (2007), Lembaga Negara Dalam Masa Transisi Demokrasi, Yogyakarta, UII Press, hlm, 76. 26 Ibid, hlm. 31. 28 Hans Kelsen, (2006), Teori Umum tentang Hukum dan Negara, Bandung, Nusamedia, hlm. 279. 26 Ibid, hlm. 31.i Independensi Kejaksaan Dalam Kamus Hukum Foc -kema Andreae yang diterjemahkan oleh Sa leh Adiwinata dkk, disebutkan, kata orgaan juga diartikan sebagai perlengkapan. Karena itu istilah lembaga negara, organ negara, dan alat perlengkapan negara seringkali diper tukarkan satu sama lain.26 Keberadaan lembaga negara merupa- kan salah satu subtansi yang penting dalam konstitusi. Karena konstitusi merupakan ba sis atau dasar yang terkuat bagi eksistensi suatu lembaga negara ketika keberadaan nya diatur langsung oleh konstitusi sebagai norma hukum dasar tertinggi. Lembaga- lembaga negara yang dibentuk dan diberi kewenangan langsung oleh konstitusi tentu memiliki prestise dan kedudukan yang lebih kuat sebagai lembaga dengan kewenangan konstitusional yang dianggap mempunyai urgensi dibandingkan dengan lembaga-lem baga lain yang keberadaannya hanya diatur berdasarkan peraturan perundang-undangan di bawah konstitusi. Adapun tujuan diadakannya lembaga- lembaga negara atau alat-alat perlengkapan negara adalah untuk menjalankan fungsi negara dan menjalankan fungsi pemerin tahan secara aktual. Dengan kata lain, lem baga-lembaga negara tersebut harus dapat membentuk satu kesatuan proses yang satu sama lain saling berhubungan dalam rangka penyelenggaraan fungsi negara.27 Salah satu persoalan yang mengemuka adalah terkait dengan tidak diaturnya lem baga kejaksaan sebagai organ konstitusi. Ditinjau dari segi historis berdasarkan UUD 1945 yang disahkan pada tanggal 18 Agustus 1945, memang tidak ditemukan satu kata pun yang menyebut institusi kejaksaan, baik dalam batang tubuh maupun penjelasannya. Demikian pula setelah UUD 1945 menga- lami empat kali perubahan di era reformasi, Dengan demikian, salah satu materi penting dan selalu ada dalam konstitusi adalah pengaturan tentang lembaga negara. Hal ini dapat dimengerti karena kekuasaan negara pada akhirnya diterjemahkan ke dalam tugas dan wewenang lembaga negara. 410 410 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) keberadaan institusi kejaksaan tetap bukan lah bagian dari lembaga negara yang diatur langsung oleh konstitusi. Hal ini menim bulkan pertanyaan, mengapa kejaksaan ke beradaannya tidak diatur oleh UUD 1945, padahal jika dibandingkan dengan institusi penegak hukum lain, yaitu kepolisian yang keberadaannya sebagai organ konstitusi dia tur langsung oleh UUD 1945 pada Pasal 30 ayat (4), yaitu: “Kepolisian Negara Republik Indonesia sebagai alat negara yang menjaga keamanan dan ketertiban masyarakat bertu gas melindungi, mengayomi, melayani ma syarakat, serta menegakkan hukum”. lembaga dimaksud misalnya adalah Ko- misi Nasional Hak Asasi Manusia (Kom nas HAM), Komisi Pemberantasan Tindak Pidana Korupsi (KPK) dan sebagainya. 30 Ibid, hlm. 15. 29 Jimly Asshiddiqie, (2008), Hubungan....Op.Cit, hlm. 14. , 31 Ibid. 29 Jimly Asshiddiqie, (2008), Hubungan....Op.Cit, hlm. 14. , 31 Ibid. Independensi Kejaksaan Lembaga-lembaga ini, seperti halnya kejak saan, meskipun tidak secara eksplisit disebut dalam UUD 1945, tetapi sama-sama memi liki constitutional importance dalam sistem konstitusional berdasarkan UUD 1945.30 Masih menurut Jimly Asshiddiqie, meskipun pengaturan dan pembentukan institusi Kejaksaan, KPK, Komnas HAM hanya didasarkan atas undang-undang, ti dak ditentukan sendiri dalam UUD, tetapi keberadaannya sebagai lembaga negara mempunyai apa yang disebut sebagai cons- titutional importance yang sama dengan lembaga-lembaga negara lainnya yang dise butkan secara eksplisit dalam UUD 1945. Sama halnya dengan keberadaan kejaksaan dan kepolisian dalam setiap sistem negara demokrasi konstitusional ataupun negara hukum yang demokratis, keduanya mempu nyai derajat kepentingan (importance) yang sama. Meski dalam UUD 1945, yang di tentukan kewenangannya hanya kepolisian, sedangkan kejaksaan tidak disebut, namun hal tersebut tidak dapat dijadikan alasan untuk menilai bahwa kepolisian lebih pent ing daripada kejaksaan. Oleh karena kedua lembaga itu sama-sama penting atau memi liki constitutional importance yang sama, sebab setiap negara yang menganut prinsip demokrasi konstitusional atau negara hukum yang demokratis, haruslah memiliki perang kat kelembagaan berupa kepolisian dan ke jaksaan sebagai lembaga-lembaga penegak hukum yang efektif.31 Apakah sebagai sama-sama penegak hukum, institusi kepolisian memiliki urgensi yang lebih penting daripada kejaksaan, se- hingga eksistensi institusi kepolisian diatur langsung oleh UUD 1945 sebagai organ konstitusi sedangkan Kejaksaan tidak. Me- nurut Jimly Asshiddiqie dalam rancangan perubahan UUD, semula tercantum peng- aturan mengenai Kejaksaan Agung. Akan tetapi, karena tidak mendapatkan kesepakatan, maka sebagai gantinya dise- pakatilah rumusan Pasal 24 ayat (3) UUD 1945, yaitu: “Badan-badan lain yang fu- ngsinya berkaitan dengan kekuasaan keha- kiman diatur dalam undang-undang”.29 Namun, karena yang disebut dalam Pasal 24 ayat (3) tersebut di atas adalah badan-badan, berarti jumlahnya lebih dari satu. Artinya, selain Kejaksaan Agung, masih ada lagi lembaga lain yang fungsinya juga berkaitan dengan kekuasaan kehakiman, yaitu yang menjalankan fungsi penyelidikan, penyidikan, dan/atau penuntutan. Lembaga- 411 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Hasanuddin Law Review Vol. 1 Issue 3, December (2015) sebagai organ konstitusi akan memiliki ke wenangan konstitusional, yaitu kewenangan yang diberikan langsung oleh konstitusi, se hingga kejaksaan memiliki legal standing yang jika dalam pelaksanaan kewenangan nya terjadi konflik kewenangan dengan or gan konstitusi lain, maka penyelesaian seng ketanya dapat diajukan kepada MK sebagai lembaga yang berwenang untuk memutus sengketa kewenangan konstitusional antar lembaga negara. 33 Hal ini ditengarai bahwa Kejaksaan mengalami intervensi politik untuk tidak melakukan penyidikan dalam menindaklanjuti rekomendasi komnasham. Tuduhan yang muncul seperti ini cukup beralasan dikarenakan Kejaksaan seperti selalu mencari dalih jika dipertanyakan bagaimana kelanjutan penanganan terhadap kasus pelanggaran HAM. Menurut Komnasham setidaknya ada sejumlah kasus pelanggaran HAM yang terhenti di Kejaksaan Agung, yaitu peristiwa Trisakti, Semanggi I dan II, peristiwa kerusuhan Mei 1998, peristiwa penculikan aktivis, peristiwa Talangsari Lampung, dan peristiwa Wasior dan Wamena. Lihat Ifdhal Kasim, 2011, Komnas HAM dan Tantangannya Dewasa Ini, Jurnal Dignitas: HAM dan Realitas Transisional, Volume VII, No. 1 Tahun 2011, Jakarta, Elsam, hlm. 83. 32 Ahmad Andriadi, (2012), Kedudukan...Op.Cit, hlm. 46. 32 Ahmad Andriadi, (2012), Kedudukan...Op.Cit, hlm. 46. 34 Berdasarkan pendapat Peter L. Strauss, check and balances dalam upaya menciptakan relasi konstitusional untuk mencegah penyalahgunaan kekuasaan di antara cabang-cabang kekuasaan negara untuk membangun keseimbangan hubungan dalam praktik penyelenggaraaan negara. Jika dalam teori pemisahan kekuasaan dan pembagian kekuasaan lebih menggambarkan kejelasan posisi setiap cabang kekuasaan negara dalam menjalankan fungsi-fungsi konstitusionalnya, sedangkan check and balances lebih menekankan kepada upaya membangun mekanisme perimbangan untuk saling kontrol antar cabang kekuasaan negara. Bagaimanapun, mekanisme check and balances hanya dapat dilaksanakan sepanjang punya pijakan konstitusional guna mencegah kemungkinan ter- jadinya penyalahgunaan kekuasaan oleh cabang- cabang kekuasaan negara. Lihat Saldi Isra, 2010, Pergeseran Fungsi Legislasi: Menguatnya Model Legislasi Parlementer Dalam Sistem Presidensial Indonesia, Jakarta, RajaGrafindo Persada, hlm. 78. Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Keempat, mempertegas kedudukan kejaksaan sebagai central of authority di bidang penuntutan. Di sini perlu ditata ke beradaan kewenangan penuntutan yang ada di KPK berhadapan dengan kewenangan yang dimiliki oleh kejaksaan. Adanya dua- lisme ini tentu tidak baik dalam membangun hubungan kelembagaan yang efektif, sehing ga potensi konflik akan terus membayangi ketika suatu fungsi atau urusan yang sama dikerjakan oleh dua lembaga atau lebih. Independensi Kejaksaan Interpretasi yang dilakukan oleh Jimly seperti di atas yang menyatakan kejaksaan mempunyai constitutional importance mes ki hanya sebagai lembaga yang dibentuk dengan undang-undang, tetap tidak dapat menutupi fakta bahwa eksistensi kejaksaan bukan sebagai bagian dari organ konstitusi. Padahal menurut Milan Hanzel, penentuan posisi lembaga penuntutan, yaitu kejaksaan dalam aturan hukum sangatlah penting, di mana pengaturan hukum posisi lembaga ini tidak hanya cukup diatur dalam aturan hu kum biasa (undang-undang), tetapi haruslah diatur dalam konstitusi. Hal ini berarti lem baga pelaksana kekuasaan penuntutan, yaitu kejaksaan adalah harus merupakan lembaga konstitusional (constitutional body). Sebe- narnya pengaturan institusi kejaksaan dalam konstitusi suatu negara bukanlah merupakan hal yang baru, karena ternyata di dunia ini terdapat hampir 90 (sembilan puluh) negara yang mengatur lembaga Kejaksaan dan/atau Jaksa Agungnya dalam Undang-Undang Dasar.32 Kedua, penguatan eksistensi kejak saan ke dalam konstitusi sekaligus dijadikan momen penataan hubungan kelembagaan dengan institusi lain yang terkait dengan kekuasaan kehakiman. Seperti hubungan an tara Kejaksaan dengan Komnas HAM dalam penyelesaian pelanggaran berat HAM yang selama ini tampak bahwa rekomendasi hasil penyelidikan dari Komnas HAM mengalami stagnasi dan mandek di kejaksaan tanpa ada- nya tanda-tanda akan dilanjutkan ke tahapan penyidikan.33 Selain itu, potensi sengketa kewenangan antarlembaga negara mungkin saja terjadi dengan adanya dualisme ke wenangan penyidikan maupun penuntutan, seperti dalam penanganan perkara tindak pidana korupsi antara Kejaksaan, Kepolisian Maka dari itu, penguatan eksistensi kejaksaan untuk diatur ke dalam konstitusi memiliki sejumlah urgensi, yaitu: Pertama, untuk memperkuat eksistensi kejaksaan menjadi lembaga negara utama (main state organ) yang keberadaannya diatur langsung oleh UUD 1945 sesuai dengan tugas dan fungsinya yang sangat urgen dalam kehidu pan bernegara sebagai lembaga penegak hukum. Pengaturan eksistensi kejaksaan ke dalam konstitusi akan memperjelas kedudu kannya sebagai organ konstitusi dalam struktur ketatanegaran Indonesia. Kejaksaan 412 412 dan KPK. Ke depannya, untuk dapat menyele saikan persoalan konflik kewenangan sebaik nya melalui mekanisme sengketa kewe- nangan yang ada di MK, bukan diselesaikan secara politis. Tentu di sini dalam konteks amandemen UUD 1945 tidak hanya ke jaksaan yang diperkuat dengan diatur oleh konstitusi melainkan juga sejumlah lembaga lain yang memiliki urgensi yang sama untuk dijadikan organ konstitusi, seperti KPK dan Komnas HAM. Agar ketika terjadi seng- keta kewenangan konstitusional, lembaga- lembaga tersebut mempunyai legal standing dalam berperkara di MK. Kelima, urgensi penguatan kejaksaan dalam konstitusi seharusnya menempat kan lembaga ini memiliki kedudukan dan hu-bungan kelembagaan yang jelas dalam struktur ketatanegaraan sebagai sebuah fungsi atau kekuasaan negara yang inde penden bukan sebagai bagian dari ekseku tif maupun yudikatif, meskipun memiliki keterkaitan terhadap keduanya. Oleh karena kekuasaan penuntutan dapat dianggap seba- gai pelaksanaan kekuasaan sendiri dalam suatu fungsi dari lembaga-lembaga negara, sehingga tidak lagi mengklasifikasikan suatu lembaga negara hanya bersandarkan kepa- da pendekatan separation of powers dalam perspektif trias politica Montesquie semata. Atau bisa juga tetap mendasarkan penglasifi kasian lembaga negara berdasarkan konsep trias politica dengan pengertian yang lebih luas, tidak hanya sebatas melihat lembaga negara terbagi antara eksekutif, legislatif dan Ketiga, untuk memperkuat indepen densi kejaksaan, karena dengan memiliki dasar hukum yang kuat sebagai organ kon stitusi sebaiknya kedudukan kejaksaan tidak ditempatkan lagi sebagai lembaga pemerin tahan yang menjadi bagian eksekutif, me lainkan menjadi lembaga negara mandiri yang direfleksikan dengan pengangkatan dan pemberhentian Jaksa Agung tidak hanya menjadi kewenangan Presiden semata seba- gai Kepala Pemerintahan melainkan meli batkan peran DPR untuk memberikan per setujuan. Hal ini merupakan praktek lazim dalam negara demokratis untuk pengangka tan atau pengisian jabatan strategis tertentu membutuhkan keterlibatan parlemen seba- gai wujud adanya checks and balances an tara eksekutif dengan legislatif.34 413 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) b) Kekuasaan legislatif, yaitu kekuasaan untuk membentuk undang-undang. Ke- kuasaan ini dilaksanakan oleh Dewan Perwakilan Rakyat (DPR). Dalam pe nyusunan peraturan perundangan ter tentu yang berkaitan dengan otonomi daerah, hubungan pusat dan daerah, pembentukan dan pemekaran serta penggabungan daerah, pengelolaan sumber daya alam dan sumber daya ekonomi lainnya, serta yang berkai tan dengan perimbangan keuangan pusat dan daerah, DPR melibatkan dan memerhatikan usulan dari Dewan Per wakilan Daerah (DPD). yudikatif secara absolut. Menurut Saldi Isra, dalam perkembangan praktik ketatanega raan modern tidak mungkin suatu cabang kekuasaan negara benar-benar terpisah dari cabang kekuasaan yang lain. Bahkan dalam pandangan John A. , . 77. 36 Anwar Sanusi (ed). (2010). Penataan Mekanisme Hubungan Antar Lembaga Negara, Jakarta, Pusat Kajian Kinerja Kelembagaan Lembaga Adminis trasi Negara, hlm. 21-23. PENUTUP Eksistensi kejaksaan dalam struktur keta- tanegaraan Indonesia memiliki posisi yang dilematis. Di satu sisi, kejaksaan adalah lembaga penegak hukum yang menjalan- kan kekuasaan penuntutan, sedangkan di sisi yang lain, merupakan bagian dari lem baga pemerintahan. Kedudukan seperti ini menyebabkan munculnya perdebatan bahwa kejaksaan sebenarnya tidak tepat dikate- gorikan sebagai bagian lembaga eksekutif, karena tugas dan fungsinya terkait dengan kekuasaan kehakiman. Dalam UU No. 16 Tahun 2004, jelas menempatkan kejaksaan di bawah eksekutif, di mana Jaksa Agung di angkat dan diberhentikan oleh Presiden. Pun dengan penegasan bahwa kejaksaan adalah lembaga pemerintahan. Pengaturan ini tentu saja memiliki dampak terhadap indepen- densi kejaksaan, sehingga memunculkan dan KPK. g) Kekuasaan konsultatif, yaitu memberi kan nasehat dan pertimbangan kepada pemerintah yang dahulu dilaksanakan oleh Dewan Pertimbangan Agung (DPA), namun dengan adanya amen demen UUD 1945, kekuasaan ini tidak berada pada level supreme lagi tetapi dilaksanakan oleh Dewan Pertim- bangan Presiden (Wantimpres). Dalam kerangka membangun dan me- nata institusi kejaksaan dalam iklim yang demokratis saat ini, untuk ke depannya pe- nguatan kejaksaan sebaiknya diatur lang sung oleh konstitusi. Hal ini dimaksudkan untuk menjadikan kejaksaan sebagai bagian dari main state organ yang mempunyai le gal standing sama seperti lembaga penegak hukum lainya, yaitu Kepolisian dan insti tusi peradilan (MA dan MK), serta untuk memerkuat dan memerjelas kedudukannya sebagai lembaga negara, memusatkan ke wenangan penuntutan berada di Kejaksaan sebagai central of authority, membenahi hubungan kelembagaan antarsesama pe- negak hukum maupun lembaga terkait dan tentu saja memerkuat independensi kejak saan sebagai lembaga mandiri yang men jalankan fungsi penuntutan dalam struktur ketatanegaraan Indonesia. h) Kekuasaan attorney dan prosecutor (pengacara dan penuntut umum nega- ra), yaitu melaksanakan kekuasaan negara dibidang penuntutan yang di laksanakan oleh Kejaksaan Agung. dan KPK. Garvey dan Aleinikoff melihat teori trias politica dalam praktik ketatanegaraan tidak mungkin memisahkan secara ketat cabang-cabang kekuasaan nega- ra di mana pembagian kekuasaan tidak diis tilahkan dengan separation atau distribution of powers, melainkan hanya berupa separa tion of functions.35 i Ke depannya, dalam mengklasifikasi kan atau melakukan pembagian kekuasaan dalam suatu negara termasuk di Indonesia, perlu dilihat adanya suatu pola hubungan yang terjadi antara lembaga-lembaga pe nyelenggara kekuasaan negara yang dibagi hanya dalam konteks separation of func tion di mana di dalamnya kejaksaan menjadi lembaga mandiri yang menjalankan fungsi penuntutan sebagai kekuasaan sendiri dalam struktur ketatanegaraan. c) Kekuasaan eksekutif, yaitu kekuasaan untuk menyelenggarakan kekuasaan pemerintahan negara. Kekuasaan ini dilaksanakan oleh Presiden. c) Kekuasaan eksekutif, yaitu kekuasaan untuk menyelenggarakan kekuasaan pemerintahan negara. Kekuasaan ini dilaksanakan oleh Presiden. d) Kekuasaan yudikatif, yaitu kekuasaan untuk menyelenggarakan kekuasaan kehakiman yang merupakan kekua saan yang merdeka untuk menyeleng garakan peradilan guna menegakkan hukum dan keadilan. Kekuasaan ini dilaksanakan oleh Mahkamah Agung (MA) serta badan peradilan yang ber- ada di bawahnya dan oleh Mahkamah Konstitusi (MK). d) Kekuasaan yudikatif, yaitu kekuasaan untuk menyelenggarakan kekuasaan kehakiman yang merupakan kekua saan yang merdeka untuk menyeleng garakan peradilan guna menegakkan hukum dan keadilan. Kekuasaan ini dilaksanakan oleh Mahkamah Agung (MA) serta badan peradilan yang ber- ada di bawahnya dan oleh Mahkamah Konstitusi (MK). Secara keseluruhan, lembaga-lembaga kekuasaan negara tersebut dalam konteks Indonesia, dapat diklasifikasikan sebagai berikut:36 a) Kekuasaan konstitutif, yaitu kekua saan untuk menetapkan dan mengubah Undang-undang Dasar Negara. Kekua saan ini diwadahi dan dilaksanakan oleh lembaga negara yang disebut se- bagai Majelis Permusyawaratan Rak- yat (MPR). e) Kekuasaan auditif, yaitu, menyeleng garakan pemeriksaan atas pengelolaan dan tanggung jawab tentang keuangan negara. Kekuasaan ini dilaksanakan oleh Badan Pemeriksa Keuangan (BPK) yang bebas dan mandiri. f) Kekuasaan moneter (otoritas moneter), yaitu menetapkan dan melaksanakan kebijakan moneter, mengatur dan men jaga kelancaran sistem pembayaran 414 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) serta memelihara kestabilan nilai ru piah. Kekuasaan ini dilaksanakan oleh Bank Indonesia (BI) sebagai bank sen tral Republik Indonesia. stigma bahwa kejaksaan hanyalah sebagai alat kekuasaan dari yang memerintah, apa lagi dibuktikan dengan pengalaman selama rezim Orde Baru, di mana institusi kejaksaan dijadikan instrumen hukum untuk melibas lawan-lawan politik penguasa pada waktu itu. BIBLIOGRAFI Anwar Sanusi (ed). (2010). Penataan Me kanisme Hubungan Antar Lembaga Negara. Jakarta: Pusat Kajian Kinerja Kelembagaan Lembaga Administrasi Negara. Dio Ashar Wicaksana. (2013). “Kedudukan Kejaksaan RI dalam Sistem Hukum Tata Negara Indonesia”. Jurnal Fiat Justitia, Vol. 1. No. 1, Maret 2013. Firmansyah Arifin dkk. (2005), Lembaga 415 Hasanuddin Law Review Vol. 1 Issue 3, December (2015) Negara dan Sengketa Kewenangan An tarlembaga Negara, Jakarta: Konsorsi um Reformasi Hukum Nasional beker jasama dengan Mahkamah Konstitusi. Negara dan Sengketa Kewenangan An tarlembaga Negara, Jakarta: Konsorsi um Reformasi Hukum Nasional beker jasama dengan Mahkamah Konstitusi. Hukum Universitas Hasanudin. Jimly Asshiddiqie. (2008). “Hubungan An tar Lembaga Negara Pasca Perubahan UUD 1945”. Bahan ceramah pada Pen didikan dan Latihan Kepemimpinan (Diklatpim) Tingkat I Angkatan XVII Lembaga Administrasi Negara. Jakarta, 30 Oktober 2008. Hans Kelsen. (2006). Teori Umum tentang Hukum dan Negara, Bandung: Nusa Media. Ifdhal Kasim. (2011). “Komnas HAM dan Tantangannya Dewasa Ini”. Jurnal Dignitas: HAM dan Realitas Transi sional, Vol. VII, No. 1. Jimly Asshiddiqie. “Lembaga-lembaga Ne- gara, Organ Konstitusional Menurut UUD 1945”. Diunduh pada laman web site: http://www.jimly.com/makalah/ namafile/50/ORGAN-ORGAN_KON STITUSI.doc, diakses pada tanggal 2 Juli 2015. Ni’matul Huda. (2007). Lembaga Negara Dalam Masa Transisi Demokrasi, Yog- yakarta: UII Press. Saldi Isra. (2010). Pergeseran Fungsi Le- gislasi: Menguatnya Model Legislasi Parlementer dalam Sistem Presidensial Indonesia, Jakarta: PT. RajaGrafindo Persada. Yusril Ihza Mahendra. “Kedudukan Kejak saan dan Posisi Jaksa Agung Dalam Sistem Presidensial Di Bawah UUD 1945”. Diunduh pada laman website: http://yusril.ihzamahendra.com/2010 /08/20/kedudukan-kejaksaan-dan-po sisi-jaksa-agung-dalam-sistem-pres idensial-di-bawah-uud-1945-oleh-prof- dr-yusril-ihza-mahendra-pendahulu an-hampir-seluruh-negara-modern- di-du/#, diakses pada tanggal 30 Juni 2015. Sumber lainnya: Ahmad Andriadi. (2012). Kedudukan Ke jaksaan dalam Sistem Ketatanegaraan Republik Indonesia (Telaah Kritis Ter hadap Undang-Undang Nomor 16 Ta hun 2004 tentang Kejaksaan Republik Indonesia). Skripsi. Makassar: Fakultas *** 416 416
https://openalex.org/W4298427762	https://dergipark.org.tr/tr/download/article-file/191226	Turkish	null	COMPARISON OF NEW GRAPHICS APPLICATION PROGRAMMING INTERFACES	DOAJ (DOAJ: Directory of Open Access Journals)	2,003	cc-by	3,768	ÖZET Bu makalede grafik yazılımları geliştirmede kullanılan uygulama geliştirme arayüzleri sınıflandırılarak kısaca tanıtılmıştır. İki boyutlu ve üç boyutlu uygulama geliştirme arayüzlerinin özellikleri belirtilmiştir. Çalışma kapsamında geliştirilen aynı işleve sahip iki yazılım (Kup3B Java2D sürümü, Kup3B Java3D sürümü) kaynak kod satır sayılarına göre karşılaştırılmışlardır. Elde edilen değerler grafiklere dönüştürülmüş ve yorumlanmıştır. Anahtar Kelimeler : Grafik yazılımları, Yazılım geliştirme, Uygulama geliştirme arayüzleri, Java2D API, Java 3D API P A M U K K A L E Ü N İ V E R S İ T E S İ M Ü H E N D İ S L İ K F A K Ü L T E S P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E M ÜHENDİ SLİK BİLİM LERİ DERGİSİ P A M U K K A L E Ü N İ V E R S İ T E S İ M Ü H E N D İ S L İ K F A K Ü L T E S P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E M ÜHENDİ SLİK BİLİM LERİ DERGİSİ P A M U K K A L E U N I V E R S I T Y E N G I N E E R I N G C O L L E G E M ÜHENDİ SLİK BİLİM LERİ DERGİSİ M ÜHENDİ SLİK BİLİM LERİ DERGİSİ J O U R N A L O F E N G I N E E R I N G S C I E N C E S ABSTRACT In this paper, Application Programming Interfaces (API) which are used for graphics software development are classified and introduced briefly. Properties of 2D and 3D API’s are specified. Programs which are implemented in this study (Kup3B Java2D version, Kup3B Java3D version) are compared by number of lines of code written. Charts are constructed by using values obtained and interpreted. Words : Graphics software, Software development, Application programming interfaces, Java 2D API, Java 3D AP YENİ GRAFİK UYGULAMA GELİŞTİRME ARAYÜZLERİNİN KARŞILAŞTIRILMASI Mustafa TÜRKSEVER, Aybars UĞUR Geliş Tarihi : 16.04.2001 Geliş Tarihi : 16.04.2001 1. GİRİŞ Her iki program da, üç boyutlu bir küpün oluşturulmasını ve etkileşimli olarak hareket ettirilmesini sağlamaktadır. İlk programda sadece iki boyutlu bir arayüz olan Java 2D kullanılmış ve üç boyut, matematiği, algoritmaları ve veri yapıları tasarlanıp kodlanarak oluşturulmuştur. İkinci programda, üç boyutlu bir arayüz olan Java 3D kullanıldığından, ve Java 3D ile hazır üç boyut matematiği, algoritmaları, veri yapıları ve üç boyutlu nesne oluşturma komutları geldiğinden, sadece içeriğin oluşturulması, programın tamamlanması için yeterli olmuştur. • Aynı özelliklere sahip iki adet program yazılmıştır. Her iki program da, üç boyutlu bir küpün oluşturulmasını ve etkileşimli olarak hareket ettirilmesini sağlamaktadır. İlk programda sadece iki boyutlu bir arayüz olan Java 2D kullanılmış ve üç boyut, matematiği, algoritmaları ve veri yapıları tasarlanıp kodlanarak oluşturulmuştur. İkinci programda, üç boyutlu bir arayüz olan Java 3D kullanıldığından, ve Java 3D ile hazır üç boyut matematiği, algoritmaları, veri yapıları ve üç boyutlu nesne oluşturma komutları geldiğinden, sadece içeriğin oluşturulması, programın tamamlanması için yeterli olmuştur. Günümüzde grafik uygulama geliştirme arayüzlerinin özellikleri genişlemiştir. İki boyutlu bir grafik uygulama geliştirme arayüzünün özellikleri aşağıdaki gibi belirtilebilir (Knudsen, 1999) : • Temel Şekiller : Temel iki boyutlu şekilleri çizdirmeyi sağlayan komutlar (nokta, çizgi, dikdörtgen, elips, yay, eğri, çokgen vb.) vardır. Diğer geometrik şekiller, temel şekillerin bileşiminden oluşur. • Bu iki yazılım, kaynak kod satır sayılarına göre karşılaştırılmıştır. Böylece, yazılımlarda üç boyutlu grafik kullanımının giderek yaygınlaştığı günümüzde, üç boyutlu grafikler içeren yazılımların geliştirilmesinde, üç boyutlu API kullanımının yazılım geliştirme sürecini ne kadar hızlandırdığı ve kolaylaştırdığı belirlenmiştir. Çalışmada elde edilen değerlerin öneminin, yazılım geliştirme maliyetlerine de yansıyacağı dikkate alındığında büyük olduğu açıktır. • Renk : Değişik amaçlarla hazır renklerin kullanımını, renklerin elde edilmesini ve renk özelliklerinin ayarlanmasını sağlayan renk komutları kullanılır. • Resim : BMP, GIF, JPEG dahil birçok resim formatındaki resmi yüklemeyi ve kaydetmeyi sağlayan komutlar yardımıyla, herhangi bir grafik programı ile veya tarayıcıdan alınarak oluşturulmuş bir resim grafik programlarında kullanılabilir hale gelmektedir. • Çizgi ve Dolgu Biçemi : Şekilleri biçemlemeyi sağlayan komutlar ve hazır özellikler, nesnelere çeşitlilik katmaktadırlar. 2. GRAFİK UYGULAMA GELİŞTİRME ARAYÜZLERİ (API) • Çizgi biçemi : Şekillerin sınırları ve çizgiler kesiksiz veya noktalı çizgi türleri ile çizilebildiği gibi herhangi bir kalınlıkta ve renkte de çizilebilir. Grafik uygulama geliştirme arayüzleri (API), iki bölümde incelenebilir. İki boyutlu grafik API’leri ve üç boyutlu grafik API’leri. İki boyutlu ve üç boyutlu grafikler içeren uygulamaların yazılmasında bu arayüzlerin kullanımı büyük olanaklar ve kolaylıklar getirmektedir. • Dolgu biçemi : Şekillerin içi sabit bir renkle, desenle, renk basamakları ile veya herhangi bir resim ile doldurulabilir. • Dolgu biçemi : Şekillerin içi sabit bir renkle, desenle, renk basamakları ile veya herhangi bir resim ile doldurulabilir. 1. GİRİŞ projelerden elde edilecek başarılarla, daha yüksek bant genişliği, servis kalitesi sağlanacak ve çok kullanıcılı, etkileşimli, gerçek zamanlı uygulamaların beklediği ortam oluşacaktır. Sonuçta üç boyutlu grafiksel içerikler oluşturmayı ve üzerinde işlemler yapmayı sağlayan yazılımların ve yazılım geliştirme ortamlarının önemi daha da artacaktır. Bilgisayar yazılım ve donanımlarında son yıllarda görülen gelişmeler, bilgisayar grafikleri ve animasyon işlemlerindeki kalite ve hızı oldukça artırmıştır. Bu şekilde günlük yaşamdan değişik bilim dallarına kadar her alanda etkisini göstermeye başlayan bilgisayar grafikleri alanına duyulan ilgi ve gereksinim giderek artmakta; bu alan standartların oluşması ve yeni kavramların eklenmesi ile her geçen gün zenginleşmektedir. Grafik yazılımları ikiye ayrılmaktadır : • Grafik Uygulama Geliştirme Arayüzleri (API’ler) • Grafik Paket Programları (Çizim, boyama, görüntü işleme, modelleme, animasyon, ışın izleme, CAD, oyun, eğitim ve diğerleri) İnternet üzerine üç boyutlu grafiksel içerik ekleme, son günlerde üzerinde çalışılan konulardandır. Internet2 ve NGI (Next Generation Internet) gibi ileri ağ teknolojilerini oluşturmaya yönelik 55 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur Üç boyutlu grafiksel yazılımların geliştirilmesinde, kullanılan grafik uygulama geliştirme arayüzünün (API) türü yönünden iki yaklaşım vardır. İki boyutlu bir arayüz kullanıp üç boyutla ilgili kodları (üç boyutlu uzayda nesneleri taşıma, üç boyutlu nesneleri köşe ve yüzey bilgilerini tutarak oluşturma, perspektif izdüşüm gibi), yazılacak uygulamaya yönelik olarak baştan oluşturmak veya doğrudan bu özellikleri sağlayan (hazır gelen) üç boyutlu bir arayüz kullanmak. yazılımların geliştirilmesinde kullanılmak üzere yeteri derecede işlev ve özellik içermektedirler. Ayrıca üç boyutlu grafikler içeren yazılımlarda da kullanılmaktadırlar. Kişisel bilgisayarlarda DOS işletim sistemi ortamında Pascal, C, C++ programları ile kullanılmış olan BGI (Borland Graphics Interface) ve Windows ortamındaki Windows API’leri birçok grafik programcısının bildiği arayüzlerdir. Günümüzde Java awt ve Java 2D API de yaygın olarak kullanılmaya başlanmıştır. Java programlama dilinin ağ merkezli olmasının getirdiği avantajlardan da yararlanılmaktadır. Yazılan grafiksel içerikli bir applet, doğrudan internet üzerine yerleştirilebilmektedir. Dünyanın farklı bölgelerindeki kullanıcılar internet üzerinden erişerek bu programları kullanabilmektedirler. Kişisel bilgisayarlarda DOS işletim sistemi ortamında Pascal, C, C++ programları ile kullanılmış olan BGI (Borland Graphics Interface) ve Windows ortamındaki Windows API’leri birçok grafik programcısının bildiği arayüzlerdir. Günümüzde Java awt ve Java 2D API de yaygın olarak kullanılmaya başlanmıştır. Java programlama dilinin ağ merkezli olmasının getirdiği avantajlardan da yararlanılmaktadır. Yazılan grafiksel içerikli bir applet, doğrudan internet üzerine yerleştirilebilmektedir. Dünyanın farklı bölgelerindeki kullanıcılar internet üzerinden erişerek bu programları kullanabilmektedirler. Bu çalışmada, • Aynı özelliklere sahip iki adet program yazılmıştır. 2. 1. İki Boyutlu grafik API’leri • Metin : Yazılar, yazıtipleri ve yazılar üzerinde biçemlemeyi sağlayan komutlar, iki boyutlu grafiklerle, grafiksel yazıların beraber kullanılabilmesini sağlar. Grafik yazılımları geliştirmede iki boyutlu grafik uygulama geliştirme arayüzleri uzun yıllardır kullanılmaktadır. İki boyutlu grafikler içeren Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur • Görüntü İşleme : İki boyutlu gelişmiş arayüzler, resimler üzerinde hazır görüntü işleme komutlarının uygulanabilmesini sağlar. donanım soyutlama katmanıdır. 1992 yılında ortaya çıkışından bu yana bilimsel programlardan görsel simülasyonlara, animasyonlardan oyunlara kadar çok geniş bir yelpazedeki uygulamanın yüksek performansla bilgisayar ortamına taşınmasını sağlamıştır. • Dönüşüm : Şekiller, resimler, yazılar küçültülüp, büyütülebilmekte, taşınabilmekte ve döndürülebilmektedir. Dördüncü kuşağa örnek olan Java 3D API, Java programlama diline üç boyutlu grafik ve görüntüleme yetenekleri kazandırmak üzere geliştirilmiştir. Java 3D API, etkileşimli üç boyutlu grafikleri içeren uygulamalar için bir dizi nesneye yönelik yüksek performanslı bir arayüz sağlar. Yazılım geliştiriciler, üç boyutlu nesneler ve görsel ortamlar oluşturmayı, bunlar üzerinde işlemler yapmayı ve davranışlarını denetlemeyi kolaylaştırıp güçlendiren yüksek düzeyli ve kolay programlama modeliyle Java 3D'nin avantajlarından yararlanırlar. Bu model, görüntüleme tekniklerinden çok yazılım içeriğine odaklanmayı sağlar. Sonuçta yazılım geliştiriciler, yüksek kaliteli, ölçeklenebilir ve platform bağımsız üç boyutlu grafikleri Java tabanlı uygulamalar ve applet'ler ile kolaylıkla bütünleştirebilir (Day, 1998; Sowizral and Nadeau, 1999). Web tarayıcılarının birçoğu Java 3D Applet'lerini doğrudan çalıştıramamakla birlikte, Java 3D API yüklendikten sonra yapılan ayarlamalarla bu sorun da ortadan kalkmaktadır. Donanımların gelişmesi ile kısa sürede Java 3D'nin yaygın bir kullanım alanı bulacağı açıktır. • Kırpma : Nesnenin bir pencere içinde kalan alanları dışındaki kısımlarını ortadan kaldırma işlemi kolaylıkla yapılabilmektedir. • “Antialiasing” : Çizimlerdeki basamaklı kenarları düzgünleştirme tekniğidir. • Saydamlık : Nesnelere geçirgenlik verilmesi üst üste gelen resim parçalarında daha gerçekçi görüntülerin oluşturulmasını sağlar. • Yazıcı : Ekrandaki resmin ve şekillerin yazıcı çıktısının alınmasını kolaylaştıran komut ve özellikler önemlidir. Bu uygulama geliştirme arayüzleri, kullanıldıkları programlama dilinin tüm özelliklerine grafik özellikleri de katmayı sağlamıştır. Ayrıca donanım ve yazılım sisteminin ve dilin grafik kullanıcı arayüzü özellikleri ile etkin pencereler ve pencere elemanları oluşturulabilmekte, fare ve klavye gibi cihazlarla başarılı bir etkileşim gerçekleştirilebilmektedir. gerçekleştirilebilmektedir. 2. 2. Üç Boyutlu Grafik API’leri Bunlar dışında VRML ve Open Inventor gibi birçok üç boyutlu grafik uygulama geliştirme arayüzü vardır. Yıllar boyunca pek çok üç boyutlu grafik standardı geliştirilmiş ve bunlara dayanan uygulama geliştirme arayüzleri kullanıma sunulmuştur. İlk grafik standardı 1977 yılında hazırlanıp, 1979 yılında düzeltilen "Core" grafik standardıdır. Bu tarihten günümüze kadar, 4 kuşak grafik uygulama geliştirme arayüzü (API) ortaya çıkmıştır (Sun Microsystems, 2001). Yüksek düzeyli üç boyutlu uygulama geliştirme arayüzlerinin sağladığı olanaklar ve özellikler şu şekilde belirtilebilir : • Üç boyutlu sanal geometrik nesneler tanımlamayı, sahneye eklemeyi, çıkarmayı ve gruplandırma işlemlerini kolaylaştıran altyapıyı sunmaktadır. Matematiksel veri tipleri, veri yapıları ve bunlar üzerinde işlem yapacak çeşitli metotlar tanımlıdır. Programcılar kendi geometrik şekillerini tanımlayabilmek için indeks tabanlı olan veya olmayan geometrileri kullanabilirler. Birinci kuşak 3B grafik API’lerinden olan “Core”’dan sonra, İkinci kuşak API’lerden PHIGS ortaya çıkmıştır. C gibi güçlü bir programlama dilinden çağrılması, iş istasyonlarında kullanılması ve katı nesnelerin sisteme eklenmiş olması önemli özelliklerindendir. OpenGL, kaliteli ve yüksek düzeyli grafikler oluşturulmasını sağlayan, iki ve üç boyutlu grafikleri destekleyen, işletim sisteminden bağımsız üçüncü kuşak bir grafik uygulama geliştirme arayüzüdür. Etkileşimli üç boyutlu grafik uygulamalarında gerekli olan yaklaşık 250 farklı komutu içerir. Birçok platformda çalışması, geniş ve iyi tanımlanmış özellikleri, hızı, sürekli gelişmesi ve ölçeklenebilir olması sayesinde oldukça yaygınlaşmıştır. Güvenilir ve kullanımı kolay bir y y g • Üç boyutlu içerik eklemeyi kolaylaştırırlar: • Üç boyutlu değişik grafik yazılımları ile oluşturulmuş dosya biçimlerindeki üç boyutlu nesnelerin eklenmesi (Day, 1999) • İki ve üç boyutlu yazılar eklenmesi • Üç boyutlu nesneler üzerinde değişik dönüşüm işlemleri ve değişik türlerde Ü • Üç boyutlu içerik eklemeyi kolaylaştırırlar: • Üç boyutlu değişik grafik yazılımları ile oluşturulmuş dosya biçimlerindeki üç boyutlu nesnelerin eklenmesi (Day, 1999) İki ü b tl l kl i • Üç boyutlu nesneler üzerinde değişik dönüşüm işlemleri ve değişik türlerde animasyonlar yaptırılmasını sağlarlar. Üç Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61 57 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur 2. 3. Üç Boyutlu Grafik Yazılımları Geliştirmede 2B ve 3B API’lerin Karşılaştırılması 3B grafik yazılımları geliştirmede 3B API’ler hazır olanaklar sağlamaktadır. Buna karşın, 3B yazılımların geliştirilmesinde, 2B API’lerin kullanımı ileri derecede matematik ve grafik bilgisi gerektirmektedir. İki boyutlu ilkeller yardımı ile üç boyutlu nesnelerin oluşturulması, oluşturulan nesneler için veri yapılarının tasarlanması, izdüşüm yöntemlerinin uygulanması, görünen yüzeylerin belirlenmesi, aydınlatma ve ışık kaynaklarının durumuna göre nesnelerin yüzeylerindeki renklerin değerlerinin hesaplanması, yapılması gereken işlemlerden sadece birkaçıdır (Watt, 2000). Üç boyutlu grafik yazılımları geliştirmek bu nedenle yazılım geliştiriciler için zor olmuştur. Donanım ve yazılımlardaki gelişmeler sonucunda üç boyutlu uygulama geliştirme arayüzlerinin kişisel bilgisayar ortamına taşınması grafik yazılımları geliştirme sürecini hızlandıracak ve kolaylaştıracaktır. Bu nedenle bu çalışma kapsamında aynı işleve sahip biri 2B API biri de 3B API kullanılarak iki program geliştirilmiştir. Kod satır sayıları dikkate alınarak karşılaştırmalar yapılmıştır. Bu çalışma kapsamında aynı işlevlere sahip fakat farklı arayüzlerle geliştirilen iki yazılım kod satır sayılarına göre karşılaştırılmışlardır. Yazılımların değişik bölümlerinin de satır sayıları dikkate alınmıştır. Doğrudan ölçütlerden olan kod satır sayılarının ölçülerek karşılaştırılması ile, grafik yazılımları geliştirme sürecindeki hızlanma ve yazılım geliştirmede harcanan çaba ve maliyetlerdeki azalma da ölçülmüş olmaktadır. 3. KUP3B YAZILIMI boyutlu nesneler üzerinde özellikle fare ve klavye gibi çevre aygıtları yardımı ile etkileşimli bir şekilde işlemler yapılabilmesini sağlayan olanaklar vardır. boyutlu nesneler üzerinde özellikle fare ve klavye gibi çevre aygıtları yardımı ile etkileşimli bir şekilde işlemler yapılabilmesini sağlayan olanaklar vardır. Bu çalışma kapsamında geliştirilen ve internet üzerine yerleştirilen Kup3B yazılımı (web tarayıcıları ile “bornova.ege.edu.tr/~ugur” internet adresinden ulaşılarak çalıştırılabilmektedir) üç temel işleve sahiptir : Bu çalışma kapsamında geliştirilen ve internet üzerine yerleştirilen Kup3B yazılımı (web tarayıcıları ile “bornova.ege.edu.tr/~ugur” internet adresinden ulaşılarak çalıştırılabilmektedir) üç temel işleve sahiptir : • Üç boyutlu nesnelerin niteliklerinin kolaylıkla belirlenmesini ve değiştirilmesini sağlarlar. Geniş bir nesne nitelikleri kütüphanesine sahiptirler. • Nesneler üzerine değişik türlerde desenler kaplanmasını kolaylaştırırlar. • Bir küp üzerinde kullanıcının temel dönüşüm işlemlerini (taşıma, döndürme ve ölçeklendirme) yapabilmesini sağlamak, • Değişik türlerde ışık kaynaklarının tanımlanmasını ve aydınlatma ve sis gibi etkilerin verilmesini kolaylaştırırlar. y p • Paralel/perspektif izdüşüm arasında geçiş yapılabilmesini sağlamak, • Kameraya yani bakış noktasına ilişkin işlemlerin yapılmasını kolaylaştırırlar. Üç boyutlu değişik görüntüleme olanakları (paralel, perspektif) gibi esnek kamera özelliklerini sunarlar. • Katı/telkafes görünüm arasında geçiş yapılabilmesini sağlamak. Yazılım ölçümü, yazılımın kalitesini anlamak ve yazılım geliştiricilerin verimliliğini belirlemek gibi amaçlarla yapılır (Sommerville, 2000). Yazılım ölçütleri fiziksel ölçütler gibi iki çeşittir : Doğrudan ölçüler ve dolaylı ölçüler. Boyut uyarlı ölçütler, yazılım mühendisliği çıktısının ve kalitesinin doğrudan ölçülmesinde kullanılır. Fonksiyon uyarlı ölçütler, dolaylı ölçütlerdir. İnsan uyarlı ölçütler, yazılım geliştiren kişilerle ilgilidir. Yazılım mühendisliği işlemindeki doğrudan ölçüler, maliyet ve harcanan çabadır. Ürünün doğrudan ölçüleri arasında, üretilen kaynak kodun satır sayısı, işletim hızı, belirli bir süre içinde tespit edilen hata sayısı sayılabilir. Ürünün dolaylı ölçüleri, fonksiyonelliği, kaliteyi, karmaşıklığı, etkinliği, güvenilirliği ve diğer özellikleri içerir. Verimlilik ölçütleri, yazılım mühendisliği işleminin çıktısına odaklanır, kalite ölçütleri, müşteri gereksinimlerinin (yazılımın kullanılabilirliği) uygunluğunun göstergesidir. Teknik ölçütler ise yazılımın mantıksal karmaşıklığı gibi yazılımın karakterine odaklanır. • Üç boyutlu grafik ve animasyonlara ses özellikleri eklemeyi kolaylaştırırlar. • Hazır geometri sıkıştırma özellikleri ile etkinlik sağlarlar. • Güvenlik ve güvenilirliği sağlayıcı özellikleri mevcuttur. • Altyapıları, grafiklerin oluşturulmasında ve diğer işlemlerde olabilecek en iyi hızı sunmaya çalışmaktadır. 2. 3. Üç Boyutlu Grafik Yazılımları Geliştirmede 2B ve 3B API’lerin Karşılaştırılması 2. 3. Üç Boyutlu Grafik Yazılımları Geliştirmede 2B ve 3B API’lerin Karşılaştırılması 4. SONUÇLAR Benzeri işlevlere sahip olan ve bu çalışma kapsamında Java 2D ve Java 3D ile geliştirilen iki yazılımın bölümlerine göre kod satır sayıları Tablo 1’de verilmiştir. Tablodaki bölümlerde dikkate alınan etkinlikler aşağıdaki gibidir; Şekil 1. Kup3B yazılımı (Java 2D sürümü) applet penceresi açılış görünümü Küpü üç boyutlu uzayda x,y,z eksenleri etrafında döndürmek için yön tuşları ile PageUp ve PageDown tuşları veya düğme panelindeki üç tane (üç eksen için) Döndür düğmesi kullanılır. • Arayüz : Ekran pencerelerinin hazırlanması, düğmeler, metin kutuları ve diğer pencere bileşenlerinin oluşturularak eklenmesi, bu tür bileşenler ile, klavye ve fare ile etkileşim için oluşacak işlem kodları Küpü üç boyutlu uzayda x,y,z eksenlerinde taşımak için yön tuşları ile PageUp ve PageDown tuşları (Ctrl tuşu basılı iken) veya düğme panelindeki üç tane (üç eksen için) Taşı düğmesi kullanılır. • Modelleme : Veri yapılarının oluşturulması, sahnenin ve üç boyutlu nesnelerin oluşturulması ve veri yapılarına yerleştirilmesi Küpü üç boyutlu uzayda x,y,z eksenlerinde ölçeklendirmek için yön tuşları ile PageUp ve PageDown tuşları (Shift tuşu basılı iken) veya düğme panelindeki üç tane (üç eksen için) Büyüt düğmesi kullanılır. • Dönüşüm : Matris çarpım metotları, taşıma, ölçeklendirme ve döndürme gibi dönüşüm işlemlerini gerçekleştiren metotlar ile dönüşüm grupları ile ilgili işlemler • Görüntüleme : Üç boyutlu nesnelerin veri yapılarından alınarak görüntülenmesi için yapılan tanımlama ve işlem kodları Paralel/Perspektif izdüşüm dönüşümü ve Katı/Telkafes görüntü dönüşümü, paneldeki düğmeler yardımı ile yapılır. • Diğer : Genel açıklamalar, standart programlama dili deyimleri, genel amaçlı veya sınıflandırılamayan deyim satırları. • Diğer : Genel açıklamalar, standart programlama dili deyimleri, genel amaçlı veya sınıflandırılamayan deyim satırları. 3. 1. Kup3B (Java2D Sürümü) Java 2D kullanılarak geliştirilen Kup3B programında dönüşüm ve geçiş işlemleri paneldeki düğmeler veya klavye tuşları yardımı ile yaptırılabilmektedir (Şekil 1). Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61 58 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur Şekil 1. Kup3B yazılımı (Java 2D sürümü) applet penceresi açılış görünümü Farenin sağ tuşuna basılarak x ve y eksenlerinde taşıma yapılabilmektedir. Alt tuşu ve farenin sol tuşu basılı iken fare yukarı/aşağı hareket ettirilerek z ekseninde taşıma gerçekleştirilmektedir. Fare, sol tuşu basılı iken değişik yönlerde hareket ettirilerek küp x, y ve z eksenleri etrafında döndürülebilmektedir. Journal of Engineering Sciences 2003 9 (1) 55-61 3. 2. Kup3B (Java3D Sürümü) Java 3D kullanılarak geliştirilen Kup3B programında dönüşüm işlemleri fare yardımı ile, geçiş işlemleri paneldeki tuşlar yardımı ile yapılabilmektedir (Şekil 2). Tablo 1. Kup3B Yazılımlarının Bölümlerine Göre Kaynak Kodlarındaki Satır Sayıları Tablo 1. Kup3B Yazılımlarının Bölümlerine Göre Kaynak Kodlarındaki Satır Sayıları Tablo 1. Kup3B Yazılımlarının Bölümlerine Göre Kaynak Kodlarındaki Satır Sayıları Kup3B Yazılımının Bölümleri (Java 2D Sürümü) Bölüm Satır Sayısı Arayüz 220 Modelleme 38 Dönüşüm 65 Görüntüleme 82 Diğer 44 Toplam 449 Kup3B Yazılımının Bölümleri (Java 3D Sürümü) Bölüm Satır Sayısı Arayüz 42 Modelleme 34 Dönüşüm 9 Görüntüleme 15 Diğer 18 Toplam 118 Şekil 2. Kup3B yazılımı (Java 3D sürümü) applet penceresi açılış görünümü Şekil 2. Kup3B yazılımı (Java 3D sürümü) applet penceresi açılış görünümü Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur Yazılımda tek nesne olduğu için, modelleme kısmında önemli bir kod azalması görülmemektedir. Şekil 3’te Java 2D, Şekil 4’te Java 3D sürümü için Kup3B kaynak kodundaki bölümlerin satır sayısı yüzdeleri gösterilmektedir. Şekil 3’te Java 2D, Şekil 4’te Java 3D sürümü için Kup3B kaynak kodundaki bölümlerin satır sayısı yüzdeleri gösterilmektedir. ğ ç kısmında önemli bir kod azalması görülmemektedir. 0 50 100 150 200 250 Arayüz Modelleme Dönüşüm Görüntüleme Diğer Java 2D Java 3D 0 100 200 300 400 500 Toplam Java 2D Java 3D Şekil 5. Java 3D kullanımının, geliştirilen Kup3B yazılımının (bölümlerine göre) kaynak kod satır sayısında sağladığı azalmayı gösteren grafik. 0 50 100 150 200 250 Arayüz Modelleme Dönüşüm Görüntüleme Diğer Java 2D Java 3D 0 100 200 300 400 500 Toplam Java 2D Java 3D KÜP ÜZERİNDE DÖNÜŞÜM İŞLEMLERİNİ GERÇEKLEŞTİREN YAZILIM (JAVA 2D YARDIMI İLE HAZIRLANAN) Arayüz 50% Modelleme 8% Dönüşüm 14% Görüntüleme 18% Diğer 10% Şekil 3. Küp3B yazılımı kaynak kodundaki bölümlerin satır sayısı yüzdeleri KÜP ÜZERİNDE DÖNÜŞÜM İŞLEMLERİNİ GERÇEKLEŞTİREN YAZILIM (JAVA 2D YARDIMI İLE HAZIRLANAN) Arayüz 50% Modelleme 8% Dönüşüm 14% Görüntüleme 18% Diğer 10% Şekil 3. Küp3B yazılımı kaynak kodundaki bölümlerin satır sayısı yüzdeleri KÜP ÜZERİNDE DÖNÜŞÜM İŞLEMLERİNİ GERÇEKLEŞTİREN YAZILIM (JAVA 3D YARDIMI İLE HAZIRLANAN) Arayüz 35% Modelleme 29% Dönüşüm 8% Görüntüleme 13% Diğer 15% Şekil 4. Küp3B yazılımı kaynak kodundaki bölümlerin satır sayısı yüzdeleri Şekil 5. Java 3D kullanımının, geliştirilen Kup3B yazılımının (bölümlerine göre) kaynak kod satır sayısında sağladığı azalmayı gösteren grafik. Tüm yazılımlarda verimin bu derece artması beklenemez. Bunu etkileyen faktörler şu şekilde sayılabilir: • Geliştirilecek yazılımın doğası, verimliliği ve yazılım geliştirme süresini etkileyecektir. Day, B. 1999. 3D Graphics Programming in Java : Part 2, Advanced Java 3D, Javaworld Ocak 1999. oluşturulabilmesini sağlamakta ve birçok 3B basit yazılım için yeterli olmaktadır. Üç boyutlu arayüzlerin ayrıca dışarıdan hazır üç boyutlu model yüklenebilmesini sağlama, üç boyutlu geometrik içerik oluşturmayı sağlama ve üç boyutlu metin oluşturma gibi önceki bölümlerde bahsedilen birçok özellikleri de vardır. Knudsen, J. 1999. Java 2D Graphics, O’Reilly, USA, 339 p. Sommerville, I. 2000. Software Engineering, 6th Edition, Addison-Wesley, USA, 693 p. Sonuçta yüksek düzeyli 3B API’ler üç boyutlu grafik yazılım geliştirme sürecini hızlandırmakta ve kolaylaştırmaktadır. Bu da, geliştirilen yazılımlardaki kaliteyi arttırmakta, maliyetleri düşürmekte ve yazılımların sürümlerinin gecikmesinden doğabilecek sorunları ortadan kaldırarak yeni fırsatlar oluşmasını sağlamaktadır. Sowizral, H. A. and Nadeau, D. R. 1999. Introduction to Programming With Java 3D, San Diego Supercomputer Center, University of California at San Diego, SIGGRAPH99 Course Notes, 603 p. Sun Microsystems, Inc., Java 3D 1.2 API Specification, www.javasoft.com/products/java- media/3D/index.html Sun Microsystems, Inc., Java 3D 1.2 API Specification, www.javasoft.com/products/java- media/3D/index.html 3. 2. Kup3B (Java3D Sürümü) Bazı grafik yazılımları doğrudan grafik, modelleme, boyama, çizim gibi işlemleri yaptıran yazılımlar olabildiği gibi, bazı yazılımlar da diğer alanlarda olup içinde grafiklerin kullanımını içeren yazılımlardır. Yazılımın içeriğine ve amacına göre bu oran değişecektir. Şekil 4. Küp3B yazılımı kaynak kodundaki bölümlerin satır sayısı yüzdeleri Aynı işlevlere sahip grafiksel yazılımların geliştirilmesinde, yazılan kod satır sayıları dikkate alındığında bu çalışma için 449/118 = 3.8 oranı ortaya çıkmaktadır. Başka bir deyişle, grafik yazılımları geliştirmek için harcanan çaba yüksek düzeyli üç boyutlu bir uygulama geliştirme arayüzünden yararlanıldığında % 74 azalarak, % 26’sına inmiştir (Şekil 5). İki boyutlu uygulama geliştirme arayüzleri yerine üç boyutluların tercih edilmesinin getireceği bu yarar maliyetlere ve yazılım geliştirme süresine de doğrudan yansıyacaktır. • Yazılımın karmaşıklık düzeyi ve grafiklerdeki gerçekçiliğin düzeyi de önemli bir faktördür. Geliştirilecek yazılımın grafiksel gerçekçilik beklentisi de bu oranı değiştirecektir. • Yüksek düzeyli 3B API’lerin ileri işlevlerinin kullanımının öğrenilmesi zordur. Yazılım geliştiricilerin bilgi ve deneyim farklılıkları harcanan çaba, yazılım geliştirme süresi ve maliyetlerdeki bu oranı etkileyecektir. Fare etkileşimi, temel nesneler ve üzerlerindeki işlemler ile görüntüleme, Java 3D API ile hazır geldiğinden, arayüz ve görüntüleme için yazılan kaynak kodu satır sayıları oldukça büyük oranlarda azalmıştır (Şekil 5). Çalışmadan elde edilen sonuçlara göre, üç boyutlu API kullanımı arayüz oluşturmak için harcanan çabayı % 81, görüntüleme için gereken çabayı % 82 ve dönüşüm işlemleri için harcanan çabayı yaklaşık % 86 azaltmıştır. Üç boyutlu nesneler için fare etkileşim özelliklerinin doğrudan gelmesi bu amaçla yazılması gereken kod satır sayısını büyük ölçüde azalttığı gibi, tüm yazılımlarda belli bir standardı da sağlamakta ve yazılım kalitesini de arttırmaktadır. Temel nesnelerin (küp, küre, koni ve silindir gibi) desteklenmesi, birçok basit sahnenin Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61 60 Yeni Grafik Uygulama Geliştirme Arayüzlerinin Karşılaştırılması, M. Türksever, A. Uğur Day, B. 1999. 3D Graphics Programming in Java : Part 2, Advanced Java 3D, Javaworld Ocak 1999. Knudsen, J. 1999. Java 2D Graphics, O’Reilly, USA, 339 p. Day, B. 1999. 3D Graphics Programming in Java : Part 2, Advanced Java 3D, Javaworld Ocak 1999. 5. KAYNAKLAR 5. KAYNAKLAR Watt, A. 2000. 3D Computer Graphics, Third Edition, Addison-Wesley, 570 p. Watt, A. 2000. 3D Computer Graphics, Third Edition, Addison-Wesley, 570 p. Day, B. 1998. 3D Graphics Programming in Java : Part 1, Java 3D, Javaworld. Aralık 1998. Mühendislik Bilimleri Dergisi 2003 9 (1) 55-61 Journal of Engineering Sciences 2003 9 (1) 55-61
https://openalex.org/W4300936509	https://www.siboncoj.ru/jour/article/download/154/156	Russian	null	SINGLE NUCLEOTIDE POLYMORPHISMS IN BREAST TUMOR AND EXPRESSION OF ABC-TRANSPORTERS AFTER NEOADJUVANT CHEMOTHERAPY	Tomsk National Research Medical Сепtеr of the Russian Academy of Sciences - Siberian Journal of Oncology	2,016	cc-by	7,119	УДК: 618.19-006:615.28:575.113 УДК: 618.19-006:615.28:575.113 М.М. Цыганов1,2, М.К. Ибрагимова1,2, Е.М. Слонимская1, Н.В. Чердынцева1,2, Н.В. Литвяков1,2 Томский НИИ онкологии, г. Томск1 Национальный исследовательский Томский государственный университет, г. Томск2 634009, г. Томск, пер. Кооперативный, 5, e-mail: tsyganovmm@yandex.ru1 Томский НИИ онкологии, г. Томск1 Национальный исследовательский Томский государственный университет, г. Томск2 634009, г. Томск, пер. Кооперативный, 5, e-mail: tsyganovmm@yandex.ru1 Аннотация В настоящее время практически не изученным остается механизм регуляции экспрессии генов АВС- транспортеров, который связан с индивидуальными особенностями организма опухоленосителя и его опухоли, определяемыми генным однонуклеотидным полиморфизмом (SNP – Single Nucleotide Poly- morphism). Проведено изучение ассоциации SNP в широкогеномном масштабе с уровнем экспрессии генов АВС-транспортеров после неоадъювантной химиотерапии (НХТ) у 68 больных с морфологически верифицированным раком молочной железы (РМЖ). При помощи количественной ПЦР с обратной транскрипцией в режиме реального времени изучена экспрессия 4 генов АВС: ABCB1, ABCC1, ABCC2, ABCG2 в операционном материале после НХТ. Для исследования SNP проводили микроматричный анализ на ДНК-чипах высокой плотности, которые содержат более 750 тыс. однонуклеотидных по- лиморфизмов. В результате биоинформатического анализа было идентифицировано 6 SNP, которые статистически значимо связаны с послеоперационным уровнем экспрессии генов АВС: ABCB1, ABCC1, ABCC2 и ABCG2. Показано, что у носителей редкого генотипа уровень экспрессии всех 4 исследо- ванных генов в опухоли после воздействия химиопрепаратов либо снижен (rs2680835, rs1951366 и rs12018988), либо повышен (rs4676478, rs6896596, rs1154121) по сравнению с носителями частого и гетерозиготного генотипов этих SNP. Обсуждены возможные механизмы влияния выявленных SNP и их генов на экспрессию АВС-транспортеров. Ключевые слова: АВС-транспортеры, рак молочной железы, однонуклеотидный полиморфизм, микроматричное исследование. Основной причиной неэффективности химио- терапии считают формирование фенотипа мно- жественной лекарственной устойчивости (МЛУ) опухоли за счет экспрессии энергозависимых бел- ков АВС-транспортеров (ABCB1, ABCB3, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2), выбрасывающих лекарственные препараты из опухолевых клеток против градиента концентрации с затратой энер- гии АТФ [9, 11]. Наши предыдущие исследования показали, что эффективность неоадъювантной хи- миотерапии (НХТ) рака молочной железы (РМЖ) связана не с исходным уровнем экспрессии АВС- транспортеров в опухоли, а с его изменением в про- цессе лечения. Если в процессе НХТ экспрессия генов АВС в опухоли снижается, то наблюдается клинический ответ на химиотерапию, в противном случае формируется фенотип множественной ле- карственной устойчивости, ответ на химиотерапию отсутствует и значительно увеличено отдаленное метастазирование [1–3, 21]. связан с индивидуальными особенностями организ- ма опухоленосителя и его опухоли, определяемыми генным однонуклеотидным полиморфизмом (SNP – Single Nucleotide Polymorphism). Известны только немногие SNP, которые ассоциированы с уровнем экспрессии АВС-транспортеров. Прежде всего, это полиморфизм самих генов АВС, которые имеют зна- чение для экспрессии генов АВС и работы транспор- теров: ABCG2 R482T или R482G, ABCB1 rs1045642, ABCC1 rs35605, GSTP1 rs1695, ABCG2 rs2725264, ABCC2 (rs1885301, rs717620 и rs3740066) и др. [5, 6, 13–15, 25–27, 29, 30]. По данным ряда авторов, экспрессия генов АВС связана и с отдельными полиморфизмами других генов [7, 9, 32]. ОДНОНУКЛЕОТИДНЫЙ ПОЛИМОРФИЗМ ОПУХОЛИ МОЛОЧНОЙ ЖЕЛЕЗЫ И ЭКСПРЕССИЯ АВС-ТРАНСПОРТЕРОВ ПОСЛЕ НЕОАДЪЮВАНТНОЙ ХИМИОТЕРАПИИ М.М. Цыганов1,2, М.К. Ибрагимова1,2, Е.М. Слонимская1, Н.В. Чердынцева1,2, Н.В. Литвяков1,2 Цыганов Матвей Михайлович, TsyganovMM@yandex.ru СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2015. № 5. С. 59–66 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2015. № 5. С. 59–66 ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ генов АВС и их регуляцией и делает актуальными исследования по выявлению генов и полиморфных локусов, участвующих в регуляции экспрессии АВС-транспортеров и формировании лекарствен- ной устойчивости опухоли. Следует отметить, что исследования связи генного полиморфизма с экспрессией генов АВС проводятся в мире в узком формате. В одном исследовании изучаются не более 100 полиморфизмов, что не позволяет в полной мере выявить индивидуальные генетиче- ские особенности организма и опухоли, связанные с экспрессией генов АВС-транспортеров. Работы по изучению связи SNP c изменением экспрессии генов АВС в процессе химиотерапии отсутствуют, а, как нами было установлено, именно изменение экспрессии АВС-транспортеров определяет эффек- тивность химиотерапии. геномном масштабе с уровнем экспрессии генов АВС-транспортеров после НХТ. Аннотация Наши предыдущие исследования позволили установить 5 SNP генов репарации, апоптоза и фолатного цикла (TP53 rs1042522, TP53 rs8073498, MTH- FR rs1801133, FGFR2 rs2981582 и ATM1 rs664143), которые связаны с регуляцией отдельных генов АВС [4]. связан с индивидуальными особенностями организ- ма опухоленосителя и его опухоли, определяемыми генным однонуклеотидным полиморфизмом (SNP – Single Nucleotide Polymorphism). Известны только немногие SNP, которые ассоциированы с уровнем экспрессии АВС-транспортеров. Прежде всего, это полиморфизм самих генов АВС, которые имеют зна- чение для экспрессии генов АВС и работы транспор- теров: ABCG2 R482T или R482G, ABCB1 rs1045642, ABCC1 rs35605, GSTP1 rs1695, ABCG2 rs2725264, ABCC2 (rs1885301, rs717620 и rs3740066) и др. [5, 6, 13–15, 25–27, 29, 30]. По данным ряда авторов, экспрессия генов АВС связана и с отдельными полиморфизмами других генов [7, 9, 32]. Наши предыдущие исследования позволили установить 5 SNP генов репарации, апоптоза и фолатного цикла (TP53 rs1042522, TP53 rs8073498, MTH- FR rs1801133, FGFR2 rs2981582 и ATM1 rs664143), которые связаны с регуляцией отдельных генов АВС [4]. Таким образом, анализ литературы свидетель- ствует о связи полиморфизма с уровнем экспрессии Практически не изученным остается механизм регуляции экспрессии АВС-транспортеров, который Цыганов Матвей Михайлович, TsyganovMM@yandex.ru Цыганов Матвей Михайлович, TsyganovMM@yandex.ru 59 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2015. № 5. С. 59–66 SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66 Результаты исследования и обсуждение Д РНК выделяли из 68 образцов после НХТ с помощью набора RNeasy Plus mini Kit (Qiagen, Germany) в соответствии с инструкцией произ- водителя. Качество и целостность РНК оценива- лись при помощи капиллярного электрофореза на приборе TapeStation (Agilent Technologies, USA). Показатель RIN составил 5,6–7,8. Для получе- ния кДНК на матрице РНК проводили реакцию обратной транскрипции с помощью набора Re- vertAid™ (Fermentas, Lithuania) со случайными гексануклеотидными праймерами в соответствии с инструкцией к набору. Остальные манипуляции, последовательность праймеров и методика оценки относительной экспрессии генов АВС описаны ра- нее [21]. В качестве результата оценивался уровень экспрессии исследуемых генов АВС: ABCB1, AB- CC1, ABCC2 и ABCG2 относительно гена-рефери GAPDH и нормальной ткани молочной железы, вычисляемый по методу Pfaffl [24] в операционном материале после НХТ. Была проанализирована связь экспрессии генов АВС-транспортеров после НХТ с SNP, которые определялись в опухоли до лечения при помощи микроматриц. Поскольку в предыдущем исследо- вании наиболее сильная коэкспрессия в процессе НХТ была показана для генов ABCB1, ABCC1, ABCC2 и ABCG2, то анализировали связь SNP с экспрессией именно этих четырех генов. Всего в биоинформатический анализ было включено 749 158 SNP на каждого пациента. В процессе ана- лиза происходила фильтрация первичных данных. В первую очередь из представленного массива дан- ных (749 158 SNP) были исключены мономорфные полиморфизмы, которые имели только один (ди- кий) или два (дикий и гетерозиготный) генотипа. Далее из анализа исключались SNP с частотами генотипов, не соответствующими ожидаемым при соблюдении равновесия Харди – Вайнберга, по- скольку в противном случае выборка по SNP оказы- вается подразделенной и ее нельзя рассматривать как единую выборку. Следующий фильтр был подобран, исходя из выбора в качестве генотипиче- ской модели – рецессивной генотипической модели (редкий генотип против гетерозиготного + частого генотипов), которая была определена согласно ин- формационному критерию Акаике. Эмпирически была подобрана необходимость наличия частоты редкого генотипа не менее 8 %. Все SNP с частотой редкого генотипа исключались из анализа. В итоге для анализа было оставлено 258 586 SNP. Для этих SNP был произведен расчёт уровня статистической значимости различий экспрессии по каждому из четырех генов АВС после НХТ – редкий генотип против гетерозиготного+частого генотипов по лог- линейной регрессии. Далее были выбраны общие для генов ABCB1, ABCC1, ABCC2 и ABCG2 SNP. В результате идентифицировано 6 полиморфиз- мов (EPHA4 (rs2680835), SCN10A (rs4676478), H2AFY (rs6896596), SLC25A21 (rs1154121), RTN1 (rs1951366), RTN1 (rs12018988)), связанных с по- слеоперационным уровнем экспрессии четырех генов АВС (ABCB1, ABCC1, ABCC2 и ABCG2) одновременно (рис. 1). Материал и методы В В исследование включены 68 больных РМЖ IIA–IIIC стадий, с морфологически верифициро- ванным диагнозом, в возрасте 28–68 лет, в среднем 53,43 ± 0,78 года (табл. 1). В соответствии с «Con- sensus Conference on Neoadjuvant Chemotherapy in Carcinoma of the Breast, April 26–28, 2003, Philadel- phia, Pennsylvania» [28] все больные получали 2–4 курса неоадъювантной химиотерапии по схемам FAC (фторурацил, доксорубицин, циклофосфан), CAX (циклофосфан, доксорубицин, кселода) или монотерапию таксотером. Через 3–5 нед после НХТ выполнялась операция, затем проводили 2 курса адъювантной химиотерапии по схеме FAC, лучевая терапия и/или гормональное лечение на- значались по показаниям. Исследование проводи- Целью исследования явилось изучение ассо- циации SNP опухоли молочной железы в широко- Таблица 1 Клинико-патологические параметры обследованных больных РМЖ Клинико-патологический параметр Число больных Возраст (лет) ≤45 21 (30,9 %) >45 47 (69,1 %) Менструальный статус Пременопауза 36 (52,9 %) Постменопауза 32 (47,1 %) Гистологический тип Инвазивный протоковый рак 58 (85,3 %) Инвазивный дольковый рак 3 (4,4 %) Медулярный рак 2 (2,9 %) Другие типы 5 (7,4 %) Размер опухоли T1 9 (13,2 %) T2 52 (76,5 %) T3 3 (4,4 %) T4 4 (5,9 %) Лимфогенное метастазирование N0 27 (39,7 %) N1 31 (45,6 %) N2 4 (5,9 %) N3 6 (8,8 %) Рецепторы эстрогена + 33 (48,5 %) – 31 (42,6 %) Нет данных 4 (5,9 %) Рецепторы прогестерона + 35 (51,5 %) – 29 (42,26 %) Нет данных 4 (5,9 %) Рецепторы эпидермального фактора роста HER2 0/+ 47 (69,1 %) ++ 10 (14,7 %) +++ 6 (8,8 %) Нет данных 5 (7,4 %) Молекулярный подтип Люминальный В 40 (59,7 %) Трижды негативный 17 (25,4 %) HER2-позитивный 10 (14,9 %) Гистологическая форма Уницентрическая 45 (66,2 %) Мультицентрическая 23 (33,8 %) Степень злокачественности 1 степень 2 (2,9 %) 2 степень 48 (70,6 %) 3 степень 6 (8,8 %) Нет данных 12 (17,6 %) Схема НАХТ CAX 21 (30,9 %) FAC 33 (48,5 %) Таксотер 14 (20,6 %) Клинико-патологические параметры обследованных больных РМЖ 60 ОДНОНУКЛЕОТИДНЫЙ ПОЛИМОРФИЗМ ОПУХОЛИ МЖ М.М. Цыганов, М.К. Ибрагимова, Е.М. Слонимская и др. М.М. Цыганов, М.К. Ибрагимова, Е.М. Слонимская и др. лось в соответствии с Хельсинкской декларацией 1964 г. (доработанной в 1975 и 1983 гг.), получено разрешение локального этического комитета Томского НИИ онкологии. Были использованы биопсийные опухолевые образцы (~10 мм3), взятые до лечения под контролем УЗИ, а также операци- онный материал (~60–70 мм3). Материал и методы В Образцы опухоли помещали в раствор RNAlater (Ambion, USA) и со- храняли при температуре –80°С (после 24-часовой инкубации при +4°С) для дальнейшего выделения РНК и ДНК. с использованием языка программирования «R» в программе R version 3.0.2. Программа предна- значена для статистической обработки большого массива данных и содержит общий пакет стати- стического анализа. Для биоинформатического анализа специально был прописан скрипт (по- следовательность команд), содержащий в себе команды форматирования данных и расчета. Для коррекции уровней значимости на множественные сравнения использовалась поправка Бонферрони. лось в соответствии с Хельсинкской декларацией 1964 г. (доработанной в 1975 и 1983 гг.), получено разрешение локального этического комитета Томского НИИ онкологии. Были использованы биопсийные опухолевые образцы (~10 мм3), взятые до лечения под контролем УЗИ, а также операци- онный материал (~60–70 мм3). Образцы опухоли помещали в раствор RNAlater (Ambion, USA) и со- храняли при температуре –80°С (после 24-часовой инкубации при +4°С) для дальнейшего выделения РНК и ДНК. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Рис. 1. Уровень статистической значимости связи SNP с экспрессией генов ABCB1, ABCC1, ABCC2 и ABCG2 после НХТ. Примечание: по оси ординат – значение десятичного логарифма уровня статистической значимости (p-level); по оси абсцисс – однонуклеотидные полиморфизмы (n = 258586 SNP). На рисунке пунктирной линией обозначено значение номинального p-level, равного 0,05, сплошной линией – значение p-level с учетом поправки Бонферрони Рис. 1. Уровень статистической значимости связи SNP с экспрессией генов ABCB1, ABCC1, ABCC2 и ABCG2 после НХТ. Примечание: по оси ординат – значение десятичного логарифма уровня статистической значимости (p-level); по оси абсцисс – однонуклеотидные полиморфизмы (n = 258586 SNP). На рисунке пунктирной линией обозначено значение номинального p-level, равного 0,05, сплошной линией – значение p-level с учетом поправки Бонферрони RTN1 может взаимодействовать с белком спастин (SPAST) [22], который, в свою очередь, опосредо- ванно влияет на экспрессию белка тубулина, что делает опухолевые клетки чувствительными к воздействию химиопрепаратов [8]. значимо ниже, чем у носителей гетерозиготного и дикого генотипов (p-level < 0,01 с учетом по- правки Бонферрони) (табл. 2). Для полиморфизмов SCN10A (rs4676478), H2AFY (rs6896596), SLC25A21 (rs1154121) было показано, что у больных с гете- розиготным и диким генотипом уровень экспрес- сии после НХТ достоверно ниже по сравнению с редким генотипом, в среднем в 2 раза (p-level<0,01 с учетом поправки Бонферрони). Таким образом, однонуклеотидный полиморфизм опухолевых клеток может оказывать влияние на уровень экс- прессии комплекса генов АВС после воздействия химиопрепаратов. значимо ниже, чем у носителей гетерозиготного и дикого генотипов (p-level < 0,01 с учетом по- правки Бонферрони) (табл. 2). Для полиморфизмов SCN10A (rs4676478), H2AFY (rs6896596), SLC25A21 (rs1154121) было показано, что у больных с гете- розиготным и диким генотипом уровень экспрес- сии после НХТ достоверно ниже по сравнению с редким генотипом, в среднем в 2 раза (p-level<0,01 с учетом поправки Бонферрони). Таким образом, однонуклеотидный полиморфизм опухолевых клеток может оказывать влияние на уровень экс- прессии комплекса генов АВС после воздействия химиопрепаратов. Для полиморфизма SCN10A (rs4676478) в базе данных http://compbio.cs.queensu.ca/F-SNP/ пока- зана функция регуляции транскрипции. Сам ген SCN10A кодирует тетродотоксин-резистентный на- триевый канал, локализуется на мембране клетки. В недавней публикации показана роль SNP этого гена в индуцированной химиотерапией гастро- интестинальной токсичности [12], в реализации которой также играют роль и полиморфизмы генов АСВ [20]. р р Механизм, благодаря которому SNP может оказывать влияние на экспрессию генов АВС, еще предстоит выявить. В настоящее время на основании литературных данных можно только высказать некоторые предположения. Функция полиморфизма EPHA4 (rs2680835), согласно базе данных http://compbio.cs.queensu.ca/F-SNP/, неиз- вестна. По поводу функций гена EPHA4 имеется некоторая информация. Результаты исследования и обсуждение Экспрессия всех четырех исследованных генов р ДНК выделяли из 68 биопсийных образцов опухолевой ткани с помощью набора QIAamp DNA mini Kit (Qiagen, Germany). Концентрацию ДНК и чистоту выделения оценивали на спектрофотоме- тре NanoDrop-2000 (Thermo Scientific, USA) (от 50 до 150 нг/мкл, А260/А280 = 2,10–2,35; А260/ А230 = 2,15–2,40). Целостность ДНК оценива- лась при помощи капиллярного электрофореза на приборе TapeStation (Agilent Technologies, USA), фрагменты ДНК имели массу более 48 kbp. SNP в опухоли изучали при помощи микрома- тричного анализа на ДНК-чипах высокой плотно- сти фирмы Affymetrix (USA) CytoScanTM HD Array (http://www.affymetrix.com/esearch/search.jsp?pd= prod520004&N=4294967292), которые содержат более 750 тыс. однонуклеотидных полиморфиз- мов. Процедуры пробоподготовки, гибридизации и сканирования проводили в соответствии с про- токолом производителя на системе Affymetrix GeneChip® Scanner 3000 7G (Affymetrix, USA). Для обработки результатов микрочипирования исполь- зовали программу Chromosome Analysis Suite 2.0 (Affymetrix, USA), которая разработана специально для анализа результатов матрицы CytoScanTM HD Array. Определение SNP генов по rs осуществляли при помощи базы данных Affymetrix (https://www. affymetrix.com/). Ассоциацию уровня экспрессии генов АВС после НХТ с SNP в опухоли оценивали Экспрессия всех четырех исследованных генов АВС у носителей редкого генотипа полиморфиз- мов EPHA4 (rs2680835), RTN1 (rs1951366) и RTN1 (rs12018988) после проведения НХТ статистически 61 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2015. № 5. С. 59–66 SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66 ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ В недавних исследованиях было показано, что целенаправленное ингибиро- вание экспрессии генов группы EPHA вызывает устойчивость опухоли молочной железы к трасту- зумабу [34], но при этом делает опухолевые клетки чувствительными к тамоксифену [10]. Учитывая то, что выведение тамоксифена из опухолевых кле- ток осуществляется двумя генами лекарственной устойчивости ABCB1 и ABCC2 [31], можно предпо- ложить, что мутантный генотип EPHA4 (rs2680835) ингибирует экспрессию этого гена. Согласно базе http://compbio.cs.queensu.ca/F- SNP/, полиморфизм гена гистонового белка H2AFY (rs6896596) находится в downstream регионе и участвует в регуляции транскрипции гена H2AFY, который регулирует HDAC (Histone Deacetylase) диацетилазу гистонов (http://www.genecards.org/cgi- bin/carddisp.pl?gene=H2AFY&keywords=H2AFY). Также недавно было показано, что HDAC1 и 2 ингибируют экспрессию генов ABCB1 и ABCC2 их продуктов в клеточных культурах colorectal adenocarcinoma и carcinoma [33]. В 2012 г. J. Radosław et al. провели микрома- тричное исследование экспрессии генов АВС- транспортеров и генов семейства транспортеров SLC. Ими была изучена роль данных генов в фор- мировании лекарственной устойчивости к таким химиопрепаратам, как метотрексат, цисплатин, доксорубицин, винкристин, топотекан, а также паклитексел – при раке яичника [18]. Оказалось, что высокий уровень экспрессии генов SLC связан с устойчивостью опухолевых клеток к метотрек- Роль гена ретикулона 1 – RTN1 – в формировании лекарственной устойчивости или взаимодействии с генами АВС остается неясной. В единичных исследованиях было показано, что продукт гена SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66 62 М.М. Цыганов, М.К. Ибрагимова, Е.М. Слонимская и др. 8. Errico A., Claudiani P., D’Addio M., Rugarli E.I. Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon // Hum. Mol. Genet. 2004. Vol. 13 (18). P. 2121–2132. ( ) j p 10. Gökmen-Polar Y., Toroni R.A., Hocevar B.A., Badve S., Zhao Q., Shen C., Bruckheimer E., Kinch M.S., Miller K.D. Dual targeting of EphA2 and ER restores tamoxifen sensitivity in ER/EphA2-positive breast cancer // Breast Cancer Res. Treat. 2011. Vol. 127 (2). P. 375–384. doi: 10.1007/ s10549-010-1004-y. 12. He Y.J., Winham S.J., Hoskins J.M., Glass S., Paul J., Brown R., Motsinger-Reif A., McLeod H.L Carboplatin/taxane-induced gastroin- testinal toxicity: a pharmacogenomics study on the SCOTROC1 trial // Pharmacogenomics J. 2015. doi: 10.1038/tpj.2015.52. 9. Gillet J.P., Gottesman M.M. Overcoming multidrug resistance in cancer: 35 years after the discovery of ABCB1 // Drug Resistance Updates. 2012. Vol. 15 (1–2). P. 2–4. doi: 10.1016/j.drup.2012.03.001. 11. Gottesman M.M., Fojo T., Bates S.E. Multidrug resistance in cancer: role of ATP–dependent transporters // Nat. Rev. Cancer. 2002. Vol. 2 (1). P. 48–58. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ОДНОНУКЛЕОТИДНЫЙ ПОЛИМОРФИЗМ ОПУХОЛИ МЖ Таблица 2 Уровень экспрессии генов ABCB1, ABCC1, ABCC2 и ABCG2 в опухоли молочной железы после НХТ в зависимости от генотипа по выявленным полиморфизмам в рецессивной модели наследования SNP Генотип Уровень экспрессии после НХТ ABCB1 ABCC1 ABCC2 ABCG2 EPHA4 (rs2680835) TT/TC 4,53 ± 1,34 1,67 ± 0,38 4,26 ± 1,23 2,87 ± 0,58 CC 3,14 ± 1,02 0,80 ± 0,13 1,32 ± 0,54 1,61 ± 0,60 RTN1 (rs1951366) AA/ AG 5,16 ± 1,33 1,790 ± 0,37 4,40 ± 1,21 3,29 ± 0,59 GG 1,94 ± 0,70 0,59 ± 0,11 1,04 ± 0,42 0,73 ± 0,26 RTN1 (rs12018988) AA/ AG 4,88 ± 1,26 1,71 ± 0,35 4,16 ± 1,14 3,14 ± 0,56 GG 2,00 ± 0,73 0,62 ± 0,16 1,10 ± 0,44 0,67 ± 0,27 SCN10A (rs4676478) CC/TC 3,99 ± 1,03 1,22 ± 0,22 3,14 ± 0,93 2,28 ± 0,41 TT 6,26 ± 3,13 3,77 ± 1,91 6,20 ± 2,29 5,11 ± 2,67 H2AFY (rs6896596) AA/ GA 3,93 ± 1,04 1,22 ± 0,22 3,18 ± 0,95 2,31 ± 0,46 GG 6,69 ± 3,22 3,60 ± 2,02 6,20 ± 2,32 5,06 ± 1,83 SLC25A21 (rs1154121) CC/CT 3,79 ± 1,03 1,20 ± 0,22 3,02 ± 0,95 2,15 ± 0,41 TT 6,40 ± 2,71 2,96 ± 1,35 5,62 ± 1,84 4,78 ± 1,84 Таблица 2 Уровень экспрессии генов ABCB1, ABCC1, ABCC2 и ABCG2 в опухоли молочной железы после НХТ в зависимости от генотипа по выявленным полиморфизмам в рецессивной модели наследования У НХТ ровень экспрессии генов ABCB1, ABCC1, ABCC2 и ABCG2 в опухоли молочной железы зависимости от генотипа по выявленным полиморфизмам в рецессивной модели нас опухоли после воздействия химиопрепаратов либо снижен (EPHA4 (rs2680835), RTN1 (rs1951366) и RTN1 (rs12018988)), либо повышен (SCN10A (rs4676478), H2AFY (rs6896596), SLC25A21 (rs1154121)) по сравнению с носителями частого и гетерозиготного генотипов этих SNP. Обсуждены возможные механизмы влияния выявленных SNP и их генов на экспрессию АВС-транспортеров. сату, и, в частности, это было показано для гена SLC19A1. Низкий уровень экспрессии других генов SLC транспортеров обусловливал формирование лекарственной устойчивости опухоли к лекар- ственным препаратам, субстратам генов АВС [16, 17, 19, 23]. Заключение Работа поддержана грантом РФФИ № НК 14- 04-31633\15 «Экспрессия генов множественной лекар- ственной устойчивости и клональные циклы эволюции опухоли молочной железы в процессе неоадъювантной химиотерапии» и программой повышения конкурен- тоспособности Национального исследовательского Томского государственного университета. В результате проведенного широкогеномного исследования полиморфизмов опухоли молочной железы были идентифицированы 6 SNP, статисти- чески значимо связанных с уровнем экспрессии генов множественной лекарственной устойчивости ABCB1, ABCC1, ABCC2 и ABCG2 после НХТ. 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Тел.: 8 (3822) 51-46-07. E-mail: nvlitv72@yandex.ru. SPIN-код: 2546-0181 M.M. Tsyganov1,2, M.K. Ibragimova1,2, E.M. Slonimskaya1, N.V. Cherdyntseva1,2, N.V. Litviakov1,2 Tomsk Саnсеr Rеsеаrсh Institute, Tomsk1 Tomsk State University, Tomsk2 5, Kooperativny Street, 634009-Tomsk, Russia, e-mail: tsyganovmm@yandex.ru1 ЛИТЕРАТУРА Functional polymorphisms of the human multidrug-resistance gene: mul- tiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo // Proc. Natl. Acad. Sci. USA. 2000. Vol. 97 (7). P. 3473–3478. химиотерапии // Медицинская генетика. 2011. Т. 10, № 10. С. 37–43. 5. Allen J.D., Jackson S.C., Schinkel A.H. A mutation hot spot in the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance // Cancer Res. 2002. Vol. 62 (8). P. 2294–2299. p the Bcrp1 (Abcg2) multidrug transporter in mouse cell lines selected for Doxorubicin resistance // Cancer Res. 2002. Vol. 62 (8). P. 2294–2299. 6. Di Francia R., Siesto R.S., Valente D., Spart D., Berretta M. Phar- macogenomics panel test for prevention toxicity in patient who receive Fluoropirimidine/Oxaliplatin-based therapy // Eur. Rev. Med. Pharmacol. Sci. 2012. Vol. 16 (9). P. 1211–1217. ( ) 15. Honjo Y., Hrycyna C.A., Yan Q.W., Medina-Pérez W.Y., Robey R.W., van de Laar A., Litman T., Dean M., Bates S.E. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP- overexpressing cells // Cancer Res. 2001. Vol. 61 (18). P. 6635–6639. 7. Einert T.R., Schmidt G., Binnig G. Diagnostics // Ann. Oncol. 2012. Vol. 23 (Suppl. 5): P. 12–22. doi:10.1093/annonc/mds161. 63 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2015. № 5. С. 59–66 ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ SPIN-код: 2340-1628 Слонимская Елена Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Чердынцева Надежда Викторовна, доктор биологических наук, профессор, заведующая лабораторией молекулярной он- р ( ) yg @y Ибрагимова Марина Константиновна, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: imk1805@yandex.ru. SPIN-код: 2340-1628 р ( ) yg @y Ибрагимова Марина Константиновна, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: imk1805@yandex.ru. SPIN-код: 2340-1628 Слонимская Елена Михайловна ок ор е ск а к рофессор за е ю а о е е е об ей о ко о То ск й НИИ Ибрагимова Марина Константиновна, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: imk1805@yandex.ru. SPIN-код: 2340-1628 Слонимская Елена Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Чердынцева Надежда Викторовна, доктор биологических наук, профессор, заведующая лабораторией молекулярной он- кологии и иммунологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-25-29. E-mail: nvch@ oncology.tomsk.ru. SPIN-код: 5344-0990 Слонимская Елена Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Чердынцева Надежда Викторовна доктор биологических наук профессор заведующая лабораторией молекулярной он- Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ , Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Слонимская Елена Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Чердынцева Надежда Викторовна, доктор биологических наук, профессор, заведующая лабораторией молекулярной он- кологии и иммунологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-25-29. E-mail: nvch@ Слонимская Елена Михайловна, доктор медицинских наук, профессор, заведующая отделением общей онкологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-код: 7763-6417 Чердынцева Надежда Викторовна, доктор биологических наук, профессор, заведующая лабораторией молекулярной он- кологии и иммунологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-25-29. E-mail: nvch@ oncology.tomsk.ru. SPIN-код: 5344-0990 онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 41 80 92. E mail: slonimskaya@rambler.ru. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Litviakov N.V., Cherdyntseva N.V., Tsyganov M.M., Denisov E.V., Garbukov E.Y., Merzliakova M.K., Volkomorov V.V., Vtorushin S.V., Zavy- alova M.V., Slonimskaya E.M., Perelmuter V.M. Changing the expression vector of multidrug resistance genes is related to neoadjuvant chemotherapy response // Cancer Chemother. Pharmacol. 2013. Vol. 71 (1). P. 153–163. doi: 10.1007/s00280-012-1992-x. 32. Vega-Gálvez A., Di Scalab K., Rodrígueza K., Lemus-Mon- dacaa R., Mirandaa M., Lópeza J., Perez-Wona M. Effect of air-drying temperature on physico-chemical properties, antioxidant capacity, colour and total phenolic content of red pepper (Capsicum annuum, L. var. Hun- garian) // Food Chemistry. 2009. Vol. 117 (4). P. 647–653. doi:10.1016/j. foodchem.2009.04.066. 22. Mannan A.U., Boehm J., Sauter S.M., Rauber A., Byrne P.C., Neesen J., Engel W. Spastin, the most commonly mutated protein in hereditary spastic paraplegia interacts with Reticulon 1 an endoplasmic reticulum protein // Neurogenetics. 2006. Vol. 7 (2). P. 93–103. 33. Xu Y., Jiang Z., Yin P., Li Q., Liu J. Role for Class I histone deacetylases in multidrug resistance // Exp. Cell Res. 2012. Vol. 318 (3). P. 177–186. doi: 10.1016/j.yexcr.2011.11.010. 23. Nakanishi T. Drug transporters as targets for cancer chemotherapy // Cancer Genomics Proteomics. 2007. Vol. 4 (3). P. 241–254.l 24. Pfaffl M.W. A new mathematical model for relative quantification in real-time RT–PCR // Nucleic Acids Res. 2001. Vol. 29 (9). P. e45. 34. Zhuang G., Brantley-Sieders D.M., Vaught D., Yu J., Xie L., Wells S., Jackson D., Muraoka-Cook R., Arteaga C., Chen J. Elevation of receptor tyrosine kinase EphA2 mediates resistance to trastuzumab therapy // Cancer Res. 2010. Vol. 70 (1). P. 299–308. doi: 10.1158/0008- 5472.CAN-09-1845. 25. Polgar O., Ozvegy-Laczka C., Robey R.W., Morisaki K., Oka- da M., Tamaki A., Koblos G., Elkind N.B., Ward Y., Dean M., Sarkadi B., Bates S.E. Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization // Biochemistry. 2006. Vol. 45 (16): P. 5251–5260. Поступила 4.07.15 Сведения об авторах Цыганов Матвей Михайлович, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: TsyganovMM@yandex.ru. SPIN-код: 1253-0240 ыганов Матвей Михайлович, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онко ссийская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: TsyganovMM@yandex.ru. SPIN-код: 1253-0240 д р ц ( ) yg @y д Ибрагимова Марина Константиновна, младший научный сотрудник лаборатории онковирусологии, Томский НИИ онкологии (г. Томск), Российская Федерация. Тел.: 8 (3822) 51-46-09. E-mail: imk1805@yandex.ru. REFERENCES 1. Litviakov N.V. Reguljacija jekspressii genov mnozhestvennoj lekarstvennoj ustojchivosti v opuholi molochnoj zhelezy pri provedenii neoadjuvantnoj himioterapii. avtoref. dissertacii … d-ra biol. nauk. Tomsk, 2014. 37 p. [in Russian] ( ) 17. Huang Y. Pharmacogenetics/genomics of membrane transport- ers in cancer chemotherapy // Cancer Metastasis Rev. 2007. Vol. 26 (1). P. 183–201. 2. Litviakov N.V. Gradient phenomenon of multidrug resistance gene expression in breast cancer during neoadjuvant chemotherapy // Sibirskij onkologicheskij zhurnal. 2013. № 4 (58). P. 5–11. [in Russian] 18. Januchowski R., Zawierucha P., Ruciński M., Andrzejewska M., Wojtowicz K., Nowicki M., Zabel M. Drug transporter expression profil- ing in chemoresistant variants of the A2780 ovarian cancer cell line // Biomed. Pharmacother. 2014. Vol. 68 (4). P. 447–453. doi: 10.1016/j. biopha.2014.02.002. 3. 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REFERENCES Di Francia R., Siesto R.S., Valente D., Spart D., Berretta M. Phar- macogenomics panel test for prevention toxicity in patient who receive Fluoropirimidine/Oxaliplatin-based therapy // Eur. Rev. Med. Pharmacol. Sci. 2012. Vol. 16 (9). P. 1211–1217. 22. Mannan A.U., Boehm J., Sauter S.M., Rauber A., Byrne P.C., Neesen J., Engel W. Spastin, the most commonly mutated protein in hereditary spastic paraplegia interacts with Reticulon 1 an endoplasmic reticulum protein // Neurogenetics. 2006. Vol. 7 (2). P. 93–103. ( ) 7. Einert T.R., Schmidt G., Binnig G. Diagnostics // Ann. Oncol. 2012. Vol. 23 (Suppl. 5): P. 12–22. doi:10.1093/annonc/mds161. 8. Errico A., Claudiani P., D’Addio M., Rugarli E.I. Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon // Hum. Mol. Genet. 2004. Vol. 13 (18). P. 2121–2132. 23. Nakanishi T. Drug transporters as targets for cancer chemotherapy // Cancer Genomics Proteomics. 2007. Vol. 4 (3). P. 241–254.l ( ) 24. Pfaffl M.W. A new mathematical model for relative quantification in real-time RT–PCR // Nucleic Acids Res. 2001. Vol. 29 (9). P. e45. 9. Gillet J.P., Gottesman M.M. Overcoming multidrug resistance in cancer: 35 years after the discovery of ABCB1 // Drug Resistance Updates. 2012. Vol. 15 (1–2). P. 2–4. doi: 10.1016/j.drup.2012.03.001. 25. Polgar O., Ozvegy-Laczka C., Robey R.W., Morisaki K., Oka- da M., Tamaki A., Koblos G., Elkind N.B., Ward Y., Dean M., Sarkadi B., Bates S.E. Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization // Biochemistry. 2006. Vol. 45 (16): P. 5251–5260. ( ) j p 10. Gökmen-Polar Y., Toroni R.A., Hocevar B.A., Badve S., Zhao Q., Shen C., Bruckheimer E., Kinch M.S., Miller K.D. Dual targeting of EphA2 and ER restores tamoxifen sensitivity in ER/EphA2-positive breast cancer // Breast Cancer Res. Treat. 2011. Vol. 127 (2). P. 375–384. doi: 10.1007/ s10549-010-1004-y. 26. Robey R., Honjo Y., Morisaki K., Nadjem T.A., Runge S., Ris- bood M., Poruchynsky M.S., Bates S.E. Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity // Br. J. Cancer. 2003. Vol. 89 (10). P. 1971–1978. 11. Gottesman M.M., Fojo T., Bates S.E. Multidrug resistance in cancer: role of ATP–dependent transporters // Nat. Rev. Cancer. 2002. Vol. 2 (1). P. 48–58. 27. Sauer G., Brehm G., Schneider S., Nielsch K., Wehrspohn R.B., Choi J., Hofmeister H., Gösele U. Highly ordered monocrystalline silver nanowire arrays // J. Abstract The mechanism of regulation of ABC transporter gene expression, which is related to individual characteristics of the tumor-bearing organism and its tumor, defined by gene single nucleotide polymorphisms (SNP – Single Nucleotide Polymorphism) has not been extensively studied. We conducted large-scale genome studies of 64 SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66 ОДНОНУКЛЕОТИДНЫЙ ПОЛИМОРФИЗМ ОПУХОЛИ МЖ М.М. Цыганов, М.К. Ибрагимова, Е.М. Слонимская и др. association of SNP with the level of ABC transporter gene expression. The study involved 68 patients with morphologically verified diagnosis of breast cancer treated with neoadjuvant chemotherapy (NAC). Using a quantitative Real-time PCR, the expression of 4 multidrug resistence (MDR) genes (ABCB1, ABCC1, ABCC2, ABCG2) was studied in surgical samples after NAC. Microarray analysis was performed using high density DNA microarrays, which contain more than 750.000 SNPs. Six SNPs significantly associated with postoperative expression levels of multidrug resistance genes, ABCB1, ABCC1, ABCC2, ABCG2, were identified. It was shown that in carriers of a rare genotype, the expression levels of all 4 examined genes in tumors after chemotherapy were either decreased (rs2680835, rs1951366 and rs12018988), or increased (rs4676478, rs6896596, rs1154121) compared to those observed in carriers of frequent and heterozygous genotypes of the SNP. The possible mechanisms of the effect of the identified SNPs and their genes on the expression of ABC transporters were discussed. association of SNP with the level of ABC transporter gene expression. The study involved 68 patients with morphologically verified diagnosis of breast cancer treated with neoadjuvant chemotherapy (NAC). Using a quantitative Real-time PCR, the expression of 4 multidrug resistence (MDR) genes (ABCB1, ABCC1, ABCC2, ABCG2) was studied in surgical samples after NAC. Microarray analysis was performed using high density DNA microarrays, which contain more than 750.000 SNPs. Six SNPs significantly associated with postoperative expression levels of multidrug resistance genes, ABCB1, ABCC1, ABCC2, ABCG2, were identified. It was shown that in carriers of a rare genotype, the expression levels of all 4 examined genes in tumors after chemotherapy were either decreased (rs2680835, rs1951366 and rs12018988), or increased (rs4676478, rs6896596, rs1154121) compared to those observed in carriers of frequent and heterozygous genotypes of the SNP. The possible mechanisms of the effect of the identified SNPs and their genes on the expression of ABC transporters were discussed. Key words: multidrug resistance genes, single nucleotide polymorphism, breast cancer, microarray research. 16. Huang Y., Sadée W. Abstract Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells // Cancer Lett. 2006. Vol. 239 (2). P. 168–182. ( ) 15. Honjo Y., Hrycyna C.A., Yan Q.W., Medina-Pérez W.Y., Robey R.W., van de Laar A., Litman T., Dean M., Bates S.E. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP- overexpressing cells // Cancer Res. 2001. Vol. 61 (18). 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Role for Class I histone deacetylases in multidrug resistance // Exp. Cell Res. 2012. Vol. 318 (3). P. 177–186. doi: 10.1016/j.yexcr.2011.11.010. SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66 32. Vega-Gálvez A., Di Scalab K., Rodrígueza K., Lemus-Mondacaa R., Mirandaa M., Lópeza J., Perez-Wona M. Effect of air-drying temperature on physico-chemical properties, antioxidant capacity, colour and total phenolic content of red pepper (Capsicum annuum, L. var. Hungarian) // Food Chemis- try. 2009. Vol. 117 (4). P. 647–653. doi:10.1016/j.foodchem.2009.04.066. 32. Vega-Gálvez A., Di Scalab K., Rodrígueza K., Lemus-Mondacaa R., Mirandaa M., Lópeza J., Perez-Wona M. Effect of air-drying temperature on physico-chemical properties, antioxidant capacity, colour and total phenolic content of red pepper (Capsicum annuum, L. var. Hungarian) // Food Chemis- try. 2009. Vol. 117 (4). P. 647–653. doi:10.1016/j.foodchem.2009.04.066. 33. Xu Y., Jiang Z., Yin P., Li Q., Liu J. Role for Class I histone deacetylases in multidrug resistance // Exp. Cell Res. 2012. Vol. 318 (3). P. 177–186. doi: 10.1016/j.yexcr.2011.11.010. ABOUT THE AUTHORS y Mikhailovich, junior research fellow, Laboratory of Viral Oncology, Tomsk Cancer Research Institute (Tomsk on. Phone: +7 (3822) 51-46-09. E-mail: TsyganovMM@yandex.ru. SPIN-code: 1253-0240 ( ) yg @y Ibragimova Marina Konstantinovna, junior research fellow, Laboratory of Viral Oncology, Tomsk Cancer Research Institute (Tomsk), Russian Federation. Phone: +7 (3822) 51-46-09. E-mail: imk1805@yandex.ru. SPIN-code: 2340-1628 ( ) @y Slonimskaya Elena Mikhailovna, MD, Professor, Head of General Oncology Department, Tomsk Cancer Research Institute (Tomsk), Russian Federation. Phone: +7 (3822) 41-80-92. E-mail: slonimskaya@rambler.ru. SPIN-code: 7763-6417 Cherdyntseva Nadezhda Viktorovna, Professor, Deputy Director for Basic Science, Head of Laboratory of Molecular Oncology and Immunology, Tomsk Cancer Research Institute (Tomsk), Russian Federation. Phone: +7 (3822) 51-53-42. E-mail: nvch@oncology. tomsk.ru. SPIN-code: 5344-0990 Litviakov Nikolay Vasilyevich, DSc, Head of Laboratory of Viral Oncology, Tomsk Cancer Research Institute (Tomsk), Russian Federation. Phone: +7 (3822) 51-46-07. E-mail: nvlitv72@yandex.ru. SPIN-code: 2546-0181 Litviakov Nikolay Vasilyevich, DSc, Head of Laboratory of Viral Oncology, Tomsk Cancer Research Institute (Tomsk), Russian Federation. Phone: +7 (3822) 51-46-07. E-mail: nvlitv72@yandex.ru. SPIN-code: 2546-0181 66 SIBERIAN JOURNAL OF ONCOLOGY. 2015. № 5. Р. 59–66
https://openalex.org/W3013911484	https://ovarianresearch.biomedcentral.com/track/pdf/10.1186/s13048-020-00634-7	English	null	Functional polymorphisms in FOXC2 gene and Epithelial ovarian Cancer susceptibility in Chinese population	Journal of ovarian research	2,020	cc-by	5,379	Abstract Background: Epithelial ovarian cancer (EOC) is highly lethal gynecological cancer. Forkhead Box Protein C2 (FOXC2) promotes occurrence and development of various malignant tumors. The present study is aimed at exploring the correlation between the polymorphism of FOXC2 and epithelial ovarian cancer susceptibility in Chinese Han population. Methods: A case-control design was used to verify the association between FOXC2 polymorphisms and epithelial ovarian cancer. The genotyping was performed using Taqman® SNP Genotyping kit by qRT-PCR. The genetic variants including rs3751794 C > T, rs1035550 A > G, rs4843163 C > G and rs4843396 C > T in FOXC2 gene were analyzed. The strength of the associations was detected using odds ratios and 95% confidence intervals. Stratification analyses showed the association between the FOXC2 gene polymorphisms rs3751794 C > T, rs4843163 C > G and rs4843396 C > T with epithelial ovarian cancer susceptibility in terms of age, metastasis status, clinical stage, pathological grade, pregnant times, pausimenia, and the expression of ER, PR, wild p53 and mutant p53. Results: Rs3751794 C > T (P = 0.0016), rs4843163 C > G (P < 0.0001) and rs4843396 C > T (P < 0.0001) were significantly associated with increased epithelial ovarian cancer risk. In stratification analyses,rs3751794 C > T, was identified to be dominant in no metastasis patients, clinical stage 4 group, middle grade pathological stage, pregnant time over 3 patients, post-menopause women, strong wild type p53 expression; rs4843163 C > G was dominant in high grade clinical stage, high grade pathological stage, post-menopause women, strong ER expression group and no mutant p53 expression group; rs4843396 C > T was dominant in high grade clinical stage, high grade pathological stage, strong ER expression group. The rs1035550 A > G was not related to epithelial ovarian cancer susceptibility. Methods: A case-control design was used to verify the association between FOXC2 polymorphisms and epithelial ovarian cancer. The genotyping was performed using Taqman® SNP Genotyping kit by qRT-PCR. The genetic variants including rs3751794 C > T, rs1035550 A > G, rs4843163 C > G and rs4843396 C > T in FOXC2 gene were analyzed. The strength of the associations was detected using odds ratios and 95% confidence intervals. Functional polymorphisms in FOXC2 gene and Epithelial ovarian Cancer susceptibility in Chinese population Zhijiao Zhou1, Xiang Ou2, Qiong Zou1, Ling Chu1, Xiyun Quan3, Yong Chen4 and Yang Liu1* Abstract * Correspondence: liuyang1_2009@163.com 1Department of Pathology, Third Xiangya Hospital,Central South University, Changsha 410013, Hunan, China Full list of author information is available at the end of the article * Correspondence: liuyang1_2009@163.com p y g _ @ 1Department of Pathology, Third Xiangya Hospital,Central South University, Changsha 410013, Hunan, China Full list of author information is available at the end of the article Abstract Stratification analyses showed the association between the FOXC2 gene polymorphisms rs3751794 C > T, rs4843163 C > G and rs4843396 C > T with epithelial ovarian cancer susceptibility in terms of age, metastasis status, clinical stage, pathological grade, pregnant times, pausimenia, and the expression of ER, PR, wild p53 and mutant p53. Results: Rs3751794 C > T (P = 0.0016), rs4843163 C > G (P < 0.0001) and rs4843396 C > T (P < 0.0001) were significantly associated with increased epithelial ovarian cancer risk. In stratification analyses,rs3751794 C > T, was identified to be dominant in no metastasis patients, clinical stage 4 group, middle grade pathological stage, pregnant time over 3 patients, post-menopause women, strong wild type p53 expression; rs4843163 C > G was dominant in high grade clinical stage, high grade pathological stage, post-menopause women, strong ER expression group and no mutant p53 expression group; rs4843396 C > T was dominant in high grade clinical stage, high grade pathological stage, strong ER expression group. The rs1035550 A > G was not related to epithelial ovarian cancer susceptibility. Conclusions: The results of the current study verified that FOXC2 gene polymorphisms were associated with increased epithelial ovarian cancer risk and suggested that FOXC2 gene polymorphisms might be a potential biomarker for epithelial ovarian cancer susceptibility. Keywords: Forkhead box protein C2, Epithelial ovarian cancer, Polymorphism © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhou et al. Journal of Ovarian Research (2020) 13:34 https://doi.org/10.1186/s13048-020-00634-7 Zhou et al. Journal of Ovarian Research (2020) 13:34 https://doi.org/10.1186/s13048-020-00634-7 Open Access Correspondence: liuyang1_2009@163.com 1Department of Pathology, Third Xiangya Hospital,Central South University, Changsha 410013, Hunan, China Full list of author information is available at the end of the article Population characteristics This study focused on the relevance between the poly- morphism of FOXC2 and epithelial ovarian cancer susceptibility. There was no significant difference in terms of age (P = 0.252) between the case and the control groups. Of them, 18, 27, 77, and 24 patients were classified as clin- ical stages I, II, III, and IV; 20, 32 and 92 patients were classified as pathological grades low, middle and high, respectively. Among these cases, 93 patients were post- menopausal, 76 patients had metastasis. According to IHC detection, the expression of ER in 4 patients was © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 2 of 6 Page 2 of 6 Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 2 of 6 Statistical analyses Departures from Hardy-Weinberg equilibrium (HWE) were evaluated for each SNP in controls by goodness-of- fit χ2 test. Two-sidedχ2 test and t test were performed, as appropriate to compare the demographic variables and allele frequencies between the cases and the control group. The odds ratio (OR), and the corresponding 95% confidence interval (CI) for each SNP were analyzed. Lo- gistic regression analysis was used to assess the correlation between SNPs and epithelial ovarian cancer susceptibility. Statistical adjustment for age was performed. The version 9.4 SAS software (SAS Institute, Cary, NC) was used to perform analysis. The significant threshold was P < 0.05. SNP genotyping and quality control The included potentially functional candidate SNPs were selected as follows: located in the exon, 5’flanking re- gion, 5’untranslated region, and 3′ untranslated region of FOXC2 gene. NCBIdb SNP database (http://www. ncbi.nlm.nih.gov/projects/SNP) and SNPinfo (http:// snpinfo.niehs.nih.gov/snpfunc.htm) online software were used to selected SNPs [17]. We chose four potentially functional SNPs in FOXC2 gene (rs3751749 C > T, rs1035550 A > G, rs4843163 C > G and rs4843396 C > T) for analysis. All of the four SNPs mapped in 5’untrans- lated region, and predicted to act as transcription factor binding sites. Genomic DNA was derived from paraffin embedding tissues or EDTA peripheral blood by using DNA extracting Kit (TianGen Biotech Co. Ltd., Beijing, China). The genotyping of all the subjects was carried out using TaqMan real-time PCR (Applied Biosystems), according to the manufacturer’s protocols. The quality control was performed as the protocol from published paper [18]. Several evidence from genome-wide association study (GWAS) showed that genetic variations were identified to associate with ovarian cancer, most of which were single nucleotide polymorphism. Some genetic variants were discovered to be shared between East Asian and European populations, but still others were specific in each population [5]. In Chinese population, genes such as WNT4 [6], PSEN1, MAML2 [7], ESR2 [8], et al. were identified to be associated with EOC. Forkhead Box Protein C2 (FOXC2), which is an im- portant member of transcription factor FOX family, plays essential role in several gene regulatory pathways. It was also showed to play role in carcinogenesis. FOXC2 was identified to induce epithelial-mesenchymal transition (EMT) in prostate cancer [9],lung cancer [10]. Overexpression of FOXC2 was related to poor prognosis of hepatocellular carcinoma [11].FOXC2 activated YAP signal pathway and enhanced the glycolysis in nasopha- ryngeal carcinoma cells [12]. Still FOXC2 was certifi- cated to promote EMT, migration and invasion in cisplatin-resistant ovarian cancer cells [13]. Mutations of DNA binding domain in FOXC2 were identified to induce Lymphoedema distichiasis syndrome [14]. It’s reported that FOXC2 polymorphism was associated with type 2 diabetes mellitus [15]. FOXC2 c.-512C > T was found to related with increased susceptibility to chronic venous diseases [16]. However, there was no evidence showed that FOXC2 polymorphisms were associated with cancers. Introduction The major clinical and biological characteristics of the patient, including age, metastasis status, clinical stage, pathological grade, pregnant times, pausimenia, and the expression of ER, PR, wild p53 and mutant p53 by immunohistochemistry (IHC) were collected. There was no significant difference in age between the case group and the control group. Ethical approved was obtained from the Institutional Review Board of the hospital. Ovarian cancer is the sixth malignant tumor in women. Since it almost doesn’t show specific symptoms at early stage, it is usually diagnosed very late, even when tumor cells spread or metastasis [1]. In 2018, it’s estimated there were more than 295 thousands new cases and 184 thousands deaths of ovarian cancer over the world [2]. Epithelial ovarian cancer (EOC) is the dominant type of ovarian cancer. The standard treatment for EOC is cytoreductive surgery combined with chemotherapy. However, most EOC patients relapse and the 5-year survival rate is no more than 35% [3]. The unsatisfying outcome of EOC treatment is attributed to late diagnosis and chemotherapy resistance [4]. Hence, there is urgent need for revealing risk factors which can be used for early diagnosis. Materials and methods Study populations A total of 150 epithelial ovarian cancer cases and 298 healthy controls from The Third Affiliated Hospital, Central South University were included in this study. Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 3 of 6 20.567, 95% CI = 10.522–40.202, P < 0.001; CC vs.GG: adjusted OR = 1.691, 95% CI = 1.034–2.765, P = 0.0363; dominant model: adjusted OR = 3.703, 95% CI = 2.408– 5.693, P < 0.0001; recessive model: adjusted OR = 15.831, 95% CI = 8.449–29.664, P < 0.0001). A positive associ- ation between rs4843396 C allele and epithelial ovarian cancer risk (TC vs. TT: adjusted OR = 20.567, 95% CI = 10.522–40.202, P < 0.001; CC vs. TT: adjusted OR = 1.691, 95% CI = 1.034–2.765, P = 0.0363; dominant model: adjusted OR = 3.628, 95% CI = 2.358–5.582, P < 0.0001; recessive model: adjusted OR = 23.546, 95% CI = 14.555–44.837, P < 0.0001). negative/mild positive, in 40 patients was strong positive; the expression of PR in 16 patients was negative/mild positive, in 32 patients was strong positive; the expres- sion of wild type p53 in 37 patients was negative/mild positive, in 29 patients was strong positive; the expres- sion of mutant p53 in 24 patients was negative/mild positive, in 36 patients was strong positive. Correlation of FOXC2 gene polymorphisms with epithelial ovarian cancer susceptibility The genotype frequencies of FOXC2 associated with epi- thelial ovarian cancer risk were shown in Table 1.Be- cause rs1035550 G > A was not accordance with HWE (< 0.05), we would not analyze the relationship between rs1035550 and epithelial ovarian cancer risk further. A positive association between rs3751749 C allele and epi- thelial ovarian cancer risk (TC vs. TT: adjusted OR = 3.415, 95% CI = 1.492–7.815, P = 0.0036; recessive model: adjusted OR = 3.707, 95% CI = 1.645–8.353, P = 0.0016). A positive association between rs4843163 C allele and epithelial ovarian cancer risk (GC vs. GG: adjusted OR = Stratification analysis The results were showed in Table 2 from stratification analyses of association between FOXC2 genotypes and epithelial ovarian cancer susceptibility, stratified by age, metastasis status, clinical stage, pathological grade, preg- nant times, pausimenia, the expression of ER, PR, wild p53 and mutant p53. For age, FOXC2 rs3751794 CC genotype was significantly associated with increased Table 1 Logistic Regression Analysis of Associations Between FOXC2 Polymorphisms and EOC susceptibility Table 1 Logistic Regression Analysis of Associations Between FOXC2 Polymorphisms and EOC susceptibility Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 4 of 6 Table 2 Stratification analysis of FOXC2 polymorphisms with EOC susceptibility Table 2 Stratification analysis of FOXC2 polymorphisms with EOC susceptibility epithelial ovarian cancer risk in both< 53 years group (adjusted OR = 5.289;95% CI = 1.331–21.013, P = 0.0180) and ≥53 years group (adjusted OR = 3.010;95% CI = 1.089–8.322, P = 0.0337); rs4843163GC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in both< 53 years group (adjusted OR = 2.761;95% CI = 1.600–4.764, P = 0.0003) and ≥53 years group (adjusted OR = 5.844;95% CI = 2.585–12.115, P < 0.0001); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in both< 53 years group (adjusted OR = 3.239;95% CI = 1.854–5.659, P < 0.0001) and ≥53 years group (adjusted OR = 4.272;95% CI = 2.160–8.447, P < 0.0001).Z CI = 2.730–24.878, P = 0.0007);rs4843163GC/CC geno- type was significantly associated with increased epithelial ovarian cancer risk in three subgroups of clinical stage (stage 2: adjusted OR = 3.312;95% CI = 1.403–7.820, P = 0.0063; stage 3: adjusted OR = 4.087;95% CI = 2.336– 7.152, P < 0.0001; stage 4: adjusted OR = 6.973;95% CI = 2.323–20.934, P = 0.0005); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in three subgroups of clinical stage (stage 2: adjusted OR = 3.165;95% CI = 1.332–7.523, P = 0.0091; stage 3: adjusted OR = 4.059;95% CI = 2.317–7.109, P < 0.0001; stage 4: adjusted OR = 9.831; 95% CI = 2.866–33.714, P = 0.0003). Discussion In the current case-control study with 150 epithelial ovarian cancer cases and 298 healthy controls from Chinese populations, we explored the potential associ- ation between FOXC2 gene polymorphisms and epithe- lial ovarian cancer susceptibility. We certificated that 3 of the 4 included polymorphisms, namely rs3751749 C > T, rs4843163 C > G and rs4843396 C > T, were associ- ated with an increased risk of epithelial ovarian cancer. So far, the current study is the first to evaluate the asso- ciation between FOXC2 polymorphisms and epithelial ovarian cancer susceptibility. , ) Furthermore, we identified that rs4843163GC/CC genotype (adjusted OR = 30.268; 95% CI = 7.187– 127.478, P < 0.0001) and rs4843396TC/CC genotype (ad- justed OR = 14.474;95% CI = 5.044–41.528, P < 0.0001) was remarkably associated with strong positive ER ex- pression. These two FOXC2 polymorphisms were sig- nificantly associated with subgroups of PR expression (rs4843163GC/CC vs. GG: negative/mild expression: ad- justed OR = 10.472;95% CI = 2.350–46.667, P = 0.0021; strong positive expression: adjusted OR = 43.792;95% CI = 5.890–325.594, P = 0.0002; rs4843396TC/CC vs. TT: negative/mild expression: adjusted OR = 6.567;95% CI = 1.845–23.370, P = 0.0037; strong positive expres- sion: adjusted OR = 21.295;95% CI = 4.986–90.953, P < 0.0001). For wild type p53 expression, rs3751794 CC genotype was significantly associated with increased epi- thelial ovarian cancer risk in strong positive group (ad- justed OR = 4.587;95% CI = 1.324–15.869, P = 0.0163); rs4843163GC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in sub- groups of wild type p53 expression (negative/mild ex- pression: adjusted OR = 24.209;95% CI = 5.759–103.466, P < 0.0001; strong positive expression: adjusted OR = 9.412;95% CI = 3.208–27.612, P < 0.0001); rs4843396TC/ CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of wild type p53 expression (negative/mild expression: adjusted OR = 15.909;95% CI = 4.774–53.014, P < 0.0001; strong positive expression: adjusted OR = 9.412;95% CI = 3.208–27.612, P = 0.0001). For mutant p53 expression, rs3751794 CC genotype (adjusted OR = 3.487;95% CI = 1.024–11.876, P = 0.0457 and rs4843163GC/CC genotype (adjusted OR = 8.877;95% CI = 3.360–23.451, P < 0.0001) was Mutations of FOXC2 in coding region could impair its transcriptional activation and DNA-binding activity in hereditary distichiasis [19, 20]. A nonsense mutation in exon was found in spinal extradural arachnoid cyst [21]. Deletion of FOXC2 could induce abnormal lymphagio- genesis and activate ERK [22]. Stratification analysis For metastasis status, FOXC2 rs3751794 CC genotype was significantly associated with increased epithelial ovarian cancer risk in no metastasis group (adjusted OR = 5.018; 95% CI = 1.973–12.762, P = 0.0007); rs4843163GC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (yes group: adjusted OR = 2.913;95% CI = 1.712–4.956 P < 0.0001; no group: adjusted OR = 4.839;95% CI = 2.641–8.868, P < 0.0001); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (yes group: adjusted OR = 3.199;95% CI = 1.857–5.510 P < 0.0001; no group: adjusted OR = 4.082;95% CI = 2.271–7.335, P < 0.0001). For pathological grade, rs3751794 CC genotype was sig- nificantly associated with increased epithelial ovarian can- cer risk in middle grade group (adjusted OR = 11.327; 95% CI = 4.166–30.792, P < 0.0001); rs4843163GC/CC genotype (adjusted OR = 11.608;95% CI = 5.777–23.323, P < 0.0001) and rs4843396TC/CC genotype (adjusted OR = 8.452;95% CI = 4.488–15.918, P < 0.0001) was significantly asso- ciated with increased epithelial ovarian cancer risk in high grade group. For pregnant times, rs3751794 CC genotype was sig- nificantly associated with increased epithelial ovarian cancer risk in > 3 times group (adjusted OR = 3.716;95% CI = 1.093–12.627, P = 0.0355); rs4843163GC/CC geno- type was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (≤3 times group: adjusted OR = 5.652;95% CI = 3.010–10.614 P < For clinical stage, FOXC2 rs3751794 CC genotype was significantly associated with increased epithelial ovarian cancer risk in stage 4 patients (adjusted OR = 7.678;95% Page 5 of 6 Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 5 of 6 Page 5 of 6 0.0001; > 3 times group: adjusted OR = 14.899;95% CI = 4.457–49.802, P < 0.0001); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (≤3 times group: adjusted OR = 5.710;95% CI = 3.041–10.725 P < 0.0001; > 3 times group: adjusted OR = 10.905;95% CI = 3.749–31.719, P < 0.0001). 0.0001; > 3 times group: adjusted OR = 14.899;95% CI = 4.457–49.802, P < 0.0001); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (≤3 times group: adjusted OR = 5.710;95% CI = 3.041–10.725 P < 0.0001; > 3 times group: adjusted OR = 10.905;95% CI = 3.749–31.719, P < 0.0001). Stratification analysis significantly associated with increased epithelial ovarian cancer risk in strong positive group; rs4843396TC/CC genotype was significantly associated with increased epi- thelial ovarian cancer risk in subgroups of mutant p53 expression (negative/mild expression: adjusted OR = 32.414;95% CI = 4.318–243.321, P = 0.0007; strong posi- tive expression: adjusted OR = 7.239;95% CI = 2.927– 17.901, P = 0.0001). , ) For pausimenia, rs3751794 CC genotype was signifi- cantly associated with increased epithelial ovarian cancer risk in postmenopausal patients (adjusted OR = 4.338; 95% CI = 1.805–10.412, P = 0.0010); rs4843163GC/CC genotype was significantly associated with increased epi- thelial ovarian cancer risk in subgroups of pausimenia (yes group: adjusted OR = 4.998;95% CI = 2.866–8.716 P < 0.0001; no group: adjusted OR = 2.303;95% CI = 1.232–4.304, P = 0.0089); rs4843396TC/CC genotype was significantly associated with increased epithelial ovarian cancer risk in subgroups of metastasis (yes group: adjusted OR = 4.422;95% CI = 2.569–7.612 P < 0.0001; no group: adjusted OR = 2.559;95% CI = 1.404–5.035, P = 0.0027). Discussion Yamada Y, et al. revealed that FOXC2 polymorphism was associated with reduced BMD susceptibility in Japanese population [23]. In addition to these diseases, there was no evidence shown mutation of FOXC2 in other diseases. In this study, we analyzed the correlation between FOXC2 polymorphisms and the patient’s metastasis sta- tus, clinical stage, pathological grade, pregnant times, pausimenia, and the expression of several EOC related proteins. And we discovered that rs3751749 C > T, rs4843163 C > G and rs4843396 C > T were associated with increased EOC risk. There is still no evidence shown that FOXC2 polymorphism is associated with cancer, even though in ovarian cancer. All of the three SNPs we detected are in upstream of FOXC2 gene. They were predicted to be function with transcription factors. In this study, we still don’t know whether rs3751749, rs4843163 of rs4843396 could affect the expression of FOXC2. In our next study, we need collect much larger sample sizes to certify the results of the current study, and the effects of these three SNPs on the expression of FOXC2 need to be discovered further in EOC cell lines. With regard to another FOXC2 polymorphism rs1035550 A > G, we couldn’t find the correlation be- tween this SNP and EOC susceptibility. Rs1035550 lo- cates in upstream of FOXC2. There is also no data shown the correlation between rs1035550 and cancers. Zhou et al. Journal of Ovarian Research (2020) 13:34 Page 6 of 6 Page 6 of 6 Still no result was shown the influence of rs1035550 on the expression of FOXC2. 6. Zhang J, Zhang P, Shen Y, Yang M, Zou H, Liu H. Relationship of WNT4 gene with the risk of epithelial ovarian Cancer: a Han Chinese population- based association study. Genet Test Mol Biomarkers. 2018;22(12):686–92. 6. Zhang J, Zhang P, Shen Y, Yang M, Zou H, Liu H. Relationship of WNT4 gene with the risk of epithelial ovarian Cancer: a Han Chinese population- based association study. Genet Test Mol Biomarkers. 2018;22(12):686–92. Although to our knowledge this study is the first to re- veal the relationship between FOXC2 polymorphisms and EOC susceptibility, several limitations should be mentioned. Firstly, there were only four SNPs were in- vestigated in the current study, more potentially func- tional SNPs in FOXC2 gene should be focused on. Secondly, the sample size should be enlarged. References 23. Yamada Y, Ando F, Shimokata H. Association of polymorphisms in forkhead box C2 and perilipin genes with bone mineral density in community- dwelling Japanese individuals. Int J Mol Med. 2006;18(1):119–27. 1. Alyahri N, Abdi S, Khan W, Elrobh M, Addar MH, Babay ZA, et al. Novel associations between BRCA1 variants C.181 T>G (Rs28897672) and ovarian Crisk in Saudi females. J Med Biochem. 2019;38(1):13–21. 2. 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Radiol Oncol. 2017;51(3):363–8. 1Department of Pathology, Third Xiangya Hospital,Central South University, Changsha 410013, Hunan, China. 2Department of Endocrinology, The First Hospital of Changsha, Changsha, Hunan, China. 3Department of Pathology, Zhuzhou Central Hospital, Zhuzhou, Hunan, China. 4Department of Clinical Laboratory, The First Hospital of Changsha, Changsha, Hunan, China. 21. Ogura Y, Fujibayashi S, Iida A, Kou I, Nakajima M, Okada E, et al. A novel FOXC2 mutation in spinal extradural arachnoid cyst. Hum Genome Var. 2015;2:15032. 21. Ogura Y, Fujibayashi S, Iida A, Kou I, Nakajima M, Okada E, et al. A novel FOXC2 mutation in spinal extradural arachnoid cyst. Hum Genome Var. 2015;2:15032. Consent for publication Agree. Consent for publication Agree. 18. Zhang J, Yang Y, Li W, Yan L, Zhang D, He J, et al. TP53 gene rs1042522 allele G decreases neuroblastoma risk: a two-Centre case-control study. J Cancer. 2019;10(2):467–71. Authors’ contributions ZJZ XO d YL f ZJZ, XO and YL performed the study and wrote the manuscript. QZ and YC provided clinical samples and analyzed clinical data. LC participated in the cases recruit of present study. XYQ carried out statistical analysis. The authors read and approved the final manuscript. 14. Bell R, Brice G, Child AH, Murday VA, Mansour S, Sandy CJ, et al. Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene. Hum Genet. 2001;108(6): 546–51. 14. Bell R, Brice G, Child AH, Murday VA, Mansour S, Sandy CJ, et al. Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene. Hum Genet. 2001;108(6): 546–51. Funding h d This study was funded by National Natural Science Foundation of China (No:81602647) and Hunan Provincial Natural Sciences Foundation of China (2016JJ4104). 15. Nian X, Zhang X, Wang Y, Li H, Li J, Liu H, et al. Correlations of FOXC2 gene expression and polymorphism with type 2 diabetes mellitus. Clin Lab. 2016; 62(5):781–91. 15. Nian X, Zhang X, Wang Y, Li H, Li J, Liu H, et al. Correlations of FOXC2 gene expression and polymorphism with type 2 diabetes mellitus. Clin Lab. 2016; 62(5):781–91. 16. Surendran S, Girijamma A, Nair R, Ramegowda KS, Nair DH, Thulaseedharan JV, et al. Forkhead box C2 promoter variant c.-512C>T is associated with increased susceptibility to chronic venous diseases. PloS One. 2014;9(3): e90682. 16. Surendran S, Girijamma A, Nair R, Ramegowda KS, Nair DH, Thulaseedharan JV, et al. Forkhead box C2 promoter variant c.-512C>T is associated with increased susceptibility to chronic venous diseases. PloS One. 2014;9(3): e90682. Received: 20 December 2019 Accepted: 12 March 2020 22. Fatima A, Wang Y, Uchida Y, Norden P, Liu T, Culver A, et al. Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation. J Clin Invest. 2016;126(7):2437–51. Competing interests Th h d l d h 19. Zhang L, He J, Han B, Lu L, Fan J, Zhang H, et al. Novel FOXC2 mutation in hereditary Distichiasis impairs DNA-binding activity and transcriptional activation. Int J Biol Sci. 2016;12(9):1114–20. 19. Zhang L, He J, Han B, Lu L, Fan J, Zhang H, et al. Novel FOXC2 mutation in hereditary Distichiasis impairs DNA-binding activity and transcriptional activation. Int J Biol Sci. 2016;12(9):1114–20. The authors declared that they have no competing interest exists. Acknowledgments Not applicable. 12. Song L, Tang H, Liao W, Luo X, Li Y, Chen T, et al. FOXC2 positively regulates YAP signaling and promotes the glycolysis of nasopharyngeal carcinoma. Exp Cell Res. 2017;357(1):17–24. 12. Song L, Tang H, Liao W, Luo X, Li Y, Chen T, et al. FOXC2 positively regulates YAP signaling and promotes the glycolysis of nasopharyngeal carcinoma. Exp Cell Res. 2017;357(1):17–24. 13. Li C, Ding H, Tian J, Wu L, Wang Y, Xing Y, et al. Forkhead box protein C2 promotes epithelial-Mesenchymal transition, migration and invasion in Cisplatin-resistant human ovarian Cancer cell line (SKOV3/CDDP). Cell Physiol Biochem. 2016;39(3):1098–110. 13. Li C, Ding H, Tian J, Wu L, Wang Y, Xing Y, et al. Forkhead box protein C2 promotes epithelial-Mesenchymal transition, migration and invasion in Cisplatin-resistant human ovarian Cancer cell line (SKOV3/CDDP). Cell Physiol Biochem. 2016;39(3):1098–110. 8. Feng Y, Peng Z, Liu W, Yang Z, Shang J, Cui L, et al. Evaluation of the epidemiological and prognosis significance of ESR2 rs3020450 polymorphism in ovarian cancer. Gene. 2019;710:316–23. 6. Zhang J, Zhang P, Shen Y, Yang M, Zou H, Liu H. Relationship of WNT4 gene with the risk of epithelial ovarian Cancer: a Han Chinese population- based association study. Genet Test Mol Biomarkers. 2018;22(12):686–92. Discussion Thirdly, patients and healthy control should be enrolled from more hospitals to avoid selection bias. 7. Xu Y, Cheng L, Dai H, Zhang R, Wang M, Shi T, et al. Variants in notch signalling pathway genes, PSEN1 and MAML2, predict overall survival in Chinese patients with epithelial ovarian cancer. J Cell Mol Med. 2018;22(10): 4975–84. 8. Feng Y, Peng Z, Liu W, Yang Z, Shang J, Cui L, et al. Evaluation of the epidemiological and prognosis significance of ESR2 rs3020450 polymorphism in ovarian cancer. Gene. 2019;710:316–23. 9. Borretzen A, Gravdal K, Haukaas SA, Beisland C, Akslen LA, Halvorsen OJ. FOXC2 expression and epithelial-mesenchymal phenotypes are associated with castration resistance, metastasis and survival in prostate cancer. J Pathol Clin Res. 2019;5(4):272–86. 9. Borretzen A, Gravdal K, Haukaas SA, Beisland C, Akslen LA, Halvorsen OJ. FOXC2 expression and epithelial-mesenchymal phenotypes are associated with castration resistance, metastasis and survival in prostate cancer. J Pathol Clin Res. 2019;5(4):272–86. 10. He Y, Xie H, Yu P, Jiang S, Wei L. FOXC2 promotes epithelial-mesenchymal transition and cisplatin resistance of non-small cell lung cancer cells. Cancer Chemother Pharmacol. 2018;82(6):1049–59. 10. He Y, Xie H, Yu P, Jiang S, Wei L. FOXC2 promotes epithelial-mesenchymal transition and cisplatin resistance of non-small cell lung cancer cells. Cancer Chemother Pharmacol. 2018;82(6):1049–59. From this study, we observed that FOXC2rs3751749 C > T, rs4843163 C > G and rs4843396 C > T were asso- ciated with increased ovarian cancer risk in Chinese population. 11. Shimoda Y, Ubukata Y, Handa T, Yokobori T, Watanabe T, Gantumur D, et al. High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma. BMC Cancer. 2018;18(1):597. 11. Shimoda Y, Ubukata Y, Handa T, Yokobori T, Watanabe T, Gantumur D, et al. High expression of forkhead box protein C2 is associated with aggressive phenotypes and poor prognosis in clinical hepatocellular carcinoma. BMC Cancer. 2018;18(1):597. 7. Xu Y, Cheng L, Dai H, Zhang R, Wang M, Shi T, et al. Variants in notch signalling pathway genes, PSEN1 and MAML2, predict overall survival in Chinese patients with epithelial ovarian cancer. J Cell Mol Med. 2018;22(10): 4975–84. 9. Borretzen A, Gravdal K, Haukaas SA, Beisland C, Akslen LA, Halvorsen OJ. FOXC2 expression and epithelial-mesenchymal phenotypes are associated with castration resistance, metastasis and survival in prostate cancer. J Pathol Clin Res. 2019;5(4):272–86. Ethics approval and consent to participate ll d f d d l h All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional review board of the hospital. 17. Wang YZ, Zhuo ZJ, Fang Y, Li L, Zhang J, He J, et al. Functional polymorphisms in hOGG1 gene and neuroblastoma risk in Chinese children. J Cancer. 2018;9(23):4521–6. Consent for publication Agree. Publisher’s Note 3. Kossai M, Leary A, Scoazec JY, Genestie C. Ovarian Cancer: a heterogeneous disease. Pathobiology. 2018;85(1–2):41–9. 3. Kossai M, Leary A, Scoazec JY, Genestie C. Ovarian Cancer: a heterogeneous disease. Pathobiology. 2018;85(1–2):41–9. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 4. Si W, Kang S, Sun H, Chen J, Cao S, Li Y. Genetic polymorphisms in hMSH2 and hMLH1 genes are associated with prognosis in epithelial ovarian cancer patients. Int J Gynecol Cancer. 2019;29(7):1148–55. 5. Lawrenson K, Song F, Hazelett DJ, Kar SP, Tyrer J, Phelan CM, et al. Genome- wide association studies identify susceptibility loci for epithelial ovarian cancer in east Asian women. Gynecol Oncol. 2019;153(2):343–55.
https://openalex.org/W4377161807	https://zenodo.org/records/7951216/files/BEST..BANKLARNING%20JISMONIY%20SHAXSLARNI%20KREDITLASH%20HOLATI%20VA%20UNI%20RIVOJLANTIRISH%20IMKONIYATLARI.pdf	Kirghiz, Kyrgyz	null	BANKLARNING JISMONIY SHAXSLARNI KREDITLASH HOLATI VA UNI RIVOJLANTIRISH IMKONIYATLARI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,098	Toshkent Moliya instituti talabasi Annotatsiya. Banklar bank tizimini tashkil etuvchi elementlar sifatida faqat uning boshqa elementlari va birinchi navbatda bank infratuzilmasi bilan o‘zaro aloqada bo‘lgandagina muvaffaqiyatli rivojlana oladi, bu esa bank hayotini ta’minlovchi elementlar majmui sifatida tushuniladi. Kreditlar jismoniy shaxslarning massaviy ijtimoiy ta’minoti va tadbirkorlik faoliyati bo’yicha turli g’oya va loyihalarni amalga oshirishlari uchun xizmat qiluvchi resurs sifatida xizmat qiladi. Jismoniy shaxslarni kreditlash muhim banka amaliyotidan hisoblanadi. Kalit so’zlar: tijorat ijtimoiy soha, bank kredit portfeli, kreditlash, banklari, aktivlar, kredit portfellari, kredit, risk. BEST SCIENTISTS -2023 -2023 BEST SCIENTISTS Olqarova Zarina Nosir qizi Toshkent Moliya instituti talabasi Olqarova Zarina Nosir qizi 2 2020 — 2025 yillarga mo‘ljallangan O‘zbekiston Respublikasining bank tizimini isloh qilish s O‘zbekiston Respublikasi Prezidentining Farmoni, 12.05.2020 yildagi PF-5992-son 1 O’zbekiston Respublikasi Prezidentining “Bank xizmatlari ommabopligini oshirish bo‘yicha qo‘shimcha chora- tadbirlar to‘g‘risida”gi № 3620 qarori. 2018 yil 23 mart. 2020 — 2025 yillarga mo ljallangan O zbekiston Respublikasining bank tizimini islo zbekiston Respublikasi Prezidentining Farmoni, 12.05.2020 yildagi PF-5992-son p g , y g ttps://lex.uz/uz/docs/4811025?ONDATE=30.12.2021&ONDATE2=18.10.2021&action=compare 1 O’zbekiston Respublikasi Prezidentining “Bank xizmatlari ommabopligini oshirish bo‘yicha qo‘shimcha 2020 — 2025 yillarga mo ljallangan O zbekiston Respublikasining bank tizimini isloh qilish strategiyasi to g risida O‘zbekiston Respublikasi Prezidentining Farmoni, 12.05.2020 yildagi PF-5992-son g g tadbirlar to‘g‘risida”gi № 3620 qarori. 2018 yil 23 mart. dbirlar to‘g‘risida”gi № 3620 qarori. 2018 yil 23 mart. 2020 2025 ill ‘lj ll O‘ b ki t R blik i i b k ti i i i i l h ili h t t i i t ‘ rlar to g risida gi № 3620 qarori. 2018 yil 23 mart. 0 2025 yillarga mo‘ljallangan O‘zbekiston Respublikasining bank tizimini isloh qilish strategiyasi to‘g‘ris KIRISH Moliyaviy sektorni izchil isloh qilish davomida qator chora-tadbirlar amalga oshirildi va natijada ilg‘or bank biznesini yuritish hamda ushbu sektorda raqobat muhitini kuchaytirish uchun zarur huquqiy shart-sharoitlar yaratildi. Biz, avvalo, chetdan kredit va sarmoyalar olib kelish bo‘yicha samarali tizim yaratishimiz, har bir kreditni aniq ishlatishni o‘rganishimiz lozim. Bu masalada yetti 1 \| 1 \| 1 \| BEST SCIENTISTS -2023 -2023 BEST SCIENTISTS o‘lchab, bir marta kesadigan, oqibatini puxta o‘ylab ish olib boradigan davr keldi” deb ta’kidlab o‘tganlari bejiz emas1. Xususan, xalqaro standartlarga muvofiq keladigan va moliyaviy sohaga xorijiy investitsiyalar kiritish uchun jozibador huquqiy muhitni yaratadigan O‘zbekiston Respublikasining «O‘zbekiston Respublikasining Markaziy banki to‘g‘risida»gi, «Banklar va bank faoliyati to‘g‘risida»gi, «Valyutani tartibga solish to‘g‘risida»gi hamda «To‘lovlar va to‘lov tizimlari to‘g‘risida»gi yangilangan qonunlari qabul qilindi. Shu bilan birga, bank sohasidagi hozirgi holat tahlili bank sektorida davlatning yuqori darajadagi aralashuvi, davlat ishtirokidagi banklarda menejment va tavakkalchiliklarni boshqarish sifatining еtarli emasligi, iqtisodiyotda moliyaviy vositachilikning past darajasi kabi bank sektorini iqtisodiy yangilanishlar va jamiyat ehtiyojlariga mos ravishda rivojlantirishga to‘sqinlik qilayotgan qator tizimli muammolar mavjudligini ko‘rsatmoqda2. Tijorat banklari jalb qilgan mablag‘larining asosiy qismini kredit berish sifatida foydalanar ekan, bunda faqatgina daromad olish uchun emas, balki mablag‘larni kredit oluvchidan to‘liq qaytarib olish masalasini oldindan belgilab olishi zarur. Chunki, bank sarmoya egasi sifatida sarmoyani emas, balki sarmoyadan foydalanish huquqini ma’lum bir shart va ustama foizlar asosida ma’lum muddatga sotadi. Ayni paytda mamlakatimizda ham ilmiy-nazariy hamda amaliyotchi olimlar tomonidan "kredit va kreditlash" tushunchasi avvalgiga nisbatan ancha ko‘proq bahs va munozaralarga sabab bo‘lmoqda. Bu albatta, bejizga emas, chunki bugungi kunda 2 \| BEST SCIENTISTS -2023 BEST SCIENTISTS -2023 -2023 respublikamiz tijorat banklarining kredit portfelida kreditlarning ulushi, uni kamaytirish choralari ko‘rilishiga qaramasdan anchagina yuqori foizni tashkil etmoqda. 3 Saeid Khajeh Dangolania. The impact of information technology in banking system (A case study in bank Keshavarzi Iran) // Procedia - social and behavioral sciences, Vol. 30, 2011. – 13-16 p. ADABIYOTLAR TAHLILI Tijorat banklarida kredit portfelini boshqarish samaradorligini oshirish masalalari bir qator xorijiy olimlarning ilmiy tadqiqotlarida o‘rganilgan va kredit portfeli tushunchasini uning mohiyatiga berilgan ta’riflarda ifodalangan. Masalan amerikalik iqtisodchilar Kris J. Barlton, Diana Mak Noton kredit portfeli - bu kreditlarni turkumlashni o‘z ichiga oladi deb ta’riflashadi. Shuningdek, N. Sokolinskaya “kredit portfeli qiska va uzoq muddatli kreditlar yig‘indisidan iborat” deb ta’riflaydi. Bu ta’rifda asosiy e’tibor kreditning muddatiga qaratilgan bu holat kredit portfelining mohiyatini to‘liq ochib bermaydi. Chunki, bank tomonidan berilgan kreditlarning muddatini belgilab qo‘yilishi va unga rioya qilinishi faqat kredit portfelining sifatini aniqlashda muhim omil bo‘lishi mumkin. O‘z o‘rnida, bank sektorining ichki va tashqi ish faoliyatlari ITlarning rivojlanishi bilan yanada rivojlanib bormoqda va bu eng katta ta’sir qilgan soha sifatida bank tizimini ko‘rsatish mumkin3. Yuqorida o‘rganilgan holatlarga asoslanib, respublikamiz tijorat banklarida jismoniy shaxslarni kreditlash sohasini boshqarish samaradorligini oshirish yo‘llari yuzasidan izlanishlar olib borilishini doimo dolzarb deb hisoblaymiz. TADQIQOT METODOLOGIYASI VA EMPIRIK TAHLIL 3 \| 3 BEST SCIENTISTS -2023 -2023 -2023 BEST SCIENTISTS Mazkur tadqiqotda statistik jadval va grafiklar, analitik taqqoslash, mantiqiy va taqqoslama tahlil, guruhlash usullari hamda mavzuga oid xorijiy va mahalliy olimlarning tadqiqot ishlaridan keng foydalanilgan. Shuningdek maqolaning nazariy va uslubiy asosi sifatida umumiqtisodiy adabiyot hamda ilmiy maqolalar, iqtisodchi olimlarning tijorat banklarida muddati o’tgan kreditlarni samarali boshqarish masalalari bo‘yicha izlanishlari, olimlar va soha vakillari bilan suhbat, ularning yozma va og‘zaki fikr-mulohazalarini tahlil qilish, ekspert baholash, jarayonlarni kuzatish, iqtisodiy hodisa va jarayonlarga tizimli yondashuv, muallif tajribalari bilan qiyosiy tahlil o‘tkazish orqali tegishli yo‘nalishlarda xulosa, taklif va tavsiyalar berilgan. Mavzuni o‘rganish jarayonida umumiqtisodiy usullar bilan bir qatorda ma’lumotlarni tizimlash bo‘yicha maxsus yondashuvlar, ya’ni taqqoslash, nazariy va amaliy materiallarni jamlash hamda tizimli tahlil kabi usullar qo‘llanilgan. 4 Azlarova Aziza Axrorovna, “Tijorat banklarida kredit portfelini samarali boshqarish masalalari” “Iqtisodiyot va innovatsion texnologiyalar” ilmiy elektron jurnali. № 6, noyabr-dekabr, 2018 yil 5-b , j p q innovatsion texnologiyalar” ilmiy elektron jurnali. № 6, noyabr-dekabr, 2018 yil 5-b Azlarova Aziza Axrorovna, “Tijorat banklarida kredit portfelini samarali boshqarish masalalari” “Iqtisodiyo 4 Azlarova Aziza Axrorovna, “Tijorat banklarida kredit portfelini samarali boshqarish masa innovatsion texnologiyalar” ilmiy elektron jurnali. № 6, noyabr-dekabr, 2018 yil 5-b 5 O`zbekistin Respublikasi Markaziy banki ma`lumotlaridan foydalanilgan. TAHLIL VA NATIJALAR Bank aktiv operatsiyalari ichida kredit asosiy o‘rinni egallaydi va bank daromadining salmoqli qismi ham aynan mazkur operatsiyalar orqali olinadi. Shu sababli bank kredit portfelining qanday shakllanishi bank faoliyatiga bevosita ta’sir ko‘rsatadi.4 Respublikamiz bank tizimini rivojlantirish, ko‘rsatilayotgan bank xizmatilarini sifati va turlarini oshirish maqsadida, O’zbekiston Respublikasi Prezidentining “Respublika bank tizimini yanada rivojlantirish va barqarorligini oshirish bo‘yicha chora-tadbirlar to‘g‘risida”gi PQ 3270 sonli qarori bilan tasdiqlangan chora- tadbirlar Dasturining ijrosi yuzasidan O’zbekiston Banklar assotsiatsiyasi tijorat 4 \| 4 Azlarova Aziza Axrorovna, “Tijorat banklarida kredit portfelini samarali boshqarish masalalari” “Iqtisodiyot va innovatsion texnologiyalar” ilmiy elektron jurnali. № 6, noyabr-dekabr, 2018 yil 5-b 4 \| BEST SCIENTISTS -2023 -2023 -2023 banklari bilan birgalikda amaldagi bank xizmatlariga belgilangan tariflar qayta ko‘rib chiqildi. 2023-yil 1-aprel holatiga ko`ra tijorat banklarida ajratilgan kreditlar miqdori 408,167 mlrd. so`mni tashkil etgan bo`lib, bu oldingi yillarga nisbatan katta o`sish bo`lganligidan dalolat beradi.(1-rasm) 1-rasm. Tijorat banklarining kredit va depozitlar to`g`risida 2023-yil 1- holatidagi statistik jadvali5 1-rasm. Tijorat banklarining kredit va depozitlar to`g`risida 2023-yil 1- 1-rasm. Tijorat banklarining kredit va depozitlar to`g`risida 2023-yil 1- holatidagi statistik jadvali5 6 www.bank.uz sayti ma’lumotlari asosida. holatidagi statistik jadvali5 Ist’emol krediti o’zining maqsadi bilan kreditning boshqa shakllaridan farq qiladi. Uning farqli belgisi - jismoniy shaxslarni kreditlash hisoblanadi. Kreditning bu shaklida kredit beruvchi sifatida maxsus kredit muassasalari bilan birga tovar va xizmatlarni sotishni amalga oshiradigan jismoniy shaxslar ham bo’lishi mumkin. 5 \| O zbekistin Respublikasi Markaziy banki ma lumotlaridan foydalanilgan. 5 \| BEST SCIENTISTS -2023 BEST SCIENTISTS -2023 Iste’mol krediti ikki shaklda: pul shaklida yoki tovar shaklida berilishi mumkin. Jismoniy shaxslarga ko’chmas mulkka egalik qilish uchun, qimmat bo’lgan davolanishni to’lash, har xil tovarlar va uy jihozlari sotib olish va boshqa ehtiyojlarni qondirish uchun ist’emol kreditlari berilishi mumkin. Pul shaklida ist’emol krediti banklar tomonidan, tovar shaklida esa tovarlar chakana savdosi jarayonida to’lov muddatini cho’zish orqali amalga oshiriladi. O’zbekistonda hozirgi kunlarda uy-joy sotib olish, uy-joy qurish uchun pul shaklidagi, uzoq muddatli ist’emol krediti va tovar shaklida avtomobil kreditga berilmoqda. 7 Shagazatov O “Ist’mol kreditining rivojlanish omillari”.// Bozor, Pul va Kredit jurnali. –T.: 2022. -№7. -6 2-rasm. Ommabop bo`lgan bank iste’mol kreditlari6 Respublikamizda aholining daromad darajasining oshayotganligi, tijorat banklarining resurs bazasini mustahkamlanayotganligi va kreditlarning foiz stavkalarini pasayayotganligi aholining tijorat banklarining iste’mol kreditlaridan 6 \| www.bank.uz sayti ma lumotlari asosida. 6 \| BEST SCIENTISTS -2023 -2023 -2023 BEST SCIENTISTS foydalanish darajasini oshirish imkonini bermoqda. Ayniqsa, maishiy asbobusukunalar xarid qilish, ta’lim olish bilan bog’liq bo’lgan iste’mol kreditlari hajmining yildan-yilga o’sishi kuzatilmoqda.7 Yuqorida qayd etilgan ijobiy jihatlar bilan birga, iste’mol kreditlarini berish amaliyotini takomillashtirish bilan bog’liq bo’lgan ayrim muammolarning mavjudligi kuzatilmoqda. Ana shunday muammmolardan biri – ayrim tijorat banklari tomonidan berilayotgan iste’mol kreditlari bahosining nisbatan yuqori ekanligidir. Hozirgi kunda O’zbekistonda aholiga iste’mol kreditlari berish tartibini amaliyotga joriy etayotgan kredit tashkilotlarining soni kundan kunga ko’payib bormoqda. Odatda, uy-joyni tanlash uchun vaqt cheklangan bo’ladi. So’ngi yillarda O’zbekiston tijorat banklari tomonidan jismoniy shaxslarga mo’ljallangan kredit mahsulotlari turi ko’payishiga qaramasdan iste’mol kreditlari eng ommabop kredit mahsuloti bo’lib turibdi (1-jadval). № Ko’rsatkichlar 2019 2020 2021 2022 Jami kreditlar portfeli 52603,1 110566,2 167287,8 210029,0 Ajratilgan kreditlar 27562,4 49540,5 100658,2 140762,4 Jismoniy shaxslarga 4698,7 6082,0 15390,1 26477,8 Shundan, ipoteka kreditlar 1856,7 2628,8 4557,2 7977,4 Iste’mol kreditlar 67,4 97,2 8102,8 11108,5 1-jadval. O’zbekiston tijorat banklarining jismoniy shaxslarni kreditlash faoliyati ko’rsatkichlari 1-jadval. O’zbekiston tijorat banklarining jismoniy shaxslarni kreditlash faoliyati ko’rsatkichlari Jadval ma’lumotlaridan ko’rish mumkinki, 2021 yilda jami 15,4 trln. so’mga yaqin kredit ajratilgan bo’lib, bu ko’rsatkich 26,5 trln. so’mga yetgan. Biroq, mazkur ajratilgan kreditlar tarkibida iste’mol kreditlarining ulushi 2021 yilda 52,6 foizni 7 \| BEST SCIENTISTS -2023 -2023 BEST SCIENTISTS -2023 tashkil etgan bo’lsa, 2022 yilda bu ko’rsatkich 42,0 foizga tushgan. Bunga bozorda overdraft, mikroqarz, kredit karta kabi yangi turdagi kredit mahsulotlarining taklif etila boshlaganligi bilan izohlanadi. XULOSA VA MUHOKAMALAR Xulosa qilib aytganda, banklarning jismoniy shaxslarni kreditlash amaliyotini takomillashtirish xususida turli mazmundagi nazariy qarashlar mavjud. Ammo ko‘pchilik iqtisodchi olimlar banklarning kreditlash amaliyotini takomillashtirishning zaruriy shartlari sifatida ipoteka kreditlarining ikkilamchi bozorining mavjud bo‘lishini, risklarni boshqarish tizimining rivojlanganligi va ipoteka kreditlarining resurs ta’minotini e’tirof etadilar. Bizning fikrimizcha mamlakatimiz tijorat banklari tomonidan jismoniy shaxslarga beriladigan kreditlarini maqsadida quyidagi takliflarni keltirish maqsadga muvofiq deb o‘ylaymiz:  banklarda aktivlarni keng diversifikatsiya qilish, kredit portfelini sog‘lomlashtirish orqali tavakkalchiliklarni samarali boshqarishga qaratilgan chora- tadbirlarni ishlab chiqish va iqtisodiy vaziyatdan kelib chiqib ularga zarur o‘zgartirishlar kiritib borish;  bank tizimiga oid qabul qilingan qarorlar hamda kredit munosabatlarini tartibga soluvchi me’yoriy hujjatlar ijrosini tashkil qilish asosida kreditlash jarayonida vujudga kelishi mumkin bo‘lgan xatarlarni baholash, o‘rganish, ularni tahlil qilish, biznes-reja ko‘rsatkichlari bajarilishini nazorat qilish;  bank kredit siyosati va kreditlash tamoyillari shartlariga rioya etilishini nazorat qilish.  bank kredit siyosati va kreditlash tamoyillari shartlariga rioya etilishini nazorat qilish. ADABIYOTLAR RO’YXATI ADABIYOTLAR RO’YXATI 8 \| BEST SCIENTISTS -2023 -2023 -2023 BEST SCIENTISTS 1. 2020-2025 yillarga mo‘ljallangan O‘zbekiston Respublikasining bank tizimini isloh qilish strategiyasi to‘g‘risida O‘zbekiston Respublikasi Prezidentining Farmoni, 12.05.2020 yildagi PF-5992-son ttps://lex.uz/uz/docs 1. 2020-2025 yillarga mo‘ljallangan O‘zbekiston Respublikasining bank tizimini isloh qilish strategiyasi to‘g‘risida O‘zbekiston Respublikasi Prezidentining Farmoni, 12.05.2020 yildagi PF-5992-son ttps://lex.uz/uz/docs 2. Vazirlar Mahkamasining «Banklarning kreditlari bo‘yicha qarzdorlik o‘z vaqtida qaytarilmagan taqdirda undiruvni qarzdorning likvidli molmulkiga qaratish tartibini tasdiqlash to‘g‘risida»gi №422 sonli Qarori, 04.12.2002 yil 3. O’zbekiston Respublikasining “Banklar va bank faoliyati to‘g‘risida”gi Qonun, T.: O’zbekiston, 1996 yil 25 aprel. 4. O’zbekiston Respublikasi Prezidentining “O’zbekiston Respublikasini yanada rivojlantirish bo‘yicha Harakatlar Strategiyasi to‘g‘risida”gi farmoni. 2017 yil 7 fevral. 5. O’zbekiston Respublikasi Markaziy bankining faoliyatini tubdan takomillashtirish chora-tadbirlari to‘g‘risidagi PF№5296 farmoni. 2018 yil 9 yanvar. 5. O’zbekiston Respublikasi Markaziy bankining faoliyatini tubdan takomillashtirish chora-tadbirlari to‘g‘risidagi PF№5296 farmoni. 2018 yil 9 yanvar. 6. O’zbekiston Respublikasi Prezidentining 2017 yil 12 sentyabrdagi “Respublika bank tizimini yanada rivojlantirish va barqarorligini oshirish bo‘yicha chora-tadbirlar to‘g‘risida”gi PQ 3270 sonli qarori. 7. Mirziyoev Sh.M. “Tanqidiy tahlil, qat’iy tartib-intizom va shaxsiy javobgarlik – har bir rahbar faoliyatining kundalik qoidasi bo‘lishi kerak”. T.: O’zbekiston. 2017 yil. 8. O’zbekiston Respublikasi Prezidenti Shavkat Mirziyoevning Oliy Majlisga Murojaatnomasi. 2017 y. 22.12. 9. O’zbekiston Respublikasi Prezidentining “Bank xizmatlari ommabopligini oshirish bo‘yicha qo‘shimcha chora-tadbirlar to‘g‘risida”gi № 3620 qarori. 2018 yil 23 mart. 9 \| 9 \| BEST SCIENTISTS -2023 -2023 10. O’zbekiston Respublikasi Prezidentining “Respublika bank tizimini yanada rivojlantirish va barqarorligini oshirish bo‘yicha chora-tadbirlar to‘g‘risida”gi Qarori bilan tasdiqlangan chora-tadbirlar Dasturi 2017 yil 12 sentyabr. 10. XULOSA VA MUHOKAMALAR O’zbekiston Respublikasi Prezidentining “Respublika bank tizimini yanada rivojlantirish va barqarorligini oshirish bo‘yicha chora-tadbirlar to‘g‘risida”gi Qarori bilan tasdiqlangan chora-tadbirlar Dasturi 2017 yil 12 sentyabr. 11. Tijorat banklarida aktivlar sifatini tasniflash va aktivlar bo‘yicha ehtimoliy yo‘qotishlarni qoplash uchun zaxiralar shakllantirish hamda ulardan foydalanish tartibi to‘g‘risidagi 2696-sonli nizom. 2015 yil 14 iyul. 12. Abdullaeva Sh.Z. Bank risklari va kreditlash. T.: Moliya, 2012, 124-6. 13. Пещанскаya И.В. Организатсиya деyaтелности коммерchеского банка: Уchебное пособие. -М.: ИНФРА-М, 2001,33-6. 14. Роуз П. Банковский менеджмент. Пер. с англ. – М.: Дело, 2019, 192-б. 14. Роуз П. Банковский менеджмент. Пер. с англ. – М.: Дело, 2019, 192-б. 15. Tijorat banklari moliyaviy resurslarini boshqarish. Sh.Abdullaeva, Z. Safarova.”Moliya” 2020 15. Tijorat banklari moliyaviy resurslarini boshqarish. Sh.Abdullaeva, Z. Safarova.”Moliya” 2020 16. Управление деyaтелностyu коммерchеского банка(банковский менеджмент)/ Под ред. д-ра экон. наук, проф. О.И.Лаврушина. – М.: Yuрист’, 2002, 456-б 17. O‘zbekiston Respublikasi Markaziy banki ma’lumotlari, 2020-2021 yillar 17. O‘zbekiston Respublikasi Markaziy banki ma’lumotlari, 2020-2021 yillar 10 \| 10 \|
https://openalex.org/W2163845031	https://sajs.co.za/article/download/9956/13663	English	null	Molecular phylogeny of Duvenhage virus	South African journal of science	2,011	cc-by	4,920	How to cite this article: Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http:// dx.doi.org/10.4102/sajs. v107i11/12.177 Research Letter Page 1 of 5 Molecular phylogeny of Duvenhage virus Authors: Charmaine van Eeden1 Wanda Markotter1 Louis H. Nel1 The Duvenhage virus (DUVV) constitutes one of the 11 species in the Lyssavirus genus and causes fatal rabies encephalitis. The virus is associated with insectivorous bat species and three human cases have been reported, all of which were linked to contact with bats. Few of these isolates have been studied and thus little is known about the phylogeny and epidemiology of this lyssavirus. Until 2007, when an isolate was made from the East African country of Kenya, all isolations of this virus had been from southern Africa. This discovery led to many questions regarding the spread and diversity of this lyssavirus. Phylogenetic analysis indicated that the DUVV isolates constitute two different lineages, in which the southern African isolates group together to form one lineage and the more recent isolate from Kenya constitutes a new, second lineage. We found that the new isolate has a genetic variation that has not yet been seen for DUVV. Not only is our lack of knowledge regarding the geographical distribution of this uniquely African virus emphasised, but we have also demonstrated the potential diversity within this genotype. Affiliation: 1Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, South Africa Introduction The Duvenhage virus (DUVV) belongs to the Lyssavirus genus, a group of single-stranded, negative-sense RNA viruses adapted to replicate in the mammalian central nervous system. All lyssaviruses cause fatal rabies encephalitis and previous classification recognised seven different genotypes (gt) based on the genetic diversity observed between isolates. This classification recently changed and the International Committee for Taxonomy of Viruses now recognises 11 species of lyssaviruses.1 Lyssavirus species are distinguished not only by their genetic diversity but also by other characteristics, such as serological profile, pathogenicity, host species and geographical distribution. The rabies virus (gt 1) (RABV) is the prototype lyssavirus and the other species are known as the rabies-related lyssaviruses (or non-rabies lyssaviruses), namely the Lagos bat virus (LBV) (gt 2), the Mokola virus (MOKV) (gt 3), the DUVV (gt 4), the European bat lyssavirus type 1 (EBLV1) (gt 5), the European bat lyssavirus type 2 (EBLV2) (gt 6), the Australian bat lyssavirus (ABLV) (gt 7)1 and the four most recently recognised species, Aravan (ARAV), Khujand (KHUV), Irkut (IRKV) and West Caucasian (WCBV) bat viruses.2,3 Of these viruses, only RABV, LBV, MOKV and DUVV have been isolated on the African continent, with LBV, MOKV and DUVV being exclusive to Africa. Antibodies that could neutralise WCBV have been identified in bats collected in Kenya,4 and, in 2009, a new lyssavirus species, Shimoni bat lyssavirus, was also isolated from Kenya.5 © 2011. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. S Afr J Sci 2011; 107(11/12) Materials and methods Viral isolates http://w typing or nucleotide sequencing procedures. Various studies have shown that DUVV is m related to EBLV1, which, although exclusive to also associated with insectivorous bat species. Bourhy et al.14 showed that EBLV1 shares mo with DUVV than with EBLV2. Also notable was EBLV1 and EBLV2 did not form a monophyletic EBLV1 and DUVV were more closely related to than EBLV1 was to EBLV2. In an investigation t the evolution of EBLV,16 it was found that EBL have evolved into at least two genetically dist groups, following geographical drift.15,16 When in the host species involved with DUVV and EBLV significant overlaps occurred with regard to distribution and co-colonisation in roosts. M and M. schreibersii, for example, have been fou between colonies and are also known to have di with each other in these mixed colonies.17 The larg and high population densities of many of these makes them well suited to the sustained transm exchange of RNA viruses,18 most likely through of infectious saliva during licking and biting.19 Several molecular epidemiological studies of RAB performed but only a few have been conducted EBLV1 and EBLV2,15,16 whilst there have been TABLE 1: Reports of Duvenhage virus (1970 – 2010). Accession number Year reported Isolate name Sp EU623437 1970 DUVVSA71 Ho EU623438 1981 DUVVSA81 Un po Mi sch AY062080 1986 DUVVZIM86 Ny EU623444 2006 DUVVSA06 H. FJ515696 2007 DUVVKENYA H. Note: Please see the full reference list of the article, Van Eeden C, Marko org/10.4102/sajs.v107i11/12.177, for more information. a, New taxonomic classification: Miniopterus natalensis.12 All three DUVV isolates from South Africa (DUVVSA71, DUVVSA81 and DUVVSA06) (Table 1) were amplified in the brains of suckling mice. The procedures were approved by the Agricultural Research Council–Onderstepoort Veterinary Institute (ethics approval reference number 15/4P001). Lyophilised passaged brain material was reconstituted in sterile phosphate buffered saline (PBS) (13.7 mmol NaCl, 0.27 mM KCl, 0.43 mM Na2HPO4.2H2O, 0.14 mM KH4PO4, pH 7.3). Two-day-old or three-day-old suckling mice received 30 μL of the reconstituted material intracranially.24 Animals were monitored and upon their deaths their brain material was removed aseptically. The direct fluorescent antibody test was used for post-mortem diagnosis of lyssavirus infection according to the standard operational procedure,25 with a polyclonal fluorescein isothiocyanate conjugated immunoglobulin (Onderstepoort Veterinary Institute, Rabies Unit, Pretoria, South Africa) used at a 1:500 dilution. Brain material that tested positive for lyssavirus was pooled and used for RNA extraction. Dates: © 2011. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. Based on phylogeny, pathogenicity and serological cross-reactivity, the lyssaviruses have been split into three phylogroups.2,3,6 Phylogroup I consists of RABV, DUVV, EBLV1, EBLV2, ABLV, Aravan, Khujand and Irkut. Phylogroup II consists of MOKV and LBV and Phylogroup III is WCBV.4 All experimental evidence to date suggests that commercial vaccines protect against only the Phylogroup I lyssaviruses and not members of Phylogroups II and III.3,7,8 Nevertheless, cross protection between some lyssavirus species is possible as a result of a similarity in glycoprotein antigenic sites towards which neutralising antibodies are directed.9 DUVV, for which there have been only five reported cases to date (Table 1), was first encountered in South Africa in 1970 following a human fatality that was associated with a contact exposure involving an unidentified bat.10 DUVV was later isolated from two insectivorous bats. One was suggested to be a member of the Miniopteridae (Miniopterus schreibersii) because of the wide distribution of this genus in the area in South Africa where the exposure occurred.11 New classification now indicates that the bat species previously identified as M. schreibersii in Africa is now considered to be Miniopterus natalensis12; the other isolation was from Nycteris thebaica in Zimbabwe in 1986.13 DUVV was again identified 36 years later in two human fatalities: in South Africa in 2006 and a few months later in Kenya in 2007. In both of these recent cases, there was an exposure to a small bat (probably insectivorous) but the bats were never found or positively identified. These isolations support the notion that the incidence of African rabies- © 2011. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License. S Afr J Sci 2011; 107(11/12) Research Letter Page 2 of 5 related lyssaviruses is underestimated as a result of poor surveillance influenced by the limited diagnostic capacities in African laboratories. Very few laboratories are capable of making a rabies diagnosis with the recommended fluorescent antibody test – which is, in any event, non-differentiating because of the broad spectrum lyssavirus conjugate used. Of these laboratories, only one national African laboratory regularly types positive specimens by monoclonal antibody typing or nucleotide sequencing procedures. Materials and methods Viral isolates Various studies have shown that DUVV is most closely related to EBLV1, which, although exclusive to Europe, is also associated with insectivorous bat species. A study by Bourhy et al.14 showed that EBLV1 shares more epitopes with DUVV than with EBLV2. Also notable was the fact that EBLV1 and EBLV2 did not form a monophyletic group,15 as EBLV1 and DUVV were more closely related to each other than EBLV1 was to EBLV2. In an investigation to determine the evolution of EBLV,16 it was found that EBLV1 isolates have evolved into at least two genetically distinguishable groups, following geographical drift.15,16 When investigating the host species involved with DUVV and EBLV1 infection, significant overlaps occurred with regard to geographic distribution and co-colonisation in roosts. Myotis myotis and M. schreibersii, for example, have been found to move between colonies and are also known to have direct contact with each other in these mixed colonies.17 The large roost sizes and high population densities of many of these bat species makes them well suited to the sustained transmission and exchange of RNA viruses,18 most likely through the transfer of infectious saliva during licking and biting.19 Dates: number of studies focusing on the African lyssavirus.21,22,23 With so little known about the molecular epidemiology of DUVV, and with the addition of two relatively recent viruses to the small pool of known DUVVs, the objective of this study was to investigate the relationship between these DUVV isolates relative to the diversity found in the Lyssavirus genus. RNA extraction, reverse transcription polymerase chain reaction and direct sequencing Several molecular epidemiological studies of RABV have been performed but only a few have been conducted on ABLV,20 EBLV1 and EBLV2,15,16 whilst there have been a limited Total RNA was extracted using a Trizol® reagent (Invitrogen, Cape Town, South Africa) according to the manufacturer’s instructions. The reverse transcription polymerase chain TABLE 1: Reports of Duvenhage virus (1970 – 2010). Accession number Year reported Isolate name Species reported Country reported Comments Reference EU623437 1970 DUVVSA71 Homo sapiens Limpopo Province, South Africa The Duvenhage virus was first isolated in February 1970, after a 31-year-old man (Mr Duvenhage) died in hospital following a 5-day illness diagnosed as clinical rabies. The source of exposure was reported to be a bat bite to the lip, which the victim had sustained at his home on the farm Tooyskraal (in Bela Bela, formerly Warmbaths), 100 km north-east of Pretoria 5 weeks earlier. The bat was not collected for identification, but circumstantial evidence suggests it may have been Schreiber’s long-fingered bat, Miniopterus schreibersiia. 10,11 EU623438 1981 DUVVSA81 Unconfirmed, possibly Miniopterus schreibersiia Limpopo Province, South Africa Isolated from an unidentified bat which was killed by a cat in the Louis Trichardt area. 11,12 AY062080 1986 DUVVZIM86 Nycteris thebaica Bulawayo, Zimbabwe Isolated from an Egyptian slit-faced bat, Nycteris thebaica, in Bulawayo, Zimbabwe during a survey for lyssaviruses. 13 EU623444 2006 DUVVSA06 H. sapiens North West Province, South Africa A 77-year-old man died from a rabies-like illness, after being scratched on the face by what appeared to be an insectivorous bat in February 2006. The victim did not seek medical attention for the scratch and became ill 27 days later and died 2 weeks thereafter. 21 FJ515696 2007 DUVVKENYA H. sapiens Tsavo West, Kenya A 34-year-old woman was scratched on the face by an unidentified bat whilst camping in Tsavo West National Park. The woman became ill and was admitted to an Amsterdam hospital in the Netherlands. After diagnosis the ‘Wisconsin rabies treatment protocol’ was initiated. However, the patient succumbed to the virus several days later. 32,33,34 Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http://dx.doi. org/10.4102/sajs.v107i11/12.177, for more information. Results Phylogenetic trees including all five DUVV isolates as well as representative isolates of other lyssavirus species (Table 2) were constructed using a 398-bp fragment of the nucleoprotein gene (nt 8–406). The neighbour-joining method indicated low bootstrap support (71% and lower) for all major clusters representing DUVV, EBLV1 and EBLV2, as well as the newly recognised species ARAV, KHUV and IRKV (Figure 1). DUVV, EBLV1 and EBLV2 all split into two lineages: EBLV1a and EBLV1b; EBLV2a and EBLV2b; and DUVV Lineage A (isolates from southern Africa – South Africa and Zimbabwe) and DUVV Lineage B (the isolate from Kenya). These groupings were supported by high bootstrap values (95% and higher). Maximum parsimony phylogenetic analysis also indicated the major clusters representing the different lyssavirus species as well as the distinct DUVV lineages (results not shown). Analysis of genetic distances between all five DUVV isolates was carried out using a well-conserved 398-nt sequence from the nucleoprotein gene (nt 8–406). The intrinsic variation between DUVV isolates from southern Africa was low, with a 97.7% – 100% nt identity, even though these isolates were isolated several years apart (ranging from 1971 to 2006). Isolate DUVVZIM86 was found to be 100% identical to DUVVSA81 for this region of the genome, although they were isolated 5 years apart in different countries. The East African isolate, DUVVKenya, had a much lower sequence identity (88.9% – 89.7%) than the other DUVV isolates, which supports the phylogenetic analysis that suggested this isolate forms part of a different lineage. DUVVKenya was shown to be most similar to DUVVSA71 (89.7%) – the first DUVV to have been isolated. RNA extraction, reverse transcription polymerase chain reaction and direct sequencing a, New taxonomic classification: Miniopterus natalensis.12 S Afr J Sci 2011; 107(11/12) http://www.sajs.co.za Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http://dx.doi. org/10.4102/sajs.v107i11/12.177, for more information. a, New taxonomic classification: Miniopterus natalensis.12 Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http://dx.doi. org/10.4102/sajs.v107i11/12.177, for more information. a, New taxonomic classification: Miniopterus natalensis.12 Research Letter Page 3 of 5 of Saitou and Nei31. The branching order of the trees was evaluated by using bootstrap analysis of 1000 pseudoreplicate data sets. Results were validated by maximum parsimony as implemented in MEGA 3.1.29 reaction (RT-PCR) was performed with the primer set described by Markotter et al.26 Complementary DNA was obtained during reverse transcription with Lys001 (25 °C for 10 min, 42 °C for 60 min and 85 °C for 5 min) in the presence of deoxyribonucleotide triphosphate (10 mM) and avian myeloblastosis virus reverse transcriptase (20 U/μL, Roche Diagnostics, Indianapolis, Indiana, USA) and subjected to 30 PCR cycles (94 °C, 30 s; 37 °C, 30 s, 72 °C for 90 s supplemented by a final extension for 7 min at 72 °C) in the presence of both Lys001 and Lys304 primers using BiolineTaq polymerase (5 U/μL, Celtic Molecular Diagnostics, Cape Town, South Africa). After gel electrophoresis, amplicons were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Fitchburg, Wisconsin, USA) according to the manufacturer’s instructions. Purified amplicons were then subjected to 25 cycles of sequencing (94 °C for 10 s and 50 °C for 5 s) and a final cycle at 60 °C for 4 min using a BigDye® Terminator v3.1 Kit (Applied Biosystems, Foster City, CA, USA) and either a sense or antisense primer. Reactions were stored at -20 °C before precipitation using the EDTA/ NaOAc/EtOH method according to the BigDye® Terminator v3.1 cycle sequencing protocol (Applied Biosystems). The precipitated reactions were submitted to the sequencing facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria and analysed on an ABI 3100 automated capillary sequencer analyser (Applied Biosystems). Phylogenetic analysis Nucleotide sequencing data were assembled using the VectorNTI 9.1.0 software package (Invitrogen) and trimmed using the BioEdit software package.27 Alignments including the DUVVKenya and DUVVZIM86 (Table 1), as well as other representative lyssavirus sequences (Table 2) available in the public domain, were then carried out using ClustalW.28 Only nt 8–406 of the nucleoprotein gene was used in this analysis for comparison with the DUVVKENYA isolate for which only a partial sequence was available in the public domain. The calculation of genetic distances and the construction of phylogenetic trees based on nucleotide sequence were performed using MEGA 3.1 software.29 Genetic distances were calculated between pairs of sequences using Kimura’s 2-parameter method,30 and based on these distances neighbour-joining trees were constructed using the method Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(1 org/10.4102/sajs.v107i11/12.177, for more information. http://www.sajs.co.za Discussion O Bootstrap values are indicated at the nodes and branch lengths are drawn to scale. p g FIGURE 1: Neighbour-joining tree of nt 8–406 of the nucleoprotein gene indicating the phylogeny of all the Duvenhage virus isolates (the rabies virus, RABV, was used as the outgroup) FIGURE 1: Neighbour-joining tree of nt 8–406 of the nucleoprotein gene indicating the phylogeny of all the Duvenhage virus isolates (the rabies virus, RABV, was used as the outgroup). FIGURE 1: Neighbour-joining tree of nt 8–406 of the nucleoprotein gene indicating the phylogeny of all the Duvenhage virus isolates ( the outgroup). DUVV. Despite the lack of widespread surveillance, our data suggest that the DUVV cycle is likely to be well established and stable in insectivorous bat species. The relationship between genomic evolution in lyssaviruses and the host’s response to infection could explain the rarity of human or other terrestrial mammal encounters with this virus. Lyssavirus genus. Based on partial nucleoprotein sequences, we confirmed a clear separation of the DUVV isolates from those of EBLV1, although these two groups are most closely related within the lyssaviruses. The DUVV isolates examined also constitute two separate lineages with the longer branch lengths suggesting that these two lineages evolved from an earlier separation than did the lineages of EBLV1. Intrinsic heterogeneity between the DUVV isolates also allows for a clear differentiation between these two lineages. Lineage A isolates, which are from southern Africa, were found to have less than 2% nucleotide variation, even though the isolates were obtained up to 36 years apart. Lineage B, at present consisting solely of the DUVV isolate from Kenya, was found to vary from the lineage A isolates, with respect to this region of the viral RNA, by 11%. Lyssavirus genus. Based on partial nucleoprotein sequences, we confirmed a clear separation of the DUVV isolates from those of EBLV1, although these two groups are most closely related within the lyssaviruses. The DUVV isolates examined also constitute two separate lineages with the longer branch lengths suggesting that these two lineages evolved from an earlier separation than did the lineages of EBLV1. Intrinsic heterogeneity between the DUVV isolates also allows for a clear differentiation between these two lineages. Lineage A isolates, which are from southern Africa, were found to have less than 2% nucleotide variation, even though the isolates were obtained up to 36 years apart. Discussion O Our investigation considered the diversity amongst all known DUVV isolates relative to other members of the TABLE 2: Lyssaviruses included in the phylogenetic analysis. Lyssavirus type Accession number Year reported Species reported Country reported Reference EBLV-1 AY863375 1995 Eptesicus serotinus Denmark 15 AY863380 2002 Sheep Denmark 15 EF157976 1968 E.serotinus Germany 35 AY863397 2000 E. serotinus France 15 AY863383 1992 E. serotinus the Netherlands 15 EBLV-2 AY863406 1986 Homo sapiens Finland 15 AY863407 1993 Myotis daubentonii Switzerland 15 EF157977 2002 H. sapiens United Kingdom 35 AY863405 1989 Myotis dasycneme the Netherlands 15 Irkut AY333112 2002 Murina leucogaster Russia 36 Aravan AY262023 1991 Myotis blythi Kyrgyzstan 36 Khujand AY863405 2001 M. daubentonii Tajikistan 36 Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http://dx.doi. org/10.4102/sajs.v107i11/12.177, for more information. Note: Please see the full reference list of the article, Van Eeden C, Markotter W, Nel LH. Molecular phylogeny of Duvenhage virus. S Afr J Sci. 2011;107(11/12), Art. #177, 5 pages. http://dx.doi. org/10.4102/sajs.v107i11/12.177, for more information. R Research Letter Genotype 5 Genotype 4 Genotype 6 Lineage A Lineage A Lineage A AY863375 AY863380 EF157976 AY863397 AY863383 DUVV Kenya EU623444 DUVVSA06 EV623437 DUVVSA71 AY062080 DUVVZIM86 EU623438 DUVVSA81 AY333112 Irkut AY262023 Aravan AY262024 Khujand AY863406 AY863407 EF157977 AY863405 M13215 RABV PV Lineage B Lineage B Lineage B 0.05 96 95 100 52 55 67 48 58 99 100 100 100 72 98 94 GenBank accession numbers are indicated for each isolate. Bootstrap values are indicated at the nodes and branch lengths are drawn to scale. FIGURE 1: Neighbour-joining tree of nt 8–406 of the nucleoprotein gene indicating the phylogeny of all the Duvenhage virus isolates (the rabies virus, RABV, was used as the outgroup). Page 4 of 5 Genotype 5 Genotype 4 Genotype 6 Lineage A Lineage A Lineage A AY863375 AY863380 EF157976 AY863397 AY863383 DUVV Kenya EU623444 DUVVSA06 EV623437 DUVVSA71 AY062080 DUVVZIM86 EU623438 DUVVSA81 AY333112 Irkut AY262023 Aravan AY262024 Khujand AY863406 AY863407 EF157977 AY863405 M13215 RABV PV Lineage B Lineage B Lineage B 96 95 100 52 55 67 48 58 99 100 100 100 72 98 94 GenBank accession numbers are indicated for each isolate. Bootstrap values are indicated at the nodes and branch leng GenBank accession numbers are indicated for each isolate. References 24. Koprowski H. The mouse inoculation test. In: Meslin FX, Kaplan MM, Koprowski H, editors. Laboratory techniques in rabies. Geneva: World Health Organization, 1996; p. 80–87. 1. International Committee for Taxonomy of Viruses. Official taxonomy: Updates since the 8th report [document on the Internet]. c2009 [cited 2008 Sept. 12]. Available from: http://talk.ictvonline.org/media/p/1208.aspx 1. International Committee for Taxonomy of Viruses. Official taxonomy: Updates since the 8th report [document on the Internet]. c2009 [cited 2008 Sept. 12]. Available from: http://talk.ictvonline.org/media/p/1208.aspx 25. Centers for Disease Control. DFA diagnosis [document on the Internet]. c2008 [cited 2009 July 24]. Available from: http://www.cdc.gov/ncidod/ dvrd/Rabies/Professional/Publications/DFA_diagnosis 2. Kuzmin IV, Orciari LA, Yohko TA, et al. Bat lyssaviruses (Aravan and Khujand) from central Asia: Phylogenetic relationships according to the N, P and G gene sequences. Virus Res. 2003;97:65–79. http://dx.doi. org/10.1016/S0168-1702(03)00217-X 26. Markotter W, Kuzmin I, Rupprecht CE. Isolation of Lagos bat virus from water mongoose. Emerg Infect Dis. 2006;12:1913–1918. PMid:17326944 27. Hall TA. BioEdit: A user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Res. 1999;41:95– 98. 3. Kuzmin IV, Hughes GJ, Botvinkin AD, Orciari LA, Rupprecht CE. Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssaviruses genotype definition. Virus Res. 2005;111:28– 43. http://dx.doi.org/10.1016/j.virusres.2005.03.008, PMid:15896400 28. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. http://dx.doi.org/10.1093/ nar/22.22.4673, PMid:7984417, PMCid:308517 4. Kuzmin IV, Neizgoda M, Franka R, et al. Possible emergence of West Caucasian bat virus in Africa. Emerg Infect Dis. 2008;14:1887–1889. http:// dx.doi.org/10.3201/eid1412.080750, PMid:19046512, PMCid:2634633 29. Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetic analysis and sequence alignment. Brief Bioinform. 2004;5:150–163. http://dx.doi.org/10.1093/bib/5.2.150, PMid:15260895 5. Kuzmin IV, Mayer AE, Niezgoda M, et al. Shimoni bat virus, a new representative of the Lyssavirus genus. Virus Res. 2010;149:197–210. http:// dx.doi.org/10.1016/j.virusres.2010.01.018, PMid:20138934 30. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;15:111–120. http://dx.doi.org/10.1007/BF01731581, PMid:7463489 6. Badrane H, Bahloul C, Perrin P, Tordo N. Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity. J Virol. 2001;75:3268–3276. http://dx.doi.org/10.1128/JVI.75.7.3268-3276.2001, PMid:11238853, PMCid:114120 31. Saitou N, Nei M. The neighbour-joining method: A new method for constructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425. PMid:3447015 7. Competing interests 18. Mackensie JS, Field HE, Guyatt KJ. Managing emerging diseases borne by fruit bats (flying foxes) with particular reference to henipaviruses and Australian bat lyssavirus. J Appl Microbiol. 2003;94:59–69. http://dx.doi. org/10.1046/j.1365-2672.94.s1.7.x We declare that we have no financial or personal relationships which may have inappropriately influenced us in writing this article. 19. Ghatak S, Banerjee R, Agarwak RK, Kapoor KN. Zoonoses and bats: A look from human health viewpoint. J Comm Dis. 2000;32:40–48. PMid:11129564 20. Guyatt KJ, Twin J, Davis P, et al. A molecular epidemiological study of Australian bat lyssavirus. J Gen Virol. 2003;84:485–496. http://dx.doi. org/10.1099/vir.0.18652-0, PMid:12560583 Discussion O Lineage B, at present consisting solely of the DUVV isolate from Kenya, was found to vary from the lineage A isolates, with respect to this region of the viral RNA, by 11%. As DUVV is present in bat populations, persons interacting with these animals should follow appropriate precautions, including vaccination. Considering the global mobility of humans and animals – and the presence of potential vector species across the globe – African lyssaviruses can easily pose a threat to any continent. It is thus of importance that laboratory, veterinary and medical personnel understand and recognise the resultant public health implications. The demonstrated separation between the Kenyan and the southern African DUVV isolates is indicative of significant spatial evolution that is clearly underestimated by current data, as it may be taken for granted that DUVV cycles are not restricted to South Africa, Zimbabwe and Kenya. It is imperative that more, active surveillance programmes are initiated in order to establish more information about the distribution, prevalence, genetic diversity and host species associated with DUVV. As a result of a number of surveillance programmes for EBLV1, many isolates have been made over the past few decades, demonstrating the significant activity of EBLV1 in bat populations in Eurasia.16 EBLV1a shows phylogenetic homogeneity between isolates across geographical regions, possibly as a result of viral traffic amongst bat populations.18 For EBLV1b, however, geographic origin plays a significant role in phylogenetic clusters, as there is less contact between bat populations18; these observations may also hold true for S Afr J Sci 2011; 107(11/12) S Afr J Sci 2011; 107(11/12) http://www.sajs.co.za http://www.sajs.co.za Page 5 of 5 Authors’ contributions 21. Paweska JT, Blumberg LH, Liebenberg C, et al. Fatal human infection with rabies-related Duvenhage virus, South Africa. Emerg Infect Dis. 2006;12:1965–1966. PMid:17326954 W.M. and L.H.N. conceived the experiments and W.M., L.H.N. and C.v.E. designed the experiments. C.v.E. performed the experiments and analysed the data. C.v.E., W.M. and L.H.N. wrote the article. W.M. and L.H.N. conceived the experiments and W.M., L.H.N. and C.v.E. designed the experiments. C.v.E. performed the experiments and analysed the data. C.v.E., W.M. and L.H.N. wrote the article. W.M. and L.H.N. conceived the experiments and W.M., L.H.N. and C.v.E. designed the experiments. C.v.E. performed the experiments and analysed the data. C.v.E., W.M. and L.H.N. wrote the article. 22. Sabeta CT, Markotter W, Mohale DK, Shumba W, Wandeler AI, Nel LH. Mokola virus in domestic mammals, South Africa. Emerg Infect Dis. 2007;13:1371–1373. PMid:18252112, PMCid:2857310 23. Markotter W, Kuzmin I, Rupprecht CE, Nel LH. Phylogeny of Lagos bat virus: Challenge for lyssavirus taxonomy. Virus Res. 2008;135:10–21. http://dx.doi.org/10.1016/j.virusres.2008.02.001, PMid:18359532 Acknowledgements 14. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic and molecular characterization of bat rabies virus in Europe. J Clin Microbiol. 1992;30:2419–2426. PMid:1401009, PMCid:265516 We would like to thank Claude T. Sabeta (Onderstepoort Veterinary Institute, Pretoria, South Africa) and Janusz Paweska (National Institute for Communicable Diseases, National Health Laboratory Services, Johannesburg, South Africa) for providing the South African DUVV isolates. 15. Davis PL, Holmes EC, Larrous F, et al. Phylogeography, population dynamics and molecular evolution of European bat lyssaviruses. J Virol. 2005;79:10487–10497. http://dx.doi.org/10.1128/JVI.79.16.10487- 10497.2005, PMid:16051841, PMCid:1182613 16. Amengual B, Whitby JE, King A, Serra Cobo J, Bourhy H. Evolution of European bat lyssaviruses. J Gen Virol. 1997;78:2319–2328. PMid:9292021 17. Serra-Cobo J, Amengual B, Abellan C, Bourhy H. European bat lyssavirus infection in Spanish bat populations. Emerg Infect Dis. 2002;8:413– 420. http://dx.doi.org/10.3201/eid0804.010263, PMid:11971777, PMCid:2730232 References Fekadu M, Shaddock JH, Sanderlin DW, Smith JS. Efficacy of rabies vaccines against Duvenhage virus isolated from European house bats (Eptesicusserotinus), classic rabies and rabies-related viruses. Vaccine. 1988;6:533–539. http://dx.doi.org/10.1016/0264-410X(88)90107-7 32. Willoughby RE, Tieves KS, Hoffman GM, et al. Survival after treatment of rabies with induction of a coma. New Engl J Med. 2005;352:2508–2514. http://dx.doi.org/10.1056/NEJMoa050382, PMid:15958806 8. Bahloul C, Jacob Y, Tordo N, Perrin P. DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses. Vaccine. 1998;16:417–425. http://dx.doi.org/10.1016/S0264- 410X(97)00204-1 33. Van Thiel P, Van Den Hoek J, Eftimov F, et al. Fatal case of human rabies (Duvenhage virus) from a bat in Kenya: The Netherlands, December 2007. Eurosurveillance. 2008;13:1. 9. Benmansour A, Leblois H, Coulon P. Antigenicity of rabies virus glycoprotein. J Virol. 1991;65:4198–4203. PMid:1712859, PMCid:248855 34. Van Thiel PAM, De Bie RMA, Eftimov F, et al. Fatal human rabies due to Duvenhage virus from a bat in Kenya: Failure of treatment with coma-induction, ketamine and antiviral drugs. PLOS Negl Trop Dis. 2009;3(7):e428. http://dx.doi.org/10.1371/journal.pntd.0000428, PMid:19636367, PMCid:2710506 10. Meredith CD, Rossouw AP, Van Praag Koch H. An unusual case of human rabies thought to be of chiropteran origin. S Afr Med J. 1971;45:767–769. PMid:5106834 35. Marston DA, McElhinney LM, Johnson N, et al. Comparative analysis of the full genome sequence of European bat lyssavirus type 1 and 2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the G-L 3’ non translated region. J Gen Virol. 2007;88:1302–1314. http://dx.doi.org/10.1099/vir.0.82692-0, PMid:17374776 11. Van der Merwe M. Bats as vectors of rabies. SAfr J Sci. 1982;78:421–422. 12. Miller-Butterworth CM, Eick G, Jacobs DS, Schoeman MC, Harley EH. Genetic and phenotypic differences between South African long-fingered bats, with global Miniopterine phylogeny. J Mammol. 2005;86:1121–1135. http://dx.doi.org/10.1644/05-MAMM-A-021R1.1 36. Kuzmin IV, Wu X, Tordo N, Rupprecht CE. Complete genomes of Aravan, Khujand, Irkut and West Caucasian bat viruses, with special attention to the polymerase gene and non-coding regions. Virus Res. 2008;136:81–90. http://dx.doi.org/10.1016/j.virusres.2008.04.021, PMid:18514350 13. Foggin CM. Rabies and rabies-related viruses in Zimbabwe: Historical, virological and ecological aspects. PhD thesis, Harare, University of Zimbabwe, 1988. S Afr J Sci 2011; 107(11/12) http://www.sajs.co.za
https://openalex.org/W3153224075	https://www.research-collection.ethz.ch/bitstream/20.500.11850/518998/2/2021.eacl-main.163.pdf	English	null	Applying the Transformer to Character-level Transduction	null	2,021	cc-by	5,038	ETH Library ETH Library 1This claim is also based on the authors’ personal commu- nication with other researchers in morphology in the corridors of conferences and through email. Code will be available at https://github.com/ shijie-wu/neural-transducer. Abstract The transformer (Vaswani et al., 2017) has been shown to outperform recurrent neural network-based sequence-to-sequence models in various word-level NLP tasks. Yet for character-level transduction tasks, e.g. mor- phological inﬂection generation and histori- cal text normalization, there are few works that outperform recurrent models using the transformer. In an empirical study, we un- cover that, in contrast to recurrent sequence- to-sequence models, the batch size plays a crucial role in the performance of the trans- former on character-level tasks, and we show that with a large enough batch size, the trans- former does indeed outperform recurrent mod- els. We also introduce a simple technique to handle feature-guided character-level trans- duction that further improves performance. With these insights, we achieve state-of-the-art performance on morphological inﬂection and historical text normalization. We also show that the transformer outperforms a strong base- line on two other character-level transduction tasks: grapheme-to-phoneme conversion and transliteration. Figure 1: Development set accuracy for 5 languages on morphological inﬂection with different batch sizes. We evince our two primary contributions: (1) we set the new state of the art morphological inﬂection using the transformer and (2) we demonstrate the transformer’s dependence on the batch size. to-sequence model with attention (Cotterell et al., 2018). This is not for lack of effort—but rather, it is the case that the transformer has consistently under- performed in experiments on average (Tang et al., 2018b).1 As anecdotal evidence of this, we note that in the 2019 SIGMORPHON shared task on cross-lingual transfer for morphological inﬂection, no participating system was based on the trans- former (McCarthy et al., 2019). Proceedings of the 16th Conference of the European Chapter of the Association for Computational Linguistics, pages 1901–1907 April 19 - 23, 2021. ©2021 Association for Computational Linguistics Shijie WuZ Ryan CotterellQ,6 Mans HuldenX ZJohns Hopkins University 6University of Cambridge QETH Z¨urich XUniversity of Colorado Boulder shijie.wu@jhu.edu ryan.cotterell@inf.ethz.ch mans.hulden@colorado.edu Shijie WuZ Ryan CotterellQ,6 Mans HuldenX ZJohns Hopkins University 6University of Cambridge QETH Z¨urich XUniversity of Colorado Boulder shijie.wu@jhu.edu ryan.cotterell@inf.ethz.ch mans.hulden@colorado.edu 16 32 64 128 256 512 Batch Size 76 78 80 82 84 86 88 90 ACC Wu and Cotterell (2019) Wu and Cotterell (2019) (Our Eval) Wu and Cotterell (2019) + LR Warmup Vanilla Transformer Feature Invariant Transformer Figure 1: Development set accuracy for 5 languages on morphological inﬂection with different batch sizes. We evince our two primary contributions: (1) we set the new state of the art morphological inﬂection using the transformer and (2) we demonstrate the transformer’s dependence on the batch size. 16 32 64 128 256 512 Batch Size 76 78 80 82 84 86 88 90 ACC Wu and Cotterell (2019) Wu and Cotterell (2019) (Our Eval) Wu and Cotterell (2019) + LR Warmup Vanilla Transformer Feature Invariant Transformer Conference Paper Conference Paper Author(s): Wu, Shijie; Cotterell, Ryan; Hulden, Mans Publication date: 2021-04 Permanent link: https://doi.org/10.3929/ethz-b-000518998 Rights / license: Creative Commons Attribution 4.0 International Originally published in: https://doi.org/10.18653/v1/2021.eacl-main.163 Author(s): Wu, Shijie; Cotterell, Ryan; Hulden, Mans g Creative Commons Attribution 4.0 International This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. 2Many successful CoNLL–SIGMORPHON shared task participants report training their models on laptop CPUs. 1 Introduction between the self-attention layer and feed-forward layer, a multi-head attention layer attends to the output of the encoder. Layer normalization (Ba et al., 2016) is applied to the output of each skip connection. Sinusoidal positional embeddings are used to incorporate positional information without the need for recurrence or convolution. Here, we describe two modiﬁcations we make to the trans- former for character-level tasks. should provide an advantage at many character- level tasks: For instance, Gehring et al. (2017) and Vaswani et al. (2017) suggest that transformers (and convolutional models in general) should be better at remembering long-range dependencies. In the case of morphology, none of these considerations seem relevant: inﬂecting a word (a) requires little capacity to model long-distance dependencies and is largely a monotonic transduction; (b) it involves no semantic disambiguation, the tokens in question being letters; (c) it is not a task for which paral- lelization during training appears to help, since training time has never been an issue in morphol- ogy tasks.2 A Smaller Transformer. As the dataset sizes in character-level transduction tasks are signiﬁcantly smaller than in machine translation, we employ a smaller transformer with N = 4 encoder-decoder layers. We use 4 self-attention heads. The em- bedding size is dmodel = 256 and the hidden size of the feed-forward layer is dFF = 1024. In the preliminary experiments, we found that using layer normalization before self-attention and the feed-forward layer performed slightly better than the original model. It is also the default setting of a popular implementation of the transformer (Vaswani et al., 2018). The transformer alone has around 7.37M parameters, excluding character em- beddings and the linear mapping before the softmax layer. We decode the model left to right in a greedy fashion. In this work, we provide state-of-the-art num- bers for morphological inﬂection and historical text normalization, a novel result in the litera- ture. We also show the transformer outperforms a strong recurrent baseline on two other character- level tasks: grapheme-to-phoneme (g2p) conver- sion and transliteration. We ﬁnd that a single hy- perparameter, batch size, is largely responsible for the previous poor results. Despite having fewer pa- rameters, the transformer outperforms the recurrent sequence-to-sequence baselines on all four tasks. We conduct a short error analysis on the task of morphological inﬂection to round out the paper. 1 Introduction The transformer (Vaswani et al., 2017) has become a popular architecture for sequence-to-sequence transduction in NLP. It has achieved state-of-the- art performance on a range of common word-level transduction tasks: neural machine translation (Bar- rault et al., 2019), question answering (Devlin et al., 2019) and abstractive summarization (Dong et al., 2019). In addition, the transformer forms the back- bone of the widely-used BERT (Devlin et al., 2019). Yet for character-level transduction tasks like mor- phological inﬂection, the dominant model has re- mained a recurrent neural network-based sequence- Character-level transduction models are often trained with less data than their word-level coun- terparts: In contrast to machine translation, where millions of training samples are available, the 2018 SIGMORPHON shared task (Cotterell et al., 2018) high-resource setting only provides ≈10k training examples per language. It is also not obvious that non-recurrent architectures such as the transformer 1901 r a e m s PST V V.PTCP <s> </s> Vanilla r a e m s PST V V.PTCP <s> </s> Feature Invariant Token Position Type 0 1 2 3 4 5 6 7 8 9 0 0 0 0 1 2 3 4 5 6 F F F C C C C C + + + + + + + + + + + + + + + + + + + + + + + + + + + + Figure 2: Handling of feature-guided character-level transduction with special position and type embeddings in the encoder. F denotes features while C denotes characters. We use morphological inﬂection as an example, inﬂecting smear into its past participle form, smeared. r a e m s PST V V.PTCP <s> </s> Vanilla Token Position Type 0 1 2 3 4 5 6 7 8 9 + + + + + + + + + + r a e m s PST V V.PTCP <s> </s> Feature Invariant 0 0 0 0 1 2 3 4 5 6 F F F C C C C C + + + + + + + + + + + + + + + + + + Figure 2: Handling of feature-guided character-level transduction with special position and type embeddings in the encoder. F denotes features while C denotes characters. We use morphological inﬂection as an example, inﬂecting smear into its past participle form, smeared. 3While the features could be encoded with a binary vector followed by MLP, it introduces a representation bottleneck for encoding features. 2 The Transformer for Characters different relative distances between a character and a set of features.3 To avoid such an inconsistency, we propose a simple remedy: We set the positional encoding of features to 0 and only start counting the positions for characters. Additionally, we add a special token to indicate whether a symbol is a word character or a feature. The right-hand side of Fig. 2 evinces how we have the same relative distance between characters and features. Figure 3: Distribution of incorrectly inﬂected forms in the test set of the inﬂection task over all 52 languages grouped by desired output word length. square root learning rate scheduler (Vaswani et al., 2017) with 4k steps during the warm-up. We train the model for 20k gradient updates and save and evaluate the model every 400 gradient updates. We select the best model out of 50 checkpoints based on development set accuracy. The number of gradi- ent updates and checkpoints are roughly the same as Wu and Cotterell (2019), the single model state of the art on the 2017 SIGMORPHON dataset. We use their model as a baseline model. For all experi- ments, we use a single predeﬁned random seed. 4We do not have access to the test set. 5 3 Empirical Findings Tasks. We consider four character-level transduc- tion tasks: morphological inﬂection, grapheme-to- phoneme conversion, transliteration, and historical text normalization. For morphological inﬂection, we use the 2017 SIGMORPHON shared task data (Cotterell et al., 2017) with 52 languages. The performance is evaluated by accuracy (ACC) and edit distance (Dist). For the g2p task, we use the unstressed CMUDict (Weide, 1998) and NETtalk (Sejnowski and Rosenberg, 1987) resources. We use the splits from Wu et al. (2018). We evaluate un- der word error rate (WER) and phoneme error rate (PER). For transliteration, we use the NEWS 2015 shared task data (Zhang et al., 2015).4 For histori- cal text normalization, we follow Bollmann (2019) and use datasets for Spanish (S´anchez-Mart´ınez et al., 2013), Icelandic and Swedish (Pettersson et al., 2013), Slovene (Scherrer and Erjavec, 2013, 2016; Ljubeˇsic et al., 2016), Hungarian and Ger- man (Pettersson, 2016).5 We evaluate using accu- racy (ACC) and character error rate of incorrect prediction (CERi). 5We do not include English due to licensing issues. 2 The Transformer for Characters Feature Invariance. Some character-level trans- duction is guided by features. For example, in the case of morphological reinﬂection, the task re- quires a set of morphological attributes that control what form a citation form is inﬂected into (see Fig. 2 for an example). However, the order of the features is irrelevant. In a recurrent neural network, features are input in some predeﬁned order as spe- cial characters and pre- or postpended to the input character sequence representing the citation form. The same is true for a vanilla transformer model, as shown on the left-hand side of Fig. 2. This leads to The Transformer. The transformer, originally described by Vaswani et al. (2017), is a self- attention-based encoder-decoder model. The en- coder has N layers, consisting of a multi-head self- attention layer and a two-layer feed-forward layer with ReLU activation, both equipped with a skip connection. The decoder has a similar structure as the encoder except that, in each decoder layer 1902 Figure 3: Distribution of incorrectly inﬂected forms in the test set of the inﬂection task over all 52 languages grouped by desired output word length. LS β2 Vanilla Feature Invariant 0 0.999 89.34 89.80 0 0.98 89.62 89.92 0.1 0.999 89.48 90.02 0.1 0.98 89.98 90.28 Table 1: Average development accuracy on morpho- logical inﬂection with different LS and β2, which de- note hyperparameter of label smoothing and Adam op- timizer respectively. LS β2 Vanilla Feature Invariant 0 0.999 89.34 89.80 0 0.98 89.62 89.92 0.1 0.999 89.48 90.02 0.1 0.98 89.98 90.28 Table 1: Average development accuracy on morpho- logical inﬂection with different LS and β2, which de- note hyperparameter of label smoothing and Adam op- timizer respectively. Table 1: Average development accuracy on morpho- logical inﬂection with different LS and β2, which de- note hyperparameter of label smoothing and Adam op- timizer respectively. different relative distances between a character and a set of features.3 To avoid such an inconsistency, we propose a simple remedy: We set the positional encoding of features to 0 and only start counting the positions for characters. Additionally, we add a special token to indicate whether a symbol is a word character or a feature. The right-hand side of Fig. 2 evinces how we have the same relative distance between characters and features. 3While the features could be encoded with a binary vector followed by MLP, it introduces a representation bottleneck for encoding features. 4We do not have access to the test set. 5We do not include English due to licensing issues. 3.1 A Controlled Hyperparameter Study Table 2: Average test performance on morphological inﬂection of Transformer against models from the liter- ature. ∗denotes model ensembling. Table 2: Average test performance on morphological inﬂection of Transformer against models from the liter- ature. ∗denotes model ensembling. WER PER ACC MFS Wu et al. (2018) 28.20 0.068 41.10 0.894 Wu and Cotterell (2019) 28.20 0.069 41.20 0.895 Transformer (Dropout = 0.3) 28.08 0.070 43.39 0.897 Transformer (Dropout = 0.1) 27.63 0.069 41.35 0.891 Table 4: Average test performance on Grapheme-to- Phoneme and dev performance on Transliteration of Transformer against models from the literature. creases steadily as the batch size is increased, sim- ilarly to what Popel and Bojar (2018) observe for machine translation. The transformer only outper- forms the recurrent baseline when the batch size is at least 128, which is much larger than batch size commonly used in recurrent models.6 Note that the model of Wu and Cotterell has 8.66M parameters, 17% more than the transformer model. To get an apples-to-apples comparison, we apply the same learning rate scheduler to Wu and Cotterell; this does not yield similar improvements and underper- forms with respect to the traditional learning rate scheduler. Our feature invariant transformer also outperforms the vanilla transformer model. We set the batch size to 400 for our main experiments. Note the batch size of 400 is especially large (4% of training data) considering the training size is only 10k. creases steadily as the batch size is increased, sim- ilarly to what Popel and Bojar (2018) observe for machine translation. The transformer only outper- forms the recurrent baseline when the batch size is at least 128, which is much larger than batch size commonly used in recurrent models.6 Note that the model of Wu and Cotterell has 8.66M parameters, 17% more than the transformer model. To get an apples-to-apples comparison, we apply the same learning rate scheduler to Wu and Cotterell; this does not yield similar improvements and underper- forms with respect to the traditional learning rate scheduler. Our feature invariant transformer also outperforms the vanilla transformer model. We set the batch size to 400 for our main experiments. Note the batch size of 400 is especially large (4% of training data) considering the training size is only 10k. Table 4: Average test performance on Grapheme-to- Phoneme and dev performance on Transliteration of Transformer against models from the literature. Morphological Inﬂection. As shown in Tab. 6It is also large in the context of character-level tasks, which typically have around 10k training examples. Batch size of 400 would imply approximately 4% of training data in a single gradient update. 3.1 A Controlled Hyperparameter Study 2, the feature invariant transformer produces state-of- the-art results on the 2017 SIGMORPHON shared tasks, improving upon ensemble-based systems by 0.27 points. We observe that as the dataset de- creases in size, a model with a larger dropout value performs slightly better. A brief tally of phenomena that are difﬁcult to learn for many machine learn- ing models, categorized along typical linguistic dimensions (such as word-internal sound changes, vowel harmony, circumﬁxation, ablaut, and umlaut phenomena) fail to reveal any consistent pattern of advantage to the transformer model. In fact, errors seem to be randomly distributed with an overall ad- vantage of the transformer model. Curiously, errors grouped along the dimension of word length reveal that as word forms grow longer, the transformer advantage shrinks (Fig. 3). Other Hyperparameters. Vaswani et al. (2017) applies label smoothing (Szegedy et al., 2016) of 0.1 to the transformer model and shows that it hurts perplexity, but improves BLEU scores for machine translation. Instead of the default 0.999 β2 for Adam, Vaswani et al. (2017) uses 0.98 and we ﬁnd that both choices beneﬁt character-level transduc- tion tasks as well (see Tab. 1). Historical Text Normalization. Tab. 3 shows that the transformer model with dropout of 0.1, as in the case of morphological inﬂection, improves upon the previous state of the art, although the model with a dropout of 0.3 yields a slightly better CERi. 3.1 A Controlled Hyperparameter Study To demonstrate the importance of hyperparame- ter tuning for the transformer on character-level tasks, we perform a small controlled hyperparame- ter study. This is important since researchers had previously failed to achieve high-performing re- sults with the transformer on character-level tasks. Here, we look at morphological inﬂection on the ﬁve languages in the 2017 SIGMORPHON dataset where submitted systems performed the worst: Latin, Faroese, French, Hungarian, and Norwegian (Nynorsk). We set the dropout to 0.3, β2 of Adam to 0.999 (the default value), and do not use label smoothing. We do not tune any other hyperparam- eter except the following three hyperparameters. The Importance of Batch Size. While recurrent models like Wu and Cotterell use a batch size of 20, halving the learning rate when stuck and employ- ing early stopping, we ﬁnd that a less aggressive learning rate scheduler, allowing the model to train longer, outperforms these hyperparameters. Fig. 1 shows that the signiﬁcant impact of batch size on the transformer. The transformer performance in- Optimization. We use Adam (Kingma and Ba, 2014) with a learning rate of 0.001 and an inverse Optimization. We use Adam (Kingma and Ba, 2014) with a learning rate of 0.001 and an inverse 1903 ACC Dist Silfverberg et al. (2017)* 92.97 0.170 Wu et al. (2018) 93.60 0.128 Wu and Cotterell (2019) 94.40 0.113 Wu and Cotterell (2019) (Our eval) 94.81 0.123 Makarov et al. (2017)* 95.12 0.100 Bergmanis et al. (2017)* 95.32 0.100 Transformer (Dropout = 0.3) 95.59 0.088 Transformer (Dropout = 0.1) 95.56 0.090 Table 2: Average test performance on morphological inﬂection of Transformer against models from the liter- ature. ∗denotes model ensembling. ACC CERi ACCs CERs i Ljubeˇsi´c et al. (2016) 91.78 0.392 90.37 0.360 Ljubeˇsi´c et al. (2016) (LM) 91.56 0.399 89.93 0.368 Bollmann (2018) 91.27 0.381 89.73 0.350 Tang et al. (2018a) 91.67 0.389 90.32 0.358 Flachs et al. (2019) - - 90.06 - Transformer (Dropout = 0.3) 91.30 0.340 89.99 0.330 Transformer (Dropout = 0.1) 91.85 0.352 90.61 0.334 Table 3: Average test performance on historical text normalization of Transformer against models from the literature. s denote subset of dataset as Flachs et al. (2019) only experiment with subset of languages. Table 3: Average test performance on historical text normalization of Transformer against models from the literature. s denote subset of dataset as Flachs et al. (2019) only experiment with subset of languages. 4 Related Work Character-level transduction is largely dominated by attention-based LSTM sequence-to-sequence (Luong et al., 2015) models (Cotterell et al., 2018). Character-level transduction tasks usually involve input-output pairs that share large substrings and alignments between these are often monotonic. Models that address the task tend to focus on ex- ploiting such structural bias. Instead of learning the alignments, Aharoni and Goldberg (2017) use external monotonic alignments from the SIGMOR- PHON 2016 shared task baseline Cotterell et al. (2016). Makarov et al. (2017) use this approach to win the CoNLL-SIGMORPHON 2017 shared task on morphological inﬂection (Cotterell et al., 2017). Wu et al. (2018) shows that explicitly model- ing alignment (hard attention) between source and target characters outperforms soft attention. Wu and Cotterell (2019) further shows that enforcing monotonicity in a hard attention model improves performance. Jimmy Lei Ba, Jamie Ryan Kiros, and Geoffrey E. Hin- ton. 2016. Layer normalization. arXiv preprint arXiv:1607.06450. Lo¨ıc Barrault, Ondˇrej Bojar, Marta R. Costa-juss`a, Christian Federmann, Mark Fishel, Yvette Gra- ham, Barry Haddow, Matthias Huck, Philipp Koehn, Shervin Malmasi, Christof Monz, Mathias M¨uller, Santanu Pal, Matt Post, and Marcos Zampieri. 2019. Findings of the 2019 conference on machine transla- tion (WMT19). In Proceedings of the Fourth Con- ference on Machine Translation (Volume 2: Shared Task Papers, Day 1), pages 1–61, Florence, Italy. As- sociation for Computational Linguistics. Toms Bergmanis, Katharina Kann, Hinrich Sch¨utze, and Sharon Goldwater. 2017. Training data aug- mentation for low-resource morphological inﬂection. In Proceedings of the CoNLL SIGMORPHON 2017 Shared Task: Universal Morphological Reinﬂection, pages 31–39, Vancouver. Association for Computa- tional Linguistics. Marcel Bollmann. 2018. Normalization of historical texts with neural network models. Ph.D. thesis, Bochum, Ruhr-Universit¨at Bochum. 3.2 New State-of-the-Art Results We train our feature invariant transformer on the four character-level tasks, exhibiting state-of-the- art results on morphological inﬂection and histori- cal text normalization. G2P and Transliteration. Tab. 4 shows that the transformer outperforms previously published strong recurrent models on two tasks despite hav- ing fewer parameters. A dropout rate of 0.3 yields 1904 ume 1: Long Papers), pages 2004–2015, Vancouver, Canada. Association for Computational Linguistics. signiﬁcantly better performance on the translitera- tion task while a dropout rate of 0.1 is stronger on the g2p task. This shows that transformers can and do outperform recurrent transducers on common character-level tasks when properly tuned. Antonios Anastasopoulos and Graham Neubig. 2019. Pushing the limits of low-resource morphological in- ﬂection. In Proceedings of the 2019 Conference on Empirical Methods in Natural Language Processing and the 9th International Joint Conference on Natu- ral Language Processing (EMNLP-IJCNLP), pages 984–996, Hong Kong, China. Association for Com- putational Linguistics. 5 Conclusion Using a large batch size and feature invariant input allows the transformer to achieve strong perfor- mance on character-level tasks. However, it is un- clear what linguistic errors the transformer makes compared to recurrent models on these tasks. Fu- ture work should analyze the errors in detail as Gorman et al. (2019) does for recurrent models. While Wu and Cotterell shows that the monotonic- ity bias beneﬁts character-level tasks, it is not evi- dent how to enforce monotonicity on multi-headed self-attention. Future work should consider how to best incorporate monotonicity into the model, either by enforcing it strictly (Wu and Cotterell, 2019) or by pretraining the model to copy (Anasta- sopoulos and Neubig, 2019). Marcel Bollmann. 2019. A large-scale comparison of historical text normalization systems. In Proceed- ings of the 2019 Conference of the North American Chapter of the Association for Computational Lin- guistics: Human Language Technologies, Volume 1 (Long and Short Papers), pages 3885–3898, Min- neapolis, Minnesota. Association for Computational Linguistics. Ryan Cotterell, Christo Kirov, John Sylak-Glassman, G´eraldine Walther, Ekaterina Vylomova, Arya D. McCarthy, Katharina Kann, Sebastian Mielke, Gar- rett Nicolai, Miikka Silfverberg, David Yarowsky, Jason Eisner, and Mans Hulden. 2018. The CoNLL– SIGMORPHON 2018 shared task: Universal mor- phological reinﬂection. In Proceedings of the CoNLL–SIGMORPHON 2018 Shared Task: Univer- sal Morphological Reinﬂection, pages 1–27, Brus- sels. Association for Computational Linguistics. Ryan Cotterell, Christo Kirov, John Sylak-Glassman, G´eraldine Walther, Ekaterina Vylomova, Patrick Xia, Manaal Faruqui, Sandra K¨ubler, David Yarowsky, Jason Eisner, and Mans Hulden. 2017. CoNLL-SIGMORPHON 2017 shared task: Univer- sal morphological reinﬂection in 52 languages. In References Roee Aharoni and Yoav Goldberg. 2017. Morphologi- cal inﬂection generation with hard monotonic atten- tion. In Proceedings of the 55th Annual Meeting of the Association for Computational Linguistics (Vol- Roee Aharoni and Yoav Goldberg. 2017. Morphologi- cal inﬂection generation with hard monotonic atten- tion. In Proceedings of the 55th Annual Meeting of the Association for Computational Linguistics (Vol- 1905 Proceedings of the CoNLL SIGMORPHON 2017 Shared Task: Universal Morphological Reinﬂection, pages 1–30, Vancouver. 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https://openalex.org/W3113052017	https://www.mdpi.com/1424-8220/20/24/7072/pdf?version=1607592150	English	null	Assessing the Performance of RGB-D Sensors for 3D Fruit Crop Canopy Characterization under Different Operating and Lighting Conditions	Sensors	2,020	cc-by	14,343	Received: 9 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Abstract: The use of 3D sensors combined with appropriate data processing and analysis has provided tools to optimise agricultural management through the application of precision agriculture. The recent development of low-cost RGB-Depth cameras has presented an opportunity to introduce 3D sensors into the agricultural community. However, due to the sensitivity of these sensors to highly illuminated environments, it is necessary to know under which conditions RGB-D sensors are capable of operating. This work presents a methodology to evaluate the performance of RGB-D sensors under diﬀerent lighting and distance conditions, considering both geometrical and spectral (colour and NIR) features. The methodology was applied to evaluate the performance of the Microsoft Kinect v2 sensor in an apple orchard. The results show that sensor resolution and precision decreased signiﬁcantly under middle to high ambient illuminance (>2000 lx). However, this eﬀect was minimised when measurements were conducted closer to the target. In contrast, illuminance levels below 50 lx aﬀected the quality of colour data and may require the use of artiﬁcial lighting. The methodology was useful for characterizing sensor performance throughout the full range of ambient conditions in commercial orchards. Although Kinect v2 was originally developed for indoor conditions, it performed well under a range of outdoor conditions. Keywords: RGB-D; depth cameras; precision agriculture; plant phenotyping; agricultural robotics; Kinect v2; time-of-ﬂight sensors; 3D sensors sensors sensors sensors www.mdpi.com/journal/sensors Assessing the Performance of RGB-D Sensors for 3D Fruit Crop Canopy Characterization under Diﬀerent Operating and Lighting Conditions Jordi Gené-Mola 1,* , Jordi Llorens 1 , Joan R. Rosell-Polo 1 , Eduard Gregorio 1, Jaume Arnó 1 , Francesc Solanelles 2 , José A. Martínez-Casasnovas 3 and Alexandre Escolà 1,* Jordi Gené-Mola 1,* , Jordi Llorens 1 , Joan R. Rosell-Polo 1 , Eduard Gregorio 1, Jaume Arnó 1 , Francesc Solanelles 2 , José A. Martínez-Casasnovas 3 and Alexandre 1 Research Group in AgroICT & Precision Agriculture, Department of Agricultural and Forest Engineering, Universitat de Lleida (UdL)–Agrotecnio Centre, Lleida, 25198 Catalonia, Spain; jordi.llorens@udl.cat (J.L.); joanramon.rosell@udl.cat (J.R.R.-P.); eduard.gregorio@udl.cat (E.G.); jaume.arno@udl.cat (J.A.) 1 Research Group in AgroICT & Precision Agriculture, Department of Agricultural and Forest Engineering, Universitat de Lleida (UdL)–Agrotecnio Centre, Lleida, 25198 Catalonia, Spain; jordi.llorens@udl.cat (J.L.); joanramon.rosell@udl.cat (J.R.R.-P.); eduard.gregorio@udl.cat (E.G.); jaume.arno@udl.cat (J.A.) 2 Department of Agriculture, Livestock, Fisheries and Food, Generalitat de Catalunya, Lleida, 25198 Catalunya, Spain; fsolanelles@gencat.cat Universitat de Lleida (UdL)–Agrotecnio Centre, Lleida, 25198 Catalonia, Spain; jordi.llorens@udl.cat (J.L.); joanramon.rosell@udl.cat (J.R.R.-P.); eduard.gregorio@udl.cat (E.G.); jaume.arno@udl.cat (J.A.) 2 Department of Agriculture, Livestock, Fisheries and Food, Generalitat de Catalunya, Lleida, 25198 Catalunya, Spain; fsolanelles@gencat.cat joanramon.rosell@udl.cat (J.R.R.-P.); eduard.gregorio@udl.cat (E.G.); jaume.arno@udl.cat (J.A.) 2 Department of Agriculture, Livestock, Fisheries and Food, Generalitat de Catalunya, Lleida, 25198 Catalunya, Spain; fsolanelles@gencat.cat y , p ; g 3 Research Group in AgroICT & Precision Agriculture, Department of Environmental and Soil Sciences, Universitat de Lleida (UdL)–Agrotecnio Centre, Lleida, 25198 Catalonia, Spain; joseantonio.martinez@udl.cat y p g 3 Research Group in AgroICT & Precision Agriculture, Department of Environmental and Soil Sciences, Universitat de Lleida (UdL)–Agrotecnio Centre, Lleida, 25198 Catalonia, Spain; joseantonio.martinez@udl.cat * Correspondence: jordi.genemola@udl.cat (J.G.-M.); alex.escola@udl.cat (A.E.) * Correspondence: jordi.genemola@udl.cat (J.G.-M.); alex.escola@udl.cat (A.E.) 1. Introduction The development of sensors capable of acquiring colour and depth data at high frame rates, known as RGB-D sensors, has represented a revolution in the way in which the environment is modelled [1]. While these devices have lower resolutions than either light detection and ranging (LiDAR) sensors [2] or structure-from-motion (SfM) techniques [3], their capacity for 3D imaging without having to move the sensor, combined with their low price, has led to them being deployed with increasing frequency. According to their speciﬁc operating principles, RGB-D sensors can be classiﬁed into structured-light (SL), time-of-ﬂight (ToF) and active stereoscopic cameras. SL cameras emit infrared laser light that generates a pattern through a diﬀraction grating that is then projected onto the target, where this light pattern is deformed. Comparisons between the original and the projected pattern allow disparity Sensors 2020, 20, 7072; doi:10.3390/s20247072 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 21 Sensors 2020, 20, 7072 maps to be generated from which it is possible to compute depth data [4]. ToF cameras are based on the emission of continuous-wave amplitude-modulated light (CW-AM). The distance from the target is determined by the phase shift between the transmitted and reﬂected signals [5]. Stereoscopic cameras provide 3D reconstruction by combining images (stereo matching) taken simultaneously using binocular or, alternatively, two monocular cameras. Active stereoscopic cameras also include a projector that emits a light pattern, which helps the process of stereo matching in low-textured scenarios [6]. [ ] In the ﬁeld of agricultural research, RGB-D sensors have been used for a number of purposes, including fruit detection [7,8], agricultural robotics [9,10] and weed detection and classiﬁcation [11,12]. The present work focuses on the use of RGB-D sensors to characterise plant geometry, an activity that seeks to obtain three-dimensional models of crops and to quantify their structural parameters. Initial work carried out in this area has mainly focused on plant phenotyping under indoor conditions using SL cameras. Chéné et al. [13] assessed the potential for using these cameras in leaf segmentation and measuring their curvature and orientation. These sensors have also been used for plant leaf segmentation under greenhouse conditions [14] and to digitize the stems and leaves of diﬀerent greenhouse tomato plants [15]. Nock et al. [16] highlighted the diﬃculties involved in detecting small branches using an SL camera. 1. Introduction This result was in accordance with [17], where authors concluded that these cameras provide more reliable results when volumetric objects (fruits) are captured. Various studies have also been carried out under ﬁeld conditions, which have shown the inability of SL cameras to measure plants in daylight [18]. A survey of the potential applications of SL sensors in agriculture and livestock was presented by [19]. The authors concluded that it is necessary to either work under low-light conditions (at sunset or dawn, or on cloudy days) or at night, with the help of artiﬁcial lighting. The eﬀect of natural lighting on SL cameras was also discussed in [20], who studied the ability of these sensors to estimate biomass in poplar plantations using diﬀerent viewing angles, while in [21] the same authors determined the structural parameters of cauliﬂower crops. The development of ToF-based RGB-D sensors capable of working outdoors has promoted their use in many 3D crop characterization studies [22]. These included trials in maize (Zea mays L.) plants to determine architectural traits, such as plant height and orientation, leaf angle, and stem diameter and position [23–26]. Accurate 3D models of vineyards have also been generated using a mobile terrestrial laser scanner (MTLS) based on a combination of a ToF camera and a real-time kinematic (RTK) global navigation satellite system (GNSS) [27]. In a close application, known initial and ﬁnal positions of vineyard rows were used to correct 3D data provided by the ToF camera [28]. In the case of tree plantations, [29] studied associated errors in RGB-D images at diﬀerent wind speeds, while [30] used depth and pseudocolour images from a ToF camera to supply a convolutional neural network for branch detection in apple trees. New RGB-D sensors based on active stereoscopy have recently been used to estimate canopy volume and bunch detection in a commercial vineyard [31]. These sensors have also been applied to the reconstruction of 3D models of fruit trees using semantic relationships between each side of the tree row [32]. Active stereoscopic cameras are generally more compact and consume less power than ToF cameras. Previous works have shown that ToF and active stereoscopic cameras are much more robust to variations in lighting than those based on structured light. 2.1. Experimental Set-Up The RGB-D sensor evaluated was the Microsoft Kinect v2. This sensor is composed of an RGB camera registered with a near infrared (NIR) ToF sensor to provide RGB, depth and NIR data according to the speciﬁcations shown in Table 1. Table 1. Microsoft Kinect v2 sensor speciﬁcations. Table 1. Microsoft Kinect v2 sensor speciﬁcations. RGB Frame Resolution 1920 × 1080 pixels RGB frame rate 30 Hz Infrared/Depth frame resolution 512 × 424 pixels Infrared/Depth frame rate 30 Hz Infrared/Depth ﬁeld of view 70◦horizontal × 60◦vertical Depth range 0.5–4.5 m 1920 × 1080 pixels 30 Hz 512 × 424 pixels 30 Hz 70◦horizontal × 60◦vertical 0.5–4.5 m Data acquisition was performed using ad hoc software developed in the C# language based on Kinect for Windows SDK v2.0. For each capture, this software saves the corresponding RGB image, NIR image and 3D point cloud. The average time required to acquire a single capture, generate the 3D point cloud and save the data, was 1.5 s, using a 64-bit operating system with 8 GB of RAM and an Intel® Core™i7-4500U processor (1.80 GHz, boosted to 2.4 GHz). In addition, a DVM 1300 lux meter (Velleman, Gavere, Belgium) was used to measure the lighting conditions of each acquired frame. This measurement was carried out by placing the lux meter in an upwards horizontal position at the centre of the captured scene. The wind speed conditions during the experiment were obtained from the Spanish Meteorological Agency (AEMET) service; data were obtained from the closest weather station, which was located 350 m away from the trial site. According to [29], wind speeds of less than 5 m s−1 do not aﬀect measurements taken by RGB-D sensors. Since the wind speed measured during the tests was lower than, or similar to this value, its eﬀect was not considered in the evaluation of the results. The generated 3D point clouds consisted of up to 217 088 points (512 × 424) with local Cartesian coordinates relative to the sensor origin, RGB data and NIR intensity (digital number, DN). Since RGB and NIR sensors have diﬀerent ﬁelds of view (FOV), the left and right RGB image boundaries were not represented in the 3D point cloud. Similarly, top and bottom points of the cloud lacked RGB data. Those points without RGB data were discarded in the colour assessment sections of the methodology. 1. Introduction Even so, and as shown in the comparative study conducted in [33], the performance of all RGB-D sensors is adversely aﬀected by direct sunlight, reducing the number of pixels with valid measurements and, thus, the number of points in 3D point clouds. Therefore, the selection of an RGB-D sensor to be used for 3D orchard characterization requires a good knowledge of the lighting range within which the sensor will operate eﬀectively and eﬃciently. It should be noted that lighting outside this range could reduce point cloud deﬁnition, leading to erroneous estimates of the structural parameters, while reduced lighting would aﬀect the quality of the RGB images. This work proposes a methodology for characterizing RGB-D sensors in terms of their performance under diﬀerent lighting conditions and at diﬀerent distances from the measured target. 3 of 21 Sensors 2020, 20, 7072 The methodology presented can be applied to any RGB-D sensor, although a Microsoft Kinect v2 (Microsoft Corporation, Redmond, WA, USA) sensor was used for validation. This is a very popular ToF camera whose introduction has had a revolutionary impact on robotics and industrial uses due to the low cost of obtaining radiometric and geometric data. The novel contributions of the present work with respect to other methodologies listed in the bibliography are the wide range of assessed lighting conditions; the use of new metrics and evaluation parameters, such as those used to evaluate spectral features (RGB colour and NIR intensities), and the provision of the processing code and the acquired dataset to facilitate the use of the present methodology in future works. The rest of the paper is structured as follows: Section 2 presents the sensor used and explains the experimental layout and the parameters used for sensor evaluation; Section 3 evaluates the Kinect v2 sensor by applying the proposed methodology; Section 4 discusses the results and compares them with those obtained in other state of the art research and, ﬁnally, the main conclusions are detailed in Section 5. The methodology presented can be applied to any RGB-D sensor, although a Microsoft Kinect v2 (Microsoft Corporation, Redmond, WA, USA) sensor was used for validation. This is a very popular ToF camera whose introduction has had a revolutionary impact on robotics and industrial uses due to the low cost of obtaining radiometric and geometric data. 1. Introduction The novel contributions of the present work with respect to other methodologies listed in the bibliography are the wide range of assessed lighting conditions; the use of new metrics and evaluation parameters, such as those used to evaluate spectral features (RGB colour and NIR intensities), and the provision of the processing code and the acquired dataset to facilitate the use of the present methodology in future works. The rest of the paper is structured as follows: Section 2 presents the sensor used and explains the experimental layout and the parameters used for sensor evaluation; Section 3 evaluates the Kinect v2 sensor by applying the proposed methodology; Section 4 discusses the results and compares them with those obtained in other state of the art research and, ﬁnally, the main conclusions are detailed in Section 5. 2.1. Experimental Set-Up All the data acquired during the experiments, called the Kinect Evaluation in Orchard conditions dataset (KEvOr Dataset), has been made publicly available at https://doi.org/10.5281/zenodo.4286460 [34]. The measurements carried out during the first experiment were taken from three sensor stations The proposed methodology includes two diﬀerent experiments. The ﬁrst aims to assess the performance of RGB-D sensors under diﬀerent illumination conditions, while the second seeks to study the performance of the sensor when measuring from diﬀerent distances to the target. All the data acquired during the experiments, called the Kinect Evaluation in Orchard conditions dataset (KEvOr Dataset), has been made publicly available at https://doi.org/10.5281/zenodo.4286460 [34]. (K2S1, K2S2 and K2S3) equipped with three different sensors. The fact that the three sensor stations, which were located at different sites in the orchard, remained static throughout the experiment was important for the acquisition and comparison of the same scenes under the different lighting conditions. K2S1 was oriented approximately to the north and located 2.5 m from the tree row axis, K2S2 was also oriented approximately to the north and located 1.5 m from the tree row axis and K2S3 was oriented approximately to the south and located 2.5 m from the tree row axis (Figure 2a). Thus, K2S1 and K2S2 measured the side of the tree row receiving direct sunlight during the experiment, while K2S3 measured the side of the tree row under indirect sunlight. This distribution allowed the illuminance conditions to be compared, as well as a study of whether the different illuminance levels had the same effect when measuring from different distances. A ranging rod was placed in each measured scene as a reference target for evaluating the accuracy of the sensors (Figure 1b). This experiment started at 12:51 h (November 6, UTC + 1) and consisted of acquiring point clouds at 28 different natural illumination levels throughout the afternoon and evening, from the highest sun illuminance until darkness. Figure 3 shows wind speed and illuminance conditions measured The measurements carried out during the ﬁrst experiment were taken from three sensor stations (K2S1, K2S2 and K2S3) equipped with three diﬀerent sensors. The fact that the three sensor stations, which were located at diﬀerent sites in the orchard, remained static throughout the experiment was important for the acquisition and comparison of the same scenes under the diﬀerent lighting conditions. 2.1. Experimental Set-Up All the data used in this work were acquired under real ﬁeld conditions at the experimental orchards of the School of Agrifood and Forestry Science and Engineering at the Universitat de Lleida. More speciﬁcally, measurements were carried out in a plot of apple trees, Malus domestica Borkh var. Fuji, at the full vegetation development stage (Figure 1a). The tree canopy was around 3 m tall and 1.5 m wide; the row spacing was 4.5 m, and the tree spacing was 0.75 m. The rows were oriented in an approximately east-west direction. Since sensor performance (resolution, accuracy, precision) 4 of 21 Sensors 2020, 20, 7072 mainly depends on the sensor itself and on the scanning conditions, and not on the fruit tree variety measured [19], it is thought that results obtained in an apple orchard should also be applicable to other fruit crops with similar vegetation characteristics. mainly depends on the sensor itself and on the scanning conditions, and not on the fruit tree variety measured [19], it is thought that results obtained in an apple orchard should also be applicable to other fruit crops with similar vegetation characteristics. Figure 1. (a) Experimental Malus domestica Borkh var. Fuji apple orchard at the full vegetation development stage where the measurements were carried out. (b) Ranging rod used to evaluate sensor accuracy by means of distances D1 and D2. Figure 1. (a) Experimental Malus domestica Borkh var. Fuji apple orchard at the full vegetation development stage where the measurements were carried out. (b) Ranging rod used to evaluate sensor accuracy by means of distances D1 and D2. Figure 1. (a) Experimental Malus domestica Borkh var. Fuji apple orchard at the full vegetation development stage where the measurements were carried out. (b) Ranging rod used to evaluate sensor accuracy by means of distances D1 and D2. Figure 1. (a) Experimental Malus domestica Borkh var. Fuji apple orchard at the full vegetation development stage where the measurements were carried out. (b) Ranging rod used to evaluate sensor accuracy by means of distances D1 and D2. The proposed methodology includes two different experiments. The first aims to assess the performance of RGB-D sensors under different illumination conditions, while the second seeks to study the performance of the sensor when measuring from different distances to the target. 2.1. Experimental Set-Up K2S1 was oriented approximately to the north and located 2.5 m from the tree row axis, K2S2 was also oriented approximately to the north and located 1.5 m from the tree row axis and K2S3 was oriented approximately to the south and located 2.5 m from the tree row axis (Figure 2a). Thus, K2S1 and K2S2 measured the side of the tree row receiving direct sunlight during the experiment, while K2S3 measured the side of the tree row under indirect sunlight. This distribution allowed the illuminance conditions to be compared, as well as a study of whether the diﬀerent illuminance levels had the same eﬀect when measuring from diﬀerent distances. A ranging rod was placed in each measured scene as a reference target for evaluating the accuracy of the sensors (Figure 1b). This experiment started at 12:51 h (November 6, UTC + 1) and consisted of acquiring point clouds at 28 diﬀerent natural illumination levels throughout the afternoon and evening, from the highest sun illuminance until darkness. Figure 3 shows wind speed and illuminance conditions measured throughout the experiment. For each of these 5 of 21 Sensors 2020, 20, 7072 28 observations, three captures (hereafter replicates) were acquired with each sensor, resulting in a total of 252 captures: 28 lighting conditions × 3 sensors × 3 replicates per sensor. Although two replicates per observation would have been enough to evaluate the sensor precision, three replicates were acquired for added security against a lack of data in the event of a corrupted ﬁle. Finally, all replicates were used to evaluate the sensor precision in a more accurate way. throughout the experiment. For each of these 28 observations, three captures (hereafter replicates) were acquired with each sensor, resulting in a total of 252 captures: 28 lighting conditions × 3 sensors × 3 replicates per sensor. Although two replicates per observation would have been enough to evaluate the sensor precision, three replicates were acquired for added security against a lack of data in the event of a corrupted file. Finally, all replicates were used to evaluate the sensor precision in a more accurate way. (a) (b) Figure 2. Experiment layout to assess (a) the effect of direct (K2S1 and K2S2) and indirect (K2S3) sunlight on the canopy on the generated point clouds; (b) the effect of the distance from the target on the generated point clouds. 2.1. Experimental Set-Up K2Si refers to each of the Kinect v2 (K2) stations. Green areas represent a top view of the tree rows, the dash-dotted line shows the axis of the row, while the thinner dotted lines that have been drawn in (b) represent canopy depth intervals (every 0.1 m). Figure 2. Experiment layout to assess (a) the eﬀect of direct (K2S1 and K2S2) and indirect (K2S3) sunlight on the canopy on the generated point clouds; (b) the eﬀect of the distance from the target on the generated point clouds. K2Si refers to each of the Kinect v2 (K2) stations. Green areas represent a top view of the tree rows, the dash-dotted line shows the axis of the row, while the thinner dotted lines that have been drawn in (b) represent canopy depth intervals (every 0.1 m). y (a) (b) (b) (a) Figure 2. Experiment layout to assess (a) the effect of direct (K2S1 and K2S2) and indirect (K2S3) sunlight on the canopy on the generated point clouds; (b) the effect of the distance from the target on the generated point clouds. K2Si refers to each of the Kinect v2 (K2) stations. Green areas represent a top view of the tree rows, the dash-dotted line shows the axis of the row, while the thinner dotted lines that have been drawn in (b) represent canopy depth intervals (every 0.1 m). Figure 2. Experiment layout to assess (a) the eﬀect of direct (K2S1 and K2S2) and indirect (K2S3) sunlight on the canopy on the generated point clouds; (b) the eﬀect of the distance from the target on the generated point clouds. K2Si refers to each of the Kinect v2 (K2) stations. Green areas represent a top view of the tree rows, the dash-dotted line shows the axis of the row, while the thinner dotted lines that have been drawn in (b) represent canopy depth intervals (every 0.1 m). Figure 3. Illuminance (left axis) and wind speed (in blue, right axis) measured throughout the experiment (UTC + 1). Grey diamonds, red squares and green triangles correspond to the illuminance of the measured scene for all K2S1, K2S2 and K2S3 observations, respectively. A regression curve has been added to each illuminance group of values to better compare its trend. Figure 3. Illuminance (left axis) and wind speed (in blue, right axis) measured throughout the experiment (UTC + 1). 2.2. Evaluation Parameters The present methodology analysed both geometrical and spectral data. Table 2 summarizes the parameters and metrics used in this evaluation. Regarding the geometrical assessment, the point cloud resolution, accuracy, precision and penetrability parameters were studied under diﬀerent illumination conditions and at diﬀerent distances from the target. On the other hand, the inﬂuence of scanning conditions on the spectral data was assessed by analysing the colour, NIR intensity and range-corrected NIR intensity (NIRc) of the acquired point clouds. All these parameters and metrics are described in detail in the following sub-sections. Table 2. Evaluation parameters and metrics. Table 2. Evaluation parameters and metrics. Parameter Metrics Units Resolution Number of points (P) points Average point cloud density (δ) points m−3 Accuracy Accuracy (Ac) mm Root mean square error (RMSE) mm Repeatability Precision error (Pr) mm Standard deviation (σ) mm Penetrability Points distribution in depth % Mean point cloud depth m Standard deviation m Colour Mean hue (H) % Mean saturation (S) % Mean brightness (V) % NIR Mean NIR intensity DN Mean NIRc intensity DN m2 A MATLAB® (R2020a, Math Works Inc., Natick, MA, USA) code was developed to analyse all the data and provide the sensor evaluation results. This code has been made publicly available at https://github.com/GRAP-UdL-AT/RGBD_sensors_evaluation_in_Orchards [35]. A MATLAB® (R2020a, Math Works Inc., Natick, MA, USA) code was developed to analyse all the data and provide the sensor evaluation results. This code has been made publicly available at https://github.com/GRAP-UdL-AT/RGBD_sensors_evaluation_in_Orchards [35]. 2.1. Experimental Set-Up A point cloud was acquired and, immediately after, the sensor was placed 1.5 m from the tree row axis to capture the same scene (K2S5) (Figure 2b). This experiment was carried out on 23 July at 20:30 (UTC + 2), when the measured scene received sunlight illuminance of 2000 lx. Due to the diﬀerent distances to the target, part of the scene measured from K2S4 was out of the K2S5 FOV. To compare the 3D models captured from both stations, the two resulting point clouds were manually registered (aligned) into the same reference systems. The point cloud acquired from station K2S4 was then cropped to match the scene captured from K2S5. 2.1. Experimental Set-Up Grey diamonds, red squares and green triangles correspond to the illuminance of the measured scene for all K2S1, K2S2 and K2S3 observations, respectively. A regression curve has been added to each illuminance group of values to better compare its trend. The second experiment was performed on a diﬀerent day and at a diﬀerent site within the orchard Kinect v2 sensor was stationed 2.5 m from the tree row axis and 1.35 m above the ground (K2S4 Figure 3. Illuminance (left axis) and wind speed (in blue, right axis) measured throughout the experiment (UTC + 1). Grey diamonds, red squares and green triangles correspond to the illuminance of the measured scene for all K2S1, K2S2 and K2S3 observations, respectively. A regression curve has been added to each illuminance group of values to better compare its trend. Figure 3. Illuminance (left axis) and wind speed (in blue, right axis) measured throughout the experiment (UTC + 1). Grey diamonds, red squares and green triangles correspond to the illuminance of the measured scene for all K2S1, K2S2 and K2S3 observations, respectively. A regression curve has been added to each illuminance group of values to better compare its trend. The second experiment was performed on a diﬀerent day and at a diﬀerent site within the orchard. A Kinect v2 sensor was stationed 2.5 m from the tree row axis and 1.35 m above the ground (K2S4). The second experiment was performed on a diﬀerent day and at a diﬀerent site within the orchard. A Kinect v2 sensor was stationed 2.5 m from the tree row axis and 1.35 m above the ground (K2S4). 6 of 21 Sensors 2020, 20, 7072 A point cloud was acquired and, immediately after, the sensor was placed 1.5 m from the tree row axis to capture the same scene (K2S5) (Figure 2b). This experiment was carried out on 23 July at 20:30 (UTC + 2), when the measured scene received sunlight illuminance of 2000 lx. Due to the diﬀerent distances to the target, part of the scene measured from K2S4 was out of the K2S5 FOV. To compare the 3D models captured from both stations, the two resulting point clouds were manually registered (aligned) into the same reference systems. The point cloud acquired from station K2S4 was then cropped to match the scene captured from K2S5. 2.2.1. Resolution Although the NIR camera which was used had a resolution of 512 × 424 pixels, the actual sensor resolution decreased due to the diﬀerent FOV between RGB and NIR cameras, and the fact that depth sensor performance is aﬀected under high lighting conditions [27]. To evaluate this issue, the total number of points P and the mean density δ were calculated for each acquired point cloud. To compute the average point cloud density δ, the algorithm counts the number of neighbouring points Ni contained in a sphere of radius R around each point of the cloud pi. The average point cloud density is then estimated by applying the following equation: δ = 1 P P X i=1 3Ni 4πR3 . (1) (1) Sensors 2020, 20, 7072 7 of 21 7 of 21 2.2.3. Repeatability The assessment of repeatability was performed by comparing the three replicates captured at each lighting condition observation. The comparison consisted of computing absolute point to point distances between each point of a cloud and its nearest neighbour in the corresponding replicated point clouds, hereafter called cloud-to-cloud distance diﬀerences. For each sensor (s) and lighting condition observation (L), the precision error (Pr) metric was computed as an average of these cloud-to-cloud distance diﬀerences: Prs@L = 1 2Pr1 + Pr2   Pr1 X i=1 d1,2,i + Pr1 X i=1 d1,3,i + Pr2 X i=1 d2,3,i   (4) (4) where Pr1 and Pr2 are the number of points in replicates 1 and 2, respectively, and dk,t,i is the distance between a given point i from replicate k and the cloud obtained with replicate t. where Pr1 and Pr2 are the number of points in replicates 1 and 2, respectively, and dk,t,i is the distance between a given point i from replicate k and the cloud obtained with replicate t. Repeatability was also evaluated in terms of the standard deviation of the cloud-to-cloud distance errors: σs@L = v u u t 1 2Pr1 + Pr2   Pr1 X i=1 (d1,2,i −Pr)2 + Pr1 X i=1 (d1,3,i −Pr)2 + Pr2 X i=1 (d2,3,i −Pr)2   (5) (5) 2.2.2. Accuracy The ranging rods shown in Figure 1b were used to evaluate sensor accuracy. The longitudinal distance D1 = 500 mm and the rod diameter D2 = 25 mm were manually measured in all K2S1 and K2S2 point clouds. This measurement was performed by computing the point to point distance of two manually selected points using CloudCompare software (Cloud Compare [GPL software] v2.11 Omnia). Since the accuracy of the measurement depended on the points manually selected, each measurement was repeated four times and averaged to obtain the average distances D1s,j and D2s,j for each point cloud. The four measurements were carried out using the ﬁrst replicate acquired for each lighting condition. Having these measurements, the average accuracy (Ac) and the root mean square error (RMSE) were computed as follows: AcD,s = 1 N XN j=1 Ds,j −D , (2) AcD,s = 1 N XN j=1 Ds,j −D , (2) RMSED,s = r 1 N XN j=1 Ds,j −D 2, (3) (2) RMSED,s = r 1 N XN j=1 Ds,j −D 2, (3) (3) where D refers to the measured distance D1 or D2, s refers to the sensor station 1 or 2, j to the diﬀerent point clouds acquired throughout the experiment and N to the total number of observations (N = 28). where D refers to the measured distance D1 or D2, s refers to the sensor station 1 or 2, j to the diﬀerent point clouds acquired throughout the experiment and N to the total number of observations (N = 28). The data acquired with sensor K2S3 were not used for the accuracy assessment since the distance j point clouds acquired throughout the experiment and N to the total number of observations (N = 28). The data acquired with sensor K2S3 were not used for the accuracy assessment since the distance from the tree row axis was the same as for K2S1 (2.5 m), and the maximum illuminance level received by the K2S3 sensor was 5500 lx. The data acquired with sensor K2S3 were not used for the accuracy assessment since the distance from the tree row axis was the same as for K2S1 (2.5 m), and the maximum illuminance level received by the K2S3 sensor was 5500 lx. 2.2.5. Colour and NIR The RGB colour data for each captured point cloud was transformed to the HSV (hue, saturation and value) space. This transformation allowed us to assess the inﬂuence of ambient light on the captured colour in terms of hue (H) related to chroma, saturation (S) and value (V) related to brightness. The average HSV values for all the points in a given cloud were calculated and compared to daylight illuminance. For the NIR intensity, a range correction was carried out in order to overcome light attenuation with distance [36]. The corrected NIR intensity (NIRc) was computed for each point by multiplying the NIR values by the squared distance from the sensor: NIRc,i = NIRi xi2 + yi2 + zi2 . (6) (6) where [xi, yi, zi] are the Cartesian coordinates of a point i with respect to the sensor. We calculated average NIR and NIRc values for all the points in a cloud, and also their distribution, using a normalised histogram. These were then compared to analyse the eﬀect of the illuminance conditions and distance from the target on the returned NIR and NIRc light. 3.1. Experiment I: Eﬀect of Daylight Illuminance 3.1. Experiment I: Eﬀect of Daylight Illuminance 2.2.4. Penetrability Using the data from the second experimental set-up (Figure 2b), we assessed the ability of the two point clouds taken from diﬀerent distances to penetrate the canopy. This was done by computing the number of points in each cloud found in diﬀerent 0.1 m sections arranged along the y-axis (Figure 2b) from the outermost part of the canopy (closer to the sensors) to the most distant part, on the other side of the row. The evaluation of penetrability includes the percentage of points found in each section in relation to the total number of points -hereafter called point distribution in depth, the mean point cloud depth and the standard deviation. Sensors 2020, 20, 7072 8 of 21 2.2.6. Statistical Analysis Statistical analysis was performed using the statistical software R (R Development Core Team, 2013) under an RSTUDIO environment (ver. 1.2.3001). The eﬀects of the diﬀerent Kinect positions and orientations plus ﬁve illumination levels (Illumination brakes at: 0, 250, 1000, 4000, 16000 and 64000 lx) were examined using two-way analyses of variance (ANOVA) followed by Tukey’s HSD (honestly signiﬁcant diﬀerence) test for multiple comparisons. Assumptions of ANOVA were checked and, according to the model’s diagnosis, Box-Cox transformation was applied. All the statistical analyses were conducted at a signiﬁcance level of α = 0.05. 3.1.1. Point Cloud Resolution The point cloud resolution remained almost stable for low illuminance levels but started to decrease signiﬁcantly for middle to high illuminance levels (>2000 lx) (Figures 4 and 5). The number of points in the cloud showed an inversely linear dependence with the ambient illuminance (Figure 4), reporting coeﬃcients of determination R2 of 0.93, 0.91 and 0.87 for K2S1, K2S2 and K2S3, respectively. The sensor station K2S1, which was placed 2.5 m from the tree row axis, showed a higher regression coeﬃcient (or slope si) s1 = −2.4 than K2S2, which was placed at a distance of 1.5 m, which reported a regression coeﬃcient of s2 = −1.7. From this, it was concluded that the illuminance eﬀect was slightly reduced when scanning closer to the target. The K2S3 sensor showed the highest regression coeﬃcient: s3 = −22. However, this sensor station was the one least aﬀected by the lighting conditions throughout the day. It reported over 60000 points in the cloud even at the time of day when the sunlight was strongest (Figure 4). The higher slope reported with K2S3 may have been due to the fact that the north face of the measured row did not receive the incidence of the sunlight directly. As a consequence, it did not experience large variations in illumination as the position of the sun changed in the course of the day (Figure 3). Even so, backlighting continued to aﬀect sensor performance. The mean point cloud density also showed an inversely linear dependence on illuminance (Figure 5), reporting R2 values of 0.95, 0.98 and 0.82, respectively. This behaviour had been expected, as the FOV of the sensors was constant and the number of points augmented as the light illuminance declined (Figure 4). Another expected result was that the average point cloud density decreased with 9 of 21 Sensors 2020, 20, 7072 distance from the target. For instance, under the worst lighting conditions (~50000 lx) K2S2 registered a higher point cloud density δ2@50klx > 4 × 105 points m−3 than K2S1 and K2S3 at low illuminance levels δ1,3@1lx < 3 × 105 points m−3. Figure 4. Evolution of the number of points in the point clouds as a function of illuminance. Figure 4. Evolution of the number of points in the point clouds as a function of illuminance. 3.1.1. Point Cloud Resolution For a qualitative assessment of the illuminance eﬀect, Figure 6 shows examples of the point clouds acquired from each sensor station at diﬀerent illuminance levels. In addition, the number of points and the point cloud density values are provided for each case (orange and blue values, respectively). Since the K2S3 sensor captured the indirectly illuminated row side, the measurements with this sensor did not achieve an ambient illumination greater than 5500 lx (Figure 3). This explains why no information is given in the K2S3–50000 lx sample. g p As can be observed in Figure 6, the K2S1 sensor was strongly inﬂuenced by high levels of illuminance, resulting in a sparse 3D model with only a limited ability to represent small objects such as fruits, trunks, branches or leaves. The high illuminance also aﬀected the data captured by the K2S2 station. In this case, the eﬀect was visible in the point cloud boundaries, where the target was further away from the sensor. Thus, although the point cloud density at high illuminance was acceptable for short range measurements (Figure 5), the lighting conditions still aﬀected the performance of the sensor by reducing its FOV. At 5000 lx, the sensor that was most aﬀected was K2S3. This eﬀect was due to the fact that the K2S3 sensor measured the indirectly illuminated side of the row. Although this side did not receive high level of illumination during the hours of strongest sunlight (5000 lx at 13:00 UTC + 1), sensor performance continued to be aﬀected due to the greater amount of backlight. Finally, when comparing the point clouds acquired at 1000 lx and those acquired at 0.1 lx, we observed no signiﬁcant diﬀerences in terms of the geometric evaluation; very similar point clouds were obtained under both lighting conditions. 10 of 21 10 of 21 Sensors 2020, 20, 7072 Figure 5. Evolution of average point cloud density as a function of illuminance. s 2020, 20, x FOR PEER REVIEW 11 of 21 Figure 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under different illuminance conditions. The orange and blue values correspond to the number of points in the cloud and the average point cloud density for each capture, respectively. 3.1.2. Point Cloud Accuracy Sensor accuracy was computed for diﬀerent lighting conditions, but not all the captures could be analysed. Under conditions of very low illuminance, the lack of colour data prevented the acquisition of reliable measurements because the coloured stripes on the ranging rod could not be diﬀerentiated. On the other hand, when the lighting levels were too high, the low point cloud resolution made it impossible to correctly identify the elements at the scene. As a result, the illuminance ranges considered for the accuracy assessment ranged from 2.3 lx to 42100 lx, and from 1 lx to 54100 lx, for K2S1 and K2S2, respectively (Table 3). Table 3. Sensor accuracy results measuring from 2.5 m (K2S1) and from 1.5 m (K2S2) of the tree row axis. In rows, the same letter indicates no signiﬁcant diﬀerences between mean accuracy values (p < 0.05). Table 3. Sensor accuracy results measuring from 2.5 m (K2S1) and from 1.5 m (K2S2) of the tree row axis. In rows, the same letter indicates no signiﬁcant diﬀerences between mean accuracy values (p < 0.05). Table 3. Sensor accuracy results measuring from 2.5 m (K2S1) and from 1.5 m (K2S2) of the tree row axis. In rows, the same letter indicates no signiﬁcant diﬀerences between mean accuracy values (p < 0.05). K2S1 K2S2 Min. illuminance to measure [lx] 2.3 1 Max. illuminance to measure [lx] 42100 54100 AccuracyD1 [mm] 3.7 a 6.1 a AccuracyD2 [mm] 7.3 a 4.4 a RMSED1 [mm] 6.6 7.5 RMSED2 [mm] 9.5 6.0 K2S1 K2S2 Min. illuminance to measure [lx] 2.3 1 Max. illuminance to measure [lx] 42100 54100 AccuracyD1 [mm] 3.7 a 6.1 a AccuracyD2 [mm] 7.3 a 4.4 a RMSED1 [mm] 6.6 7.5 RMSED2 [mm] 9.5 6.0 After analysing the accuracy results over the speciﬁed ranges, we found no relationship between the illuminance of the scene and the accuracy of the sensor and concluded that sensor accuracy was not aﬀected by the lighting conditions. Table 3 reports the mean accuracy and the RMSE obtained for measuring distances D1 and D2. K2S1 showed slightly better performance measuring longer distances (D1 = 500 mm), while K2S2 was slightly more accurate when measuring shorter distances (D2 = 25 mm), but no signiﬁcant diﬀerences were observed. 3.1.1. Point Cloud Resolution As can be observed in Figure 6, the K2S1 sensor was strongly influenced by high levels of minance, resulting in a sparse 3D model with only a limited ability to represent small objects such uits, trunks, branches or leaves. The high illuminance also affected the data captured by the K2S2 on. In this case, the effect was visible in the point cloud boundaries, where the target was further 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under diﬀerent illuminan ions. The orange and blue values correspond to the number of points in the cloud and t ge point cloud density for each capture, respectively. Figure 5. Evolution of average point cloud density as a function of illuminance. ensors 2020, 20, x FOR PEER REVIEW 11 of Fi 5 E l ti f i t l d d it f ti f ill i S 2020 20 FOR PEER REVIEW 11 Figure 5. Evolution of average point cloud density as a function of illuminance. 20, 20, x FOR PEER REVIEW 11 Figure 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under different illuminance conditions. The orange and blue values correspond to the number of points in the cloud and the average point cloud density for each capture, respectively. As can be observed in Figure 6, the K2S1 sensor was strongly influenced by high levels of Figure 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under diﬀerent illuminance conditions. The orange and blue values correspond to the number of points in the cloud and the average point cloud density for each capture, respectively. Figure 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under different illuminance conditions. The orange and blue values correspond to the number of points in the cloud and the average point cloud density for each capture, respectively. A b b d i Fi 6 th K2S1 t l i fl d b hi h l l f Figure 6. Point clouds acquired from sensor stations K2S1, K2S2 and K2S3 under diﬀerent illuminance conditions. The orange and blue values correspond to the number of points in the cloud and the average point cloud density for each capture, respectively. 11 of 21 11 of 21 Sensors 2020, 20, 7072 3.1.2. Point Cloud Accuracy Based on the small diﬀerences between the two sensor stations (K2S1 and K2S2), we concluded that there was no clear relationship between sensor accuracy and distances between sensors and row axis of 1.5 m and 2.5 m. 3.1.3. Repeatability of the Measurements Mean values of precision errors and standard deviation at each illumination level with their mean statistical diﬀerences. Station K2S3 was not tested at above 16000 lx. In rows, for each parameter, the same letter (Latin letters for precision error and Greek letters for standard deviation) indicates no signiﬁcant diﬀerences between the mean accuracy values (p < 0.05). p g Figure 7. Evolution of point cloud precision as a function of the illuminance conditions throughout the experiment (UTC + 1). The centres of the grey, red and green squares, respectively, indicate the precision errors, 𝑃𝑟, at K2S1 (a), K2S2 (b) and K2S3 (c), while the whiskers correspond to the േ Figure 7. Evolution of point cloud precision as a function of the illuminance conditions throughout the experiment (UTC + 1). The centres of the grey, red and green squares, respectively, indicate the precision errors, Pr, at K2S1 (a), K2S2 (b) and K2S3 (c), while the whiskers correspond to the ± standard deviation. standard deviation. Table 4. Mean values of precision errors and standard deviation at each illumination level with their mean statistical diﬀerences. Station K2S3 was not tested at above 16000 lx. In rows, for each parameter, the same letter (Latin letters for precision error and Greek letters for standard deviation) indicates no signiﬁcant diﬀerences between the mean accuracy values (p < 0.05). standard deviation. Table 4. Mean values of precision errors and standard deviation at each illumination level with their mean statistical diﬀerences. Station K2S3 was not tested at above 16000 lx. In rows, for each parameter, the same letter (Latin letters for precision error and Greek letters for standard deviation) indicates no signiﬁcant diﬀerences between the mean accuracy values (p < 0.05). Class Lux Range [lx] Precision Error [mm] Standard Deviation [mm] K2S1 K2S2 K2S3 K2S1 K2S2 K2S3 1 0 to 250 8.54 a 4.62 b 7.86 a 15.90 α 11.82 β 15.80 α 2 250 to 1000 10.76 a 7.64 b 6.74 c 13.53 β 13.49 β 19.35 α 3 1000 to 4000 11.76 a 7.89 b 12.50 a 20.84 β 16.38 β 24.78 α 4 4000 to 16000 13.43 a 8.61 b 14.14 a 26.17 β 23.28 γ 30.18 α 5 16000 to 64000 25.86 a 9.88 b – 54.72 α 31.75 β – 3.1.4. Spectral results: Color and NIR 3.1.4. 3.1.3. Repeatability of the Measurements Repeatability results also showed a dependency on lighting conditions (Figure 7 and Table 4). The most aﬀected sensor station was K2S1, which showed an increase in the precision error with brighter lighting conditions from Pr1@1lx = 8.2 mm to Pr1@55klx = 44 mm. This station also experienced the highest increase in distance error standard deviation: from σ1@1lx = 21.5 mm to σ1@55klx = 98.4 mm. These diﬀerences could also be seen in the mean precision error and mean standard deviation in each illumination class (Table 4). Sensor station K2S2 presented the best precision values across the illumination range, and also the least variation in these results. Within the ranges 0 lx to 250 lx (Class 1) and 1000 to 16000 lx (Class 3 and 4), the precision of the K2S3 results was statistically the same as that for K2S1 (Table 4). Despite this similar behaviour, as the illuminance values throughout the day were higher for K2S1 than for K2S3, the latter was less aﬀected by the time of day with most sunlight: from 12:51 h to 16 h (UTC + 1) (Figure 7). Finally, the precision of K2S2 was the least aﬀected by the lighting conditions. Measuring the scene from a shorter distance signiﬁcantly improved the precision of the results under high illuminance conditions. This can be clearly observed by comparing K2S2 and K2S1 (Figure 7): at 55000 lx, Pr2@55klx = 10.6 mm and σ2@55klx = 35.2 mm. This eﬀect was also observed when comparing stations K2S2 and K2S3, but to a lesser extent. Sensors 2020, 20, 7072 standard deviatio i i l 12 of 21 the best recision values across the illumination range, and also the least variation in these results. Figure 7. Evolution of point cloud precision as a function of the illuminance conditions throughout the experiment (UTC + 1). The centres of the grey, red and green squares, respectively, indicate the precision errors, 𝑃𝑟, at K2S1 (a), K2S2 (b) and K2S3 (c), while the whiskers correspond to the േ standard deviation. Figure 7. Evolution of point cloud precision as a function of the illuminance conditions throughout the experiment (UTC + 1). The centres of the grey, red and green squares, respectively, indicate the precision errors, Pr, at K2S1 (a), K2S2 (b) and K2S3 (c), while the whiskers correspond to the ± standard deviation. Table 4. 3.1.3. Repeatability of the Measurements Figure 9. Colour images acquired from the K2S1, K2S2 and K2S3 sensor stations under different illuminance conditions. Pink, orange and grey values correspond to the mean hue (H), saturation (S), and brightness (V) indices of the corresponding point clouds. Figure 9. Colour images acquired from the K2S1, K2S2 and K2S3 sensor stations under diﬀerent illuminance conditions. Pink, orange and grey values correspond to the mean hue (H), saturation (S), and brightness (V) indices of the corresponding point clouds. For lighting conditions below 50 lx (from 18:00 onwards), the illuminance was too low to be corrected by adjusting the sensor exposure. As a result, V started to decrease significantly. This effect can be clearly seen in Figure 8, but it can also be qualitatively observed by comparing the images in Fi 9 i hi h h d h i il V l l 1000 l d 50 l b h i ifi l Hue (H) showed similar behaviour to the V curve (Figure 8). From this it was concluded that images acquired at illuminance levels of less than 50 lx could not be postprocessed to correct low brightness because the image chroma was also aﬀected under such lighting conditions. For lighting conditions below 50 lx (from 18:00 onwards), the illuminance was too low to be corrected by adjusting the sensor exposure. As a result, V started to decrease significantly. This effect can be clearly seen in Figure 8, but it can also be qualitatively observed by comparing the images in Fi 9 i hi h h d h i il V l l 1000 l d 50 l b h i ifi l Hue (H) showed similar behaviour to the V curve (Figure 8). From this it was concluded that images acquired at illuminance levels of less than 50 lx could not be postprocessed to correct low brightness because the image chroma was also aﬀected under such lighting conditions. Figure 9 in which the measured scene shows similar V levels at 1000 lx and 50 lx, but they significantly decrease with lower ambient illuminance of 20 lx and 0.1 lx. Hue (H) showed similar behaviour to the V curve (Figure 8). From this it was concluded that images acquired at illuminance levels of less than 50 lx could not be postprocessed to correct low brightness because the image chroma was also affected under such lighting conditions. 3.1.3. Repeatability of the Measurements Spectral results: Color and NIR The colour camera integrated into the tested RGB-D device showed a good level of adaptation to diﬀerent lighting conditions above 50 lx. Between 16:30 h and 18:00 h, the mean image brightness (V) increased at all three sensor stations (Figure 8). This behaviour contrasted with the natural lighting conditions, which decreased from 3000 lx to 50 lx (approximately) during this period. Automatic adjustments of the colour sensor explain the diﬀerence between the ambient illuminance and the V value. Since the sensor automatically adjusts the image exposure to maintain a good level of colour brightness in the target area, the background image becomes highly exposed (or even burnt) when it measures a shadowed scene. As a result, the highly exposed background increased the mean V. This eﬀect can be observed by comparing the images at 50000 lx and at 1000 lx, in Figure 9. For lighting conditions below 50 lx (from 18:00 onwards), the illuminance was too low to be corrected by adjusting the sensor exposure. As a result, V started to decrease signiﬁcantly. This eﬀect can be clearly seen in Figure 8, but it can also be qualitatively observed by comparing the images in Figure 9 in which the measured scene shows similar V levels at 1000 lx and 50 lx, but they signiﬁcantly decrease with lower ambient illuminance of 20 lx and 0.1 lx. 13 of 21 13 of 21 Sensors 2020, 20, 7072 Figure 8. Evolution of the colour indices HSV, NIR and NIRc intensities and illuminance throughout the experiment. The plots of (a–c) correspond to the results obtained from sensor stations K2S1, K2S2 and K2S3, respectively. Figure 8. Evolution of the colour indices HSV, NIR and NIRc intensities and illuminance throughout the experiment. The plots of (a–c) correspond to the results obtained from sensor stations K2S1, K2S2 and K2S3, respectively. 14 of 21 2S2 Sensors 2020, 20, 7072 the experiment and K2S3 resp Figure 9. Colour images acquired from the K2S1, K2S2 and K2S3 sensor stations under different illuminance conditions. Pink, orange and grey values correspond to the mean hue (H), saturation (S), and brightness (V) indices of the corresponding point clouds. Figure 9. Colour images acquired from the K2S1, K2S2 and K2S3 sensor stations under diﬀerent illuminance conditions. Pink, orange and grey values correspond to the mean hue (H), saturation (S), and brightness (V) indices of the corresponding point clouds. 3.2. Experiment II: Eﬀect of the Distance from the Target The geometrical parameter most affected by t density which showed an increase of 200 5 % when m The geometrical parameter most aﬀected by the distance from the target was point cloud density, which showed an increase of 200.5 % when measured from a distance of 1.5 m, compared to the results obtained from 2.5 m (Table 5). These results are consistent with those reported in the ﬁrst experiment (Section 3.1.1, Figure 5), which showed a signiﬁcant diﬀerence between sensor stations K2S1 (2.5 m) and K2S2 (1.5 m) throughout the day. y, , p the results obtained from 2.5 m (Table 5). These results are consistent with those reported in the first experiment (Section 3.1.1, Figure 5), which showed a significant difference between sensor stations K2S1 (2.5 m) and K2S2 (1.5 m) throughout the day. Table 5. Geometric and spectral results at different distances from the tree row axis: 2.5 m and 1.5 m. Table 5. Geometric and spectral results at diﬀerent distances from the tree row axis: 2.5 m and 1.5 m. K2S4 (2.5 m) K2S5 (1.5 m) Diﬀerence Point cloud density [points m−3] 215 × 103 646 × 103 200.5 % Penetrability [m] 0.922 0.772 16.3 % Hue (H) [%] 46.5 45.9 1.3 % Saturation (S) [%] 25.5 27.6 8.2 % Brightness (V) [%] 41.1 53.6 30.4 % Mean NIR intensity [DN] 1205 3996 231.6 % Mean NIRc intensity [DN m2] 6526 6404 1.9 % K2S4 (2.5 m) K2S5 (1.5 m) Difference Point cloud density [points m−3] 215 × 103 646 × 103 200.5 % Penetrability [m] 0.922 0.772 16.3 % Hue (H) [%] 46.5 45.9 1.3 % Saturation (S) [%] 25.5 27.6 8.2 % Brightness (V) [%] 41.1 53.6 30.4 % Mean NIR intensity [DN] 1205 3996 231.6 % Mean NIRc intensity [DN m2] 6526 6404 1.9 % Table 5. Geometric and spectral results at diﬀerent distances from the tree row axis: 2.5 m and 1.5 m. K2S4 (2 5 m) K2S5 (1 5 m) Difference While point cloud density decreased with distance, point cloud penetrability improved. As can be observed in Figure 10, points measured from 1.5 m were shallowly distributed compared with those measured from 2.5 m, which were able to penetrate the canopy more deeply. As a result, the mean depth distribution (penetrability) increased from 0.772 m at 1.5 m to 0.922 m at 2.5 m (Table 5). 3.1.3. Repeatability of the Measurements For the sake of simplicity in the visualization of the plots, the mean NIR and NIR௖ (corrected) values plotted in Figure 8 were scaled within the range of 0 to 1. This was done by dividing the NIR and NIR௖ values by a factor of 15000, which coincides with the maximum NIR௖ value reported during the experiment. Looking at Figure 8a (K2S1 sensor), the NIR௖,ଵ increased significantly at high ill i l l i idi ith th ill i h th h d l l ti For the sake of simplicity in the visualization of the plots, the mean NIR and NIRc (corrected) values plotted in Figure 8 were scaled within the range of 0 to 1. This was done by dividing the NIR and NIRc values by a factor of 15000, which coincides with the maximum NIRc value reported during the experiment. Looking at Figure 8a (K2S1 sensor), the NIRc,1 increased signiﬁcantly at high illuminance levels, coinciding with the illuminance range where the sensor showed low resolution values (Section 3.1.1). Since NIRc is related to an object’s reﬂectance [37], it can be concluded that the higher the ambient illuminance, the greater the object’s reﬂectance needs to be measured by the sensor. This eﬀect was minimized when measuring from shorter distances (1.5 m), as can be observed in Figure 8b (K2S2 sensor). illuminance levels, coinciding with the illuminance range where the sensor showed low resolution values (Section 3.1.1). Since NIR௖ is related to an object’s reflectance [37], it can be concluded that the higher the ambient illuminance, the greater the object’s reflectance needs to be measured by the K2S1 and K2S3 showed similar NIR values: between 0.05 and 0.1 (scaled values). This contrasts with the values reported by K2S2, whose NIR values ranged from 0.2 to 0.25. This diﬀerence was due to the inverse-square law, i.e., the attenuation of the intensity of light coming from a source expressed as the inverse of the square of the distance from that source. This eﬀect was remedied with the range Sensors 2020, 20, 7072 as the inverse of th correction As can 15 of 21 he range er range correction. As can be observed in Figure 8, although NIR1 and NIR3 are lower than NIR2, after range correction, all three values NIRc,1, NIRc,1 and NIRc,1 are similar (between 0.4 and 0.5). ୡ,ଵ ୡ,ଵ ୡ,ଵ ( ) 3.2. 3.1.3. Repeatability of the Measurements Experiment II: Effect of the Distance from the Target 3.2. Experiment II: Eﬀect of the Distance from the Target The geometrical parameter most affected by the distance from the target was point cloud density, which showed an increase of 200 5 % when measured from a distance of 1 5 m, compared to 3.2. Experiment II: Eﬀect of the Distance from the Target The geometrical parameter most affected by the distance from the target was point cloud density, which showed an increase of 200.5 % when measured from a distance of 1.5 m, compared to 4. Discussion 4. Discussion The first experiment showed that high illumination levels affected the geometrical data acquisition performance while low illumination levels affected the colour data. In the case of geometrical performance, the point cloud resolution and repeatability were the parameters most affected. The number of measured points and the point cloud density both decreased significantly for middle to high illuminance levels (>2000 lx). In contrast, too little illuminance levels affected the quality of the colour data, with performance being poor when lighting levels were below 50 lx. In order to obtain reliable measurements, for both geometrical and spectral data, the lighting level should, therefore, be within the range of 50 to 2000 lx. The ﬁrst experiment showed that high illumination levels aﬀected the geometrical data acquisition performance while low illumination levels aﬀected the colour data. In the case of geometrical performance, the point cloud resolution and repeatability were the parameters most aﬀected. The number of measured points and the point cloud density both decreased signiﬁcantly for middle to high illuminance levels (>2000 lx). In contrast, too little illuminance levels aﬀected the quality of the colour data, with performance being poor when lighting levels were below 50 lx. In order to obtain reliable measurements, for both geometrical and spectral data, the lighting level should, therefore, be within the range of 50 to 2000 lx. should, therefore, be within the range of 50 to 2000 lx. The objectives and agronomic applications of RGB-D sensors are highly varied, and high levels of performance are not always required for both geometrical and spectral data. In consequence, different optimal working conditions may be established depending on the application. For instance, if the objective is to find only the geometric characteristics of an orchard [38], or to measure the porosity of its canopy [39], it should be a priority to capture as many points as possible, even if this entails sacrificing the colour data at each point. In such a case, the measurements should be performed in the absence of, or with only low levels, of light. 3.2. Experiment II: Eﬀect of the Distance from the Target The geometrical parameter most affected by t density which showed an increase of 200 5 % when m The authors attribute this diﬀerence to the fact that the closer the target was to the sensor, the larger were the shadows from the NIR light emitted by the sensor, due to its greater interference with the outer elements of the canopy. While point cloud density decreased with distance, point cloud penetrability improved. As can be observed in Figure 10, points measured from 1.5 m were shallowly distributed compared with those measured from 2.5 m, which were able to penetrate the canopy more deeply. As a result, the mean depth distribution (penetrability) increased from 0.772 m at 1.5 m to 0.922 m at 2.5 m (Table 5). The authors attribute this difference to the fact that the closer the target was to the sensor, the larger were the shadows from the NIR light emitted by the sensor, due to its greater interference with the outer elements of the canopy. Figure 10. Penetrability of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. Vertical dash-dotted lines illustrate the mean depth values, computed as the mean of points’ depth distribution. With respect to colour, brightness (V) was the colour parameter that was most aﬀected by distance showed an increase of 30.4 % when measured from a distance of 1.5 m compared with result Figure 10. Penetrability of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. Vertical dash-dotted lines illustrate the mean depth values, computed as the mean of points’ depth distribution. With respect to colour, brightness (V) was the colour parameter that was most aﬀected by distance It showed an increase of 30.4 % when measured from a distance of 1.5 m compared with results Sensors 2020, 20, 7072 With respect distance It showe 16 of 21 cted by ed with obtained from 2.5 m (Table 5). However, since the hue (H) and saturation (S) values did not present any major diﬀerences between 1.5 m and 2.5 m measures, the diﬀerence in brightness could be corrected by scaling RGB channels with a scaling factor >1. ( ) ( ) ( ) present any major differences between 1.5 m and 2.5 m measures, the difference in brightness could be corrected by scaling RGB channels with a scaling factor > 1. 3.2. Experiment II: Eﬀect of the Distance from the Target The geometrical parameter most affected by t density which showed an increase of 200 5 % when m As for the NIR intensity values, due to the inverse-square law attenuation of NIR light intensity, As for the NIR intensity values, due to the inverse-square law attenuation of NIR light intensity, the NIR intensity distribution at 1.5 m diﬀered signiﬁcantly from that at 2.5 m (Figure 11a). The point cloud acquired at 2.5 m showed a mean NIR intensity of 1205 (DN) with a standard deviation of 509 (DN), while at 1.5 m the mean NIR was 3996 (DN) and the standard deviation was 2000 (DN). Since light attenuation depends on the square of the distance [37], the distance eﬀect was overcome after applying range correction (Equation (6)) and obtaining similar mean NIRc values at both of the distances tested (Figure 11b). This same eﬀect was also observed under diﬀerent lighting conditions in the ﬁrst experiment (Section 3.1.4, Figure 8). the NIR intensity distribution at 1.5 m differed significantly from that at 2.5 m (Figure 11a). The point cloud acquired at 2.5 m showed a mean NIR intensity of 1205 (DN) with a standard deviation of 509 (DN), while at 1.5 m the mean NIR was 3996 (DN) and the standard deviation was 2000 (DN). Since light attenuation depends on the square of the distance [37], the distance effect was overcome after applying range correction (Equation (6)) and obtaining similar mean NIRc values at both of the distances tested (Figure 11b). This same effect was also observed under different lighting conditions in the first experiment (Section 3.1.4, Figure 8). (a) (b) Figure 11. Distribution of the backscattered NIR (a) and NIRc (b) intensities of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. Figure 11. Distribution of the backscattered NIR (a) and NIRc (b) intensities of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. (b) (a) (b) (a) Figure 11. Distribution of the backscattered NIR (a) and NIRc (b) intensities of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. Figure 11. Distribution of the backscattered NIR (a) and NIRc (b) intensities of point clouds acquired from 1.5 m (orange) and 2.5 m (blue). The colour brown corresponds to the orange and blue bars overlapping. 4. Discussion 4. Discussion On the other hand, if the main goal is to classify tree elements (such as leaves, fruits, trunks, branches) and/or to assess their condition (e.g., state of fruit ripeness, or water stress) from leaf colour, obtaining sensor conditions that can provide The objectives and agronomic applications of RGB-D sensors are highly varied, and high levels of performance are not always required for both geometrical and spectral data. In consequence, diﬀerent optimal working conditions may be established depending on the application. For instance, if the objective is to ﬁnd only the geometric characteristics of an orchard [38], or to measure the porosity of its canopy [39], it should be a priority to capture as many points as possible, even if this entails sacriﬁcing the colour data at each point. In such a case, the measurements should be performed in the absence of, or with only low levels, of light. On the other hand, if the main goal is to classify tree elements (such as leaves, fruits, trunks, branches) and/or to assess their condition (e.g., state of fruit ripeness, or water stress) from leaf colour, obtaining sensor conditions that can provide good colour quality while, at the same time, still maintaining a signiﬁcant number of points should be prioritized. In this case, low illuminance should be avoided. When both colour and geometric features are important, the hours of the day guaranteeing optimal lighting conditions are very limited. Even so, scanning conditions can be improved by adapting and optimising the experimental layout. One solution could be to plan the captures so that the sensor 17 of 21 Sensors 2020, 20, 7072 only measures the indirectly illuminated side of each row when solar illumination is too intense. This was evaluated using sensor station K2S3, which showed acceptable geometrical performance throughout the day, reporting mean point cloud densities that were greater than 1.5 × 105 points m−3. Another solution would be to reduce the distance between the sensor and the target. By placing the sensor 1.5 m from the tree row axis (K2S2 and K2S5), the average point cloud density increased, registering mean density values of more than 4 × 105 points m−3 throughout the experiment. The main disadvantage of scanning from a shorter distance is that the sensor FOV is reduced signiﬁcantly. 4. Discussion 4. Discussion This issue could be solved by using an array of sensors distributed at diﬀerent heights with a certain degree of overlap and using them to obtain a single point cloud for the whole captured scene [40,41]. Finally, it would be possible to extend the period with adequate lighting conditions by using artiﬁcial lighting. This has been considered in many agricultural applications that require high-quality colour data, such as automatic fruit detection [42], weed detection [43] and with harvesting robots [44], among others. Besides extending the period with adequate illumination, the use of artiﬁcial lighting optimises the illumination, thereby reducing any undesirable eﬀects such as shadows or specular reﬂections. In [45], the authors used an over-the-row machine to cover the measured scene, preventing high illuminance from direct sunlight and obtaining better control of the light by means of artiﬁcial lighting. This solution could provide optimal lighting conditions throughout the day, but would require a bulky and unwieldy scanning machine. Although all the tests carried out in this work involved Kinect v2, the method could be applied to evaluate any other RGB-D sensor. Other authors have evaluated the performance of diﬀerent RGB-D sensors under diﬀerent measuring conditions. In [46], ten RGB-D sensors were evaluated considering the inﬂuence of diﬀerent target materials, lighting conditions and interference from other sensors, but all of those tests were carried out under indoor lighting conditions at under 535 lx. Similarly to the present work, the Kinect v2 sensor evaluated in [46] did not show any signiﬁcant diﬀerences in accuracy or precision at distances of between 2.5 m and 1.5 m under low illuminance conditions. In [47], three diﬀerent RGB-D sensors were tested. Outdoor experiments evaluated the sensors from the geometric point of view, considering diﬀerent materials, shapes, distances, angles of incidence and light intensities ranging from 15000 lx to 100000 lx. Despite there being fewer points in high illuminance environments, the results showed that the Kinect v2 performed well to ﬁt planes and cylinders on geometrical surfaces. These results were consistent with those reported in the present work, as a relatively small number of points tended to be enough to ﬁt a geometrical shape. Despite having a low point cloud resolution, the accuracy of the sensor was not aﬀected by high lighting conditions. Similarly, in [33], four RGB-D sensors were tested to measure young corn plants and tomatoes. 5. Conclusions This work presents a methodology for assessing RGB-D sensors used in fruit tree orchards within the framework of precision agriculture. This was used to evaluate the performance of the Kinect v2 sensor under diﬀerent scanning conditions. Two diﬀerent experiments were carried out. The ﬁrst evaluated sensor performance under diﬀerent lighting conditions. The results showed that the point cloud resolution remained stable at low illuminance levels, but decreased signiﬁcantly at middle to high illuminance levels (>2000 lx). This illuminance eﬀect was slightly reduced when measurements were taken closer to the target (canopy), but short-range measurements also reduced sensor FOV, thereby limiting the capacity to characterize trees with a single sensor. Although high illuminance conditions reduced the number of points obtained, the accuracy of the remaining points was not aﬀected. In contrast, since the points obtained at high illuminance diﬀered between replicates, the precision of the measurements was aﬀected at times of high exposure to sunlight. The colour data showed a good adaptation to diﬀerent lighting conditions above 50 lx, but below this level the illuminance was too low to be corrected by the sensor exposure adjustment. The second experiment aimed to assess sensor performance at diﬀerent distances from the target. The results showed that point cloud resolution decreased with distance. This eﬀect was mainly due to the higher FOV of the sensor and the constant number of 3D points measured. In addition, as NIR light intensity decreases quadratically with distance when measuring from long distances, the NIR camera could not distinguish the sensor-emitted light. This attenuation of the light was clearly observed when comparing the NIR intensities obtained from distances of 1.5 m and 2.5 m. To overcome this attenuation of the light, we range-corrected the NIR light and obtained a similar distribution of NIRc intensities when correcting both captures (1.5 m and 2.5 m). The proposed method was able to evaluate RGB-D sensors in terms of geometrical and spectral features and to consider the diﬀerent scanning conditions found in commercial orchards. Although the RGB-D sensor that we tested was originally developed to work under indoor conditions, it has been shown that it can also be used under certain speciﬁc outdoor conditions. The optimal lighting conditions for using the Kinect v2 in agricultural environments range from 50 lx to 2000 lx. However, this range could be expanded for agricultural applications that do not require full sensor performance. 4. Discussion 4. Discussion The scanning conditions considered included diﬀerent distances from the target, ranging from 0.2 m to 1.5 m, and four illuminance intensities. The geometrical parameters measured were the ﬁll rate and the RMSE obtained when measuring tomatoes. The RMSE results reported in [33] for Kinect v2 showed an increase with distance, which contrasts with the results obtained in the present work and in [46], in which no signiﬁcant diﬀerence was observed between the accuracy measured at 1.5 m and at 2.5 m. However, these results are not fully comparable since the ranges of distance tested in the two works were diﬀerent. The precision of the measurements could have been aﬀected by the movement of leaves and branches under high wind conditions. The highest wind speeds were recorded from 15:30 (UTC + 1) to 17:00 (UTC + 1) (Figure 3), with values ranging from 5 to 6 m s−1. Even so, no signiﬁcant diﬀerences were observed in the precision error metric during this period. These results were similar to those reported in [29], from which it could be concluded that sensor performance was not aﬀected by wind speeds under 5 m s−1. The proposed methodology includes a comprehensive evaluation of RGB-D sensors and oﬀers certain advantages over methodologies used in previous works [33,46,47]. Firstly, this methodology was speciﬁcally designed to evaluate RGB-D sensors in orchard environments. It considers a wide range of lighting conditions, two diﬀerent sensor orientations and distances from the row axis of 1.5 m and 2.5 m, which is similar to the spacing between the tree row axis and the centre of the alleyway in 18 of 21 18 of 21 Sensors 2020, 20, 7072 most commercial orchards. In addition to the geometrical assessment, the present work also includes an evaluation of the colour and NIR intensities, which are important factors in many agriculture applications. Finally, to facilitate the use of the present methodology in future works, with other sensors and under other conditions, the KEvOr dataset and the processing code have been made publicly available at [34] and [35], respectively. Author Contributions: Conceptualization, A.E. and J.R.R.-P.; methodology, A.E., J.R.R.-P., J.A., J.G.-M., F.S. and J.L.; software, J.G.-M. and J.R.R.-P.; formal analysis, A.E., J.A., J.R.R.-P., J.G.-M. and J.L.; investigation, J.G.-M., J.L., A.E., E.G. and J.R.R.-P.; data curation, A.E., J.G.-M., J.L. and J.R.R.-P.; writing—original draft preparation, J.G.-M., A.E., E.G. and J.A.M.-C.; writing—review and editing, J.L., A.E., J.R.R.-P., E.G., J.G.-M. and F.S.; visualization, J.G.-M., J.L., E.G. and J.R.R.-P.; project administration, J.R.R.-P., J.A.M.-C. All authors have read and agreed to the published version of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: The authors would like to express their gratitude to Manel Ribes Dasi and Xavier Tor their contributions in the data processing. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness and the Ministry of Science, Innovation and Universities through the program Plan Estatal I+D+i Orientada a los Retos de la Sociedad, grant numbers AGL2013-48297-C2-2-R and RTI2018-094222-B-I00, respectively. References 1. Henry, P.; Krainin, M.; Herbst, E.; Ren, X.; Fox, D. RGB-D mapping: Using Kinect-style depth cameras for dense 3D modeling of indoor environments. Int. J. Rob. Res. 2012, 31, 647–663. [CrossRef] 1. Henry, P.; Krainin, M.; Herbst, E.; Ren, X.; Fox, D. RGB-D mapping: Using Kinect-style depth cameras for dense 3D modeling of indoor environments. Int. J. Rob. Res. 2012, 31, 647–663. [CrossRef] 2. Sanz, R.; Llorens, J.; Escolà, A.; Arnó, J.; Planas, S.; Román, C.; Rosell-Polo, J.R. LIDAR and non-LIDAR-based canopy parameters to estimate the leaf area in fruit trees and vineyard. Agric. For. Meteorol. 2018, 260, 229–239. [CrossRef] 3. Gené-Mola, J.; Sanz-Cortiella, R.; Rosell-Polo, J.R.; Morros, J.-R.; Ruiz-Hidalgo, J.; Vilaplana, V.; Gregorio, E. Fruit detection and 3D location using instance segmentation neural networks and structure-from-motion photogrammetry. Comput. Electron. Agric. 2020, 169. [CrossRef] 4. Sarbolandi, H.; Leﬂoch, D.; Kolb, A. Kinect range sensing: Structured-light versus Time-of-Flight Comput. Vis. Image Underst. 2015. [CrossRef] 5. Dal Mutto, C.; Zanuttigh, P.; Cortelazzo, G. Time-of-Flight Cameras and Microsoft KinectTM; Springer Science & Business Media: New York, NY, USA, 2012. 6. Giancola, S.; Valenti, M.; Sala, R. A survey on 3D cameras: Metrological comparison of time-of-ﬂight, structured-light and active stereoscopy technologies. In Springer Briefs in Computer Science; Springer: Berlin/Heidelberg, Germany, 2018. 7. Gené-Mola, J.; Vilaplana, V.; Rosell-Polo, J.R.; Morros, J.R.; Ruiz-Hidalgo, J.; Gregorio, E. Multi-modal deep learning for Fuji apple detection using RGB-D cameras and their radiometric capabilities. Comput. Electron. Agric. 2019, 162, 689–698. [CrossRef] p g 8. Nguyen, T.T.; Vandevoorde, K.; Wouters, N.; Kayacan, E.; De Baerdemaeker, J.G.; Saeys, W. Detection of red and bicoloured apples on tree with an RGB-D camera. Biosyst. Eng. 2016, 146, 33–44. [CrossRef] 9. Nissimov, S.; Goldberger, J.; Alchanatis, V. Obstacle detection in a greenhouse environment using the Kinect sensor. Comput. Electron. Agric. 2015, 113, 104–115. [CrossRef] 10. Xiong, Y.; Peng, C.; Grimstad, L.; From, P.J.; Isler, V. Development and ﬁeld evaluation of a strawberry harvesting robot with a cable-driven gripper. Comput. Electron. Agric. 2019, 157, 392–402. [CrossRef] 11. 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Author Contributions: Conceptualization, A.E. and J.R.R.-P.; methodology, A.E., J.R.R.-P., J.A., J.G.-M., F.S. and J.L.; software, J.G.-M. and J.R.R.-P.; formal analysis, A.E., J.A., J.R.R.-P., J.G.-M. and J.L.; investigation, J.G.-M., J.L., A.E., E.G. and J.R.R.-P.; data curation, A.E., J.G.-M., J.L. and J.R.R.-P.; writing—original draft preparation, J.G.-M., A.E., E.G. and J.A.M.-C.; writing—review and editing, J.L., A.E., J.R.R.-P., E.G., J.G.-M. and F.S.; visualization, J.G.-M., J.L., E.G. and J.R.R.-P.; project administration, J.R.R.-P., J.A.M.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness and the Ministry of Science, Innovation and Universities through the program Plan Estatal I+D+i Orientada a los Retos de la Sociedad, grant numbers AGL2013-48297-C2-2-R and RTI2018-094222-B-I00, respectively. Acknowledgments: The authors would like to express their gratitude to Manel Ribes Dasi and Xavier Torrent for their contributions in the data processing. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 19 of 21 19 of 21 Sensors 2020, 20, 7072 References Chéné, Y.; Rousseau, D.; Lucidarme, P.; Bertheloot, J.; Caﬃer, V.; Morel, P.; Belin, É.; Chapeau-Blondeau, F. On the use of depth camera for 3D phenotyping of entire plants. Comput. Electron. Agric. 2012, 82, 122–127. [CrossRef] 14. Xia, C.; Wang, L.; Chung, B.K.; Lee, J.M. In situ 3D segmentation of individual plant leaves using a RGB-D camera for agricultural automation. Sensors 2015, 15, 20463–20479. [CrossRef] [PubMed] 15. Li, D.; Xu, L.; Tan, C.; Goodman, E.D.; Fu, D.; Xin, L. Digitization and visualization of greenhouse tomato plants in indoor environments. Sensors 2015, 15, 4019–4051. [CrossRef] [PubMed] 16. 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W3022421248.txt	https://www.nature.com/articles/s41467-017-01107-0.pdf	en		Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration	Nature communications	2,017	cc-by	14,387	ARTICLE DOI: 10.1038/s41467-017-01107-0 OPEN Asymmetric localization of DLC1 deﬁnes avian trunk neural crest polarity for directional delamination and migration Jessica Aijia Liu1, Yanxia Rao1, May Pui Lai Cheung1, Man-Ning Hui2, Ming-Hoi Wu1, Lo-Kong Chan3, Irene Oi-Lin Ng3, Ben Niu1, Kathryn S.E. Cheah1, Rakesh Sharma4, Louis Hodgson 5 & Martin Cheung1 Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its ﬂuctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC speciﬁers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization. 1 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 2 Department of Obstetrics and Gynaecology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 3 State Key Laboratory for Liver Research and Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 4 Proteomics and Metabolomics Core Facility, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. 5 Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Jessica Aijia Liu and Martin Cheung contributed equally to this work. Correspondence and requests for materials should be addressed to M.C. (email: mcheung9@hku.hk) NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 D elamination and migration of neural crest cells (NCCs) is one of the deﬁning embryonic events in vertebrate development and dysregulation of these processes leads to various human diseases1. Multipotent NCCs originate in the dorsal neural tube and implement a transcriptional programme to initiate an epithelial-mesenchymal transition (EMT) that involves alteration of cytoskeletal structure, loss of cell-cell adhesion and apical-basal polarity to convert an epithelial cell into a mesenchymal motile phenotype2. At the onset of delamination, NCCs acquire front-back polarity that is essential for directional migration into the periphery where they differentiate into neurons and glia3. However, characteristic molecular circuits regulating NCC morphodynamics at this stage are not known. The RHO family of small GTPases acts as molecular switches, cycling between an active GTP-bound and an inactive GDPbound state. RHO GTPases play key roles in regulating cytoskeletal dynamics, polarization and adhesion during cell migration4–6. Previous studies demonstrated that functional requirements for the levels of RHO signaling in EMT differ between species or cranial vs. trunk NCCs7–11. However, conﬂicting studies using distinct RHO inhibitors treatment at the same axial level have not clearly deﬁned whether RHO plays a positive or negative role in trunk NC delamination8–10. These differing reports likely reﬂect the fact that RHO signaling is highly context dependent where precise spatiotemporal dynamics of RHO activity determines cellular phenotypes. Previous studies have shown that localized RHO activation in a discrete apical region of premigratory cranial NCCs is essential for detachment from the neuroepithelium12. So far, there is not yet a clear picture of RHO activity in emigrating trunk NCCs in favor of a speciﬁc GTPase at subcellular scales. Moreover, how this dynamic cartography of the RHO GTPase activity is spatially regulated and coordinated with NC polarity and migratory behavior are unknown. We thus examined the regulatory principles that link activity of RHO signaling and their regulators to trunk NCC polarization and directional migration. In this study, using a ﬂuorescence resonance energy transfer (FRET)-based biosensor for RHOA in chick embryos and live-cell imaging in vitro, we demonstrate that high RHOA activity in the cytoplasm deﬁnes the prospective rear of delaminating and migratory trunk NCCs while ﬂuctuating RHOA activity in the membrane protrusion at the cell front. This differential RHOA activity is regulated by asymmetric localization of the RhoGAP Deleted in liver cancer 1 (DLC1) in the cytoplasm at the cell front. Gain- and loss-of-function studies reveal that appropriate level of DLC1 activity is essential for the establishment of NC polarity through spatial restriction of RHOA activity at the back, which is prerequisite for directional delamination and migration. Importantly, this differential distribution of DLC1 depends on its binding partner, NEDD9 and their association is required for the establishment of polarized rather than the total level of RHOA activity. Last, DLC1 and NEDD9 genes are subject to transcriptional regulation by NC speciﬁers SOX10 and SOX9 respectively. Together, these results reveal a SOX9/SOX10NEDD9/DLC1-RHOA regulatory axis to orient trunk NCCs in the direction of movement. Results Asymmetric active RHOA indicates NC migratory polarization. Although subcellular localization of RHOA activity has been shown to be crucial in determining polarity of different cell types13, whether RHOA also engages in establishing trunk NC polarity is largely unknown. To investigate the spatiotemporal dynamics of RHOA signaling in delaminating and migrating 2 NCCs, we electroporated caudal neural tube of chick embryos in Hamburger and Hamilton stages14 (st) 11–12 with a RHOA single chain FRET-based biosensor that was previously used to detect localized RHOA activation in ﬁbroblasts15. At 24 h post-transfection (hpt), electroporated embryos at st 15–16 were harvested for FRET analysis on cross-section of the thoracic neural tube where NC delamination and migration are ongoing. FRET imaging and measurement of the FRET index between the back and front of NCCs revealed high RHOA activity in the cytoplasm of the cell rear relative to low or ﬂuctuating RHOA activity in membrane protrusions at the leading front of delaminating/early- and late-migrating NCCs, which are marked by NC speciﬁers, SOX9 and SOX10, respectively (Fig. 1a–d). In contrast, moderate level of RHOA activity is detected and uniformly distributed throughout neuroepithelial cells in the neural tube (Supplementary Fig. 1a, b). These results indicate that NCCs display differential RHOA activity in subcellular localization as they undergo directional delamination and migration. To examine the dynamics of RHOA activity in live NCCs, we electroporated neural tube explants with FRET biosensor and performed time-lapse imaging of NCCs undergoing directional delamination onto ﬁbronectin-coated dishes. Consistent with in vivo observations, imaging results revealed that RHOA activity was highly enriched at the cell rear and also dynamically localized in membrane protrusions at the leading edge of polarized NCCs emigrating from the explants (Fig. 1e and Supplementary Movie 1). To determine the persistence of RHOA activity, we measured the FRET index between the back and front of polarized NCCs over time4. We found that RHOA activity was persistently high at the back while ﬂuctuating levels were observed at the front during directional migration over time (Fig. 1f). Analysis of the total FRET signal showed that RHOA activity was higher at the back than to the front (Fig. 1g). Clustering of the FRET index ratio between the back and front over a 20 min of live cell imaging conﬁrmed the maintenance of polarized RHOA activity during migration (Fig. 1h). To further interrogate whether this differential RHOA activity is maintained as NCCs change in migratory direction, emigrating NCCs from neural tube explants were exposed to beads coated with stromal cell derived factor 1 (SDF-1), which is a chemoattractant for trunk NCCs16, to mimic the in vivo environment. We observed initial polarization of RHOA activity along the front-back axis and the subsequent re-localization of high RHOA to the prospective cell rear was synchronized with the establishment of new membrane protrusions at the cell front as NCCs underwent directional change in response to SDF-1 (Fig. 1i and j and Supplementary Movie 2). Quantiﬁcation of the FRET index between the back and front over time revealed the maintenance of high RHOA activity at the cell rear even when NCCs changed their direction of movement (Fig. 1k). Thus, pre-existing asymmetrical localization of RHOA activity deﬁnes the cell’s eventual direction of polarization. To further correlate asymmetric RHOA activity with cell polarization, we examined RHOA dynamics in a population of emigrating NCCs, which undergo front-back switch in response to SDF-1. Time-lapse FRET imaging showed that NCC with a front-back polarized morphology gradually acquired elevated RHOA activity at the front (A) which eventually became the back of the cell following cell repolarization together with the formation of a new membrane protrusion at the front pointing toward SDF-1 (B) (Fig. 1l and m and Supplementary Movie 3). Quantiﬁcation of the FRET activity between A and B over time revealed that redistribution of differential RHOA activity preceded initiation of a gradual front and back switch (Fig. 1n). Altogether, in vitro neural tube explant studies further consolidate in vivo observations that existing asymmetry of RHOA activity sometimes NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 Deleted in liver cancer 1 (DLC1), which has been shown to play a key role in the inhibition of tumor growth and metastasis through suppression of RHO signaling17, 18. The chick has three major transcriptional isoforms of the DLC1 gene (Fig. 2a). Comparative sequence analysis revealed that their amino acid sequences exhibit extensive overall homology with their counterparts in both human and mouse, with the greatest indicates the future back-front polarity and directional migration of trunk NCCs. DLC1 is asymmetrically expressed in migratory NCCs. In search for genes involved in regulating polarized activity of RHOA, we identiﬁed RhoGTPase-activating protein (RhoGAP), FRETSOX9DAPI c 3.05 FRET ratio back/FRET ratio front 2.70 d Polarized neural crest cell FRETSOX10DAPI 1 Late migrating NCCs Back 380 s 0s 722 s ns ns 2.0 1.5 1.0 0.5 ting ting ting ina migra migra lam De Early Late Front 1596 s 1102 s 2.5 1862 s 1 1.8 e FRETSOX9DAPI 1 2.35 Early migrating NCCs b 1 Delaminating NCCs a g Directional migration Front 1.4 1.2 0 1.0 t ck on Ba 500 1000 1500 Time (s) Fr Sample ID 20 min 1520 s FRET ratio back/FRET ratio front 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 Sample ID 1.5 Front Time 0 0s * 2.0 1.0 Back i 2.5 >2.03 1.39 20 min 0.9 0 2660 s 2280 s Sample ID h 1.6 1 2 3 4 5 6 7 8 9 10 11 12 Back Directional migration >1.43 1.0 0s 3420 s 20 min 0.82 3990 s 2.4 FRET ratio back/FRET ratio front Polarized neural crest cell Total FRET ratio (norm) f Front Front Back 1 Back k Change of migratory direction Front Front Back Back l 0s FRET ratio back/FRET ratio front SDF-1-coated beads A Front 2.5 1.5 1.0 1520 s 760 s Change of migratory direction 2.0 1000 2000 3000 4000 Time (s) 2242 s 3002 s 3724 s 2.4 j A Back Back B Front 1 B m Accumulation of Front Back Front to back switch Back Front SDF-1-coated beads NATURE COMMUNICATIONS \| 8: 1185 FRET ratio A/FRET ratio B n Re-polarized NCC high RHOA activity Re-polarized NCC 2.5 2.0 1.5 1.0 0 1000 2000 3000 4000 Time (s) \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 similarities in the four functional domains, the sterile α motif domain (SAM), the focal adhesion targeting (FAT) region, the RhoGAP and the steroidogenic acute regulatory domain (START) (Supplementary Fig. 2a, b). We ﬁrst examined DLC1 expression from st 8 to 14 chick embryos using a riboprobe against the conserved RhoGAP domain of the three isoforms (Fig. 2a). DLC1 mRNA was initially detected at st 8 and 9 in the premigratory and delaminating cranial NCCs that co-express with NC speciﬁer, SOX9, and the migratory NC marker, HNK-1 (Fig. 2d, e). DLC1 expression was maintained in migratory cranial NCCs in the midbrain, hindbrain regions and in the frontonasal process from st 11–14 (Fig. 2d). In the posterior trunk from st 11 to 13, DLC1 exhibited a similar expression pattern to SOX9 and other NC speciﬁers, SOX10 and SNAIL2, in early migrating NCCs (Fig. 2d, f). Coinciding with the rostral to caudal gradient of NC development, DLC1 was ﬁrst initiated in the premigratory and maintained in the migratory cranial NC population followed by expression in early migratory trunk NCCs. To further delineate which isoforms were expressed in the trunk NCCs, we took advantage of a SOX10 enhancer (SOX10-E1) to drive EGFP reporter expression speciﬁcally in vagal/trunk NCCs19. This allowed us to enrich this population of cells by ﬂuorescence activated cell sorting (FACS) for qPCR analysis with isoformspeciﬁc primers (Fig. 2b). The results showed that DLC1 isoform 3 was highly expressed in sorted EGFP+ cells compared to low levels expression of DLC1 isoforms 1 and 2 (Fig. 2c), indicating that isoform 3 is predominantly expressed in trunk NCCs. For simplicity, we therefore named isoform 3 as DLC1 for the rest of the analyses. Strikingly, immunoﬂuorescence analysis of neural tube explants showed that DLC1 exhibited asymmetric localization in the cytoplasm at the front of emigrating and migrating NCCs (Fig. 2g–j) but was not colocalized with the focal adhesion kinase (FAK) as shown in previous studies20 (Fig. 2k). Similarly, when V5-tagged DLC1 was ectopically expressed at low level in chick neural tube, we observed preferential cytoplasmic localization of DLC1 to the front of delaminating and early migrating NCCs (Supplementary Fig. 3a). To quantify asymmetric expression of DLC1, we measured signal intensity by line scan analysis along the cell body from different protrusions at the leading edge (Fig. 2h). The results revealed that DLC1 expression was enriched at the cell front in the cytoplasm between membrane protrusions and nucleus, whereas expression was low in the nucleus and at the back, indicating that DLC1 exhibited polarized expression at the onset of NC delamination (Fig. 2l). We then compared co-localization patterns between RHOA activity and DLC1 ﬂuorescence intensity from the leading cell edge to perinuclear region of cytoplasm in different groups of cells (Fig. 2m), and conﬁrmed a negative correlation with high DLC1 expression in the region of low RHOA activity and vice versa (Fig. 2n, o), suggesting that DLC1 may be involved in establishing polarized RHOA activity in delaminating NCCs. DLC1 regulates polarized RHOA activity and NC migration. To further investigate whether DLC1 regulates spatial restriction of RHOA activity for the establishment of NC polarity and directional migration, we overexpressed FRET biosensor together with DLC1, which harbors a strong RhoGAP activity to inhibit active form of RHOA-GTP expression as determined by RHOA pull-down activation assay, into the caudal hemineural tube of st 10–11 chick embryos (Fig. 3a, b). At 24hpt, analysis of FRET signals on sections showed that RHOA activity was reduced or barely detectable in DLC1 overexpressing NCCs, which exhibited lack of front-back polarity axis (Fig. 3c). In addition, overexpression of DLC1 did not induce ectopic expression of SOX9, SOX10 and HNK-1 in the neuroepithelium, suggesting that DLC1 is not sufﬁcient to trigger NC formation. However, expression of these markers in migratory NCCs was reduced on the transfected side (Fig. 3e–h and u). Strikingly, neuroepithelial cells overexpressing DLC1 were observed delaminating not only from the basal surface of the dorsal neural tube where laminin was lost but also into the lumen (Fig. 3i, j). Consistently, N-Cadherin (N-Cad) expression was lost at the apical surface of dorsal neuroepithelium (Fig. 3k, l), indicating that overexpression of DLC1 disrupted apical-basal polarity of dorsal neural tube cells. These data suggest that high level of DLC1 expression reduced RHOA activity, resulting in aberrant polarity of premigratory NCCs that delaminate into the neural tube lumen instead of following their normal migratory route. To examine whether inhibition of DLC1 function affected RHOA activity, NCC fate, polarity and migratory behavior, we generated a dominant negative version of DLC1 (DN-DLC1), consisting of an amino-terminal region of DLC1 without the RhoGAP and START domains (Fig. 3a). This construct was previously shown to impair DLC1 association with its partner factor, resulting in inhibition of cell motility in vitro21. By contrast to DLC1, overexpression of DN-DLC1 resulted in increased level of RHOA-GTP (Fig. 3b). To further demonstrate a direct inhibitory effect of DN-DLC1 on DLC1, we co-electroporated DN-DLC1 with DLC1 and found the level of RHOA-GTP was slightly reduced compared to the control and DLC1 (Fig. 3b), indicating that DN-DLC1 indeed functions as a dominant negative to inhibit endogenous RhoGAP activity of DLC1. Consistently, overexpression of DN-DLC1 in vivo resulted in a marked elevation of RHOA activity, which was evenly distributed throughout the cytoplasm of delaminating NCCs without distinct subcellular localization and cells appeared round Fig. 1 Asymmetric RHOA activity correlates with NC polarity and directed migration. Processed FRET signal images of delaminating a, early migrating b and late migrating NCCs c. The magniﬁed area and selected cells are marked with dotted squares. White arrowheads indicate FRET signal. Scale bars, 50 µm. d Schematic representation of asymmetric RHOA activity in a polarized NCC. Quantiﬁcation of the ratio of the FRET index between the back and front of delaminating (n = 107), early migrating (n = 123) and late migrating NCCs (n = 196). 25 embryos were analyzed. Mean ± s.e.m. Student’s t-test, ns, not signiﬁcant. e Processed FRET signal images of time-lapse series of delaminating NCCs. White arrowheads indicate RHOA activity at the cell front. Scale bar, 50 µm. f Schematic representation of asymmetric RHOA activity in a polarized NCC undergoing directional migration. Quantiﬁcation of the ratio of the FRET index between the back and front over time. n = 81 cells from 26 explants were analyzed. g Quantiﬁcation of the total FRET index in the indicated region. Mean±s.e.m.; n = 81; Student’s t test; p < 0.0001. h Heat maps representing the FRET index as a function of time at the back or at the front, and the FRET ratio between the back and front of 12 selected NCCs from 12 explants. i Processed FRET signal images of time-lapse series of a migrating NCC. White arrows indicate the change of migratory direction toward SDF-1. Scale bar, 50 µm. j Schematic representation of RHOA activity distribution in a polarized NCC undergoing directional change of movement toward SDF-1 coated beads. k Quantiﬁcation of the ratio of the FRET index between the back and front of migratory NCCs. n = 81 cells from 21 explants were analyzed. l Example of a stationary NCC undergoing front and back switch in response to SDF-1. n = 72 from 17 explants were analyzed. White arrowheads indicate initial accumulation of high RHOA activity in the cell front. White arrows indicate direction of cell repolarization. Scale bar, 50 µm. m Schematic representation of the example shown in Fig. 1l. n Quantiﬁcation of the ratio of the FRET index between A and B of the example shown in Fig. 1l. Mean ± s.e.m 4 NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 b c Relative fold change a SAM FAT Rho GAP 1532 aa START Isoform 2 SAM FAT Rho GAP START 1120 aa Isoform 3 SAM FAT Rho GAP START 1086 aa FSC-H DLC1 Isoform 1 250 K 200 K 150 K 100 K 50 K 0 FSC-H SOX10 enhancer (E1)-GFP GFP– 2 3 4 0 10 10 10 10 FITC-A 5 250 K 200 K 150 K 100 K 50 K 0 GFP+ 1.62% 2 3 4 0 10 10 10 10 FITC-A 5 20 * 15 10 5 0 m for Iso 1 m for 2 Iso m for 3 Iso e d Sense control e f DLC1 HNK-1 SOX9 f g st 9 st 11 st 14 st 13 DLC1+Phalloidin+DAPI DLC1 SOX9 SNAIL2 SOX10 DLC1 Migratory direction DLC1 4000 Neural tube explant Neural tube explant Neural tube explant 395 h DLC1 i Migratory direction DLC1 j 4000 395 l i k j FAK+DLC1+DAPI 3500 3000 2500 2000 1500 1000 500 0 Dlc1 DAPI 0 m n Front Fluorescence intensity (a.u.) FRET ratio 3.0 2.5 2.0 1.5 1.0 0.5 0 5 10 15 20 25 30 Distance (μm) 50 75 Distance (μm) Cell edge 1 25 o RHOA activity 1.9 Back Front Fluorescence intensity (a.u.) i Signal intensity st 8 Signal intensity st 9 Back 3500 3000 2500 2000 1500 1000 500 0 0 5 10 15 20 25 30 Distance (μm) Fig. 2 Asymmetric localization of DLC1 negatively correlates with RHOA activity. a Schematic representation of three chick DLC1 isoforms harboring four conserved regions: the sterile α motif (SAM) domain, the focal adhesion targeting (FAT) domain, the Rho GTPase activating (RhoGAP) domain and the steroidogenic acute regulatory (START) domain. b Flow cytometry enriched trunk NCCs (~ 1.62%) labeled by SOX10 enhancer (E1)-driven GFP expression. c mRNA expression levels of DLC1 isoforms in sorted NCCs. Mean ± s.e.m. Student’s t-test, **p < 0.0001 d Sense riboprobe for DLC1 serves as a negative control. In situ hybridization of DLC1 from stages (st) 8–14 chicken embryos. Scale bar, 150 µm. e Cross section at the cranial region of a st 9 chick embryo stained with DLC1 (black line) and immunoﬂuorescence for SOX9 and HNK-1 on consecutive sections. f Cross section at the trunk region of a st 11 chick embryo stained with DLC1 (black line) and immunoﬂuorescence for SOX9, SOX10 and SNAIL2 on consecutive sections. Scale bars, 50μm. Immunoﬂuorescence for DLC1 and phalloidin on delaminating g and early migratory NCCs h from neural tube explants and nuclei are stained with DAPI. DLC1 channels are also shown in pseudocolor. White dotted lines outline the border of neural plate explant. i, j Magniﬁcation of the boxed regions with enriched DLC1 expression shown in pseudocolor. k Immunoﬂuorescence for FAK and DLC1 on migratory NCCs and nuclei are stained with DAPI. l Line scans analysis showing the average ﬂuorescence intensity of DLC1 along the white dotted line in h. n = 47. m Representative image for the measurement of RHOA activity along the white line. Scale bars, 20μm. n Quantiﬁcation of RHOA activity (n = 26) and o DLC1 ﬂuorescence intensity (n = 47) from the leading cell front to the perinuclear region of cytoplasm NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 DN-DLC1 SAM FAT Rho GAP START EP: RHOA-GTP 25 20 Total RHOA 25 20 FAT Relative fold change SAM Ctrl DLC1 migratory NCCs expressing HNK-1 compared to the contralateral side (Fig. 3m–p and u). Consistently, the amount of early migratory NCCs expressing SOX9 and SOX10 were reduced on the transfected side but their expression in the premigratory domain remains unaltered, indicating that NC identity was not affected by overexpression of DN-DLC1 (Fig. 3q, r). In contrast to DLC1, the majority of NCCs expressing DN-DLC1 remained in DLC1 b DN-DLC1 a DLC1+DN-DLC1 in shape (Fig. 3c). Quantitative analysis of total FRET activity in vivo revealed that, compared to vector control, the overall levels of RHOA activation were signiﬁcantly increased (Student’s t-test, p < 0.0001) and reduced (Student’s t-test, p < 0.001) in NCCs overexpressing DN-DLC1 and DLC1, respectively (Fig. 3d). Analysis of NC markers revealed that overexpression of DN-DLC1 resulted in markedly fewer ns 1.8 * 1.2 0.6 0.0 rl Ct C1 C1 C1 DL +DL DL 1 LC -D DN DN c d 1 EP: DLC1 FRETSOX9DAPI GFPSOX10 e f Laminin GFPLaminin i GFPHNK-1 HNK-1 g h N-Cad GFPN-Cad k j * 10 9 8 7 6 5 4 3 2 1 0 1 1 rl Ct DLC LC D N D FRETSOX9DAPI GFPSOX9 EP: DLC1 (24 hpt) 8.3 Total FRET ratio(norm) 1.35 EP: DN-DLC1 1 l GFPHNK-1 n GFPSOX9 q o p GFPLaminin GFPN-Cad GFPSOX10 r s t u No. of emigrating GFP+ NCCsexpressing HNK-1 EP: DN-DLC1 (24 hpt) m 10 5 0 rl 1 Ct NATURE COMMUNICATIONS \| 8: 1185 LC -D DN 6 15 C1 DL \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 the neuroepithelium without disrupting apical-basal polarity (Fig. 3s, t). To further demonstrate that DLC1 is required for NC polarity and delamination, we electroporated a ﬂuoresceintagged morpholino targeting DLC1 isoform 3 (DLC1-MO) into the caudal hemineural tube of st 10–11 embryos. At 24 hpt, DLC1 protein was diminished using higher dosage of DLC1-MO, whereas its expression remained unaltered in embryos treated with control-MO (Ctrl-MO) and vector alone (Supplementary Fig. 4a). Similar reduction of DLC1 expression was observed in DLC1-MO-treated cultured NCCs, which appeared round in shape without distinct front-back polarity like DN-DLC1 expressing cells (Supplementary Fig. 4b and Fig. 3c). In agreement with this, cells expressing DLC1-MO resulted in reduced expression of FOXD3+ migrating NCCs compared to the untransfected side and the Ctrl-MO-treated embryos (Supplementary Fig. 4c). These results demonstrate functional requirements for DLC1 in the establishment of NC polarity and delamination. Altogether these in vivo studies indicate that appropriate level of DLC1 activity is required for the spatial restriction of RHOA activity, the establishment of NC polarity and directional delamination of NCCs. DLC1 spatially restricts RHOA for directed NC migration. To further evaluate how alteration of DLC1 activity affects the dynamics of NC migratory behavior in more details, we performed in vitro time-lapse imaging of delaminating NCCs from the neural tube explants electroporated with vector control, DLC1 and DN-DLC1 and examined the effects of cell polarity based on the degree of membrane protrusions22, 23. Since the formation of membrane protrusions is driven by actin dynamics, NCCs were also transfected with Lifeact-mCherry to label actin cytoskeleton for quantiﬁcation of the angles between membrane protrusion and migratory direction (Fig. 4a). As expected, GFP expressing cells migrated out of the explants, and exhibited a characteristic pattern of membrane protrusions at the cell front toward the direction of migration (Fig. 4a and Supplementary Movie 4). By contrast, NCCs overexpressing DLC1 lost frontback polarization with randomly oriented and erratic ﬁnger-like projections and exhibited loss of directionality (Fig. 4a and Supplementary Movie 5). Membrane protrusions in the form of blebbing were observed in DN-DLC1 expressing cells, which exhibited limited motility (Fig. 4a and Supplementary Movie 6). We then measured a set of migration parameters to determine how alteration of NC polarity by manipulation of DLC1 activity affected migratory behavior. The travel distance, persistence and net speed of cell migration were reduced in both DLC1 and DN-DLC1 overexpressing NCCs compared to the vector control and non-GFP expressing cells (Fig. 4b–e), indicating lack of directionality. The total speed of migrating NCCs expressing DN-DLC1 was markedly lower than that of DLC1 overexpressing cells, which migrated at a comparable speed to the control and non-GFP expressing cells (Fig. 4f). These results indicate that overexpression and dominant-negative inhibition of DLC1 activity disrupted NCC polarization and directional migration. We then evaluated their effects on RHOA activity in cultured NCCs transfected with FRET-biosensor in the presence of SDF-1 to determine whether polarized morphology and migratory behavior of NCCs could be restored by a guidance signal (Fig. 4g). Consistent with in vivo observations, time-lapse imaging and the FRET index between the back and front revealed that overexpression of DLC1 and DN-DLC1 resulted in marked reduction and elevation of RHOA activity throughout the cell respectively compared to the vector control, which exhibited a reproducible polarized distribution of RHOA activity with persistent high FRET signal at the back and migrated toward SDF-1 (Fig. 4h, i and Supplementary Movies 7–9). In addition, we observed a similar lack of cell polarity and directional movement in both treatments, suggesting that addition of SDF-1 in neural tube explant culture was not able to restore the morphological and migration defects caused by aberrant RHOA signaling (Fig. 4h). Altogether, these in vitro studies further consolidate in vivo results that delaminating NCCs require precise spatial activity of DLC1 for restricting RHOA activity high at the back that is essential for the acquisition of polarized NCC morphology and motility toward the source of chemoattractant. NEDD9 is required for the asymmetric localization of DLC1. Previous studies showed that DLC1 interacts with other factors to modulate its RhoGAP function17. To identify protein factors associated with DLC1, we performed immunoprecipitation with DLC1 antibody speciﬁc for isoform 3 using chick neural tube protein lysates. A proteomic analysis revealed different functional categories of proteins enriched by DLC1 (Fig. 5a, Supplementary Fig. 5a, b and Supplementary Data 1). The Ingenuity Pathway Knowledge base analysis showed that the majority of these proteins were involved in EIF2 signaling, remodeling of epithelial adherens junctions, and regulation of actin-based motility by RHO (Fig. 5b and Supplementary Data 2). Consistently, interactome network indicated that proteins related to cell motility and cytoskeleton regulation were highly associated with DLC1 (Fig. 5c). NEDD9, a member of the Cas family of scaffolding proteins, was chosen for further study because it has been shown to be involved in NC motility24 although the Fig. 3 DLC1 spatially restricts RHOA activity and regulates NC migratory behavior. a Schematic diagram of a full-length DLC1 and the dominant-negative DLC1 (DN-DLC1) containing the C-terminally truncated fragment of DLC1 without the RhoGAP and START domains. b Immunoblot for RHOA-GTP on protein lysates extracted from neural tubes electroporated with the indicated constructs at 24 hpt. Total RHOA is used as a loading control. Bars represent results from densitometric analysis (mean ± s.e.m., n = 3 independent experiments). Student’s t-test, p < 0.05; ns, not signiﬁcant. c Processed FRET signal images of cross-sections from embryos electroporated with the FRET probe plus DLC1 or DN-DLC1. NCCs are marked by SOX9 immunoﬂuorescence and nuclei are stained with DAPI. The magniﬁed area and selected cells are marked with dotted squares. White arrowheads indicate FRET signal. Scale bars, 20 µm. d Quantiﬁcation of the total FRET index in control (n = 425/25 embryos), NCCs expressing DN-DLC1 (n = 148/21 embryos) and DLC1 (n = 131/16 embryos). Student’s t-test, p < 0.001; p < 0.0001. e–l Immunoﬂuorescence for SOX9, SOX10, HNK-1, Laminin and N-Cad on transverse sections of embryos electroporated (EP) with DLC1 at 24 hpt (n = 12). Insets show the merge images of GFP and endogenous SOX9 or SOX10 expression. White triangle indicates endogenous HNK-1 expression in migratory NCCs, whereas open triangles indicate reduced expression of HNK-1, Laminin and N-Cad on the transfected side of the neural tube. Scale bars, 50μm. m, n Immunoﬂuorescence for HNK-1 in an embryo electroporated with DN-DLC1 at 24 hpt (n = 17). Scale bar, 150μm. o, p Cross-section at the level of the white line in m and n. White triangles indicate endogenous HNK-1 expression in migratory NCCs, whereas open triangles indicate reduced HNK-1 expression in the transfected side. q–t Immunoﬂuorescence for SOX9, SOX10, N-Cad and Laminin on transverse sections of embryos electroporated with DN-DLC1 at 24 hpt (n = 17). Insets show merge images of green and red channels. Scale bar, 50μm. u Quantiﬁcation of the number of emigrating GFP+ cells expressing HNK-1 in the transfected side of the neural tube electroporated with the indicated constructs. Average of cells counted from at least 30 sections of 10 embryos per treatment. Mean ± s.e.m. Student’s t-test p < 0.001 NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 Indeed, ectopic expression of DN-DLC1 was also able to co-immunoprecipitate with NEDD9. Further domain mapping revealed that the C-terminally truncated version of DN-DLC1 comprising of SAM and FAT domains [DN-DLC1 (320)]25 where most interaction partners of DLC1 bind could associate with mechanism of action is not known. Co-immunoprecipitation conﬁrmed an association of endogenous DLC1 and NEDD9 proteins in the embryonic neural tubes (Fig. 5d), raising the possibility that DN-DLC1 may interfere with the DLC1-NEDD9 interaction based on its mode of action from a previous study21. a 10 min Ctrl 0 min 30 min 20 min 90° 40 min DLC1 –50 0 –25 25 180° 50 0° 90° DN-Dlc1 –50 –25 0 25 180° 50 0° 90° 0° 0.2 ns 40 –50 –25 0 1 LC D 1 LC G -D N ns 50 FP 0 30 20 10 0 FP 1 1 -G LC on D FP LC G -D D N N FP -G on N FRET ratio B/FRET ratio A 100 es Non si -G ng F ce Plls f 10 1 1 200 pr ns 20 LC N D 200 2.0 Ctrl DN-DLC1 DLC1 1.5 * Bead 150 Distance (μm) 30 D FP LC -D FP on -G G i 100 –200 0 Back (side B) 50 –100 300 1 0.4 N 0 –200 –150 –100 –50 –50 1 0.6 1.0 0.5 0 1 50 20 0 Speed (μm/h) Net speed (μm/h) Persistence 40 Front (side A) 500 1000 1500 2000 Time (s) 2.5 0s 228 s 570 s 874 s 1178 s 1862 s 0s 228 s 570 s 874 s 1102 s 1862 s 228 s 570 s 874 s 1178 s 1862 s DLC1 Ctrl h 100 –150 e * * 0.0 g 200 Distance (μm) Distance (μm) ns 150 ex –180 Migratory distance relative to explant border (μm) –130 –200 0.8 100 –80 –150 d 50 LC –100 –200 –150 –100 –50 0 –30 * * ns D 400 LC 300 FP 200 20 -D 100 150 70 400 N 0 –400 –300 –200 –100 0 –50 200 120 D 50 Distance (μm) Distance (μm) 100 c DLC1 170 D DN-DLC1 150 G GFP 200 Distance (μm) b 25 50 180° 1 7.2 DN-DLC1 0s 8 NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 NEDD9, whereas SAM domain alone did not show interaction with NEDD9 (Fig. 5e). Consistently, overexpression of DN-DLC1 or DN-DLC1 (320) but not the SAM domain alone was able to reduce NC delamination (Supplementary Fig. 6a). These results suggest that NEDD9 could be sequestered from its endogenous interaction with DLC1 through binding to the FAT domain of DN-DLC1. In situ hybridization on consecutive sections show that NEDD9 mRNA was detected in the premigratory and early migratory trunk NCCs of st 11 embryos in a similar manner to DLC1, SOX9 and SOX10. In addition, NEDD9 expression was also notable in the ﬂoor plate. The distribution of NEDD9 transcripts resemble its protein expression which was detected in the cytoplasm of the premigratory and emigrating NCCs in an overlapping manner with nuclear localization of SOX9 and SOX10 proteins, indicating co-localization of these factors at the onset of NC delamination (Fig. 5f). Immunoﬂuorescence analysis indicated that NEDD9 exhibited a similar pattern to DLC1 with polarized localization between the leading edge and the nucleus of emigrating NCCs (Fig. 5g). We then examined whether the spatial expression of DLC1 is altered in cells treated with NEDD9 morpholino (NEDD9-MO), which reduced levels of NEDD9 protein in delaminating NCCs, compared to the untransfected side and embryos treated with control-MO (Ctrl-MO) (Supplementary Fig. 7a). NEDD9-MO resulted in aberrant and elongated morphology of NCCs without discernible front-back polarity (Fig. 5h). Importantly DLC1 protein exhibited random distribution throughout the NEDD9-MO transfected cells as shown by line scan analysis while polarized DLC1 expression remained unaltered in Ctrl-MO-treated cells (Fig. 5h, i), suggesting that the asymmetric localization of DLC1 is regulated by association with its binding partner NEDD9. Association of DLC1 with NEDD9 restricts active RhoA. Since NEDD9 has been implicated in regulating Rho GTPases in cancer cells26, the above ﬁndings raise two possibilities. The loss of NEDD9 function might lead to aberrant RHOA activity resulting in loss of cell polarity. Alternatively, mislocalization of DLC1 is the primary cause for the dysregulated NCC polarity in NEDD9MO. To distinguish these possibilities, we ﬁrst examined RHOA activity in delaminating NCCs expressing NEDD9-MO in vivo and in neural tube explants treated with SDF-1 (Fig. 6a, c). Surprisingly quantiﬁcation of the total FRET activity in cells treated with NEDD9-MO in vivo was comparable to the vector control and NEDD9 overexpressing cells (Fig. 6b), suggesting that loss of NEDD9 function and increased level of NEDD9 expression have no direct impact on total RHOA activity. However, we observed mislocalization of RHOA activity around the nucleus of NEDD9-MO-treated cell, which exhibited aberrant front-back polarization and lack of directionality in migration (Fig. 6a, c and Supplementary Movie 10). Consistently the FRET index between the back and front of NEDD9-MO-treated cells did not exhibit differential character compared to the control and NEDD9 overexpressing cells (Fig. 6b–d, f). These data suggest that lack of RHOA polarity in the absence of NEDD9 function could be attributed to the dysregulated localization of DLC1, which disrupted precise spatial RHOA restriction without altering the total level of RHOA activity. This prompted us to further examine whether association of DLC1 with NEDD9 is functionally required for the establishment of RHOA polarity. We then overexpressed DN-DLC1 to impair interaction of endogenous DLC1 with NEDD9, rendering DLC1 incapable for spatial restriction of RHOA activity at the cell rear. If that was the case, polarized RHOA activity could be restored by overexpression of NEDD9 in DN-DLC1 expressing cells. Indeed, the differential distribution of RHOA activity between the back and front in DNDLC1 + NEDD9 expressing cells in vivo was restored with the FRET index comparable to the control and NEDD9 overexpression alone, but its total FRET signal remained higher than other treatments (Fig. 6a, b). Clustering of the FRET index between the back and front of cultured NCCs expressing DNDLC1 + NEDD9 also showed restoration of differential RHOA activity like NEDD9 overexpressing cells, and cells exhibited polarized morphology and directional migration (Fig. 6c, e, f and Supplementary Movies 11 and 12). In agreement with this, restoration of NC delamination was observed in embryos electroporated with DN-DLC1 + NEDD9 as opposed to a marked reduction of SOX10+ and HNK-1+ cells in the transfected side of embryos electroporated with DN-DLC1 or NEDD9-MO, whereas overexpression of NEDD9 alone did not affect NC delamination (Fig. 6g, h). Collectively, these data show that interaction of DLC1 with NEDD9 is essential for the spatial restriction of RHOA activity at the back of the cell and the leading edge that is prerequisite for the establishment of a front-rear polarity axis and directional delamination and migration of trunk NCCs. SOXE factors regulate DLC1 and NEDD9 genes expression. The SOXE transcription factors SOX9 and SOX10 play important roles in regulating trunk NC speciﬁcation and delamination27, 28. The overlapping expression of DLC1, NEDD9, SOX9 and SOX10 in premigratory and early migratory NCCs prompted us to examine whether DLC1 and NEDD9 genes expression are subjected to the transcriptional regulation by SOX9 and/or SOX10. The results revealed that SOX9 or SOX10 was sufﬁcient to induce ectopic expression of DLC1 throughout the neural tube at 12–24 hpt but not 6 hpt (Fig. 7a and Supplementary Fig. 8a, b). Considering SOX9 induced SOX10 expression29, DLC1 was not induced in the neural tube electroporated with SOX9 plus SOX10-MO at 24 hpt (Supplementary Fig. 8c). Consistently, downregulation of DLC1 expression was observed in SOX10-MO treated embryos, compared to Ctrl-MO (Fig. 7b). These results show that SOX9 induces SOX10 to regulate DLC1 expression. To further investigate whether DLC1 could mediate SOX9 and SOX10-induced NC delamination and migration, we electroporated SOX9 or SOX10 plus DN-DLC1 and examined their Fig. 4 Appropriate level of DLC1 activity is required for directional migration of NCCs. a Time-lapse imaging showing the protrusion dynamics of emigrating NCCs expressing GFP control, DLC1 and DN-DLC1. White arrows indicate protrusions direction. Polar histogram plots showing distribution of the angle between actin protrusions and subsequent migration direction from all the time series. n = 22/10 explants for GFP control, n = 20/12 explants for DLC1, n = 25/12 explants for DN-DLC1. Scale bar, 20 μm. b Migration tracks of 17 representative NCCs expressing GFP control, DN-DLC1 and DLC1 are shown. Quantiﬁcation of the total migratory distance c, persistence d, net speed e and total speed f of NCCs electroporated with the indicated constructs. GFP and non-GFP cells serve as controls. Mean±s.e.m. Student’s t-test, *p < 0.0001; p < 0.001; ns, not signiﬁcant. g Schematic diagram showing the back (side B) and front side (side A) of a neural crest cell migrating toward SDF-1 coated beads. Scale bar, 50 μm. h Processed FRET signal images of time-lapse series of emigrating NCCs from neural tube explants treated with vector control (n = 111/16 explants), DLC1 (n = 93/17 explants) and DN-DLC1 (n = 88/13 explants). Scale bars, 50 μm. i Quantiﬁcation of the ratio of the FRET index between the back (B) and front (A) of the cluster for the indicated conditions. Mean±s.e.m. Student’s t-test, *p < 0.0001 NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 impact on NC motility using FOXD3 as a migratory NC marker (Fig. 7c, d). While overexpression of SOX9 or SOX10 promoted robust NC delamination and migration, electroporation of SOX9 + DN-DLC1 resulted in almost complete abrogation of NC delamination and migration (Fig. 7c, d). The effects were relatively mild in embryos electroporated with SOX10 + DN-DLC1 with reduced FOXD3+ migrating NCCs compared to the a c IgG DLC1 untransfected side (Fig. 7d). Together, these data demonstrate that SOX10 promoted NC migration through regulation of DLC1. We then examined whether SOX9 and/or SOX10 regulates NEDD9 expression. Electroporation of SOX9 resulted in ectopic expression of NEDD9 at 6 and 12 hpt throughout the dorsalventral axis of the neural tube compared to non-electroporated side (Fig. 7e and Supplementary Fig. 8d). In addition, SOX9 Cell motility/cytoskeleton regulation Action types Activation Inhibition Binding 69 236 Catalysis 121 Phenotype Posttranslational modification Reaction Transcriptional regulation b Top canonical pathways Overlaps with dataset –log(P value) No overlaps with dataset EIF2 signaling 194 Epithelial adherens junction signaling 146 Actin cytoskeleton signaling 228 Regulation of eIF4 and p70S6K signaling 157 mTOR signaling 199 ILK signaling 196 Tight junction signaling 167 Remodeling of epithelial adherens junctions 68 Regulation of Actin-based motility by Rho 91 d 120 110 100 90 80 70 60 50 40 30 20 0 10 Percentage f IP:IgG IP:DLC1 NT 130 αNEDD9 100 130 αDLC1 100 e DN-DLC1 SAM FAT DN-DLC1 (320) SAM FAT DN-DLC1 (SAM) SAM NEDD9 DLC1 SOX9 SOX10 NEDD9 SOX9 SOX10 Merge NEDD9 4000 75 55 αV5 395 Signal intensity a NEDD9+Phalloidin+DAPI 3500 3000 2500 2000 1500 1000 500 0 Fluorescence intensity (a.u.) g pCIG DN-DLC1 (SAM) DN-DLC1 (320) EP: DN-DLC1 NT 25 NEDD9 DAPI 0 130 100 75 i DLC1+Phalloidin+NEDD9-MO DLC1 Signal intensity 395 NATURE COMMUNICATIONS \| 8: 1185 Fluorescence intensity (a.u.) DLC1 4000 10 50 Distance (μm) h DLC1+Phalloidin+Ctrl-MO 25 IP: αV5 αNEDD9 3500 3000 2500 2000 1500 1000 500 0 DLC1 DAPI 0 25 50 75 Distance (μm) \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 expressing cells were co-localized with ectopic NEDD9 protein expression, indicating that SOX9 induces NEDD9 expression in a cell-autonomous manner (Supplementary Fig. 8d). In agreement with this, depletion of SOX9 expression in SOX9-MO treated embryos resulted in downregulation of NEDD9 at 24 hpt compared to Ctrl-MO (Fig. 7f), indicating that SOX9 is required for NEDD9 expression. By contrast, depletion of SOX10 protein expression by Sox10-MO did not affect SOX9 and NEDD9 expression levels, and NEDD9 expression was still induced in SOX9 + SOX10-MO expressing cells (Supplementary Fig. 8e, f), suggesting that SOX10 is not required for NEDD9 expression. Consistently, overexpression of SOX10 was not able to induce ectopic NEDD9 expression though it triggered HNK-1 expression at 12 hpt (Supplementary Fig. 8g). Chromatin immunoprecipitation and reporter assays showed that NEDD9 is a direct transcriptional target of SOX9 but not SOX10 through binding to NEDD9 enhancer sequences but not promoter region (Fig. 7g, h and Supplementary Fig. 9a–d). Thus, these results indicate a speciﬁc role for SOX9 in the direct transcriptional regulation of NEDD9 expression. Accordingly, epistasis analysis revealed that cells expressing SOX9 + NEDD9-MO remained in the neuroepithelium and the number of HNK-1+ migratory NCCs on the transfected side was reduced compared to the vector control and embryos electroporated with SOX9 or SOX9 + Ctrl-MO (Fig. 7j, k). Taken together, these results show that SOX9 promotes NC delamination and migration by direct transcriptional activation of NEDD9 expression. Discussion During EMT, premigratory NCCs lost apical-basal polarity to delaminate from the dorsal neural tube followed by acquisition of a front-rear polarity axis that is pre-requisite for directional migration to their correct destinations for differentiation30, 31. While several studies have revealed the molecular control for regulating the cellular events during cranial NC EMT and directional migration7, 12, 32–34, candidate factors and the underlying mechanisms for trunk NCCs to acquire polarization and directional migration are not yet well understood. Our study reveals a SOXE-DLC1/NEDD9-RHOA regulatory axis to establish avian trunk NCC polarity for directional delamination and migration (Fig. 8). Small Rho GTPases spatiotemporally coordinate cell polarization and migration in variety types of cell4–6, 15. Previous studies in trunk NCCs utilized the Rho-binding domain of Rhotekin for the detection of active GTP-bound forms of RHO proteins in the membrane10. In addition, functional studies have largely relied on the use of distinct Rho inhibitors with differential potencies7, 8 or on expression of dominant-negative11. Although these studies have demonstrated the involvement of RHO signaling in NC motility, these approaches are potentially non-speciﬁc targeting RHOA, RHOB and RHOC activities or their downstream effectors resulting in either promoting or inhibiting NC delamination8–10. These conﬂicting ﬁndings reﬂect the fact that precise subcellular localization and activity of Rho GTPases control different cellular events, which cannot be precisely controlled in loss-of-function experiments. Moreover, which speciﬁc Rho GTPases are involved in establishing NC polarity remains elusive. Our study for the ﬁrst time reveals a unique spatiotemporal proﬁle of RHOA GTPase activity in delaminating and migrating trunk NCCs using FRET biosensor in chick embryos and neural tube explants. We provide two major insights in this study. First, we found that the majority of RHOA active GTP-bound form is localized in the cytoplasm at the cell back and also dynamically expressed at the leading edge that coincides with acquisition of a back-to-front polarity axis aligned with the prospective vector of migration. Second, the persistence of high RHOA activity in cytoplasm correlates with the eventual cell rear as NCCs undergo re-polarization concomitant with the change in migratory direction in response to chemoattractant, SDF-1. Thus, the asymmetric localization of RHOA activity is a key molecular signal that mediates cell orientation to the direction of movement. In contrast, previous FRET analysis in Xenopus embryos revealed that RHOA activity is localized to the cell periphery and elevated at the site of collision of two cranial NCCs leading to contact inhibition of locomotion32. NCCs arising from different axial levels may account for the differences in the subcellular localization of RHOA activity but its level of activity is similarly required for directional migration of both cranial and trunk NCCs. While cranial NCCs adopt contact inhibition of locomotion to govern their collective migratory behavior, trunk NCCs migrate as single cell chains and interact extensively with neighboring cells35. A recent report demonstrated that leader cells are crucial for orchestrating the orderly movement of trunk NCCs through cell-cell contact with follower cells36. Based on these studies together with our results, it is tempting to speculate that leader cells may provide guidance signals through dynamic interaction with the followers to maintain differential RHOA activity and front-back polarity axis for directed migratory patterns of trunk NCCs. Further analysis will be required to address this issue. The cytoplasmic localization at the rear-cell body suggest that RHOA might reside in the endoplasmic reticulum or endosomal vesicles as described previously37, serving as reservoir of active RHOA to be translocated to the leading edge of migrating cells. In addition, this pool of active RHOA has also been shown to regulate cellular contractility via microtubule depolymerization38, while dynamic activation of RHOA at the leading edge controls actin ﬁber assembly and focal adhesion dynamics to coordinate both extension and retraction of membrane protrusions at the cellular front39. The mechanisms of RHOA activation in both sub-cellular regions of delaminating NCCs are largely unknown. Fig. 5 NEDD9 as DLC1 interacting protein is required for the polarized expression of DLC1. a Venn diagram showing the number of proteins enriched by DLC1. b DLC1-associated proteins involved in major canonical pathways as assessed by the Ingenuity Pathway Knowledge base. c Interactome network of DLC1 and its associated proteins involved in cell motility and cytoskeleton regulation. d Immunoprecipitation (IP) was performed in neural tube lysate using anti-DLC1, and IgG as control. Immunoprecipitated proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and Western blotting was performed with anti-NEDD9 and anti-DLC1 antibodies. e Schematic showing the design of the C-terminally truncated DN-DLC1 constructs. IP with anti-V5 was performed in lysates extracted from neural tubes electroporated with the indicated constructs followed by western blot with anti-V5 or anti-NEDD9. White arrowhead indicates the speciﬁc protein band of DN-DLC1 (SAM). f In situ hybridization for NEDD9, DLC1, SOX9 and SOX10 on transverse sections of st 11 chick embryo. Immunoﬂuorescence for NEDD9, SOX9 and SOX10 on transverse sections of st 11 chick embryo. Scale bar: 50 µm. g Immunoﬂuorescence for NEDD9 and phalloidin on emigrating NCCs from neural tube explants and nuclei are stained with DAPI. Polarized expression of NEDD9 is shown in pseudocolour. Line scan analysis showing the average ﬂuorescence intensity of NEDD9 along the white dotted line (n = 103/11 explants). h Immunoﬂuorescence for DLC1 and phalloidin on NCCs expressing control morpholino (Ctrl-MO) (n = 65/11 explants) or NEDD9MO (n = 112/12 explants). Distribution of DLC1 expression in both treatments are shown in pseudocolour. Scale bars, 20 µm. i Line scan analysis showing the average ﬂuorescence intensity of DLC1 in NEDD9-MO-treated NCCs (n = 112/12 explants) NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 It has been shown that the RHO guanine nucleotide exchange factor (GEF) Trio mediates protrusive activity of cranial NCCs through Rho GTPase activation40 while in cell lines, GEF-H1 mediates RHOA activation in the perinuclear region38 and leading edge41. Whether Trio or GEF-H1 contributes to RhoA 2.7 EP: NEDD9-MO FRETSOX9DAPI FRETSOX9DAPI FRETSOX9DAPI 4 Total FRET ratio (norm) EP: NEDD9 b FRET ratio back/ FRET ratio front 1 EP: DN-DLC1+NEDD9 a activation in trunk NCCs remains to be determined. Nevertheless, our data reveal the asymmetric localization and activity of RhoA deﬁne the back and front polarity of NCCs for directional migration, and is spatially restricted by the polarized expression of a RhoGAP protein, DLC1. 3 2 1 0 NEDD9-MO 1.0 0.5 2.0 1 0s 342 s 722 s 1102 s 1482 s 1862 s 342 s 722 s 1102 s 1482 s 1862 s 266 s 570 s 874 s 1178 s 1444 s Side B DN-DLC1+NEDD9 Side A 0s Back Front NEDD9 0s Back Front 0.9 20 min f >2.4 20 min 1.61 Sample ID Back NEDD9 20 min 0.9 0 Time 0 20 min 20 min 0.9 0 20 min0.82 FRET ratio back/FRET ratio front >1.8 >2.4 1.61 1.0 0 Front 12 11 10 9 8 7 6 5 4 3 2 1 12 11 10 9 8 7 6 5 4 3 2 1 12 11 10 9 8 7 6 5 4 3 2 1 g 1.0 0.82 20 min 0 h GFPSOX10’3UTR GFPSOX10’3UTR EP:DN-DLC1+NEDD9 EP:NEDD9-MO GFPSOX10’3UTR EP: NEDD9 EP:DN-DLC1 GFPSOX10’3UTR NATURE COMMUNICATIONS \| 8: 1185 * 20 15 10 5 0 NEDD9 Time 0 FRET ratio back/FRET ratio front >1.8 Front 12 11 10 9 8 7 6 5 4 3 2 1 DN-DLC1+ NEDD9 Sample ID Back 12 11 10 9 8 7 6 5 4 3 2 1 0.82 20 min 0 NEDD9-MO 0 1.0 Ctrl 1.61 >1.8 12 11 10 9 8 7 6 5 4 3 2 1 DN-DLC1 e 20 min FRET ratio B/FRET ratio A >2.4 No. of emigrating GFP+ NCCs-expressing HNK-1 Time 0 DN-DLC1+NEDD9 Front(side A) 12 11 10 9 8 7 6 5 4 3 2 1 Sample ID Sample ID NEDD9-MO Back(side B) 12 11 10 9 8 7 6 5 4 3 2 1 Sample ID d 12 Ctrl NEDD9-MO DN-DLC1+NEDD9 NEDD9 * * 1.5 Sample ID c 2.0 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 DLC1 has been well characterized as a tumor suppressor protein, suppressing tumor growth and metastasis through its RhoGAP domain to inhibit RHO signaling activity17. By contrast, overexpression of DLC1 resulted in inhibition of RHOA signaling and loss of apical-basal polarity in neural progenitor cells that caused their inﬁltration into the lumen of the neural tube. These ﬁndings coincide with an essential requirement of RHOA for the maintenance of neuroepithelial integrity42. In addition, the inﬁltration phenomenon was restricted to the dorsal neural progenitors where RHOA is predominantly expressed8. However, exogenous expression of DLC1 gene did not change the inﬁltrated and non-migratory population of neural progenitors into NCC fate, indicating that DLC1 is not sufﬁcient to induce NC speciﬁcation. On the other hand, overexpression of DN-DLC1 to antagonize endogenous DLC1 activity resulted in increased RHOA activity associated with an inhibition of NC delamination, consistent with a previous report that the active form of RHO tends to maintain NCCs in the epithelial state10. Both in vivo and explant studies at the single cell level by FRET further demonstrated that disruption of RHOA polarity and activity (either reduced or excessive amount) by overexpression of DLC1 or DNDLC1, respectively impeded the directionality and migratory behavior of NCCs that underlie the importance of asymmetric localization and proper level of DLC1 expression for the spatial restriction of RHOA activity to dictate NC front-back polarity. These ﬁndings are in line with the notion that the precise localization of DLC1 protein determines the spatiotemporal RHO activation pattern17. Moreover, our ﬁndings reveal an unexpected requirement for DLC1 in regulating NC delamination that is different from being a metastasis suppressor gene18, implying a differential function of DLC1 between developmental and pathological context. The underlying mechanisms conferring the differences remain to be determined. The ability of DLC1 to restrict location-speciﬁc RHO signaling depends on its subcellular location. DLC1 is recruited by speciﬁc factors to different cellular sites. In most cases, DLC1 is recruited to the membrane periphery, focal adhesions and adherens junctions that are involved in cell migration events17. In contrast, our data revealed that DLC1 did not show this subcellular localization but was associated with cytoplasmic NEDD9 protein expressing in a polarized manner that determines the asymmetric and cytoplasmic localization of DLC1. Previous studies showed that NEDD9 functions as an adaptor protein to assemble protein complex containing Src and FAK for inhibition of RHO/ROCK signaling in order to promote mesenchymal invasion of NCderived melanoma cells43. In addition, NEDD9 has also been shown to be required for NC delamination24 consistent with the notion that cell migratory machinery is conserved between NC and melanoma44 but whether Src and FAK are involved in NEDD9-mediated RHO signaling restriction to regulate NC motility remains to be determined. Here, we provide new insight into NEDD9 function by demonstrating that NEDD9 is not required for the regulation of total level of RHOA activity but its interaction with DLC1 is important for the establishment of RHOA polarity between the front and back for directed migratory behavior of trunk NCCs. SOX9 and its downstream gene SOX10 are crucial not only for the establishment of NC identity but also for cell migration28, 45, indicating that these two events are intimately linked. However, the transcriptional targets for SOX9 and SOX10 to regulate NC motility remain elusive. Here, we identiﬁed NEDD9 and DLC1 as the key mediators of SOX9 and SOX10 in controlling NC delamination and migration, respectively. Although SOX10 is both sufﬁcient and necessary for DLC1 gene expression, ectopic DLC1 expression was not detected until 12 hpt suggesting that SOX10 might activate DLC1 expression indirectly through another factors, which remain to be identiﬁed. In addition, electroporation of SOX9 + DN-DLC1 resulted in a marked reduction of FOXD3 expression in the early migratory NCCs to a greater extent than that of SOX10 + DN-DLC1. Considering SOX9 functions upstream of SOX1029, these results imply that SOX9 and SOX10 might induce similar but not identical molecular events and the differences could modulate the antagonistic interaction between DN-DLC1 and ectopic DLC1 expression, resulting in differential inhibitory effects on NC motility. In contrast to DLC1, SOX9 but not SOX10 speciﬁcally activates NEDD9 expression through binding to its enhancer regions. In addition, epistasis analysis reveal that NEDD9 is not required for mediating the ability of SOX9 to establish NC identity but is essential for NC delamination and migration consistent with previous studies in which downregulation of NEDD9 resulted in reduced number of migratory NCCs24. In conclusion, our ﬁndings provide mechanistic insight into how the transcriptional program of NC speciﬁers governs directional NC migratory behavior. Methods Expression vectors and morpholinos. Full-length chick SOX9, SOX10, DLC1, NEDD9 and DN-DLC1 cDNAs were inserted upstream of an internal ribosomal entry site (IRES) and a nuclear localization sequence (nls)-tagged EGFP in a pCIG expression vector (a gift from A. McMahon, University of Southern California). A single-chain RHOA biosensor contains a fragment of Rhotekin that binds only to activated RHOA and is attached to RHOA as part of the same protein chain46. Full length DLC1 and DN-DLC1 were cloned into IFP2.0-C1 expression vector (a gift from C-H. Yu, the University of Hong Kong) for co-electroporation with FRET biosensor. Morpholinos (MO) for SOX9, NEDD9, SOX10, DLC1 and their corresponding MO-controls (Ctrl) were tagged with or without 3′ ﬂuorescein and obtained from Gene Tools and their sequences are: SOX9-MO 5′-GGGTCTAGGAGATTCATGCGAGAAA-3′, SOX9-MO-Ctrl 5′-ATGGCCTCGGAGCTGGAGAGCCTCA-3′, NEDD9-MO 5′-TAAGATTCTTGTACTTCATGGCTGC-3′, NEDD9-MO-Ctrl 5′-TAACATTGTTCTACTTGATCGCTGC-3′, SOX10-MO 5′-CCGACAGATCTTGGTCATCAGCCAT-3′ SOX10-MO-Ctrl 5′-CCCACACATCTTGCTCATGACCCAT-3′. DLC1-MO 5′-TGGCCTCGATTTGAGTGAGGATCAT-3′ DLC1-MO-Ctrl 5′-TGCCCTCCATTTCAGTCAGCATCAT-3′ The ﬁnal molar concentration of each morpholino oligonucleotide used was 0.75 mM. Fig. 6 Interaction between NEDD9 and DLC1 is required for RHOA polarity and directed NC migration. a Processed FRET signal images of cross-sections from embryos electroporated with the FRET probe plus NEDD9-MO (n = 146/17 embryos), NEDD9 (n = 168/13 embryos) or DN-DLC1 + NEDD9 (n = 152/ 13 embryos). NCCs are marked by SOX9 immunoﬂuorescence and nuclei are stained with DAPI. The magniﬁed area and selected cells are marked with dotted squares. White arrowheads indicate FRET signal. Scale bars: 20μm. b Quantiﬁcation of the total FRET index of NCCs electroporated with the indicated constructs. Quantiﬁcation of the ratio of the FRET index between the back and front of NCCs electroporated with the indicated constructs (control, n = 425/25 embryos). Mean±s.e.m.; Student’s t-test, p < 0.001; Bonferroni multiple comparison test, p < 0.0001 c Processed FRET signal images of time-lapse series of migrating NCCs expressing NEDD9-MO (n = 97/15 explants), DN-DLC1 + NEDD9-MO (n = 92/19 explants) and NEDD9 (n = 106/16 explants). Scale bars: 20μm. d Heat maps representing the FRET index as a function of time at the back or front, and the FRET ratio between the back and front of 12 selected NCCs expressing NEDD9-MO, e DN-DLC1 + NEDD9, and f NEDD9. g In situ hybridization of SOX10 3’UTR in embryos electroporated with DN-DLC1 (n = 9), NEDD9 (n = 10), NEDD9-MO (n = 10) and DN-DLC1 + NEDD9 (n = 9). Black arrowheads indicate reduced SOX103’UTR expression in the transfected sides. Scale bar, 10 µm. h Quantiﬁcation of the number of GFP+ NCCs expressing HNK-1+ in embryos electroporated with the indicated constructs. Mean±s.e.m. Bonferroni multiple comparison test, p < 0.001; p < 0.0001 NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 Electroporation and neural tube explant culture. In ovo electroporation and neural tube explants culture were performed as previously described27, 29. Plasmid DNA or FRET biosensor for the detection of active RHOA was injected into the lumen of HH st 11–12 neural tubes, electrodes were placed on either side of the neural tube, and electroporation was carried out using a BTX electroporator delivering ﬁve 50-ms pulses of 30 V. Electroporated embryos were allowed to Chick embryos. Fertilized chick eggs were obtained from Jinan Poultry Co. (Tin Hang Technology) and were incubated at 38 °C in a humidiﬁed incubator (Brinsea Octagon 250 incubator). Embryos were staged according to Hamburger and Hamilton (HH)14. The Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong has approved all animal experiments. a b EP:SOX10 (12 hpt) GFP EP:Ctrl-MO EP:SOX10-MO DLC1 DLC1 c DLC1 d EP:SOX9 GFP EP:SOX10 EP:SOX9+DN-DLC1 FOXD3 GFP GFP FOXD3 EP:SOX10+DN-DLC1 FOXD3 GFP FOXD3 f e NEDD9 GFPNEDD9 EP:SOX9-MO EP:Ctrl-MO EP:SOX9 (6 hpt) NEDD9 SOX9 NEDD9 SOX9 NEDD9 g 9.5 kb 45 kb E1 G 35 * 7 6 5 4 3 2 1 SO X SO 9 X1 0 0 X9 X1 FP 0 Relative fold change 10 FP 20 6 5 4 3 2 1 G Relative fold change *** 30 E2 k 20 15 10 5 SOX10’3’UTR Laminin GFPLaminin NATURE COMMUNICATIONS \| 8: 1185 -M O M O trl 9- +C D X9 SO GFPHNK1 X9 +N ED G X9 0 FP GFPLaminin 25 SO Laminin * 30 SO EP:SOX9+NEDD9-MO Sox103’UTR No. of emigrating GFP+ NCCs-expressing HNK-1 EP:SOX9 ns GFPHNK1 14 *** SO j 40 G Relative fold change * SO αI g G * Luciferase reporter assay E1 Promoter 0 6 5 4 3 2 1 X9 αS O * Relative expression levels Relative expression levels 5 4 3 2 1 0 B3 αI gG αS O X αS 9 O X1 0 αI gG αS O X αS 9 O X1 0 Relative expression levels 6 5 4 3 2 1 * i ChIP-qPCR assay B2 B1 X1 h B3 FP SO X9 SO X1 0 B2 αS O B1 * NEDD9 enhancers NEDD9 promoter SOX9 binding sites E2 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 develop for 24 h post-transfection (hpt) before being processed for FRET analysis, RhoA activation assays, immunoﬂuorescence and in situ hybridization. For neural tube explant culture, electroporated embryos were harvested at 2 hpt, and neural tubes at the level of recently formed three somites were carefully dissected following Dispase (Roche) treatment, placed onto the ﬁbronectin-coated dishes and cultured in F12-based medium with L-glutamine (Gibco), 1% N2 supplement (Gibco), and 1% penicillin-streptomycin (Gibco) for 24 h at 37 °C and 5% CO2. Heparin-acrylic beads were washed multiple times in PBS and soaked in 50 ng/ml SDF-1 (R&D Systems) before placing adjacent to the adhered neural tubes. Subsequently, the neural tube explants were ﬁxed for 30 min in 4% paraformaldehyde (PFA), immunostained with mouse anti-DLC1 (1:200; Santa Cruz SC-271915 or 1:500; BD Transduction Lab 612020), mouse anti-NEDD9 (1:1000; Abcam ab18056), rabbit anti-GFP (1:1000; Abcam ab6556), mouse antiFAK (1:500; BD Transduction Lab 610088) and Alexa Fluor 568 Phalloidin (1:250; Thermo Fisher Scientiﬁc A22283) and mounted in VectaShield mounting medium with DAPI (Vector Laboratories H-1200) and photographed using a Carl Zeiss LSM 710/780 laser scanning confocal microscope in the Faculty Core Facility in Li Ka Shing Faculty of Medicine of the University of Hong Kong. Cell sorting and quantitative PCR. After electroporation with Sox10-E1:EGFP, embryos were incubate at 37 °C until HH13 and screened for robust expression of EGFP. Embryos with weak GFP expression or at incorrect stages of development were discarded. Dissected trunks (HH13) were washed in cold PBS and dissociated with trypsin. Cells were subsequently washed, passed through a 40-μm cell strainer (BD Biosciences) and resuspended in FASC buffer at 1 × 105 cells/ml. GFP + cells were then sorted using a BD FACS Aria I Cell Sorter (BD Biosciences) with 7-AAD exclusion to eliminate dead and damaged cells. GFP positive cells were collected for quantitative PCR analysis, which was performed on an ABI PRISM Applied BioSystems 7000 Sequence Detection System using Power SYBY® Green Master Mix (Thermo Fisher Scientiﬁc). We designed primers speciﬁc for each Dlc1 isoform with the Primer3Plus Program: chick DLC1 isoform 1, forward 5′-aatgaggaagccgaaacgtc-3′ and reverse 5′-tcagatttcctgcgctgttg-3′; chick DLC1 isoform 2, forward 5′-gaaagcccctctgagaagat-3′ and reverse 5′-ccagataacatccaaggctc-3′; chick DLC1 isoform 3 forward 5′-aagcttccttggcctcgatt-3′ and reverse 5′-tctcggaggcagcagtgag-3′; chick GAPDH forward 5′-attcctccacctttgatgcg-3′ and reverse 5′-tggaccatcaagtccacaac-3′. Real-time PCR efﬁciencies were determined for all sets of designed primers. Expression levels of DLC1 isoforms were normalized to the expression of GAPDH, and fold changes in mRNA expression were calculated by the 2−ΔΔCt method. Signiﬁcant changes in expression levels between isoforms were determined by unpaired Student’s t-test. In situ hybridization and immunohistochemistry. Electroporated embryos were harvested and ﬁxed for 1 h at 4 °C in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), cryoprotected with 30% sucrose in PB and sectioned at 10 µm. Immunohistochemical localization of proteins on cryosections were performed as previously described45. Antibodies against the following proteins were used: sheep anti-GFP (1:1000; AbD Serotec 4745-1051), guinea pig anti-SOX9 (1:1000), rabbit anti-SOX10 (1:2000, gifts from V. Lee, STEMCELL Technologies), rat anti-N-Cad (1:500; Zymed 13-2100), anti-NEDD9, mouse anti-HNK-1 (1:100; BD Biosciences 347390), rabbit anti-SNAIL2 (1:800, Cell Signaling 9585), and rabbit anti-Laminin (1:1000; Sigma L9393). A Carl Zeiss LSM 780 Meta laser scanning confocal microscope acquired images. Whole-mount in situ hybridization using NBT/BCIP (Roche) detection was performed as described45. The following anti-sense riboprobes were used: chick SOX10 3′UTR27, chick FOXD3 (I.M.A.G.E. ID 418507), chick NEDD9 and chick DLC1. RhoA activation assay. Following 24 hpt, 10 well-transfected neural tubes were dissected and processed for RhoA activation assays using the Biochem Kit from Cytoskeleton, Inc. Assay based on the manufacturer’s instructions. In brief, equal amounts of protein from neural tube extracts were immunoprecipitated with Rhotekin-RBD followed by blotting with mouse anti-RHOA (1:1000). Horseradish peroxidase-conjugated anti-mouse immunoglobulin G (GE Healthcare) was used for the second reaction at 1:5000 dilutions. Immunocomplexes were visualized by enhanced chemiluminescence (ECL), using an ECL kit (GE Healthcare). The intensity of protein band from each sample relative to the control was quantiﬁed with ImageJ. Time-lapse imaging of emigrating NCCs. Time-lapse imaging of NCCs emigrating from neural tube explants was performed on a Zeiss LSM 780 Meta laser scanning confocal microscope equipped with an incubator capable of maintaining 37 °C temperature, 95% relative humidity and 5% CO2. For cell migratory distance/speed /persistence assay, images were acquired with 10X (1.4 ampliﬁcation) objective. Three to four successive images of different explants were collected every 15 min for a total period of 16 h at one time. For acquiring lifeact-mcherry and RhoA biosensor images, the 40X oil DIC lens with 1.6–1.7 ampliﬁcation were applied. Activation levels of RHOA in living cells were measured by calculating the ratio of citrine-YFP FRET (emission from CFP) over CFP emission. All images for ratiometric FRET measurements were line (1) scanned at speed 4 (1024 × 1024 frame) simultaneously to avoid artefacts from cell movement between sequential CFP and FRET image acquisitions. Agron laser 458 nm were applied with maximum pinhole and laser power was set as 2 to avoid bleaching. For CFP acquisition, detector 480/40, gain (Master) 700, digital offset 0 and digital gain 2 were set up. For FRET, detector 540/30, gain (Master) 700, digital offset 0 and digital gain 1 were set up. Auto-focus was set up during time-lapse imaging. Image analysis. To track distance traveled by the transfected cells over time, the Metamorph software (universal imaging) with manual tracking function was used. The distance (µm) was measured from the initial cell location near the border of the explant to the ﬁnal destination. The net speed was determined by the distance traveled divided by the time of each path (h). Persistence of the cells was calculated by dividing distance, as a straight line, between the point of origin and the end point by the total distance traveled (µm). Results represent the mean ± SEM of at least 50 transfected cells from 6 explants. Signiﬁcance was examined by Student’s t-test, and a P value of 0.05 or less being considered signiﬁcant. For polar histograms plot, the angle between NCC protrusions direction and single NCC migratory direction was calculated. Any actin extensions more than 4.5 µm were deﬁned as protrusions, ADAPT plugin of ImageJ were applied for protrusion detection47. For FRET analysis, background subtraction, cell segmentation and ratio calculation were performed as previous described5, 6. Metamorph software (universal imaging) was applied for image analysis. Images were ﬁrst processed with local background subtraction. The background intensity or region was deﬁned as a set of background pixels excluding pixels with signiﬁcant signal from cells or other ﬂuorescent objects. The sum of the CFP and FRET images (after background subtraction) was then used to compute cell masks (“Threshold Image” command in Metamorph). The sum of the two channels was used for better signal to noise than either individual channel, and its value is less sensitive to differences in FRET efﬁciency. Cells were ﬁrst crudely identiﬁed as large non-background objects with values equal to 1 and 0 elsewhere outside of the cell. Binary masks were computed locally for each cell. Each FRET ratio value was computed as the sum of the FRET intensities divided by the sum of CFP intensities over a window. No bleed through is required as CFP and Citrine-YFP are equimolar in any given pixel for this biosensor. For visual representations of ratiometeric images, pseudocolor was applied to all ratio images. A scaling factor of 1000 was speciﬁed as a multiplication Fig. 7 SOXE factors regulate DLC1 and NEDD9 expression. a In situ hybridization for DLC1 on transverse sections of embryos electroporated with SOX10 at 12 hpt (n = 9). Scale bar, 50μm. b In situ hybridization for DLC1 on embryos electroporated with Ctrl-MO (n = 10) or SOX10-MO (n = 10) at 24hpt. Black arrows indicate loss of DLC1 expression on the transfected side of the neural tube. Scale bar, 10μm. c In situ hybridization for FOXD3 on embryos electroporated with SOX9 (n = 9) or SOX9 + DN-DLC1 (n = 9) at 24hpt. Scale bar, 20 µm d In situ hybridization for FOXD3 on embryos electroporated with SOX10 (n = 10) or SOX10 + DN-DLC1 (n = 10) at 24hpt. Scale bar, 10 µm. e Immunoﬂuorescence for NEDD9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with SOX9 (n = 12) at 6 hpt. Scale bar, 50 µm. f Immunoﬂuorescence for SOX9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with Ctrl-MO (n = 9) or SOX9-MO (n = 9). White arrowhead indicates loss of SOX9 expression on the transfected side. Scale bar: 50 µm. g Schematic diagram showing the seven exons (black) of chick NEDD9 gene with promoter region in blue, −9.5 kb enhancer 1 (E1 harboring two putative SOX9 binding motifs, B1 and B2, and + 45 kb enhancer 2 (E2) containing a putative SOX9 binding motif, B3. h Fold enrichments of three independent ChIP-qPCR assays for SOX9, SOX10 and IgG (control) antibodies on B1, B2 and B3. Data represented as fold enrichments for putative SOX9 binding motifs relative to control. Mean±s.e.m. Bonferroni multiple comparison test, p < 0.05, p < 0.001, p < 0.0001. i Fold activation of three independent in vivo luciferase assays for reporter constructs carrying NEDD9-promoter, E1 or E2. Mean±s.e.m. Bonferroni multiple comparison test, p < 0.001, **p < 0.0001. j Immunoﬂuorescence for HNK-1, Laminin and in situ hybridization for SOX103’UTR on transverse sections of embryos electroporated with SOX9 or SOX9 + NEDD9-MO at 24hpt. White arrowhead indicates loss of Laminin expression on the basement membrane of the transfected neural tube. Scale bar: 50 µm. k Quantiﬁcation of the number of emigrating GFP+ NCCs expressing HNK-1 in embryos electroporated with the indicated constructs. Mean±s.e.m. Bonferroni multiple comparison test, p < 0.05; **p < 0.001; ns, not signiﬁcant NATURE COMMUNICATIONS \| 8: 1185 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 15 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-017-01107-0 a b SOX9 Polarized NCC (control) c SOX9 SOX10 SOX10 Front NEDD9 DLC1 Depolarized NCC (DN-Dlc1) Cofactor DNDLC1 d SOX9 SOX10 DLC1 DNDLC1 Back SOX9 Depolarized NCC (NEDD9-MO) SOX10 NEDD9 NEDD9 DLC1 Cofactor RHOAGTP Depolarized NCC (DLC1) NEDD9 DLC1 DLC1 DLC1 RHOAGTP RHOA -GTP RHOA -GTP Fig. 8 Summary of the effects on RHOA activity and NC polarity following manipulation of DLC1 and NEDD9 functions. a SOX9 induces SOX10 to activate DLC1 expression (dotted arrows indicate transactivation is likely to be indirect), while SOX9 directly transactivates NEDD9 expression through binding to its enhancer regions. NEDD9 is required for the polarized expression of DLC1 and their interaction is crucial for the establishment of high RHOA activity at the back and low at the front that directs polarized NC morphology and motility. b Overexpression of DN-DLC1 disrupts the interaction between endogenous DLC1-NEDD9 and other cofactors through sequestration, resulting in elevation of RHOA activity throughout the cell and unpolarized NC morphology with defective motility. c Excessive amount of DLC1 inhibits RHOA activity throughout the cell, resulting in lack of polarized NC morphology and loss of directionality. d Expression of NEDD9-MO leads to lack of polarized DLC1 expression that in turn disrupts asymmetric distribution of active RHOA between the back and front of NCC which exhibits unpolarized morphology and limited motility factor for all the ratio calculation. The ratio values were normalized to the lower scale value (1.0). To avoid low intensity pixels, bottom 5% of the total histogram distribution was excluded. Finally, ratiometeric images were subjected to a Gaussian ﬁlter of width 2 pixels (0.32 μm) to reduce pixel noise. Immunoprecipitation and mass spectrometry. To identify DLC1-associated proteins for mass spectrometry, 10 chick neural tubes were micro-dissected and lysed using Pierce MS-compatible magnetic IP kit (90409). Pre-clearing of embryonic neural tube lysates was performed by incubating with protein A/G magnetic beads for 16 h at 4 °C, followed by overnight immunoprecipitation with rabbit anti-DLC1 (Abcam Ab104764) or rabbit anti-IgG as a negative control. The protein-antibody complex was then incubated with protein A/G magnetic beads for 4 h at 4 °C. The immunoprecipitated protein complex was then eluted by acidic buffer. Protein lysate was subject to Liquid chromatography-mass spectrometery analysis at the Centre for Genomic Sciences, the University of Hong Kong. For immunoprecipitation, the DLC1-immunoprecipitated protein complex and the total input of protein lysate were immunoblotted with mouse anti-V5 (Invitrogen R96025), mouse anti-Nedd9 and mouse anti-Dlc1. All uncropped western blots can be found in Supplementary Fig. 10. Chromatin immunoprecipitation. ChIP was performed based on the protocol19. Dorsal neural tubes were dissected in Ringer’s solution from 20 st 11–12 chick embryos and transferred to 1 ml isotonic buffer [0.5% Triton X-100, 10 mM TrisHCl, pH 7.5, 3 mM CaCl2, 0.25 M sucrose, protease inhibitor table (Complete Protease Inhibitor EDTA-free, Roche), 1 mM DTT, and 0.2 mM PMSF] on ice. Tissue was homogenized and cells cross-linked by adding formaldehyde to a ﬁnal concentration of 1% and nutated for 10 min at room temperature. Glycine (ﬁnal concentration of 125 mM) was added to stop the cross-linking reaction. The crosslinked cells were washed three times and cell pellets were re-suspended in isotonic buffer and nuclei isolated using homogenizer, washed, and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) for 10 min. The lysate was then diluted 3-fold with ChIP dilution buffer (0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1 mM DTT, 0.4 mM PMSF, and protease inhibitors) and one half of chromatin was sheared into ~ 200 to 500 bp DNA fragments using Diagenode Bioruptor® Plus sonicator. The sonicated chromatin was immunoprecipitated with Dynal bead protein A (Invitrogen) pre-absorbed with anti-SOX9, anti-SOX10 or control rabbit anti-IgG (Abcam). Samples were washed, eluted and reverse cross-linked. The precipitated DNA fragments were puriﬁed and quantiﬁed by qPCR with primers speciﬁc for NEDD9-p, E1 and E2 regions (Supplementary Fig. 6d). Each sample was run in triplicate and the results were quantiﬁed using the ΔΔCt method. Analysis was done using Applied Biosystem’s instructions. In vivo luciferase assay. The luciferase reporter assay was performed based on the manufacturer’s protocol from Dual-Luciferase® Reporter Assay System (Promega). Brieﬂy, NEDD9-p, E1- or E2 enhancers driven luciferase reporters were mixed with Renilla and SOX9 or SOX10 expression construct and electroporated into the chick neural tube. The trunk parts of eight well-transfected chick embryos from each treatment were harvested 24 hpt, lysed and assayed for ﬁreﬂy luciferase activity (Microplate Luminometer LB96V, EG&G BERTHOLD). Experiments were performed in triplicate. Statistical analysis was performed using Student’s t-test. Data availability. The authors declare that all data supporting the ﬁndings of this study are available within the article and its supplementary information ﬁles or from the corresponding author upon reasonable request. 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Asymmetric localization of DLC1 deﬁnes avian trunk neural crest polarity for directional delamination and migration. ﬁgshare. 10.6084/m9. ﬁgshare.5324431.v1 (2017). Acknowledgements We thank J. Briscoe for helpful discussion and comments on the manuscript, J. Guo, C. Lai and C.-H. Yu for their assistance and advice on FRET imaging and analysis, and M. Way for providing LifeAct-mCherry construct. Irene Ng is Loke Yew Professor in Pathology. This work was supported by grants from the Research Grants Council and University Grants Council of Hong Kong (ECS_27100314), (X_HKU708/14), (GRF_17110715) to M.C. and J.AI.L. and (AoE/M-04/04), (T12-708/12-N) to K.S.E.C. Author contributions J.AI.L. and M.C. conceived and designed the experiments and wrote the manuscript. J.AI.L. and M.C. performed and analyzed most of the experiments, except in situ hybridization, immunoﬂuorescence, cell sorting and quantitative PCR, which were carried out by Y.X.R. and M.P.L.C. ChIP and reporter assays were carried out by M.N.H. and M-H.W. In silico analysis of NEDD9 regulatory regions was carried out by B.N. DLC1 constructs were provided by L.K.C. and I.O.L.N. K.S.E.C. designed ChIP and reporter assays. R.S. performed shotgun proteomics analysis. L.H. generated the FRET construct and analyzed data. Additional information Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01107-0. Competing interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2017 \| DOI: 10.1038/s41467-017-01107-0 \| www.nature.com/naturecommunications 17
https://openalex.org/W3155234175	http://www.ecoeet.com/pdf-135447-63795?filename=Potential of Production.pdf	English	null	Potential of Production of Energy Crops in Ukraine and their Processing on Solid Biofuels	Ecological Engineering & Environmental Technology	2,021	cc-by	7,078	Ecological Engineering & Environmental Technology Received: 2021.03.02 Accepted: 2021.03.22 Published: 2021.04.06 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 https://doi.org/10.12912/27197050/135447 ISSN 2719-7050, License CC-BY 4.0 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 https://doi.org/10.12912/27197050/135447 ISSN 2719-7050, License CC-BY 4.0 ABSTRACT Renewable energy sources in Ukraine account for only 4% of the total energy consumption today. At the same time, Ukraine has favorable climatic conditions and fertile soils, as well as areas of agricultural land, which make it possible to meet the demand for food products both for domestic consumption and for export. The tendencies towards the depletion of traditional fuels and their rise in price determine the diversification of the fuel and energy sector and the search for reserves for the production of their own environmentally friendly energy. The paper de- scribes the characteristics of energy crops for biofuel production. The advantages of growing bioenergy crops were presented. The characteristics of energy crops in relation to growing conditions were determined. Ukraine has a great potential for growing the most popular energy crops: miscanthus, switchgrass, energy willow, poplar without endangering food security but this potential has not been realized yet. eywords: energy crops, solid biofuels, bioenergy policy, miscanthus, switchgrass, energy willow, poplar. 1 Department of Management and Law, Vinnytsia National Agrarian University, Ukraine * Corresponding author’s e-mail: natalka.vinn@gmail.com Department of Management and Law, Vinnytsia National Agrarian University, Ukraine * Corresponding author’s e-mail: natalka.vinn@gmail.com * Corresponding author’s e-mail: natalka.vinn@gmail.com * Corresponding author’s e-mail: natalka.vinn@gmail.com Potential of Production of Energy Crops in Ukraine and their Processing on Solid Biofuels Grigoriy Kaletnik1, Natalia Pryshliak1, Dina Tokarchuk1 Grigoriy Kaletnik1, Natalia Pryshliak1, Dina Tokarchuk1 Grigoriy Kaletnik1, Natalia Pryshliak1*, Dina Tokarchuk1 Introduction by-products of animal husbandry, which require disposal in an environmentally sustainable man- ner. In addition, manure is a good substrate for biogas production, as it is easily mixed with oth- er feedstock, such as corn silage, plant residues and others [Tokarchuk et al., 2020]. The rise in energy prices and the degradation of the ecological condition of the environment as a result of the growing consumption of fos- sil fuels are prompting humanity to use biomass for energy needs – biofuel production. World experience convinces that the production of bio- fuels provides benefits for the economy of each country, in particular, it makes it possible to cre- ate new jobs not only in rural areas, but also in industrial centers, as well as improves the envi- ronmental situation in the country, regions, etc. [Pryshliak, Tokarchuk, 2020]. Plant biomass is used for production of vari- ous types of biofuels. Two yields can be harvested annually from each field: food and energy. Along with the gradual refusal of food crops for biofuel production, plant biomass energy has become one of the most dynamic aspects of the modern global energy market, on the basis of which gov- ernments of the world’s leading countries seek to adopt the existing renewable energy policies and regulatory mechanisms to stimulate the de- velopment of non-food biomass production. The European bioenergy policy is changing dynami- cally, following the trend of sustainable develop- ment. Ukraine, having a significant agricultural potential and following the European vector of development, strongly depends on the European tendencies [Trypolska, Kyryzyuk, 2018]. Agriculture has been considered as one of the priority sectors including energy production direction in the research of Kaletnik, Honcha- ruk, Okhota, 2020. It is the main industry, pro- ducing biomass. The biomass of animal origin includes manure and bird droppings; in turn, the biomass of plant origin includes plant waste and energy crops. Unlike other types of biomass, such as energy crops, manure is produced as 59 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 In January 2020, the Ministry of Energy and Environmental Protection of Ukraine present- ed the Concept of “Green Energy Transition of Ukraine until 2050” [Green Energy Transition of Ukraine until 2050]. The concept aims to achieve a climate-neutral economy by 2070. Introduction Among the main directions of decarbonization of the econo- my, including energy as its important component, the following are identified: the development of renewable energy sources (RES) in combination with an increase in energy efficiency; reducing the consumption of carbon-intensive energy re- sources to zero and maximizing the use of RES so that the agricultural and forestry sector can switch to full self-sufficiency in energy resources; increasing sustainable production of biomass, biofuels and other RES to support the implemen- tation of the “green” transition in other sectors of the economy. Biomass is currently seen as a promising renewable energy source, which can be sustainably utilized in the production of fuels and electric energy, adding no carbon dioxide to the environment [Ramos et al., 2018]. surface washout and soil degradation. In addition, these plants are characterized by low production costs and do not require significant use of fertil- izers and pesticides. This will allow the cultiva- tion of energy plants on lands removed from the crop rotation. If the full potential of the use of untapped lands of Ukraine is estimated, in 2018 it amounted to 4 million hectares [State Statistics Service of Ukraine], which can be defined as the theoretical potential of growing energy crops. With its full use, it is possible to replace about 20 billion m3 of natural gas. The technically achiev- able potential is 2 million hectares of land, which is equivalent to the replacement of 10 billion m3 of gas or 34% of consumption (according to JSC “Naftogaz of Ukraine” Ukraine consumed 29.8 billion m3 of natural gas in 2019, [JSC “Naftogaz of Ukraine”]). In monetary terms, the use of these lands for growing energy crops will allow import substitution worth about $1.8 billion. fi Nowadays, the efficiency of using agricul- tural feedstock for bioenergy production in dis- cussed widely. A significant number of scientific publications are devoted to the study of the effi- ciency of growing agricultural and energy crops. Kulyk et al. (2020) investigated the economic and energy efficiency of biomass production, the output of solid biofuel, its energy intensity and energy output. The input and output of energy are two important factors used to determine the en- ergetic and ecological of a fuel or its production technology. Scholz et al. Introduction (1998) have investigated the usefulness of a number of different methods for the production of five biofuels which can be produced in agriculture and calculated their en- ergy balance. Among the renewable energy sources, bio- mass can be used to meet a variety of energy needs, including generating electricity, heating homes, fuelling vehicles, and providing process heat for industrial facilities. Biomass energy also generates less air emissions, reduces the amount of waste sent to landfills, and decreases our reliance on foreign oil [Janiszewska, Ossowska, 2020]. The fuels derived from energy crops are not only potentially renewable, but are also reason- ably similar in origin to fossil fuels (which also originate from biomass) to provide a direct re- placement. Bioenergy crops are easy to store, transport, affordable, and can be converted to a wide range of energy sources using the existing and new conversion technologies, and thus could be a powerful alternative to traditional fuels in the twenty-first century. More and more countries around the world are beginning to prefer high- performance energy crops with high biomass yields and high cellulose content as raw materi- als for biofuel production. Such crops include miscanthus (Miscanthus), switchgrass (Panicum- virgatum), willow (Salix), poplar (Populus L.). Unpretentiousness to soil and climatic conditions of cultivation allows growing these perennial phytoenergetic plants on unproductive soils with rugged relief. In addition to the actual energy benefit, this will help to preserve the soil from water erosion, reduce the nutrient losses with The goal of the study is the review of poten- tial types of bioenergy raw materials and the ef- ficiency of their cultivation and processing into solid biofuels. THE CHARACTERISTICS OF ENERGY CROPS The high cost of fossil fuels and the devel- opment of technological progress have enabled the emergence of energy systems on biomass, which allow obtaining energy directly or indi- rectly through combustion, pyrolysis or gasifica- tion. These systems are becoming more efficient, more reliable and cleaner. The number of boiler houses operating on renewable energy sources, 60 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 grown on degraded lands. Among the EU coun- tries, Italy is in the lead in terms of the land ar- eas used for energy plantations with 57 thousand hectares (the largest area in Europe). In Poland, 13 thousand hectares are allocated for this pur- pose, in Sweden – 12 thousand hectares, in Ger- many – 11 thousand hectares, in Denmark – 10 thousand hectares, whereas in Finland – 8 thou- sand hectares [Eurostat, 2020]. In addition, the cultivation of energy crops on low-yielding lands will not compete with the possibility of growing food crops on them and will meet the criteria of sustainability, in accordance with the require- ments of Directive 2009/28/EC (Fig. 3). mainly solid fuel boilers, began to grow rapidly in Ukraine after a sharp rise in gas prices in 2014. Constantly growing demand has led to the prob- lem of obtaining raw materials at affordable pric- es. The availability of local resources in a specific area is a prerequisite for a successful transition to solid biofuels and the development of bioenergy in general. In some Western European countries, in Scandinavian countries (Finland, Sweden and Norway), Poland and Denmark, an effective sub- stitute for solid forest biomass has been found – energy crops, wood (willow, poplar, paulownia, etc.) and grass (miscanthus, switchgrass, etc.). The interpretation of the category “energy crops” by scientists differs, which is the reason for the dif- ficulties in approaches to stimulating their produc- tion and use. The most widely used is the defini- tion of energy crops as the plants that are specially grown for use directly as fuel or for biofuel pro- duction. Thus, these crops are used in energy farm- ing – a new type of agriculture [Koçar and Civaş, 2013]. The general characteristics of energy crops for biofuel production are shown in Figure 1. The cultivation and use of special plants for biofuel purposes is an effective measure for the efficient functioning of rural areas. THE CHARACTERISTICS OF ENERGY CROPS These plants are called energy crops, they are mainly peren- nial, are well adapted to the growing conditions, and are capable of forming a high yield of bio- mass. The biomass of energy crops is charac- terized by a low content of chemical elements, contains a significant amount of lignin and cel- lulose, sugar and starch in some plants, and is an excellent feedstock for the production of energy- intensive biofuels. The yield of energy crops is the main factor when choosing the type of crop to grow in a particular area and directly depends on the climatic, soil and other conditions. Crops have different water requirements and can differ significantly in terms of their frost and drought resistance (Table 1). The energy efficiency of agricultural crops should be optimized in an era of growing demand for energy, including renewable energy (Stolar- ski et al., 2019). The use of energy crops as raw materials for biofuel production has a number of advantages, which include: the possibility of natural gas substitution, reducing the cost of en- ergy, land reclamation, decarbonization, reducing emissions, economic growth (Fig. 2). Unlike food crops, energy crops do not have special requirements for the soil, they can be Among energy crops, in terms of biomass yield potential, energy productivity level, a complex of Fig. 1. Characteristics of energy crops on the basis of [Chaika, Yasnolob, 2017, Heletukha et al., 2017] Fig. 1. Characteristics of energy crops on the basis of [Chaika, Yasnolob, 2017, Heletukha et al., 2017] Fig. 1. Characteristics of energy crops on the basis of [Chaika, Yasnolob, 2017, Heletukha et al., 2017] 61 61 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 Fig. 2. Advantages of growing energy plants on the basis of [Hanegraaf and Biewinga, 1998; Zegada-Lizarazu and Monti, 2011; Fernando et al., 2010] Fig. 3. Sustainability requirements to biofuels according to [Directive 2009/28/EC] Fig. 2. Advantages of growing energy plants on the basis of [Hanegraaf and Biewinga, 1998; Zegada-Lizarazu and Monti 2011; Fernando et al 2010] Fig. 2. Advantages of growing energy plants on the basis of [Hanegraaf and Biewinga, 1998; Zegada-Lizarazu and Monti, 2011; Fernando et al., 2010] Fig. 3. Sustainability requirements to biofuels according to [Directive 2009/28/EC] Fig. 3. Sustainability requirements to biofuels according to [Directive 2009/28/EC] Fig. 3. Sustainability requirements to biofuels according to [Directive 2009 Fig. 3. Sustainability requirements to biofuels according to [Directive 2009/28/EC] Table 1. THE CHARACTERISTICS OF ENERGY CROPS Characteristics of energy crops in relation to the growing conditions based on [Alexopoulou et al. 2012] Crop Temperature, °С Need for water Frost resistance Drought resistance Seed germination Plant growth min max Fast-growing tree crops Willow - 0.1 30.0 high high low Poplar - 0.1 30.0 medium medium medium Eucalyptus - 5.0 30.0 high low high Perennial herbaceous crops Switchgrass >15 10.0 35.0 medium high medium / high Miscanthus >8 10.0 40.0 medium / high low low Artichoke islanskij >5 5.0 35.0 low low high (Panicumvirgatum L.), giant miscanthus (Mis- canthusgiganteus), energy willow (Salix L.) and energy poplar (Populus). The choice of an energy crop for industrial cultivation depends on several adaptive properties, economically useful traits and productivity potential and the possibility of growing on non-agricultural lands, the follow- ing energy crops are distinguished: switchgrass 62 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 Table 2. Features of growing bioenergy crops, based on [Ganzhenko, 2017] Energy crop Soil requirements, рН Precipitation, mm / year Depth of groundwater Life cycle, years Periodicity of harvesting Willow 5.0–7.0 650–700 up to 2 m 20–25 1 time in 2–3 years Poplar 6 >600 up to 2 m 20–25 1 time in 2–3 years Miscanthus 5.5–7.5 500–700 4 m Up to 20 annually Switchgrass 5.5–7.0 380–760 5 m 10–15 annually Among a wide range of high-yielding crops, energy willow has significant advantages: a wide range of genetic diversity; easy reproduction; tol- erance to a wide range of site conditions; CO2- neutrality; 1 ha of energy willow corresponds to approximately 4700 liters of oil; 1 hectare yields from 15 to 25 tons of wood chips per year; willow requires minimal pesticide use and can be grown organically without much difficulty; it cleans waste water; grows by about 2 meters per year - and up to 10 cm per day can be harvested many times, every 2-4 years; 1 liter of oil = 2.5 kg of dry wood chips = 4.5 kg of cod with a water content of 50% energy willow produces about 20 times more energy than is required for growing and harvest- ing; in comparison, wheat has an energy efficiency of only eight times the cost [Stolarski, et al., 2020; Nordborg et al., 2018; Amichev et al., 2018]. natural factors: soil type, soil moisture/rainfall, landscape type. THE CHARACTERISTICS OF ENERGY CROPS The requirements for cultivation and the peculiarities of the growing season of the main energy crops are shown in Table 2. When choosing a site for a plantation, one of the main parameters that needs to be assessed is the soil. Usually, energy crops grow well on medium to heavy loamy soils, well aerated, with acidity in the range of pH 5-7.5. The ability of the soil to retain moisture is also important; there- fore, cultivation in light sandy soils is not rec- ommended due to the possible water availability problems. On the other hand, the choice for the area of floodplains and sensitive wetlands can complicate the work of heavy machinery, espe- cially for planting and harvesting. Soil compac- tion can have a negative effect on wet soils, so the use of heavy machinery for such soils is rec- ommended either in very dry periods or when the soil is frozen. Shallow (i.e. with a thin layer of humus) soils are also not recommended due to low yields. Greater water needs of energy crops compared to agricultural ones can help reduce the leakage of dangerous amounts of nutrients into nearby reservoirs or groundwater in the case of growing energy crops as buffer zones in the areas with intensive agriculture. The technology of growing energy willow includes: The technology of growing energy willow includes: •• pruning every two to three years using special machines in winter;i •• growing for five years, before it gives the best harvest, then it can be harvested until the trees reach 20-25 years, after which they will need to be uprooted and new ones planted; •• harvesting is usually performed in winter, it is also suitable for levelling the sowing season, i.e. the farmer moves part of his usual field work from summer / autumn to winter; Energy poplar (Populus) Poplar (Populus) – willow family (Salicace- ae). It is a close relative of the willow, which has also found its way into bioenergy. Like willow, it is grown in Western Europe for heating. In our climatic conditions, among all trees, poplar grows faster than willow, under similar condi- tions. For growth, it requires a lot of moisture and light, so the greatest biomass yield will be under the conditions close to those in river val- leys [Pryshliak, 2021]. p [ g ] The main agroenergetic characteristics are as follows: the frequency of harvesting – annu- ally from the second year of the growing season, the period of use of the plantation – 15-20 years; minimum pending requirements; high tempera- ture resistance. The environmental benefits of growing switchgrass biomass are: no need to use pesticides, it fights against soil erosion, helps pre- serve natural conditions, improve soil structure and reduce the greenhouse gas emissions. The switchgrass biomass structure has typical com- ponents for biofuel feedstock: about 50% carbon, 43% oxygen and 6% hydrogen. Dry biomass has a low ash content – up to 2-4% compared to the low content of potassium and sodium in combina- tion with increased content of calcium and mag- nesium, contributes to a high combustion tem- perature and reduces the likelihood of slagging during combustion in boilers. The cost price of switchgrass biomass in different countries ranges from 15 to 40 EUR/t dry matter [Buzovsky, Vyt- vytska, Skrypnychenko, 2008]. The technology of growing energy poplar re- quires compliance with the following agro-tech- nological requirements: planting density – up to 9000 pcs/ha, and the optimal length of the seed- ling – 25 cm, when planting at least one bud of the seedling should remain above the ground. The plant is planted only in the spring. 10 tons of pop- lar cod replaces 2500 m3 of natural gas. The main agro-energy characteristics of pop- lar: yield: 40-60 t/ha, every 3-5 years; annual growth: 16 t/ha; productivity: 20-25 years; the heat of combustion is 18 MJ/kg. Recently, due to the relatively rapid growth and formation of biomass, poplar plantations are increasingly used as a regenerative energy source for biofuel production. Its wood is quite lightweight and is widely used for technical purposes. Poplar absorbs a large amount of carbon dioxide, owing to which one can obtain an excellent environmentally friendly fuel. Energy willow (Salix L.) The energy willow is the most widespread crop in the world. This is due to the fact that the willow genotype is one of the richest after rice, and this makes it possible to create new varieties and hybrids for different purposes. The produc- tivity of willow, according to experts, is 10-15 t/ha (under favourable soil and climatic condi- tions, the yield increases to 25-30 t/ha) of dry weight per year, exceeds the productivity of tra- ditional forest plantations by 14 times [Kulyk et al., 2020]. With sufficient soil moisture, only 1% of humus is needed for the plant to receive the required amount of nutrients and in the future to ensure good yields. •• using specialized equipment for planting and harvesting. In Europe, small energy willow companies often operate without their own equipment – for certain operations they hire a company that has all the necessary machines. This model works in Po- land, Germany, Denmark, Sweden. Every Euro- pean country has the companies that have special attachments for combines, and they turn to them when it is time to harvest energy willow. Energy willow is an environmentally friendly feedstock used as a renewable solid biofuel of or- ganic origin, which, when burned in boilers, does 63 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 biomass during this period can be harvested every 3-6 years [Khivrych et al., 2011]. not affect the carbon balance in the atmosphere. It is a type of solid biofuel, suitable for industrial production of heat and electricity at a price that is half that of using gas. The energy willow has a positive effect on the ecology and the environ- ment. One hectare of plantation absorbs more than 200 tons of CO2 from the air in three years. The calorific value of willow is about 18 MJ/kg of dry matter. One ton of willow with a humidity of 40% provides 1 Gcal of heat [Marchenko, 2012]. Willow does not emit harmful products dur- ing combustion and has a high heat transfer: 1 ton of dry biomass replaces more than 500 m3 of natural gas or 700 kg of brown coal. The biomass can be used to produce both solid granular bio- fuel in the form of briquettes and pellets, and, if appropriate equipment is available, in the case of oxygen-free combustion, synthesis gas. not affect the carbon balance in the atmosphere. Switchgrass (Panicumvirgatum) Switchgrass is a thermophilic perennial plant that naturally grows in North America along 45- 55° N longitude. Switchgrass is one of the prom- ising perennial plants for biofuel production. The plant has erect stems of different colours, which reach 0.5-2.7 m in height, propagates by seeds and rhizome. Inflorescence – open raceme 15-50 cm long. Its powerful root system can reach up to 3 m in depth [Moser, Vogel, 1995]. Energy willow (Salix L.) It is a type of solid biofuel, suitable for industrial production of heat and electricity at a price that is half that of using gas. The energy willow has a positive effect on the ecology and the environ- ment. One hectare of plantation absorbs more than 200 tons of CO2 from the air in three years. The calorific value of willow is about 18 MJ/kg of dry matter. One ton of willow with a humidity of 40% provides 1 Gcal of heat [Marchenko, 2012]. Fuel pellets and briquettes for burning in boil- ers are made of poplar wood and woodworking waste; part of the wood is burned in its raw form. The use of poplar biomass as an energy raw mate- rial can significantly save money and at the same time not harm nature, because the combustion of poplar does not emit toxic components into the atmosphere. Willow does not emit harmful products dur- ing combustion and has a high heat transfer: 1 ton of dry biomass replaces more than 500 m3 of natural gas or 700 kg of brown coal. The biomass can be used to produce both solid granular bio- fuel in the form of briquettes and pellets, and, if appropriate equipment is available, in the case of oxygen-free combustion, synthesis gas. Energy poplar (Populus) In industrial plantings, the yield of dry mass of poplar is up to 6-12 t / ha. Poplar plantations remain productive for 15-20 years or more, and Growing switchgrass for biomass is very profitable from an economic point of view. The crop has a low cost and practically no risks when growing. The switchgrass business is profitable relatively quickly. The plant gives a high yield with very little investment, which means that the money spent will quickly pay off. Switchgrass, as an alternative energy source, is most often used as a solid fuel for boilers. It can be burned 64 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 unprocessed, or it can be made into fuel briquettes or pellets. Thus, 1 ton of fuel pellets from switch- grass is equivalent to 1.14 tonnes of wood, 490 m3 of natural gas, 399 kg of diesel fuel, 437 kg of fuel oil, 779 kg of coal, 418 kg of oil. of biomass, starting from the third year of the growing season. At the initial stage, when land- ing, special technical means are required. The planters that are used to plant potatoes can also be used. Depending on the soil and climatic con- ditions, the recommended planting density is ap- proximately 18.5 thousand rhizomes per 1 ha. In order to harvest the vegetative mass of Miscan- thus, one can use the classic silage technique. fi Miscanthus (Miscanthus) Giant Miscanthus – a close relative of sugar cane, is directly related to the genus of herba- ceous perennial plants of the bluegrass family (cereals). Giant miscanthus is characterized by higher biomass yields and higher energy effi- ciency of biomass production [Dubis, Jankowski, Załuski, 2019)]. Other advantages of Miscanthus include [Kiesel., Wagner, & Lewandowski, 2017; McCalmont et al., 2017]:i Economic calculations of the efficiency of growing miscanthus on one hectare showed that the most invested funds are in the first year – 2046,8 US dollars (USD). However, from the second year onward, there is an opportunity to make a profit (Fig. 5). Due to the high yield of dry biomass (up to 25 t/ha), high calorific value (5 kWh / kg or 18 MJ/ kg (pellets)), low natural humidity of the stems in the collection (up to 15%), miscanthus gigan- teus is one of the most efficient plants for biofuel production compared to other crops. When the miscanthus biomass burns, less carbon dioxide is released than it was absorbed by plants during photosynthesis, so the use of miscanthus biofuel will not contribute to the greenhouse effect. Its •• can grow for twenty-five years in one place; •• the biomass can be obtained annually; •• accumulates ten tons of underground biomass; •• provides year-round employment; •• accumulates ten tons of underground •• provides year-round employment; •• provides year-round employment; •• provides phytoremediation; •• provides biodiversity. •• provides biodiversity. The technology of growing Miscanthus gi- ganteus (Fig. 4) provides for the annual collection Fig. 4. Technology of growing giant miscanthus, on the basis [Katelevskyi, 2020] Fig. 4. Technology of growing giant miscanthus, on the basis [Katelevskyi, 2020] Fig. 5. Economic calculations of the efficiency of growing miscanthus on one hectare, USD Fig. 5. Economic calculations of the efficiency of growing miscanthus on one hectare, USD Fig. 5. Economic calculations of the efficiency of growing miscanthus on one hectare, US 65 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 There are several cost-effective technologies for the preparation and processing of biomass for solid fuel in the world today. The most com- mon are pressing, resulting in pellets, briquettes or granules. The process of producing solid fuel from biomass is quite simple and usually includes the following technological operations (Fig. 7). stems contain 64-71% cellulose, which provides a high energy value. Miscanthus (Miscanthus) The total yield of solid biofuel from 1 hectare of plantation is 20-25 tons. In order to support the choice of the most suitable biomass crops for a specific environment and end use des- tination, besides biomass yield, biomass quality should also be considered [Amaducci et al., 2017]. The use of biomass for fuel production is as- sociated with certain difficulties in comparison with traditional fuels. This is due to the fact that biomass is characterized by: lower density and calorific value; seasonality of formation; addi- tional moisture content; a variety of thermochem- ical characteristics and chemical composition, depending on the type of biological raw materi- als, weather and climatic conditions and agro- technological factors; large dimensions and cost of systems for storage, transportation, preparation and supply of fuel to a copper. The SWOT analy- sis of using energy crops for biofuel production is given in Table 3. f PRODUCTION AND USE OF SOLID BIOFUELS The most important fuel and technological characteristic of biomass used as a solid biofuel is its calorific value, which depends on many fac- tors: genetic characteristics of energy plants, en- vironmental impact, storage conditions, humidity, etc. The practical calculations of the efficiency of using solid biofuel are carried out using the val- ues the lowest calorific value in the working state of the fuel, which is formed when burning a unit mass of fuel. The comparative characteristics of energy plants for the production of solid biofuel are given in Table 3. There are different variants for growing and further use of energy crops as fuel, which in- cludes such options as growing crops and their sale to enterprises, producing solid biofuels; use for energy autonomy (direct combustion or pro- duction of briquettes, pellets, granules and their The quality of biofuels is determined by the three phases that the biomass for energy purposes undergoes (Fig. 6). Table 3. Comparative characteristics of energy plants for the production of solid biofuels, according to data of [Kulyk et al., 2020] Crop Yield of dry mass (t/ha) / year Heat of combustion, MJ/kg of dry weight Energy production, GJ/ha Water content at harvest, % Ash, % Miscanthus 8.0–32.0 17.5 311.9–419.0 15.0 3.7 Switchgrass 9.0–18.0 17.0 266.8–312.2 15.0 6.0 Willow 8.0–20.0 18.5 280.0–315.0 53.0 2.0 Poplar 9.0–16.0 18.7 1700–300.0 49.0 1.5 Fig. 6. Phases of biomass passage, on the basis [Blum et al., 2010; Elbersen, 2001] ve characteristics of energy plants for the production of solid biofuels, according to data of Comparative characteristics of energy plants for the production of solid biofuels, according to data of al., 2020] Fig. 6. Phases of biomass passage, on the basis [Blum et al., 2010; Elbersen, 2001] Fig. 6. Phases of biomass passage, on the basis [Blum et al., 2010; Elbersen, 2001] 66 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 Fig. 8. Options for using energy crops (sales/energy autonomy) Fig. 8. Options for using energy crops (sales/energy autonomy) Table 4. Economic efficiency of willow cultivation and its use for the production of solid biofuels on the farm (the theoretical area of plantations on the farm is 300 hectares) combustion), and sale of secondary energy prod- ucts on the market (Fig. 8). fi The calculation of economic efficiency of en- ergy crops on the example of energy willow and its use for the production of solid biofuels for energy autonomy and sale on the farm (from 5 years of cultivation) showed that under the cur- rent Ukrainian market conditions, it is more prof- itable for companies to sell products than to use for energy autonomy (Table 4). Indicator Indicator value Dry mass yield, t/ha 23.0 Dry biomass yield, t/year 6900.0 Solid biofuel yield (10% moisture), t/year 62100 Self-sufficiency profitability, % 67.3 Average cost of 1 ton of solid biofuel, USD 103.6 Profit per 1 ton of solid biofuel, thousands USD 25.1 Sale profitability level, % 72.5 Growing energy crops needs the state sup- port. Compensation to farmers reaches 50% of the cost of planting in the EU, in addition, there Fig. 9. Directions for the use of energy crops, according to the data from [Kalinichenko et al., 2 67 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 enterprises for the cultivation and processing of energy crops with the participation of energy ser- vice companies will make it possible to provide stable supply of the transformed energy resource to consumers. are tax incentives. It is advisable in Ukraine to introduce exemptions from rent for unproductive and abandoned land, which are put into circula- tion for growing energy crops for 5 years, i.e. un- til the receipt of revenue; the application of tax benefits for VAT and income tax; simplified land allocation. In addition to biofuel benefits, energy crops have the following uses (Fig. 9). fi The total production of pellets in Ukraine in 2020 amounted to about 3.6 million tons of oil equivalent at 494 enterprises (Fig. 11). Currently, a large share of pellets and briquettes are exported from Ukraine to European countries due to the insufficient demand in the domestic market and higher prices for products in foreign markets. An efficient model for the production and en- ergy conversion of biomass provides for the cul- tivation of energy crops, the production of bio- mass, its processing and the supply of energy to consumers. At the same time, it is recommended to adhere to a closed waste-free production cycle – the supply of energy. For the rational use of bio- fuels from biomass of energy crops (obtained on marginal lands) and providing alternative energy to consumers in rural areas, the following scheme is proposed (Fig. 10). REFERENCES 1. Alexopoulou E, Christou M, Eleftheriadis I.D. Role of 4F cropping in determining future biomass poten- tials, including sustainability and policy related is- sues. Biomass Department of CRES. 2012. Retrived from http://www.biomassfutures.eu/public_docs/ final_deliverables/WP3/D3.2%20Role%20of%20 4F%20crops.pdf. 2. Amaducci, S., Facciotoo, G., Bergante, S., Perego, A., Serra, P., Ferraini, A., & Chimento, C. 2017. Biomass production and energy balance of herba- ceous and woody crops on marginal soils in the Po Valley. Global Change Biology Bioenergy, 9, 31–45. 3. Amichev, B.Y., Volk, T.A., Hangs, R.D., Bélanger, N., Vujanovic, V., Van Rees, K.C.J. 2018. Growth, survival, and yields of 30 short-rotation willow cul- tivars on the Canadian Prairies: 2nd rotation impli- cations. New Forests, 49, 649–665. The technologies for growing energy willow, poplar and miscanthus differ, but they share the ability to grow on unproductive degraded lands. There are various options for the cultivation and subsequent use of bioenergy crops - the sale of raw materials (biofuel) or use for energy autono- my (direct combustion, production and combus- tion of solid biofuel). It is advisable for enter- prises to carry out the calculations of the most economically profitable option in accordance with specific business conditions and take into account market conditions. 4. Blum Ya.B., Geletukha G.G., Grigoryuk I.P. et al. 2010. New technologies of bioenergy conversion. К: “Agrar Media Group”, 326 p. 5. Buzovsky E.A., Vytvytska O.D., Skrypnychenko V.A. 2008. Unconventional energy sources – the re- quirements of the time. Scientific Bulletin of the Na- tional Agrarian University of Ukraine, 119. 289–294. 6. Chaika T.O, Yasnolob I.O. 2017. Ecological, socio- economic advantages of growing energy crops. Eco- nomics of Agro-Industrial Complex, 12, 28-34. 7. Directive 2009/28/EC of the European Parliament and of the Council of 23 April 2009 on the promotion of the use of energy from renewable sources. Re- trieved from https://eur-lex.europa.eu/legal-content/ EN/ALL/?uri=CELEX%3A32009L0028. One of the reasons that hinder the development of growing bioenergy crops in Ukraine is the im- perfection of the regulatory legal regulation of the renewable energy industry in Ukraine. In addition, the lack of mechanisms for providing incentives and subsidies to the companies that are ready to in- vest in “green energy” creates significant financial burdens for the investor at the initial stage of plan- tation laying and, in turn, significantly slows down the development of such a business in Ukraine.i 8. Dubis, B., Jankowski, K.J., Załuski, D. 2019. RECAPITULATION Bioenergy crops are an important component of the bioenergy potential of Ukraine. Their cul- tivation has a number of positive effects, includ- ing: replacement of natural gas, which will help improve the balance of payments of our state; the The creation of an infrastructure that pro- vides the attraction of the resource of agricultural Fig. 10. An integrated logistic model for the production, processing and use of biomass from bioenergy crops, according to the data from [Kulyk et al., 2019; Kulyk et al., 2020] Fig. 10. An integrated logistic model for the production, processing and use of biomass from bioenergy crops, according to the data from [Kulyk et al., 2019; Kulyk et al., 2020] Fig. 11. Dynamics of solid biofuel production in Ukraine [Energy Strategy of Ukraine until 2035] 68 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 possibility of reducing the heat tariff for the popu- lation by 10%; land reclamation and restoration; decarbonization of the economy and the beginning of the transition to a bioeconomy based on the use of biological resources as energy carriers; econom- ic growth in rural areas due to the creation of new jobs, an increase in revenues to local budgets in the form of taxes from the enterprises engaged in the cultivation of energy crops and their processing into biofuels, etc. At the same time, it is important to create an infrastructure for the cultivation, pro- cessing and use of bioenergy raw materials, which provides for the attraction of agricultural resources for the cultivation of energy crops, the collection and processing of biomass, the supply of the trans- formed energy resource to consumers. REFERENCES Bio- mass production and energy balance of Miscan- thus over a period of 11 years: A case study in a large‐scale farm in Poland. 2019. GCB Bioenergy, 11,1187–1201. 9. Elbersen H.W. 2001. Switchgrass variety choice in Europe. Aspects of Applied Biology, 65. 21–28. Significant agricultural land areas and geo- graphic location make Ukraine one of the most attractive countries in Europe for the sustainable cultivation of energy crops without harming rec- reational or nature conservation areas. We need an initiative on the part of the state, the creation of favourable conditions at the legislative level and the attraction of investors, to promote the devel- opment of the production of solid biofuels from bioenergy raw materials. The use of an efficient logistic scheme for the production and use of bio- fuels from biomass of energy crops will provide the population with alternative energy and con- tribute to the sustainable development of the bio- energy sector in rural areas. 10. Energy Strategy of Ukraine till 2035. Retrieved from http://mpe.kmu.gov.ua/minugol/doccatalog/ document?id=245213112. 11. Fernando, A. L., Duarte, M. P., Almeida, J., Boléo, S., & Mendes, B. 2010. Environmental impact as- sessment of energy crops cultivation in Europe. Bio- fuels, Bioproducts and Biorefining, 4(6),594-604. 12. Ganzhenko O.M. Features of cultivation and use of energy crops. Presentation. 2017. Retrieved from https://saee.gov.ua/uk/news/1751. 13. Green Energy Transition of Ukraine until 2050, Ministry of Energy and Environmental Protec- tion of Ukraine], 2020. Retrieved from https://bit. ly/2tR0P7n. 69 Ecological Engineering & Environmental Technology 2021, 22(3), 59–70 development. Journal of Environmental Manage- ment & Tourism, 11(5), 1040-1053. 14. Hanegraaf, M.C. & Biewinga, E.E. 1998. Assessing the ecological and economic sustainability of energy crops. Biomass and Bioenergy, 15(4-5), 345-355. 26. Marchenko V. 2012. Energy crops in Ukraine. Agro- expert, 9, 114-117. 15. Heletukha, H., Drahniev, S., Kucheruk, P., Mat- vieiev, Yu. 2017. A practical guide to the use of bio- mass as a fuel in the municipal sector of Ukraine (for representatives of the agro-industrial complex)]. Retrieved from http://bioenergy.in.ua/media/filer_ public/f5/9c/f59c3f7f-8eca-4b6d-94cd-ffda1150f3 ae/ biofin.pdf. 27. McCalmont, J.P., Hastings, A., McNamara, N.P., Richter, G.M., Robson, P., Donnison, I.S., & Clif- ton-Brown, J.C. 2017. Environmental costs and benefits of growing Miscanthus for bioenergy in the UK. Global Change Biology Bioenergy, 9, 489–507. 28. Moser L.E. and Vogel K.P. 1995. Switchgrass, big bluestem, and indiangrass. An introduction to grass- land agriculture, 1, 409-420. 16. Janiszewska, D. & Ossowska, L. 2020. REFERENCES Biomass as the most popular renewable energy source in EU. Retrieved from https://www.um.edu.mt/library/oar/ handle/123456789/58012. 29. Nordborg, M., Berndes, G., Dimitriou, I., Henriks- son, A., Mola-Yudego, B., Rosenqvist, H. 2018. En- ergy analysis of willow production for bioenergy in Sweden. Sustainable Energy Review, 93, 473–482. 17. Kaletnik, G., Honcharuk, I., & Okhota, Y. 2020. The Waste-Free Production Development for the Energy Autonomy Formation of Ukrainian Agricultural En- terprises. Journal Of Environmental Management And Tourism, 11(3), 513-522. 30. Official website of JSC “Naftohaz of Ukraine”. 2020. Retrieved from https://www.naftogaz.com. 31. Official website of the Eurostat. (2020). Retrieved from https://ec.europa.eu/eurostat/data/database. 18. Kalinichenko А., Kalinichenko О., Kulyk М. 2017. 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Renewable and Sustainable Energy Reviews, 81, 380-398. 20. Khivrych O.B., Kvaka V.M., Kaskiv V.V., Mamai- sur V.V., Makarenko A.S. 2011. Energy plants as an alternative to traditional fuels. Agrobiology, 6, 153-157. 35. Scholz, V., Berg, W., & Kaulfuss, P. 1998. Energy balance of solid biofuels. Journal of Agricultural Engineering Research, 71(3), 263-272. 21. Kiesel, A., Wagner, M., & Lewandowski, I. 2017. Environmental performance of miscanthus, switch- grass and maize: Can C4 perennials increase the sustainability of biogas production? Sustainability, 9, 1–20. 36. Stolarski, M. J., Niksa, D., Krzyżaniak, M., Tworkowski, J., & Szczukowski, S. 2019. Willow productivity from small- and large-scale experimen- tal plantations in Poland from 2000 to 2017. Renewa- ble and Sustainable Energy Reviews, 101, 461–475. 22. Koçar, G., and Civaş, N. 2013. An overview of bio- fuels from energy crops: Current status and future prospects. Renewable and Sustainable Energy Re- views, 28, 900-916. 37. 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https://openalex.org/W4360827075	https://jmla.mlanet.org/ojs/jmla/article/download/1590/2147	English	null	Standards of practice for hospital libraries and librarians, 2022: Medical Library Association Hospital Libraries Caucus Standards Task Force	Journal of the Medical Library Association	2,023	cc-by	7,088	Standards of practice for hospital libraries and librarians, 2022: Medical Library Association Hospital Libraries Caucus Standards Task Force Jill Tarabula; Donna S. Gibson; Bridget Jivanelli; J. Michael Lindsay; Ana Macias; Sondhaya McGowan; Lori Mills; Louise McLaughlin The Hospital Library Caucus of the Medical Library Association (MLA) follows the practice established in 1953 of developing quality indicators and best practices in the newly developing and fast-changing world of hospital libraries. As these libraries increased in number and prominence, the Joint Commission on the Accreditation of Hospitals (JCAHO) included in 1978 a hospital library standard developed in collaboration with MLA. Subsequent changes in JCAHO, then The Joint Commission (TJC) knowledge management criteria as well as technological changes in the curation and delivery of evidence-based resources influenced standards changes over the years. The 2022 standards mark the most recent edition, replacing the 2007 standards. Keywords: Librarians; libraries; hospital library standards; professional role; library services/standards; personnel staffing and scheduling/standards; evidence-based information; virtual library services BACKGROUND (IT) teams, and accrediting bodies to ensure that libraries have the resources and services to effectively meet the hospital's KBI needs, to support evidence-based practices, quality and safety program development, and improved patient outcomes. The Medical Library Association's (MLA's) “Standards for Hospital Libraries” were developed in 2002 as a guide for hospital administrators, librarians, and accrediting bodies to ensure that hospitals have the resources and services to effectively meet their needs for knowledge-based information (KBI) [1]. In approving the original and subsequent revised versions of the standards, the MLA Board of Directors recommended that the Standards Committee of the Hospital Libraries Section continually evaluate the standards and revise them as necessary to reflect changes in the health care environment and MLA priorities. This document reflects the most recent update of these standards. as an organizational benchmark to be used to meet the KBI needs for continuous access to point of care resources, to improve access to documented best practices that improve patient outcomes and contribute to quality and performance improvement initiatives, while being mindful of budget, space, and staffing constraints. While many of the library practices remain constant over the years, the Committee updated or added content to 399 399 399 SPECIAL REPORT SPECIAL REPORT DOI: dx.doi.org/10.5195/jmla.2022.1590 EXECUTIVE SUMMARY • demonstrate the commitment to diversity, equity, and inclusion in all health care environments. MLA's “Standards of Practice for Hospital Libraries and Librarians, 2022” have been revised to reflect the technological, environmental, and budgetary changes so visible in hospitals today, with updates to meet the dynamic needs of 21st century hospital libraries and the institutions they serve. The document serves a two-fold purpose: • create consistency through the use of the broader term “hospital” or “health sciences library” in place of “medical library.” • bring to light the increasing involvement of librarians with IT applications and teams, including KBI source links in electronic health records (EHR), mobile application support, and • as a guide for hospital senior and department- level leaders, librarians, information technology • as a guide for hospital senior and department level leaders, librarians, information technology , , gy DOI: dx.doi.org/10.5195/jmla.2022.1590 DOI: dx.doi.org/10.5195/jmla.2022.1590 Standard 4: The health sciences librarian, as the principal KBI professional in the organization, collaborates with the information technology team to ensure online resources and services are functional and available at the point of need, including in the EHR. interactions with electronic resource products and vendors. interactions with electronic resource products and vendors. • broaden the concept of library space to include physical and virtual libraries. • highlight new research available for calculating staffing ratios and coordinating staffing with library service levels. Standard 5: Evidence provides the scientific basis linking KBI and improved patient care; enhanced patient education; support for performance improvement projects; patient safety functions; and student, nursing, medical, graduate, and continuing medical education. • expand roles in teaching, researching, and appraising evidence-based literature across clinical specialties and to hospital staff at every level. Standard 6: The librarian provides evidence of an ongoing assessment of the information needs of the organization, and the development and implementation of a plan to provide appropriate resources, services, and technology to meet those identified needs. • emphasize the significance of the librarian having an advanced degree and AHIP certification • expand suggested duties of the librarian to include o support for publishing and adhering to copyright laws. Standard 7: The librarian actively promotes KBI services and resources to all user groups and provides documented evidence thereof. o developing a needs assessment and strategic plan for the library. g p y o planning physical and virtual library spaces including an intra- or internet presence. Standard 8: All KBI functions are performed in compliance with applicable federal, state, and local laws and regulations including but not limited to copyright, HIPAA (patient privacy and confidentiality), and vendor licensing agreements. • reference additional materials to support these recommended, evidence-based library practices. The 2022 “MLA Statement Calling on Hospital and Health System Executives to Fund Libraries and Library Staff,” co-signed by multiple credentialing and health care institutions, adds additional support to the maintenance of library services. The health sciences librarian is positioned to play a key role in the hospital. The ubiquitous nature of the internet and primarily online collections, existing and disruptive technologies, and evolving means of communication by medical, nursing, and allied health staffs, patients, and the community require new strategies, strategic planning, allocation of adequate resources, and selection and evaluation of appropriate information resources and technologies. INTRODUCTION Following the publication of the Standards in 2002, a revised version was published in 2005 [1, 2]. In March of 2005, the National Network of Libraries of Medicine (NNLM) Hospital Internet Access Task Force issued a final report and made several recommendations, one of which was to work with the HLS Standards Committee to add a technology standard to the “Standards for Hospital Libraries.” The new standard defined the minimum levels of technology needed for hospital libraries to function in their role as providers of KBI resources. In May 2007, Standard 11 was approved by the MLA Board at its annual meeting. As well, the MLA educational policy statement and the Competencies for Lifelong Learning and Professional Success were added to the Standards document. Other pertinent updates were made to reflect changes in resources and terminology in use. Additionally, the MLA Board recommended that the revised standards be called “Standards for Hospital DOI: dx.doi.org/10.5195/jmla.2022.1590 It is the hope of the Hospital Library Standards Committee that the material herein advances these efforts. Standard 9: KBI resources are available to staff twenty- four hours a day, seven days a week. Standard 10: The term library refers to the department— physical, virtual or a combination thereof—and includes a qualified librarian and support professionals, services, and resources that when put together, best meet the information needs of the health care organization. Standard 11: IT resources are available to support the library’s mission of providing KBI resources and services. SYNOPSIS OF STANDARDS OF PRACTICE Standard 1: The librarian serves as the primary department head responsible for managing resources and services to meet the KBI needs of the organization. The library has its own budget, and the library director/manager, as a department head, reports to the senior management of the organization. Standard 2: Health care research and reference information systems and services are directed by a qualified librarian. Academy of Health Information Professionals (AHIP) membership is preferred. Standard 3: Informed by MLA’s commitment to fostering diversity, equity, and inclusion, library staffing formulas and service level agreements guide human resource allocation. Additionally, the MLA Board recommended that the revised standards be called “Standards for Hospital Journal of the Medical Library Association jmla.mlanet.org jmla.mlanet.org 110 (4) October 2022 Tarabula et al. 400 Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 401 Libraries 2007” and that the publication history be indicated [3]. Intent. Librarian qualifications. Providing medical, nursing, and clinical information requires specific knowledge and skills related to understanding information management and technologies, meeting rapidly changing user needs in a health care setting and provision of information services and resources aligning with the organization’s mission, vision, goals, and strategic plan. Reliance on a commercial electronic resource for clinical information cannot substitute for a qualified medical librarian. In October 2019, a discussion ensued regarding a review and update of the existing “Standards for Hospital Libraries 2007.” The Hospital Libraries Task Force was formed, with members volunteering to review specific standards for currency and application to modern practices and technologies. Despite COVID-19’s impact and after numerous virtual meetings, a final draft was prepared for presentation to the Hospital Library Caucus in the Spring of 2022. A qualified librarian is a person who has earned a Master’s degree from a: DETAILED STANDARDS OF PRACTICE AND INTENT • program accredited by the American Library Association (ALA) or its successor accrediting organization [10] or Standard 1: The librarian serves as the primary department head responsible for managing resources and services to meet the KBI needs of the organization. The library has its own budget, and the library director/manager, as a department head, reports to the senior management of the organization [3, 4]. Standard 1: The librarian serves as the primary department head responsible for managing resources and services to meet the KBI needs of the organization. The library has its own budget, and the library • program in library and information studies accredited or recognized by the appropriate national body of another country As a health information professional, the health sciences librarian possesses unique knowledge, skills and abilities that have been identified as professional competencies. Evidence of continued advancement of these competencies is membership in MLA’s Academy of Health Information Professionals (AHIP) [11]. The MLA has developed Competencies for Lifelong Learning and Professional Success outlining the essential professional skills and abilities that are expected of health sciences librarians. Competency areas include information services and management, instruction, leadership, evidence-based practice, research, and professionalism [12]. Intent. Library as department. As reflected in Joint Commission resources, access to KBI is an information management requirement for a hospital or health system [5–7]. To enable the development of systems, resources, and services to meet this requirement, the needs, concerns, and contribution of the library must be communicated to decision makers at the highest levels in the organization. Departmental status with the librarian in a director/manager level leadership position facilitates this connection. Librarian interaction with other departmental leaders and administrators fosters a deeper understanding of the information needs of the organization. This interaction can also facilitate access to institutional resources and data necessary for providing information to satisfy the needs of hospital senior leaders, clinicians, patients, and family members [8]. Provision of health information occurs in a variety of settings including hospitals and health care centers, research, and academic/medical, nursing, and allied health schools. Library and information services are developed that meet the strategic information needs of the individual or group being served. The role of the health sciences librarian may include but is not limited to [13–14]: In 2022, the MLA issued a statement adding additional support to the provision and maintenance of curated library services. 110 (4) October 2022 110 (4) October 2022 110 (4) October 2022 Journal of the Medical Library Association Journal of the Medical Library Association jmla.mlanet.org jmla.mlanet.org Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 DETAILED STANDARDS OF PRACTICE AND INTENT • developing, providing and promoting library services to support access to biomedical literature including: [Excerpt] "Hospital and health system executives must take concrete action to support clinical and administrative staff, patients and their families, and communities by funding hospital library services led by a professional librarian. Working together, hospitals, health care systems, and librarians are positioned to provide the resources, access, and support your health care teams can depend upon to provide evidence-based practice services that will support healthy patient outcomes." o mediated literature searches o document delivery/interlibrary loan (ILL) o document delivery/interlibrary loan o current awareness services • responding to consumer health requests related to patient care, health and disease, medications, and community resources • selecting, evaluating, licensing/purchasing and promoting resources for incorporation into the physical or electronic collections National associations and institutions have endorsed the full statement supporting hospital library departments [9]. National associations and institutions have endorsed the full statement supporting hospital library departments [9]. • development and maintenance of an online presence, to include remote access Standard 2: A qualified librarian directs medical research and reference information systems and services. Academy of Health Information Professionals membership is preferred. • individual and institutional support for copyright compliance and publication DOI: dx.doi.org/10.5195/jmla.2022.1590 The amount of staffing throughout the system should be at least at the level specified in the library staffing formula for minimum level of service (bronze or silver), taking all components and needs of the health care system into account. Whether each hospital or institution is treated separately in determining staffing levels, or the system is taken as a whole, is left to the judgment of the health information professional and administrators. It stands to reason that librarians providing services at the gold level, such as support for systematic reviews and other knowledge synthesis, or research data management, etc. (or higher in the case of libraries providing services not listed in the HSICT document), will require additional staff. The important point is that staffing is sufficient to serve the number of users and is appropriate for the level of service required to meet the needs of the organization. The grid in Table 1 may be used to determine appropriate staffing needed by level of service [16]. • providing new/existing employee orientation to resources and services • providing new/existing employee orientation to resources and services • strategic planning for library operations to support the mission of the institution • budgeting for library operations; identifying partnerships and consortia purchasing opportunities • hiring, training, and evaluating the performance of the library staff These roles and services are explored further in the following standards. Standard 3: MLA’s commitment to diversity, equity, and inclusion should form the foundation for determining staffing levels and service level agreements. MLA fosters excellence and is committed to diversity, equity, and inclusion in professional practice, leadership of health sciences libraries, and for information professionals, now and in the future [15]. Table 1 Staffing grid by HSICT level of service Number of institutional staff* Number of FTE health information professionals Staff Sq. DOI: dx.doi.org/10.5195/jmla.2022.1590 Root† Bronze Silver Gold 400 20 1.24 1.55 1.85 625 25 1.55 1.93 2.32 900 30 1.85 2.32 2.78 1225 35 2.16 2.70 3.24 1600 40 2.47 3.09 3.71 2025 45 2.78 3.48 4.17 2500 50 3.09 3.86 4.63 3025 55 3.40 4.25 5.10 3600 60 3.71 4.63 5.56 4225 65 4.08 5.02 6.03 4900 70 4.33 5.41 6.49 5625 75 4.63 5.79 6.95 6400 80 4.94 6.18 7.42 *Includes all active medical staff, as well as health care personnel on service contract †Calculated using square roots in increments of 5 from 20 to 80 (20x20=400; 25x25=625, etc.) The following formulae are used to calculate number of library staff needed per level of service: Table 1 Staffing grid by HSICT level of service Table 1 Staffing grid by HSICT level of service Intent. Staffing. An adequately staffed library is necessary to fully serve the KBI needs of the total institutional staff. Intent. Staffing. An adequately staffed library is necessary to fully serve the KBI needs of the total institutional staff. Consideration for library staffing needs to include an understanding of the user community’s information needs, new and potential users, and outreach endeavors that utilize/require library services. Library operations and workload are driven by the staff size, clinical research activities, and mission and complexity of the institution. As stated in Standard 1, interaction of the librarian with other departmental leaders and administrators fosters a deeper understanding of the information needs of the organization. Working together, librarians, administrators, and stakeholders should decide upon the levels of library services when formulating a service level agreement. We recommend that each hospital library utilize the grid in Table 1 presented in the Canadian Health Libraries Association (CHLA) Standards that considers Van Moorsel’s “golden ratio,” MLA’s 2007 Standards, and the chart developed by the Health Science Information Consortium of Toronto (HSICT) [16, 17, 3, 18]. The latter serves as a guide to outline possible services based on bronze, silver, and gold levels [Appendix A]. Attention must be called to Van Moorsel’s use of the “mean” number (2,865) of FTE’s as calculated from his survey results. Calibration using this method for gauging staffing requirements for relatively large or relatively small libraries must be carefully considered [18]. Updates to both Canadian works are in the planning stage. DOI: dx.doi.org/10.5195/jmla.2022.1590 Future benchmarking studies could dive deeper by collecting specific data that would correlating the number of staff support per individual library service. The 2019 study by Spencer, Mamo, and Billman published in February 2021 is a step in this direction [19]. The following formulae are used to calculate number of library staff needed per level of service: The following formulae are used to calculate number of library staff needed per level of service: 110 (4) October 2022 jmla.mlanet.org 110 (4) October 2022 Journal of the Medical Library Association jmla.mlanet.org Tarabula et al. 402 Frequent provision of information on which performance improvement and patient safety decisions are based; routing current literature to appropriate individuals, committees, and teams relevant to the hospital’s quality indicators, top chronic conditions/diagnoses, performance improvement projects, patient safety goals and/or identified problem areas [25]. This information provides a starting point for determining what service level is desired and what staffing will be required to support those needs at each institution [Appendix A]. In establishing library staffing size, consideration must also be given to the service level, hours of operations, and expectations agreed upon by the hospital administrator/hospital leadership team, stakeholders, and library director or information professional. Just having a staffing formula is not enough to accurately determine how best to serve the organization. Patient education: Active membership of the librarian on patient education teams; consultation with team concerning selection, creation, and quality filtering of sources for patient education materials; provision of (or facilitation of access to) patient education materials for clinical staff that are diverse, culturally and age-appropriate and understandable; provision and marketing of library services directly to patients and families; and instruction in utilizing online resources for patient education [23]. Standard 4: The health sciences librarian, as the principal KBI professional in the organization, collaborates with the information technology team to ensure online resources and services are functional and available at the point of need, including in the EHR. Intent. Information technology role. Because KBI resources have made a substantial shift to online formats, it is imperative that the information technology team and the librarian communicate regularly regarding technology needs and changes, including but not limited to authentication tools, networking, systems, integration in the EHR, etc. Migrations, upgrades, and vendor-based changes can impact access to costly licensed resources that can hinder if not prevent access altogether. The librarian and the information technology team must work closely to maintain consistent access and may need to troubleshoot issues to ensure library users have access to all the organization’s KBI resources while onsite using standard desktop hardware or at the bedside using a mobile device or working remotely [13, 21, 22]. Journal of the Medical Library Association Journal of the Medical Library Association Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 403 Bronze √total FTE institution / 10 (1.61803399) [1.0 (16.180339) is an accurate alternative.] Silver √total FTE institution x 1.25 / 16.1803399 Gold √total FTE institution x 1.5 / 16.1803399 Bronze √total FTE institution / 10 (1.61803399) [1.0 (16.180339) is an accurate alternative.] Standard 5: Evidence provides the scientific basis linking KBI and improved patient care; enhanced patient education; support for performance improvement projects; patient safety functions; and student, medical, nursing, graduate and continuing medical education. Intent: Patient care and safety. The librarian serves all user groups in the hospital and outlying clinics as applicable. The functions listed in Standard 2 are among the most dependent on evidence. Examples of connections and evidence include: Additional staffing is necessary if the librarian provides enhanced services (e.g., at the silver or gold levels) or services usually performed by other departments, or supports entities other than just a hospital, such as, but not limited to, the examples listed below: • research data management • publication and institutional repository support • systematic review support • primary responsibility for audiovisual equipment and other information technology • substantial responsibility for web programming, hospital-wide intranet or internet site • coordination of, or clerical support for, continuing medical education program • networked system with multiple libraries • support of entities other than hospital (e.g., research institutes, education programs) • Patient care: Frequent provision of evidenced- based information to support patient care decisions, integration of evidenced-based resources to support clinical and hospital staff providing information at the point of care, supporting morning rounds, and providing case- specific literature needed in support of rounds or other related activities [20, 23, 24]. • research data management • publication and institutional repository support • systematic review support • primary responsibility for audiovisual equipment and other information technology • substantial responsibility for web programming, hospital-wide intranet or internet site • coordination of, or clerical support for, continuing medical education program • networked system with multiple libraries • support of entities other than hospital (e.g., research institutes, education programs) • Performance improvement and patient safety: Active membership of the librarian on performance improvement committees and patient safety teams. Education of hospital’s clinical, nursing, and medical staff: Active membership of the librarian on teams or committees concerned with diversity, equity and inclusion initiatives and clinical, nursing and medical educational functions; provide regular input regarding online resources and materials to support planning and preparation of educational activities; education of hospital clinical, nursing, and medical staff in accessing, retrieving, searching online resources; instruction in appraising and evaluating evidence-based information and peer reviewed resources; support and encourage the use of technology, medical mobile applications and • inclusion of library services and resources in orientation of interns and residents (if applicable), nurses, and new medical and hospital staff members, Intent. Evaluations of KBI needs. The librarian uses a variety of tools and techniques, both formal and informal, direct and indirect, to assess the information needs of the hospital and medical staff. In response, resources and services are made available to meet those identified needs. Techniques may include, but are not limited to, focus groups, library committees, surveys, analysis of usage patterns, budget and strategic planning, inventory of collections, and one-on-one conversations with health care leaders regarding clinical and organizational information needs [29–33]. • activities in observance of National Library Week and/or National Medical Librarians Month, • participation in information fairs, skills fairs, Research Day, or Match Day activities • promotion of existing current awareness services or proactive provision of these services, • presentations to groups on what the library can offer them. The librarian assures access to a current and authoritative collection of information that can be used for evidence- based practice, scholarly activity, and continuing education. Key resources, such as print, e-resources, multimedia, databases, etc., can be identified by review products such as Doody’s Core Titles or by seeking recommendations from hospital staff or the national hospital library community. Resource-sharing agreements and membership in consortia should be utilized to enable efficient provision of additional resources. Standard 8: All KBI functions are performed in compliance with applicable federal, state, and local laws and regulations including but not limited to copyright, HIPAA (patient privacy and confidentiality), and vendor licensing agreements [35, 36]. Standard 8: All KBI functions are performed in compliance with applicable federal, state, and local laws and regulations including but not limited to copyright, HIPAA (patient privacy and confidentiality), and vendor licensing agreements [35, 36]. Intent. Regulatory compliance. Interlibrary loan, document delivery, and onsite and remote access to subscription resources, as well as copying, faxing and printing of biomedical literature must adhere to vendor license agreements, publishing contracts, and federal and/or international copyright legislation. In addition, the hospital library adheres to the Health Information Portability and Accountability Act (HIPAA) and patient confidentiality when assisting patients, families, health care providers and others who may use the library or any of its services or resources [34, 35]. Services should include expert literature searching, document delivery, and interlibrary loan. 110 (4) October 2022 jmla.mlanet.org Journal of the Medical Library Association Tarabula et al. 404 DOI: dx.doi.org/10.5195/jmla.2022.1590 Standard 7: The library actively promotes KBI services and resources to all user groups and provides documented evidence thereof. Standard 7: The library actively promotes KBI services and resources to all user groups and provides documented evidence thereof. other resources to support education and training; identification of print and electronic resources to support individualized learning and educational development; participate in continuing medical education (CME), graduate medical education (GME), clinical nursing education(CNE) [26, 27]. Intent. Service promotion. Promotion increases user awareness and efficient use of the services and resources available [34]. The library serves not only clinical staff, but may serve others, including: Intent. Service promotion. Promotion increases user awareness and efficient use of the services and resources available [34]. The library serves not only clinical staff, but may serve others, including: • Clinical education (nursing) and resident/medical students; provision of specific literature to support grand rounds and related activities; and provision of access and instruction to virtual learning and other technology [28]. • Clinical education (nursing) and resident/medical students; provision of specific literature to support grand rounds and related activities; and provision of access and instruction to virtual learning and other technology [28]. • administrative and managerial staff, • research staff, • staff in off-site locations, • students in affiliated programs, • patients and their families, and • other groups as applicable • administrative and managerial staff, Standard 6: The librarian provides evidence of an ongoing assessment of the information needs of the organization and the development and implementation of a plan to provide appropriate resources, services, and technology to meet those identified needs. Promotion of services may take the form of: • announcements to hospital and/or medical staff of new services, resources, or offerings, • announcements to hospital and/or medical staff of new services, resources, or offerings, • inclusion of library services and resources in orientation of interns and residents (if applicable), nurses, and new medical and hospital staff members, • activities in observance of National Library Week and/or National Medical Librarians Month, • participation in information fairs, skills fairs, Research Day, or Match Day activities • promotion of existing current awareness services or proactive provision of these services, • plan for reaching library nonusers, and • presentations to groups on what the library can offer them. A recent benchmarking survey may be useful for determining what services other hospital libraries provide. Services should be ongoing and consider timeliness of delivery and user satisfaction [19]. Technology considerations include providing and maintaining a library catalog and/or discovery service to identify and locate library holdings; providing and maintaining a mechanism for remote access of library resources; and integration of library resources within EHR, intranet, and/or public website for easy access Standard 9: Service availability. KBI resources are available to clinical staff twenty-four hours a day, seven days a week. Intent. Clinical decision-making occurs at all hours, so access to KBI must be continuously available. This is reinforced by the Joint Commission’s element of performance for the Information Management standard IM.03.01.01: “The hospital provides access to knowledge- A strategic plan can help a librarian analyze trends and anticipate future needs. A plan for providing information needs to take into account the values of the broader organization. Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 405 users' needs to access essential web-based KBI resources from the point of need are balanced with the network security needs and firewall policies of the institution. Examples of essential information technology resources include: based information resources 24 hours a day, 7 days a week” [5, 7]. The Accreditation Council for Graduate Medical Education also requires that “Residents must have ready access to specialty-specific and other appropriate reference material in print or electronic format” (I.D.3.) [26]. • access to the internet sufficient to use email, DOCLINE, OCLC, PubMed, and any commercial databases and full-text resources to which the library may subscribe, Continuous access to resources may take multiple forms, depending on the size and structure of the institution. A broad selection of resources should be made available in a physical library and/or through electronic means. Physical resources should be made continually accessible to clinical staff, and the library should provide onsite and remote access to electronic resources. • access should be convenient for all users in the library's institutions twenty-four hours, seven days a week; remote access should be available as licenses permit, p • specialized library software that can describe and track library resources and their use (e.g., catalog, circulation, serials control, and/or an integrated library system) appropriate to the library's collection and services; this software can be hosted locally or remotely, and • access to high-bandwidth communication technologies (e.g., full-motion video, video streaming, and webcasting) appropriate to the library's services and its institution's educational programs. • specialized library software that can describe and track library resources and their use (e.g., catalog, circulation, serials control, and/or an integrated library system) appropriate to the library's collection and services; this software can be hosted locally or remotely, and Standard 10: The term library refers to the department— physical, virtual or a combination thereof—and includes a qualified librarian and support professionals, services, and resources that when put together, best meet the information needs of the health care organization. Intent. Library as space. The term library has evolved and should reflect the dynamics of the health information world. Given the ever-changing needs of a health care organization, a library must be nimble and flex to meet the needs of users. Traditionally, the library was a physical space located onsite with a print collection. Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 Today a library can be a single physical location that provides resources, staff, and services to one or more facilities, or it could be a completely virtual presence, or any combination of the two. A library that meets the needs of the organization it serves, whether physical, virtual, or a combination of the two is a library that is likely to be well- utilized. • access to high-bandwidth communication technologies (e.g., full-motion video, video streaming, and webcasting) appropriate to the library's services and its institution's educational programs. • access to high-bandwidth communication technologies (e.g., full-motion video, video streaming, and webcasting) appropriate to the library's services and its institution's educational programs. CONCLUSION As the team worked, we acknowledged that we stood in the shadow of those who went before us. The original Hospital Libraries Section of MLA saw the need for cohesiveness in setting out a philosophy of services unique to those serving the medical profession. Standards established in response to those set forth by the medical associations forged a bond assuring physicians of easy access to authoritative literature. As the fields of medical sciences progressed, so did the need for updated hospital library standards. In the case of a physical library, the space should be large enough to accommodate walk-in users and the library staff, any in-house collection(s), and an appropriate number of computer or other IT hardware workstations. Space should permit demonstration of and access to resources and ample seating for users. A separate office for staff ensures privacy of communication among library staff and confidentiality for persons requesting information [37]. The team also stood on the shoulders of our contemporaries. As we looked back and around, we recognized with professional pride how the term “health sciences librarians” easily expanded from serving physicians to including nurses, pharmacists, physical and allied health therapists, technicians, and ultimately administrators. As colleagues, we generously shared our time, wisdom, and experience with each other in revising the 2007 standards. We also accept that new research and knowledge will ultimately require continued and timely revisions. Standard 11: IT resources are available to support the library’s mission of providing KBI resources and services. Intent. IT resources. Adequate IT resources are essential in the provision of up-to-date KBI resources and services. The library must have hardware and library-specific software applications to perform basic functions related to acquiring, organizing, retrieving, and delivering KBI resources to support the institution's mission. The library also must have internet connections with sufficient speed, performance, and bandwidth to access the many web- based resources now available [6, 7, 11–13, 21]. The Task Force includes representatives from diverse fields with varying levels of experience. Our passion for using our talents to make the best and the latest knowledge available to our health care teams knows no bounds and aims for inclusivity. 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Bleich MR, Brown R. Evidence-based leadership practice and the role of the librarian. J Contin Educ Nurs. 2019 Dec 1;50(12):537-539. doi: 10.3928/00220124-20191115-03. • Appendix D: Glossary 31. Oelschlegel S, Grabeel KL, Tester E, Heidel RE, Russomanno J. Librarians promoting changes in the health care delivery system through systematic assessment. Med Ref Serv Q. 2018 Apr-Jun;37(2):142-152. DOI: 10.1080/02763869.2018.1439216. AUTHORS’ AFFILIATIONS Jill Tarabula, jtarabula@cvph.org, https://orcid.org/0000-0003- 1037-5888, Regional Medical Librarian, Champlain Valley Physicians Hospital (UVMHN), Plattsburgh, NY. 32. Harnegie MP. Evaluation of library usage and attitudes of residents and fellows: Results of 017–2019 surveys, J Hosp Librariansh. 2021;21(2):141-157. DOI: https://doi.org/10.1080/15323269.2021.1899781 Donna S. Gibson, GibsonD@mskcc.org, https://orcid.org/0000- 0003-3333-6742, Memorial Sloan Kettering Cancer Center, New York, NY. 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Health Insurance Portability and Accountability Act of 1996 (HIPAA). [Internet]. Atlanta, GA; CDC; rev 2018 [cited 17 Standards of practice for hospital libraries and librarians DOI: dx.doi.org/10.5195/jmla.2022.1590 407 110 (4) October 2022 Journal of the Medical Library Association jmla.mlanet.org Standards of practice for hospital libraries and librarians 4 DOI: dx.doi.org/10.5195/jmla.2022.1590 DOI: dx.doi.org/10.5195/jmla.2022.1590 Sondhaya McGowan, Mcgowan.sunny@scrippshealth.org, https://orcid.org/0000-0003-3736-7503, Scripps Mercy Hospital, San Diego, CA. Lori Mills, lori.mills@mclaren.org, Librarian, IPCE Coordinator, McLaren Macomb, Mt Clemens, MI. Louise McLaughlin, louise.mclaughlin@womans.org, https://orcid.org/0000-0002-4995-517X, Health Sciences Library (retired), Woman’s Hospital, Baton Rouge, LA. Received November 2021; accepted May 2022 Sondhaya McGowan, Mcgowan.sunny@scrippshealth.org, https://orcid.org/0000-0003-3736-7503, Scripps Mercy Hospital, San Diego, CA. Lori Mills, lori.mills@mclaren.org, Librarian, IPCE Coordinator, McLaren Macomb, Mt Clemens, MI. Louise McLaughlin, louise.mclaughlin@womans.org, https://orcid.org/0000-0002-4995-517X, Health Sciences Library (retired), Woman’s Hospital, Baton Rouge, LA. Received November 2021; accepted May 2022 Lori Mills, lori.mills@mclaren.org, Librarian, IPCE Coordinator, McLaren Macomb, Mt Clemens, MI. Louise McLaughlin, louise.mclaughlin@womans.org, https://orcid.org/0000-0002-4995-517X, Health Sciences Library (retired), Woman’s Hospital, Baton Rouge, LA. Received November 2021; accepted May 2022 Received November 2021; accepted May 2022 ISSN 1558-9439 (Online) Journal of the Medical Library Association 110 (4) October 2022 110 (4) October 2022 jmla.mlanet.org Tarabula et al. 408 DOI: dx.doi.org/10.5195/jmla.2022.1590 jmla.mlanet.org Journal of the Medical Library Association 110 (4) October 2022 Journal of the Medical Library Association
https://openalex.org/W2488079809	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0160121&type=printable	English	null	The Effect of Altered Loading on Mandibular Condylar Cartilage	PloS one	2,016	cc-by	9,357	Raman Kaul1, Mara H. O’Brien1, Eliane Dutra1, Alexandro Lima1, Achint Utreja2, Sumit Yadav1* 1 Division of Orthodontics, University of Connecticut Health Center, Farmington, United States of America, 2 Division of Orthodontics, Indiana University Purdue University Indianapolis, Indianapolis, United States of America * Yadav_sumit17@yahoo.com Results Our flow cytometery data showed that altered loading resulted in a significant increase in a number of Col2a1-positive (blue) and Col10a1-positive (red) expressing cells. The gene expression analysis showed significant increase in expression of BMP2, Col10a1 and Sox 9 in the altered loading group. There was a significant increase in the bone volume fraction and trabecular thickness, but a decrease in the trabecular spacing of the subchondral bone with the altered loading. Morphometric measurements revealed increased mandibular length, increased condylar length and increased cartilage width with altered loading. Our histology showed increased mineralization/calcification of the MCC with 5 days of loading. An unexpected observation was an increase in expression of tartrate resistant acid phos- phatase activity in the fibrocartilaginous region with loading. Abstract Objective a1111 The purpose of this study was to delineate the cellular, mechanical and morphometric effects of altered loading on the mandibular condylar cartilage (MCC) and subchondral bone. We hypothesized that altered loading will induce differentiation of cells by accelerat- ing the lineage progression of the MCC. OPEN ACCESS OPEN ACCESS Citation: Kaul R, O’Brien MH, Dutra E, Lima A, Utreja A, Yadav S (2016) The Effect of Altered Loading on Mandibular Condylar Cartilage. PLoS ONE 11(7): e0160121. doi:10.1371/journal. pone.0160121 Editor: Alejandro Almarza, University of Pittsburgh, UNITED STATES Received: April 16, 2016 Accepted: July 12, 2016 Published: July 29, 2016 Copyright: © 2016 Kaul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All the relevant data are within the paper. Funding: The study was partially funded by American Association of Orthodontic Foundation and partially by startup funds of Sumit Yadav. Competing Interests: The authors have declared that no competing interests exist. Four-week-old male Dkk3 XCol2A1XCol10A1 mice were randomly divided into two groups: (1) Loaded-Altered loading of MCC was induced by forced mouth opening using a custom- made spring; (2) Control-served as an unloaded group. Mice were euthanized and flow cytometery based cell analysis, micro-CT, gene expression analysis, histology and morpho- metric measurements were done to assess the response. RESEARCH ARTICLE RESEARCH ARTICLE The Effect of Altered Loading on Mandibular Condylar Cartilage Raman Kaul1, Mara H. O’Brien1, Eliane Dutra1, Alexandro Lima1, Achint Utreja2, Sumit Yadav1* * Yadav_sumit17@yahoo.com Introduction The temporomandibular joint (TMJ) is a complex load bearing joint in the craniofacial com- plex. Numerous investigators have shown that the TMJ is a load bearing joint and the unique- ness of the mandibular condylar cartilage (MCC) lies in its ability to be remodeled by The temporomandibular joint (TMJ) is a complex load bearing joint in the craniofacial com- plex. Numerous investigators have shown that the TMJ is a load bearing joint and the unique- ness of the mandibular condylar cartilage (MCC) lies in its ability to be remodeled by mechanical loading[1–3]. While reduced loading and overloading causes cartilage degradation, a moderate level of activity is necessary for maintenance of cartilage integrity and joint homeo- stasis. Mechanical stimulation of the MCC has been shown to generate biochemical signals, which increases the anabolic activity of chondrocytes[4, 5]. mechanical loading[1–3]. While reduced loading and overloading causes cartilage degradation, a moderate level of activity is necessary for maintenance of cartilage integrity and joint homeo- stasis. Mechanical stimulation of the MCC has been shown to generate biochemical signals, which increases the anabolic activity of chondrocytes[4, 5]. The MCC is composed of four different zones: the fibrous, proliferative, pre-hypertro- phic and hypertrophic[6–8]. The proliferative zone separates the fibrocartilaginous part of MCC with the more mature hyaline cartilage (pre-hypertrophic and hypertrophic zones of the MCC)[8]. The MCC is a load bearing musculoskeletal tissue, which distributes stress [9, 10]. The nature and duration of applied loads determine the biomechanical properties of the MCC. Small changes in the integrity, composition, or organization of cellular com- ponents of the cartilage will alter the matrix production and may eventually alter its mechanical properties. The literature lacks cellular details regarding altered loading of the MCC and none of the published studies have clearly elucidated the cellular morphometry and associated changes in the morphology of the cartilage and associated subchondral bone. The Dickkopf family of genes encodes secreted proteins that primarily act as antagonists of the Wnt/β-catenin signaling pathway. Of the four main Dkk family members in vertebrates (Dkk 1,2,3,4), Dkk3 differs from the rest, both structurally and in chromosomal location, indi- cating a functional divergence into two Dkk subfamilies[11–13]. The role of Dkk3, specifically in cartilage development, has not been investigated; however, upregulation of the gene was observed during pathological states. Conclusion Altered loading leads to mineralization of fibrocartilage and drives the lineage towards differ- entiation/maturation. Competing Interests: The authors have declared that no competing interests exist. 1 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Loading and Mandibular Condylar Cartilage PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Introduction A comparison of gene expression profiles between normal and osteoarthritic cartilage demonstrated consistently upregulated Dkk3 levels in osteoarthritis, suggesting its involvement in disease progression[14]. Addressing the question of whether Dkk3 has a pro- or anti-inflammatory effect in this situation, it was recently shown that Dkk3 inhibits matrix degradation by inflammatory cytokines in osteoarthritis, thus protecting the cartilage. Our study employs a combination of strategies (Fluorescence activated cell sorting analy- ses, micro-CT, histology, morphometric measurements and gene expression) to study the effect of altered loading on the MCC. The primary objective is to study the effects of mechan- otransduction at the cellular level after altered mechanical loading. The objectives were achieved by determining the spatial and temporal changes in Dkk3-eGFP, Col2a1-CFP, and Col10a1-RFP transgene expression, tissue remodeling (tartrate resistance acid phosphatase, TRAP), enzymatic indicator of mineralization (alkaline phosphatase, AP) and cartilage pro- teoglycan distribution (toluidine blue staining) after altered loading. We believe understand- ing the cellular changes due to altered mechanical loading of the MCC may contribute to our understanding of the mechanism underlying condylar growth modification in response orthodontic forces. In this research we utilized the forced mouth-opening model published by Sobue et al with minor modifications [5]. Our objectives were to study the 1) the cellular changes in the MCC with the altered loading; 2) tissue level changes in the subchondral bone and mineralized cartilage. Our null hypothesis is that there is no difference in cellular (cell proliferation and cell types), structural and morphometric characterization in the MCC and subchondral bone in the altered loaded model as compared to the healthy animals (control group). 2 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Loading and Mandibular Condylar Cartilage Fig 1. Altered loading due to forced mouth opening. The compressive force on the mandibular condylar cartilage due to forced mouth opening. The loading of the MCC is symmetrical between the right and the left side of the TMJ. Fig 1. Altered loading due to forced mouth opening. The compressive force on the mandibular condylar cartilage due to forced mouth opening. The loading of the MCC is symmetrical between the right and the left side of the TMJ. doi:10.1371/journal.pone.0160121.g001 doi:10.1371/journal.pone.0160121.g001 Materials and Methods GFP reporter mice The Institutional Animal Care Committee of the University of Connecticut Health Center approved this animal study. We used 4 week old (postnatally), male, triple transgenic mice (Dkk3 X Col2a1 X Col10a1) on a CD-1 background for the study. The Dkk3-eGFP transgene was obtained from the MMRRC repository (MMRRC: MGI: 4846992) (http://www.mmrrc. org). Dkk3-eGFP transgene was developed from a bacterial artificial chromosome (BAC) con- taining eGFP in the first exon of the murine Dkk3 gene. The two other GFP transgenes (Col2a1-GFPcyan and Col10a1-RFPcherry) used in this study has been previously described [15, 16]. All three GFP reporters were bred to make triple transgenic mice used in this study [17]. After the experimental procedure animals were killed with CO2 asphyxiation; the MCC along with sub-chondral bone was quickly dissected free by cutting the muscular attachment without scrapping the cartilage of the condyle. Micro-CT Mineralized cartilage and subchondral bone was analyzed using micro-computerized tomogra- phy (SCANCO Medical AG, Brüttisellen, Switzerland). The samples were scanned in liquid, one at a time, with high resolution in a 16mm holder. Serial tomographic projections were acquired at 55kV and 145μA, with a voxel size of 6μm and 1000 projections per rotation col- lected at 300000μs. The DICOM images were transferred, segmented and reconstructed using the mimics software (Materialise, Belgium). In order to distinguish calcified tissue from non- calcified tissue, an automated algorithm using local threshold segmented the reconstructed grey scale images. Bone mineral density (BMD (mg/cc)), bone volume fraction (BVF (%)), tra- becular thickness (Tb.Th (um)), and trabecular spacing (Tb.Sp (um)) were determined. Histomorphometry The MCC of the TMJ along with the sub-chondral bone were fixed for 24hours in 10% forma- lin and were placed in 30% sucrose overnight and embedded in cryomedium (Thermo Shan- don, Pittsburgh, PA) using disposable base molds (Thermo Shandon, Pittsburgh, PA). The medial surfaces of the samples were embedded against the base of the mold and were parallel to the floor of the mold. Specimens were stored at -20°C or -80°C before they were sectioned using Leica cryostat (Nussloch, Germany). Sections were 5 to 7μm in thickness and were trans- ferred to slides using a tape transfer method[18]. Sequential sections were mounted using 50% glycerol buffered in PBS and were stored in the dark at 4°C. Sections were examined with an observer ZI fluorescent microscope (Carl Zeiss, Thornwood, NY, USA) using appropriate fil- ters (Chroma Technology, Bellow Falls, VT, USA). Loading and Mandibular Condylar Cartilage intraperitoneally. Further they were injected with 5-ethnyl-2’-deoxyuridine (30mg/kg body weight) intraperitoneally, 24 hours before euthanization. The mice in the experimental and control groups were euthanized 24 hours after the force application on the 5th day. Flow Activated Cell Analyses Nine mice in each experimental and control groups were used in this part of our study. Either the left or right MCC were used for the flow activated cell analyses. Immediately after euthani- zation the MCC was dissected under a dissection microscope and only the MCC was collected. Cells from the MCC were harvested by digestion with collagenase D (4mg/ml) (Roche Diag- nostics, Mannheim, Germany) and dispase (4mg/ml) (Gibco, Grand Island, NY). Single cell suspensions were prepared by re-suspending cell pellets in 2 ml of fixed staining medium (HBSS+10mML of HEPES + 2%FBS) and passing through an 18-gauge needle followed by fil- tration through a 70μm strainer. Single cell suspensions were analyzed on an LSRII flow cytometer (BD Biosciences, San Jose, California) and analyzed using BD FACS Diva analysis software (BD Biosciences). Gates for single cells and debris exclusion were made based on light scatter properties. For each sam- ple, a minimum of 50,000 events was collected. Green fluorescent protein was excited using a 50mW 488nM laser and fluorescence detected from 505-550nM. Cyan fluorescent protein was excited using a 100mW 405nM laser and fluorescence detected from 425-475nM. mCherry was excited using a 100mW 561nm laser and fluorescence detected from 600-620nM. Experimental Procedure The mice were divided into 2 groups: (1) Experimental Group (n = 15): Altered loading through forced mouth opening continuous for 1hr/day for 5 days (Fig 1); (2) Control Group (n = 15): Unloaded group with incisor trimming (mandibular) every alternate day (Table 1). The mice in experimental and control groups were anesthetized with a mixture of ketamine (90mg/kg) and Xylazine (13mg/kg). In the experimental group, the mice were loaded with the custom made springs as described by Sobue et al[5]. However, our springs were made from Connecticut New Archform (CNA) wire (0.32inch) and applied 0.5N of force as measure by the Correx tension gauge (Haag-Streit, Bern, Switzerland). All the mice were injected with aliz- arin (3μg/kg body weight) on the third day and calcein (3μg/kg body weight) on the fifth day Table 1. Number of Animals. Mouse Tota(n) Age(weeks) FlowActivatedCellSorting (n) Real-time PCR(n) Micro CT(n) Histolog(n) Experimental(Dkk3xCol2a1xCol10a1) 15 3–4 9condyles 9condyles 12condyles 12condyles Control(Dkk3xCol2a1xCol10a1) 15 3–4 9condyles 9condyles 12condyles 12condyles doi:10.1371/journal.pone.0160121.t001 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 3 / 17 Table 1. Number of Animals. Mouse Tota(n) Age(weeks) FlowActivatedCellSorting (n) Real-time PCR(n) Micro CT(n) Histolog(n) Experimental(Dkk3xCol2a1xCol10a1) 15 3–4 9condyles 9condyles 12condyles 12condyles Control(Dkk3xCol2a1xCol10a1) 15 3–4 9condyles 9condyles 12condyles 12condyles doi:10.1371/journal.pone.0160121.t001 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 3 / 17 Table 1. Number of Animals. Table 1. Number of Animals. 3 / 17 Histological Staining Our histological sections were stained following a previously described protocol [19]. The 5μm- 7μm MCC sections remain adherent to glass slides through all of the process of staining and imaging. The first step was to image for GFP signals Dkk3 (green), Col2a1 (blue) and Col10a1 (red) and bone labels alizarin complexone (red) and calcein (green). Baseline imaging of the sections was performed with the observer ZI fluorescent microscope (Carl Zeiss, Thorn- wood, NY, USA) using a yellow fluorescent protein filter (eYFP, Chroma Cat 49003ET, EX: 500/20, EM: 535/30), a cyan fluorescent protein filter (CFP, Chroma Cat 49001ET, EX: 436/20, EM: 480/40), and a RFPcherry filter that was also used for detecting alizarin complexone stain- ing (mCherry, Chroma Cat 49009ET, EX: 560/40, EM: 630/75). In the next step, the coverslip was removed by soaking in PBS and the sections from both the altered loading group and unloaded group were stained for EdU (Life Technologies, Grand Island, NY) and imaged. Sub- sequently, sections were stained for Tartrate Resistant Acid Phosphatase (TRAP) using ELF97 (Life Tech, E6589), which generates a yellow fluorescent signal. After imaging for TRAP, the coverslip was removed and the same slide was stained for Alkaline Phosphatase (AP) activity using a fluorescent fast red substrate (Sigma, #F8764-5G) and DAPI (Mol Probes #D-1306) and re-imaged. Finally the slide was rinsed in distilled water, stained with toluidine blue, and reimaged. RNA extraction and real-time PCR Nine mice (9 condyles) in each experimental and control groups were used in this part of our study. Either the left or right MCC were used for the RNA extraction and gene expression anal- yses. The MCC along with the subchondral bone was minced and total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, CA, USA) using manufacturer’s protocol and as described previously[16]. The total RNA obtained was converted to cDNA using the ABI High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manu- facturer’s protocol. Real-time PCR was performed for the expression of different genes in sepa- rate wells (singleplex assay) of 96-well plates in a reaction volume of 20μl. The relative expression in a test sample compared to a reference calibrator sample (^^Ct method) was used for the data analysis. The primers for target genes were purchased from the Applied Biosystem. Gene expression analyses were performed for Bone Morphogenic Protein 2 (BMP2)[20], SRY- box containing gene 9 (Sox9)[21], Indian Hedgehog (Ihh) [20], Collagen type 2 (Col2a1)[21], Collagen type 10 (Col10a1)[21], Sclerostin (Sost)[22] and Glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) as the internal control. The data were collected from three independent pooled samples (n = 9). Nine mice (9 condyles) in each experimental and control groups were used in this part of our study. Either the left or right MCC were used for the RNA extraction and gene expression anal- yses. The MCC along with the subchondral bone was minced and total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies, CA, USA) using manufacturer’s protocol and as described previously[16]. The total RNA obtained was converted to cDNA using the ABI High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manu- facturer’s protocol. Real-time PCR was performed for the expression of different genes in sepa- rate wells (singleplex assay) of 96-well plates in a reaction volume of 20μl. The relative Morphological Measurement Radiographs of the mandibles were taken with a MX20 Radiography System (Faxitron X-Ray LLC, Lincolnshire, IL, USA) at a 26Kv for 5 seconds. We performed morphometric 4 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Loading and Mandibular Condylar Cartilage measurements on x-rays of mice subjected to altered loading and compared it with control tri- ple transgenic mice. The parameters measured and compared were: 1) mandibular length (con- dylion to incisor process); 2) Condyle head length (the perpendicular distance from condylion to a line traced from the sigmoid notch to the deepest point in the concavity of the mandibular ramus) and; 3) Condyle head width (distance from the most anterior to the most posterior point of the condylar articular surface). Measurements were made using Digimizer1 Image software (MedCalc Software, Mariakerke, Belgium). Each measurement was done in triplicate and then the average was calculated. Altered Loading Causes Increase in chondrocytes proliferation and differentiation/maturation Our histology showed that Dkk3-positive green cells are present in the superficial layer of fibrocartilaginous zone of MCC (Fig 2A). The Col2a1-positive blue and Col10a1-positive red cells are present in the pre-hypertrophic and hypertrophic zone of the MCC, respectively (Fig 2A). Furthermore, Col10a1 cells were present at the tidemark and in the mineralized region of the cartilage. The alizarin complexone and calcein bone labels were not distinct in the MCC, but were completely separated from each other in subchondral bone (Fig 2A). The flow analysis revealed that there was a significant increase in the number of Col2a1-positive (p<0.05) and Col10a1-positive (p<0.05) expressing cells with altered loading (Fig 2B). However, our gene expression, analysis only showed significantly increased expression of Col10a1 (p<0.05) and Sox9 (p<0.05) in the loading group when compared to the control group (Fig 3). Furthermore, there was a decrease in the number of Dkk3-positive expressing cells with altered loading, but was not significantly different from the control group (Fig 2B). Furthermore there was increase in the number of EdU positive cells in the loaded group (65.5% increase) signifies increase in proliferation (Fig 2C, 2D and 2E). Statistical Analysis Descriptive statistics were used to examine the distribution of bone volume fraction, tissue den- sity, trabecular thickness, trabecular spacing, morphometric measurements (mandibular length, condylar length, condylar width), EdU positive cells, histomorphometric image analy- ses and gene expression analyses. A one Sample Kolmogorov-Smirnov test was used to exam- ine the normality of data distribution. Outcomes were compared between the loaded and the unloaded group. Statistically significant differences among means were determined by unpaired t-test, with post hoc analysis by Mann-Whitney U test. All statistical tests were two sided and a p-value of <0.05 was deemed to be statistically significant. Statistical analyses were computed using Graph Pad Prism (San Diego, CA, USA) Loading and Mandibular Condylar Cartilage EdU positive pixels over DAPI positive pixels. We examined TRAP activity in the MCC and subchondral bone by counting the number of yellow pixels (generated by ELF97) and dividing it by the total number of pixels in the subchondral bone region. Alkaline Phosphatase (AP) staining was quantified by measuring the distance from the most superficial cellular layer of MCC to the AP stain. Finally, sections were stained with Toluidine Blue (TB) to evaluate pro- teoglycan secretion within the MCC. TB stained area and TB distance mapping (in five differ- ent locations in the MCC) were also evaluated using adobe Photoshop (San Jose, CA) Image Quantification Cell proliferation in the MCC was quantified by measuring the EdU and DAPI positive pixels in the superficial and proliferative zone of the MCC and then calculating the percentage of 5 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Altered Compressive Loading Causes Increase in Bone Volume and Trabecular Thickness The ex-vivo micro-CT protocol provided sufficient spatial resolution for quantitative compari- son of the structure and morphology of the mineralized cartilage and subchondral bone between the experimental and the control group. The analysis revealed a significant increase in the bone volume fraction (10.56%) (p<0.05) with altered loading (Fig 4A). This was paralleled by an increase in trabecular thickness (p<0.05) and decrease in trabecular spacing (p<0.05) in the loaded group when compared to the control group (Fig 4B and 4C). The trabecular thick- ness increased by 6.07% and there was a 9.02% decrease in the trabecular spacing in the altered loading group. 6 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Loading and Mandibular Condylar Cartilage Fig 2. Altered loading leads to increased proliferation and differentiation. (2A): The histological section of mandibular condylar cartilage from a 4 week- old-mice (Dkk3xCol2a1xCol10a1). Dkk3-positive (green) cells are present in the superficial zone/ fibrocartilaginous zone of MCC, Col2A1-positive (blue) cells are present in the pre-hypertrophic region of mature cartilage and Col10A1-positive (red) cells are present in the hypertrophic zone, adjacent to tidemark. Distinct bone labels in the subchondral bone. (2B): Flow cytometry based cell analysis (percentage of total positive cells) (: p<0.05 = significant difference between the altered loading and unloaded (control) group. (2C): EdU labeled proliferating cells were significantly higher in the loaded group when compared to control group (: p<0.05 = significant difference between the altered loading and control group). (2D): Sagittal section of MCC in the control Fig 2. Altered loading leads to increased proliferation and differentiation. (2A): The histological section of mandibular condylar cartilage from a 4 week- old-mice (Dkk3xCol2a1xCol10a1). Dkk3-positive (green) cells are present in the superficial zone/ fibrocartilaginous zone of MCC, Col2A1-positive (blue) cells are present in the pre-hypertrophic region of mature cartilage and Col10A1-positive (red) cells are present in the hypertrophic zone, adjacent to tidemark. Distinct bone labels in the subchondral bone. (2B): Flow cytometry based cell analysis (percentage of total positive cells) (: p<0.05 = significant difference between the altered loading and unloaded (control) group. (2C): EdU labeled proliferating cells were significantly higher in the loaded group when compared to control group (: p<0.05 = significant difference between the altered loading and control group). (2D): Sagittal section of MCC in the control Fig 2. Altered loading leads to increased proliferation and differentiation. (2A): The histological section of mandibular condylar cartilage from a 4 week- old-mice (Dkk3xCol2a1xCol10a1). PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Loading and Mandibular Condylar Cartilage group showing EdU positive cells in the superficial layer and proliferating layer. (2E): Sagittal section of MCC in the altered loaded group showing more number of EdU positive cells in the superficial layer and proliferating layer. Histograms represent means ± SD. Scale bar = 500μm. group showing EdU positive cells in the superficial layer and proliferating layer. (2E): Sagittal section of MCC in the altered loaded group showing more number of EdU positive cells in the superficial layer and proliferating layer. Histograms represent means ± SD. Scale bar = 500μm. group showing EdU positive cells in the superficial layer and proliferating layer. (2E): Sagittal section of MCC in the altered loaded group showing more number of EdU positive cells in the superficial layer and proliferating layer. Histograms represent means ± SD. Scale bar = 500μm. doi:10.1371/journal.pone.0160121.g002 doi:10.1371/journal.pone.0160121.g002 Altered Compressive Loading Causes Increase in Bone Volume and Trabecular Thickness Dkk3-positive (green) cells are present in the superficial zone/ fibrocartilaginous zone of MCC, Col2A1-positive (blue) cells are present in the pre-hypertrophic region of mature cartilage and Col10A1-positive (red) cells are present in the hypertrophic zone, adjacent to tidemark. Distinct bone labels in the subchondral bone. (2B): Flow cytometry based cell analysis (percentage of total positive cells) (: p<0.05 = significant difference between the altered loading and unloaded (control) group. (2C): EdU labeled proliferating cells were significantly higher in the loaded group when compared to control group (: p<0 05 significant difference between the altered loading and control group) (2D): Sagittal section of MCC in the control Fig 2. Altered loading leads to increased proliferation and differentiation. (2A): The histological section of mandibular condylar cartilage from a 4 week- old-mice (Dkk3xCol2a1xCol10a1). Dkk3-positive (green) cells are present in the superficial zone/ fibrocartilaginous zone of MCC, Col2A1-positive (blue) cells are present in the pre-hypertrophic region of mature cartilage and Col10A1-positive (red) cells are present in the hypertrophic zone, adjacent to tidemark. Distinct bone labels in the subchondral bone. (2B): Flow cytometry based cell analysis (percentage of total positive cells) (: p<0.05 = significant difference between the altered loading and unloaded (control) group. (2C): EdU labeled proliferating cells were significantly higher in the loaded group when compared to control group (: p<0.05 = significant difference between the altered loading and control group). (2D): Sagittal section of MCC in the control PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 7 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Altered Loading Causes Increase in Mandibular Length, Condyle Head Length and Cartilage Width Altered compressive loading of the TMJ did cause an increase in morphometric parameters after 5 days of loading. The mandibular length in the loaded group (16.08 ± 0.21) was signifi- cantly greater (p<0.05) than the unloaded group (15.08 ± 0.53) (Fig 4D and 4E). There was a 6.67% increase in the length of the mandible with the 5 days of altered loading. Similarly, con- dyle head length was significantly greater (p<0.05) in the loaded group (4.01 ± 0.04) when compared to unloaded group (3.88 ± 0.09) (Fig 4D and 4F). Moreover, the condyle width was Fig 3. Altered loading leads to increase in proliferation (Sox9) and differentiation markers (Col10a1 and BMP2). 4-weeks-old male mice were exposed to altered and normal loading. Real time PCR analysis was performed for SRY-box containing gene 9(Sox9), Collagen 2 (Col2a1), Collagen X (Col10a1), Bone Morphogenic Protein 2 (BMP2), Indian Hedgehog (Ihh) and Sclerostin (SOST). Significant increase (p<0.05) were seen for Sox9 and Col10a1 expression. Histograms represent means ± SD. doi:10.1371/journal.pone.0160121.g003 Fig 3. Altered loading leads to increase in proliferation (Sox9) and differentiation markers (Col10a1 and BMP2). 4-weeks-old male mice were Fig 3. Altered loading leads to increase in proliferation (Sox9) and differentiation markers (Col10a1 and BMP2). 4-weeks-old male mice were exposed to altered and normal loading. Real time PCR analysis was performed for SRY-box containing gene 9(Sox9), Collagen 2 (Col2a1), Collagen X (Col10a1), Bone Morphogenic Protein 2 (BMP2), Indian Hedgehog (Ihh) and Sclerostin (SOST). Significant increase (p<0.05) were seen for Sox9 and Col10a1 expression. Histograms represent means ± SD. doi:10 1371/journal pone 0160121 g003 doi:10.1371/journal.pone.0160121.g003 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 8 / 17 Loading and Mandibular Condylar Cartilage PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 9 / 17 Loading and Mandibular Condylar Cartilage Fig 4. Altered loading of MCC increases the bone volume and trabecular thickness. Micro-CT and morphometric measurements of altered compressive loading and unloaded groups. (4A) micro-CT analysis of bone volume fraction, (4B) micro-CT analysis of trabecular thickness and, (4C) micro-CT analysis of trabecular spacing, (4D) Representative image of the morphometric parameters measured in the experimental and control groups, (4E) Mandibular length on the 2D x-ray (faxitron), (4F) Condylar head length on the 2D x-ray (faxitron) and, (4G) Condyle width on the 2D x-ray (faxitron). (: p<0.05 = significant difference between the altered loading and control group. Histograms represent means ± SD. Altered Loading Causes Increase in Mandibular Length, Condyle Head Length and Cartilage Width doi:10 1371/journal pone 0160121 g004 doi:10.1371/journal.pone.0160121.g004 also significantly more (p<0.05) in the loaded group (2.44 ± 0.10) then unloaded group (2.03 ± 0.15) (Fig 4D and 4G). The condyle head length and condyle width increased by 5.15% and 20.19% in the loaded group when compared to the unloaded group. Altered Compressive Loading Causes Increase in Mineral apposition The altered compressive loading resulted in increased mineralized matrix apposition and increase in calcified cartilage, which was evident by TRAP (Fig 5A, 5B and 5K), alkaline phos- phatase (Fig 5G, 5H and 5L) and toluidine blue staining (Fig 6A, 6B, 6F and 6G). In the altered loading (Fig 5A and 5B) and control group (Fig 5D and 5E) the TRAP positive cells were pres- ent in the proliferative zone of the MCC. However, the TRAP positive cells were much more in the loaded group (Fig 5B), when compared to control group (Fig 5E). We believe that these TRAP positive cells were not osteoclasts/macrophage specific, because the TRAP in the carti- lage has a punctate intracellular distribution within a cell in contrast to the homogeneous dis- tribution of TRAP within multinucleated osteoclastic cells in the subchondral bone. However, we were not able to detect any differences in the TRAP staining of the subchondral bone between the loaded (Fig 5C) and the Control group (Fig 5F). Similarly, the AP stain was present in the proliferative zone and more towards the fibrocarti- lage layer in the loaded group (Fig 5G and 5H), when compared to the unloaded group (Fig 5I and 5J). This finding was further confirmed by the increase in Toluidine blue staining through- out the mineralized region of the fibrocartilage in the altered compressive loading group (Fig 6A and 6B). PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Discussion Mandibular condylar cartilage is affected by mechanical environment and it has been specu- lated that under stimulation and over stimulation can induce degenerative changes[23]. Adap- tive remodeling of MCC is necessary for the homeostasis and proper functioning of the TMJ. In this study we have used the forced mouth-opening model to alter the mechanical environ- ment of MCC of the TMJ. The altered loading in our study has resulted in specific and consis- tent differences in the MCC and subchondral bone. We used triple transgenic mice (Dkk3 X Col2a1 X Col10a1) as we wanted to study the cellular effects of altered loading on fibrocartilage and mature cartilage. Dkk3 is primarily expressed in fibrocartilaginous zone of the MCC while Col2a1 and Col10a1 are expressed in the pre-hypertrophic and hypertrophic zone of mature cartilage. We believe that the use of the triple colored transgenic mice and the histological fea- tures can contribute to an improved understanding of the molecular and cellular sequence of events after altered loading. The mandible is considered to be a class III lever; the condyle acts as a fulcrum, the masticatory muscles as applied force and bite pressure as a resistance[24–27]. Historically, loading of the TMJ is understood to be compressive in nature. i.e. the force applied at the loading surfaces of the MCC is compressive (Fig 1). Experimental and computer model- ing studies have indicated that MCC is subjected to both compressive and tensile forces, and the forces vary significantly among different species and therefore is difficult to explain the pre- cise forces on the MCC when the mechanical environment is altered[28–32]. In our study we PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 10 / 17 Loading and Mandibular Condylar Cartilage Fig 5. Altered loading increases TRAP activity and mineralization. The histological changes (TRAP & AP) within the mandibular condylar cartilage in the altered loaded and control groups. Fig 5A–5C shows staining for TRAP in the altered loaded group, (5A) TRAP stained sagittal section of the MCC along with Fig 5. Altered loading increases TRAP activity and mineralization. The histological changes (TRAP & AP) within the mandibular condylar cartilage in the altered loaded and control groups. Fig 5A–5C shows staining for TRAP in the altered loaded group, (5A) TRAP stained sagittal section of the MCC along with PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 11 / 17 Loading and Mandibular Condylar Cartilage the subchondral bone, (5B) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC. Number and the intensity of the TRAP positive cells in the proliferative zone of MCC are more in the loaded group, when compared to the control group, (5C) TRAP positive cells in the subchondral bone of the loaded group. Fig 5D–5F show staining for TRAP positive cells in control group, (5D) TRAP stained sagittal section of the MCC along with the subchondral bone in the control group, (5E) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC, (5F) TRAP positive cells in the subchondral bone of the control group. (5G-5H) AP staining in the altered loaded group, (5G) AP staining in MCC and subchondral bone of loaded group, (5H) AP staining in the proliferative and fibrocartilaginous zone of the MCC. (5I-5J) AP staining in the control group, (5I) AP staining in the MCC and subchondral bone of the control group, (5J) AP staining in the proliferative zone of MCC but not in the fibrocartilaginous region of MCC. (5K) Quantification of TRAP positive pixels, (5L) Quantification of distance map. Histograms represent means ± SD. Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. the subchondral bone, (5B) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC. Number and the intensity of the TRAP positive cells in the proliferative zone of MCC are more in the loaded group, when compared to the control group, (5C) TRAP positive cells in the subchondral bone of the loaded group. Fig 5D–5F show staining for TRAP positive cells in control group, (5D) TRAP stained sagittal section of the MCC along with the subchondral bone in the control group, (5E) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC, (5F) TRAP positive cells in the subchondral bone of the control group. (5G-5H) AP staining in the altered loaded group, (5G) AP staining in MCC and subchondral bone of loaded group, (5H) AP staining in the proliferative and fibrocartilaginous zone of the MCC. (5I-5J) AP staining in the control group, (5I) AP staining in the MCC and subchondral bone of the control group, (5J) AP staining in the proliferative zone of MCC but not in the fibrocartilaginous region of MCC. (5K) Quantification of TRAP positive pixels, (5L) Quantification of distance map. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. (5H) AP staining in the proliferative and fibrocartilaginous zone of the MCC. (5I-5J) AP staining in the control group, (5I) AP staining in the MCC and subchondral bone of the control group, (5J) AP staining in the proliferative zone of MCC but not in the fibrocartilaginous region of MCC. (5K) Quantification of TRAP positive pixels, (5L) Quantification of distance map. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. doi:10.1371/journal.pone.0160121.g005 Fig 6. Altered loading increases proteoglycan accumulation. The histological changes (TB Staining) within the mandibular condylar cartilage in the altered compressive loaded and control groups. Fig 6A–6B shows TB staining in the altered loaded group. Strong staining for proteoglycans is observed. The thickness of the mineralized cartilage is more. (6A) TB stained sagittal section of the MCC along with the subchondral bone, (6B) Intense proteoglycan staining in the fibrocartilaginous zone of the MCC, (6C-6D) TB staining in the altered unloaded group shows weaker staining when compared to the loaded group and less thickness of the mineralized cartilage. (6C) TB staining of the sagittal section of the MCC along with the subchondral bone in the unloaded group, (6D) thickness of mineralized fibrocartilage is less. (6F) Quantification of TB distance map, (6G) Quantification of TB stained area. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. doi:10.1371/journal.pone.0160121.g006 doi:10.1371/journal.pone.0160121.g005 Fig 6. Altered loading increases proteoglycan accumulation. The histological changes (TB Staining) within the mandibular condylar cartilage in the altered compressive loaded and control groups. Fig 6A–6B shows TB staining in the altered loaded group. Strong staining for proteoglycans is observed. The thickness of the mineralized cartilage is more. (6A) TB stained sagittal section of the MCC along with the subchondral bone, (6B) Intense proteoglycan staining in the fibrocartilaginous zone of the MCC, (6C-6D) TB staining in the altered unloaded group shows weaker staining when compared to the loaded group and less thickness of the mineralized cartilage. (6C) TB staining of the sagittal section of the MCC along with the subchondral bone in the unloaded group, (6D) thickness of mineralized fibrocartilage is less. the subchondral bone, (5B) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC. Number and the intensity of the TRAP positive cells in the proliferative zone of MCC are more in the loaded group, when compared to the control group, (5C) TRAP positive cells in the subchondral bone of the loaded group. Fig 5D–5F show staining for TRAP positive cells in control group, (5D) TRAP stained sagittal section of the MCC along with the subchondral bone in the control group, (5E) TRAP positive cells (punctate appearance) cells in the proliferative zone of the MCC, (5F) TRAP positive cells in the subchondral bone of the control group. (5G-5H) AP staining in the altered loaded group, (5G) AP staining in MCC and subchondral bone of loaded group, (5H) AP staining in the proliferative and fibrocartilaginous zone of the MCC. (5I-5J) AP staining in the control group, (5I) AP staining in the MCC and subchondral bone of the control group, (5J) AP staining in the proliferative zone of MCC but not in the fibrocartilaginous region of MCC. (5K) Quantification of TRAP positive pixels, (5L) Quantification of distance map. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. Loading and Mandibular Condylar Cartilage have chosen 0.5N of force, as Sobue et al. have showed that force less than 0.5N has less ana- bolic effect on the MCC[5]. We applied the static force only for 5 days, as Lai et al. have showed that shorter duration of static compression (1hour each day for 14 days) on growth plate are anabolic[33]. Mandibular condylar cartilage has a heterogeneous cell population and consists of fibroblast like cells, fibrochondrocytes and chondrocytes[34]. The nature and behavior of these cells remains poorly understood during altered loading. The MCC plays a major role in absorbing the mechanical forces exerted by various oral functions and para-functions. It has been shown that loading of the MCC within the adaptive capacity may stimulate remodeling and increased synthesis of the extracellular matrix. Our flow activated cell analysis showed an increased num- ber of Col2a1blue cells (30% increased when compared to control) and Col10a1red cells (100% increased when compared to control) with altered loading of MCC. Numerous in vitro studies in MCC and articular cartilage have shown an increase in cell differentiation and increased proteoglycan synthesis after mechanically loading the articular chondrocytes [35–39]. Simi- larly, Yang et al. in their in vitro study with articular chondrocytes showed a two-fold increase in type X collagen with mechanical loading[40]. Moreover, Sobue et al. showed an increase in Col2a1 and Col10a1 with 0.5N of compressive load on the MCC in mice [5]. Our experiments show similar results and it may be due to accelerated maturation/lineage progression with altered compressive load. Furthermore, we show a decrease in the number of Dkk3-positive (green) cells with loading and it may be due to finite number of Dkk3 cells present in the fibro- cartilaginous zone of the MCC, and which may progress to type 1 collagen expressing cells thus accelerating the differentiation/lineage progression (data not shown). Dkk3 expression has been upregulated in osteoarthritis, suggesting its role in the pathogenesis of the diseases. The altered compressive loading in the subchondral bone caused increased mineralization, increased bone volume fraction, increased trabecular thickness and decreased trabecular spac- ing. It has been postulated that subchondral bone adapts to applied load/stress. A sequential and coordinated response of modeling and remodeling control this adaptive response. Bone formation is hypothesized to be driven by the magnitude, rate, and duration of applied bone strain[41]. PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 (6F) Quantification of TB distance map, (6G) Quantification of TB stained area. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. doi:10.1371/journal.pone.0160121.g006 Fig 6. Altered loading increases proteoglycan accumulation. The histological changes (TB Staining) within the mandibular condylar cartilage in the altered compressive loaded and control groups. Fig 6A–6B shows TB staining in the altered loaded group. Strong staining for proteoglycans is observed. The thickness of the mineralized cartilage is more. (6A) TB stained sagittal section of the MCC along with the subchondral bone, (6B) Intense proteoglycan staining in the fibrocartilaginous zone of the MCC, (6C-6D) TB staining in the altered unloaded group shows weaker staining when compared to the loaded group and less thickness of the mineralized cartilage. (6C) TB staining of the sagittal section of the MCC along with the subchondral bone in the unloaded group, (6D) thickness of mineralized fibrocartilage is less. (6F) Quantification of TB distance map, (6G) Quantification of TB stained area. Histograms represent means ± SD. * Statistically significant difference between the altered loaded side and the control group. Scale bar = 500μm. doi:10.1371/journal.pone.0160121.g006 doi:10.1371/journal.pone.0160121.g006 doi:10.1371/journal.pone.0160121.g006 12 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 Conclusions In conclusion altered loading of the MCC leads to: In conclusion altered loading of the MCC leads to: 1. Increased cellular proliferation (number of EDU positive cells) and differentiation. 2. Increased mandibular length, condylar head length and condylar width. 3. Increased subchondral bone volume, increased trabecular thickness and decreased trabecu- lar spacing. 4. Increased mineralization as evident by the TRAP, alkaline phosphatase and toluidine blue staining Acknowledgments We would like to thank Dr. David Rowe and Jianping Huang for providing the triple transgenic mice. Loading and Mandibular Condylar Cartilage synthesis[48]. In the MCC the toluidine blue staining is found in the pre-hypertrophic and hypertrophic zone. With altered loading the toluidine blue stain approaches the articular sur- face of the MCC, where as in the unloaded group it was in the pre-hypertrophic and hypertro- phic zones of MCC. Concomitant with the increase in toluidine blue staining (proteoglycan accumulation), there was increase in the number of Col10a1 cells and Col2a1 cells. We were surprised to see the TRAP activity in the cells within the uncalcified fibrocartilage. The TRAP activity was primarily in the proliferative zone of MCC and was more intense in the loaded group, when compared to the unloaded group. The punctate appearance of TRAP has been observed previously in the histology of cartilage and macrophages[49]. However, the role of TRAP in remodeling of the fibrocartilage prior to mineralization is still not clear. Similarly, we noted increased in AP staining in the altered compressive loading group. The AP activity in the altered loading group was synonymous with our micro-CT results, which showed increase in bone volume fraction in the loaded group when compared to unloaded group. Limitations and future directions: Our study is the first study using combination of strate- gies to study the effect of altered loading on MCC and subchondral bone. The limitation of this study was our inability to accurately define the force felt by the MCC. However, TMJ is a complex joint and scientific literature lacks the force range, which could stimulate adaptive remodeling of the MCC. Another limitation of our study could be use of male mice as TMJ disorders are more prevalent in female mice, but numerous studies have been done in the past on female mice and we wanted to study whether the mandibular growth could be modified using altered loading model. Furthermore, there are equal numbers of male and female patients with small mandible. Our future studies are focusing on applying the altered load in an adult animal model and sacrificing the animals at different time points to study the concept of adaptive remodeling. Author Contributions Conceived and designed the experiments: RK SY ED AU. Performed the experiments: RK SY ED MHO AL AU. Analyzed the data: RK SY ED MHO AL AU. Contributed reagents/materi- als/analysis tools: RK SY ED MHO AL AU. Wrote the paper: RK SY ED MHO AL AU. Our result shows an increase in bone volume and trabecular thickness with the static 0.5N load and a plausible reason could be that the strain felt by the subchondral bone cells was above the maximum strain threshold, thus leading to bone formation. The mecha- nism by which bone strain induces bone formation is unknown, however, microcrack propaga- tion through the bone matrix has been shown to stimulate bone remodeling[42, 43]. Our results are contrary to Sobue et al. as they showed a decrease in bone volume and trabecular thickness with loading. These differences could possibly be explained by different strains of mice, as well as different gender and age [5]. Our morphometric measurement showed increased mandibular length, increased condylar length and increased mandibular condyle width. These increases in morphometric parameters with altered loading can be due to increased proliferation of chondrocytes and increased secre- tion of pericellular and extracellular matrix. Sobue et al. have shown increased proliferation with 0.5N of static compressive force on the MCC[5]. Similarly, Pirttiniemi et al. and Chen et al. showed that reduced loading leads to a lower number of proliferating chondrocytes and a thinner cartilaginous layer[4, 44, 45]. Our data shows that static compressive forces can be used to modify the growth of the mandible, however, whether the effects of the static force on the MCC are transitory, needs to be further evaluated. Our histology shows increased mineralized matrix (toluidine blue & alkaline phosphatase staining) with altered loading, when compared to unloaded group. In vitro studies have dem- onstrated that static compressive loading leads to increased proteoglycan synthesis[46, 47]. Conversely, decreased loading of the MCC of the joint results in decreased proteoglycan PLOS ONE \| DOI:10.1371/journal.pone.0160121 July 29, 2016 13 / 17 References 1. Beek M, Koolstra JH, van Ruijven LJ, van Eijden TM. 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https://openalex.org/W2563795363	https://europepmc.org/articles/pmc5348119?pdf=render	English	null	Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR	Biomolecular detection and quantification	2,017	cc-by	9,218	Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR Alexander Templar, Desmond M. Schoﬁeld, Darren N. Nesbeth ∗ The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1E 6BT, UK Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 Contents lists available at ScienceDirect Abbreviations: PCR, Polymerase chain reaction; HCD, high cell density; qPCR, quantitative PCR; SF, shake ﬂask; wcw, wet cell weight; WCB, working cell bank; LRE, linear regression of efﬁciency; OCF, optical calibration factor. ∗Corresponding author. E-mail address: d.nesbeth@ucl.ac.uk (D.N. Nesbeth). ttp://d .do .o g/ 0. 0 6/j.bdq. 0 6. .00 2214-7535/© 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). a r t i c l e i n f o Article history: Received 5 October 2015 Received in revised form 29 September 2016 Accepted 1 December 2016 Available online 29 December 2016 Handled by Jim Huggett Keywords: Pcr Synthetic biology Standard curve Standardisation Linear regression Efﬁciency We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the per- formance of the Linear Regression of Efﬁciency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biol- ogy standard. We ﬁrstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is ampliﬁed. Ampliﬁcation efﬁciency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake ﬂask cultivation. We then compared LRE qPCR to the Standard Curve method of absolute qPCR (SC qPCR). LRE qPCR method matched the performance of the SC qPCR when used to measure 417–4.17 × 107 copies of the BirA target sequence present in a shake ﬂask-derived cell sonicates sample, and for 97–9.7 × 105 copies in the equivalent bioreactor-derived sample. A plasmid-encoded T7 bacteriophage sequence was next used to compare the methods. In the presence of cell sonicates from samples of up to OD600 = 160, LRE qPCR outperformed SC qPCR in the range of 1.54 × 108–1.54 × 1010 copies of the T7 target sequence and matched SC qPCR over 1.54 × 104–1.54 × 107 copies. These data suggest the CAL1 standard, com- bined with the LRE qPCR method, represents an attractive choice as a synthetic biology qPCR standard that performs well even when unpuriﬁed industrial samples are used as the source of template material. © 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). PCR Experiments’ or ‘MIQE’ guidelines proposed by Bustin et al. [5]. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.bdq.2016.12.001 2214-7535/© 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR The amount of PCR product (amplicon) present during each reaction cycle can be determined by measuring in real time [9] the light emission from a ﬂuorescent reporter dye that binds to double-stranded DNA (dsDNA). The kinetics of the appearance of ﬂuorescence over time can then be used to infer the amount of template cDNA that was initially present [20]. A key metric for this procedure is the number of cycles required for ﬂuorescence to exceed a set threshold. This cycle number is known as the quan- tiﬁcation cycle (Cq). The less cycles required for ﬂuorescence to reach Cq, the more template was present in the starting material. For relative qPCR the principle data gathered is the fold-difference in abundance of the well-characterised, reference mRNA in com- parison to the mRNA of unknown abundance. Perhaps the most widely known use of real time PCR, also known as quantitative PCR (qPCR), is as a tool to measure the relative abundance of a given messenger RNA (mRNA) transcript. In this ‘rel- ative qPCR’ approach reverse transcriptase (RT) is used to convert a population of mRNA molecules to single stranded complimen- tary DNA (cDNA) molecules. A bespoke primer pair is then used to amplify cDNA corresponding to an mRNA whose abundance is well characterised. Further primer pairs are used to amplify cDNA corresponding to mRNA molecules whose abundance is unknown. Ideally, primers for qPCR will be designed in accordance with the ‘Minimum Information for Publication of Quantitative Real-Time While relative qPCR has been immensely valuable in helping researchers gain fundamental biological insights, it is arguably less well suited to the aim of synthetic biology, which is to render bio- logical systems more amenable to rigorous engineering methods. Fortunately for synthetic biologists it is possible to derive abso- lute measurements using qPCR [23]. Serially diluted standards of known concentration produce a linear relationship between the Cq value and the logarithm of the initial amount of total template DNA A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 22 pproaches and cell cultivation. A) i) Illustration of the ﬂuorescence data proﬁle for a conventional qPCR experiment. Serial dilutions of template ar earance of ﬂuorescence plotted as a function of cycle number. 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR Fluorescent data points for three dilutions of template are indicated by black, grey and w ) data points to convey increasing template dilution. Typically four or more are used in actual qPCR experiments. The point at which each ﬂuoresc Cq is logged (cycles a, b and c). ii) Cq number is proportional to the log of the DNA concentration in puriﬁed target sequence samples of known con provides a standard curve for calibration of Cq data gathered from samples of unknown target sequence concentration. B) i) For LRE qPCR these is to perform a template dilution series or set a Cq threshold. Instead the ﬂourescence data set is analysed as a classic Boltzmann sigmoid function Fig. 1. PCR approaches and cell cultivation. A) i) Illustration of the ﬂuorescence data proﬁle for a conventional qPCR experiment. Serial dilutions of template are made and real time appearance of ﬂuorescence plotted as a function of cycle number. Fluorescent data points for three dilutions of template are indicated by black, grey and white (with black border) data points to convey increasing template dilution. Typically four or more are used in actual qPCR experiments. The point at which each ﬂuorescence signal reaches the Cq is logged (cycles a, b and c). ii) Cq number is proportional to the log of the DNA concentration in puriﬁed target sequence samples of known concentration. This data set provides a standard curve for calibration of Cq data gathered from samples of unknown target sequence concentration. B) i) For LRE qPCR these is no inherent requirement to perform a template dilution series or set a Cq threshold. Instead the ﬂourescence data set is analysed as a classic Boltzmann sigmoid function such that a R approaches and cell cultivation. A) i) Illustration of the ﬂuorescence data proﬁle for a conventional qPCR experiment. Serial dilutions of template are made Fig. 1. PCR approaches and cell cultivation. A) i) Illustration of the ﬂuorescence data proﬁle for a conventional qPCR experiment. Serial dilutions of template are made and real time appearance of ﬂuorescence plotted as a function of cycle number. Fluorescent data points for three dilutions of template are indicated by black, grey and white (with black border) data points to convey increasing template dilution. Typically four or more are used in actual qPCR experiments. The point at which each ﬂuorescence signal reaches the Cq is logged (cycles a, b and c). 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR ii) Cq number is proportional to the log of the DNA concentration in puriﬁed target sequence samples of known concentration. This data set provides a standard curve for calibration of Cq data gathered from samples of unknown target sequence concentration. B) i) For LRE qPCR these is no inherent requirement to perform a template dilution series or set a Cq threshold. Instead the ﬂourescence data set is analysed as a classic Boltzmann sigmoid function such that a A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 23 (Fig. 1 A). Comparison with such a standard curve (SC) allows exact inference of the number of template molecules that were originally present in a given sample. contaminant organism genome. Gel electrophoresis is then used to score the absence or presence of an amplicon band as an indicator of the presence or absence of the contaminant organism within the sample [12]. The reliability of absolute, standard curve qPCR (SC qPCR) depends on the efﬁciency of template ampliﬁcation for the target and the standard curve, both of which must be evaluated. Most often, the SC is comprised of the same primer pair and template that are used for the experimental samples in which the amount of template is not known. Such a standard curve is ideally performed alongside every experiment, increasing the time taken to perform the assay, and is unique to each target so is inherently unsuited to global standardisation. SC qPCR also assumes constant ampliﬁ- cation efﬁciency across all template dilutions, but in reality this is seldom observed [26]. Factors such as gene expression [13], plasmid copy number [16] and the dosage of deﬁned loci within a genome [22,11] can all impact industrial performance of engineered cells. All these factors can potentially be monitored using qPCR, given a sufﬁciently rapid procedure. Bacteriophage can compromise virtually any industrial process involving bacteria [33], even those with comprehensively refactored genomes [14] and as such rapid and accurate detection of bacteriophage within industrial conditions is highly desirable. Sample processing [15] represents a signiﬁcant proportion of the time taken to perform most qPCR protocols and is widely believed to be necessary for removal of constituents that may inhibit the reaction and its accuracy. Sample processing for PCR typically involves removal of all non-DNA macromolecules from a given sample by physicochemical means, either by a standard protocol or with commercial kits. sub-set of data points is identiﬁed at the midpoint of the ampliﬁcation proﬁle, known as the LRE zone (in grey). ii) Fluorescence data points within the LRE zone (indicated by asterisks) have a linear relationship to a value deﬁned by Rutledge and Stewart [28] as cycle efﬁciency (Ec). iii) Rutledge and Stewart [28] also relate these LRE zone data points to the original mass of template present, expressed as a ﬂuorescence value (F-zero). F-zero can be converted to template DNA mass using an OCF, for which CAL1 has been identiﬁed as the superior candidate. C) Primers (black triangles), detailed in Table 1, were used to amplify i) the designated CAL1 locus with the lambda bacteriophage genome, ii) target DNA within the BirA locus of the E. coli genome and iii) a bacteriophage sequence present in the plasmid pPROX1 as a proxy for pathogen detection. Expected amplicon size (bp) is indicated under the bar at the bottom of each panel. D) A 40 mL culture of E. coli W3110 production strain grown in LB was used to inoculate 360 mL deﬁned media in a 5L shake-ﬂask. An uninduced sample was taken at the start of stationary phase growth in shake ﬂasks (black ﬁlled square) for PCR experiments. E) 10% of shake ﬂask culture was used to inoculate 3.6 L deﬁned media in a New Brunswick 7L bioreactor. In bioreactor cultivation, IPTG was added to induce transgene expression at 34 h post-inoculation (grey ﬁlled square) and a sample taken 2 h post-induction (black ﬁlled square) for PCR experiments. Agitiation, grey line, and dissolved oxygen tension (DOT), dashed line, were plotted alongside cell growth. Both cell growth data sets are representative of n = 3 experiments. 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR Absolute measurements could enable a move away from relative measures of gene expres- sion units [39], enabling cell status to play a greater role in quantifying performance of synthetic gene networks. Using absolute units for measurement makes standardisation possible by replacing target-by-target standard curves with a sin- gle, well-deﬁned universal standard. The CAL1 reaction, consisting of a pair of 27 bp primers speciﬁc for ampliﬁcation of a 151 bp amplicon from lambda bacteriophage genomic DNA (Fig. 1C), was shown by Rutledge and Stewart [28] to exhibit ampliﬁcation and ﬂuorescence performance with a high degree of reliability over multiple experiments in a 4 month period. As such the CAL1 reac- tion is a strong candidate for adoption as a universal qPCR standard. 1.3. Aims of this study In this study we compare performance of LRE qPCR and SC qPCR methods using i) material from shake ﬂask and high cell density bioreactor cultivation and ii) measuring a genomic locus and a plasmid-encoded bacteriophage sequence as a targets. We will also discuss the suitability of LRE qPCR as a synthetic biology standard for qPCR. A single universal quantitative standard for qPCR could bet- ter enable monitoring of inter-run performance and benchmarking for comparison of sample preparation procedures, reaction meth- ods, instrumentation, and data processing. Absolute measurements could enable a move away from relative measures of gene expres- sion units [39], enabling cell status to play a greater role in quantifying performance of synthetic gene networks. 2. Materials and methods All reagents were of molecular biology grade unless other- wise stated. All stocks, solutions and reagents were prepared with molecular biology grade water (Millipore, Billerica, USA), conﬁrmed DNA and ribonuclease free by the supplier. Oligonu- cleotides were synthesised by Euroﬁns MWG Operon (Acton, UK, www.euroﬁnsdna.com). 1.1. LRE qPCR and the CAL1 reaction as a synthetic biology standard for qPCR Rutledge and Stewart [28] proposed a universal standard for absolute qPCR as an element of their Linear Regression of Efﬁciency method of absolute qPCR (LRE qPCR). LRE qPCR does not require a target-speciﬁc standard curve, assume constant ampliﬁcation efﬁ- ciency across all template dilutions or involve determination of Cq values (Fig. 1B). In LRE qPCR, original template quantity is inferred instead by applying a Boltzmann sigmoidal statistical framework to raw ﬂuorescence data in the central area of an ampliﬁcation proﬁle [38]. Other linear regression approaches to analysis of qPCR ﬂuores- cence data have been developed, a selection of which are discussed by [24]. However, in the case of LRE qPCR, Rutledge and Stewart [28] also took pains to demonstrate that the ﬂuorescence intensity of SYBR Green I [37] generated during real-time PCR is not impacted by either amplicon size or guanine:cytosine (GC) base pair (bp) con- tent. Spandidos et al. [32] also reported this property of SYBR Green I. This means that absolute ﬂuorescence units (FU) can be correlated with base pair production (FU/bp). The trade-off of sample preparation for PCR is that the duration of the procedures can restrict application to off-line, retrospec- tive analyses. Furthermore, some commercial sample preparation kits may introduce error through loss of target material [18], co- puriﬁcation of inhibitors [30] or introduction of contaminant DNA [29]. Shortening or foregoing sample preparation in a manner that preserves the accuracy of PCR-based assays could signiﬁcantly reduce assay time-scales. Combining this with recent develop- ments in ‘ultra-rapid’ thermal cycling speeds (XxpressTM PCR, London, UK) [7] and electrophoresis tech- nology (FlashGelTM, Lonza, Basel, Switzerland) [8] could make PCR a realistic analytical procedure for at-line bioprocess monitoring. Using absolute units for measurement makes standardisation possible by replacing target-by-target standard curves with a sin- gle, well-deﬁned universal standard. The CAL1 reaction, consisting of a pair of 27 bp primers speciﬁc for ampliﬁcation of a 151 bp amplicon from lambda bacteriophage genomic DNA (Fig. 1C), was shown by Rutledge and Stewart [28] to exhibit ampliﬁcation and ﬂuorescence performance with a high degree of reliability over multiple experiments in a 4 month period. As such the CAL1 reac- tion is a strong candidate for adoption as a universal qPCR standard. A single universal quantitative standard for qPCR could bet- ter enable monitoring of inter-run performance and benchmarking for comparison of sample preparation procedures, reaction meth- ods, instrumentation, and data processing. 2.1. Cultivation of E. coli The sequence speciﬁcity and accuracy [17] of PCR have made it an effective assay for detection of bioprocess contamination, most commonly for mycoplasma in mammalian host chassis cultivation [1], but also of bacteriophage in E. coli cultivation [36]. Typically a sample is removed from cultivation and tested using end point PCR (e-pPCR) with primers speciﬁc for a target sequence present in the An E. coli W3110 production strain that harbours the 3010 bp plasmid pTTOD-A33 which encodes a recombinant Fab’ fragment [19] inducible by addition of isopropyl -d-1- thiogalactopyranoside (IPTG) was grown in Luria Bertani (LB) A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 24 uence of disrupted E. coli cells on LRE and SC qPCR. Real time PCR was performed using template material either from bioreactor sonicates (gr or puriﬁed DNA (black squares and lines) from OD600 = 2.5 shake ﬂask (A) and OD600 = 160 bioreactor (B) cultivation. Spectrophotometric estimatio undiluted, puriﬁed DNA sample derived from shake ﬂask material contained 112 ng total DNA (2.17 × 107 copies BirA target) and a 5 L sonicated 215 ng total (4.17 × 107 copies BirA target). For bioreactor material, 3.9 g (7.57 × 108 copies BirA target) and 5 g (9.7 × 108 copies BirA target) res resent in a 5 L puriﬁed gDNA and 5 L cell sonicate samples respectively. Lines indicate iterative least-square ﬁtting of a linear function to data poin ﬁed with 100 ± 10% efﬁciency, at a conﬁdence level of R2 > 0.99. Error bars show standard error across n = 3 analytical repeats. C) Copy number estimatio Fig. 2. Inﬂuence of disrupted E. coli cells on LRE and SC qPCR. Real time PCR was performed using template material either from bioreactor sonicates (grey triangles and lines) or puriﬁed DNA (black squares and lines) from OD600 = 2.5 shake ﬂask (A) and OD600 = 160 bioreactor (B) cultivation. Spectrophotometric estimation indicated that a 5 L undiluted, puriﬁed DNA sample derived from shake ﬂask material contained 112 ng total DNA (2.17 × 107 copies BirA target) and a 5 L sonicated cell sample contained 215 ng total (4.17 × 107 copies BirA target). For bioreactor material, 3.9 g (7.57 × 108 copies BirA target) and 5 g (9.7 × 108 copies BirA target) respectively of DNA was present in a 5 L puriﬁed gDNA and 5 L cell sonicate samples respectively. ﬂask derived sample was determined by three spectrophotometric measurements (grey circles) which were linearly extrapolated (dashed lines) and plotted alongside copy numbers determined by SC qPCR (circles) and LRE qPCR (rhomboids) methods. D) Copy number estimation in a bioreactor derived sample was determined by three spectrophotometric measurements (grey circles), linear extrapolation of that date (dashed lines) and plotted alongside SC qPCR (circles) and LRE qPCR (rhomboids) data. E) XY plot (grey triangles) comparison of SC qPCR and LRE qPCR shake ﬂask data sets from graph A. F) XY plot (grey circles) comparison of SC qPCR and LRE qPCR bioreactor data sets from graph B. G) Bland-Altman analysis of graph C plots the difference between the X and Y data points (grey triangles) and the overall average difference between the X and Y data (dark dashed lines). 1.96 x the standard deviation (+/−) of this bias (grey dashed lines) is also plotted to indicate the upper and lower limits of statistical signiﬁcance [4]. H) Equivalent Bland-Altman analysis of bioreactor data (grey circles) from graph D. 2.3. Cell disruption Shake ﬂask and bioreactor samples were sonicated using the procedure below to determine typical DNA estimations by spec- trophotometry and densitometry. After this initial scoping study, the volume of sample, from 400 L–4 mL, required to provide the ﬁnal estimated DNA concentrations indicated in Fig. 2B, C and Fig. 3 was centrifuged at 10,000 RPM for three minutes and re-suspended in 400 L dH2O. A Soniprep 150 sonicator (MSE, London, UK) was used to subject samples to three cycles of the following treatment: 10 s pulses of 100% amplitude sonication followed by 10 s rest, for a total duration of 60 s, 30 s of which is the total period during which cells were subjected to sonication. For PCR experiments with bac- teriophage target, bioreactor sonicate was diluted with dH2O to an equivalent OD600 of 5 or 50, using the original of OD600 of 160 to inform the volumetric calculations. 2.2. Total nucleic acid puriﬁcation DNA was puriﬁed by the method below from the shake ﬂask and bioreactor samples to determine typical DNA measurements by spectrophotometry. After this initial scoping study samples ranging in volume from 400 L–8 mL were used to provide the DNA concen- tration in the undiluted template reactions indicated in Figs. 2 and 3. Samples were centrifuged at 10,000 RPM for 3 min, re-suspended in 400 L lysis buffer (2% Triton X100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA) and freeze-thawed twice by incubating at −80 ◦C for 3 min and 95 ◦C for 1 min. Total nucleic acid was puri- ﬁed using a standard phenol/ethanol extraction procedure [35] and the puriﬁed DNA was resuspended in 400 L 10 mM Tris (pH 7.5) for both shake ﬂask and bioreactor-derived samples. Six aliquots of puriﬁed DNA were made and stored at −20 ◦C. A given aliquot was thawed once for experimentation and any unused portion of the aliquot discarded. The proxy plasmid pPROX1 was puriﬁed by standard plasmid DNA puriﬁcation using a Key Prep mini prep kit (Anachem, Luton, UK). A 300 bp proprietary bacteriophage target sequence is present within a pUC57 backbone in the 3010 bp plasmid pPROX1 (Fig. 1C) and 21 bp primers were used for amplicon production. The use of a proxy sequence in this way enables investigation of detection of many pathogen types without the need to risk infection of other cultivation experiments being performed in the same facility. After sample processing, pPROX1 was added at known con- centration and the ability of PCR methods to detect or quantify the bacteriophage sequence was tested. The BirA locus (Gene ID: 12934397) of E. coli W3110 strain was chosen as a single copy genomic target locus (Fig. 1C, Table 1). CAL1 primers, ampliﬁcation target and PCR conditions set out by Rutledge and Stewart [28] were used to calibrate LRE qPCR experiments. Agarose gel electrophore- sis showed all three reactions produced only amplicon of expected size. Table 1 an Optical Calibration Factor (OCF) suitable for use as a global standard reaction for LRE qPCR. We use the CAL1 standard and apply the LRE qPCR derivation of ﬂuorescent data using a Java program developed and maintained by Rutledge [27]. Primer sequences (Table 1) were designed in accordance with MIQE guidelines [5] and screened in silico for speciﬁcity and poten- tial for self-annealing using the National Center for Biotechnology Information (NCBI) ‘primer blast’ tool (http://www.ncbi.nlm.nih. gov/tools/primer-blast/accessed 22.03.15) and the ‘PCR primer stats’ tool (http://www.bioinformatics.org/sms2/pcr primer stats. html accessed 22.03.15) respectively. 2.5. DNA mass estimation by spectrophotometry and densitometry DNA mass in nucleic acid puriﬁed samples and sonicated mate- rial was estimated by spectrophotometry, using a Nanodrop 1000 spectrophotometer (Thermo Scientiﬁc, Waltham, MA, USA). Soni- cated samples were also analysed on a 1% agarose gel stained with ethidium bromide. Stained DNA was visualised using a GelDoc 2000 (BioRad, Hercules, CA, USA) and Quantity One software version 4.6.8 (BioRad, Hercules, CA, USA). ImageJ software (version 1.46r, National Institutes of Health, Bethesda, USA) was used to select a region of the gel image, either a lane or a band, containing a known mass of DNA and a brightness value captured. A selected region of the same size and shape was then used to capture the bright- ness level in a region of unknown DNA concentration on the same gel. Background noise was subtracted using ImageJ ‘subtract back- ground’ command, details of which can be found at this support website − http://imagej.net/Rolling Ball Background Subtraction. 2.1. Cultivation of E. coli Lines indicate iterative least-square ﬁtting of a linear function to data points until a set was identiﬁed with 100 ± 10% efﬁciency, at a conﬁdence level of R2 > 0.99. Error bars show standard error across n = 3 analytical repeats. C) Copy number estimation in a shake A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 25 Table 1 Oligonucleotide primers for PCR. Target Primers Sequence E. coli BirA gene BirA Fwd ATCCACCCCTGATTAACGAC Rev BirA CGGAAGTATTACGCAAGCTG Lambda OCF region Cal 1 Fwd AGACGAATGCCAGGTCATCTGAAACAG Rev CAL1 CTTTTGCTCTGCGATGCTGATACCG 300 bp bacteriophage sequence Forward 21mer Reverse 21mer Table 1 Oligonucleotide primers for PCR. medium. 40 mL of this LB inoculum was used to inoculate 360 mL chemically deﬁned media [2] in a 5L shake-ﬂask to OD600 = 2.5 (Fig. 1D), typical of the end-point of seed train cultivation used to provide inoculum for growth in bioreactors. 10% of this shake ﬂask material was used to inoculate 3.6 L deﬁned media in a New Brunswick 7L bioreactor. “A fed batch protocol was used as previ- ously described by Balasundaram et al. [2] until OD600 = 130 and a spike in dissolved oxygen tension (DOT) were reached, indicat- ing complete consumption of the starting batch of glycerol”. From this point on fed-batch mode was applied along with IPTG addi- tion to induce Fab’ fragment expression (Fig. 1E). An experimental sample was taken two hours post-induction, at OD600 = 160, during early idiophase growth when bacteriophage contamination can be highly costly. 2.6. Quantitative PCR assembly and data capture 2.6. Quantitative PCR assembly and data capture Assuming an E. coli W3110 genome size of 4,646,332 bp plus 20 replicons/cell of the 6480 bp plasmid pTTOD-A33, host genomic DNA (gDNA) should constitute 97.2% of total host cell DNA (total size of DNA per cell of 4,775,932 bp). We used these assumptions to convert DNA concentration levels, derived from spectrophotome- try, into target sequence copy numbers. Assuming an E. coli W3110 genome size of 4,646,332 bp plus 20 replicons/cell of the 6480 bp plasmid pTTOD-A33, host genomic DNA (gDNA) should constitute 97.2% of total host cell DNA (total size of DNA per cell of 4,775,932 bp). We used these assumptions to convert DNA concentration levels, derived from spectrophotome- try, into target sequence copy numbers. Reactions were carried out in a total volume of 20 L, with each reaction containing 10 L of 2 x SsoAdvanced SYBR Green Supermix (BioRad, Hercules, CA, USA), 5 L of material containing template DNA and 1 L of each primer to give a ﬁnal concentration of 500 nM. Reactions were performed in a CFX Connect Real-time PCR Detec- tion System (Bio-Rad, Hercules, CA, USA) with a cover heated to 105 ◦C. Each reaction was run at a total of 40 cycles, with the same cycling conditions as above. Cq values were generated using Bio- Rad CFX manager 3.0 (BioRad, Hercules, CA, USA) and exported to Microsoft Excel 2010 for analysis. For BirA quantiﬁcation exper- iments white Hard-Shell ® Low-Proﬁle Thin-Wall 96-Well Skirted PCR plates (Bio-Rad, #HSP9601) were used and for bacteriophage quantiﬁcation clear MultiplateTM Low-Proﬁle 96-Well Unskirted PCR plates (Bio-Rad, #MLL9601) were used (Supplementary Fig. 1). A sample containing 217 ng of puriﬁed DNA from shake ﬂask material (Fig. 2A) exhibited acceptable ampliﬁcation efﬁciency across only 3 reactions (2.17 × 106–2.17 × 104 copies of the BirA target − estimated by spectrophotometry) from a dilution series of 7 reactions. By contrast, 3.9 g DNA puriﬁed from bioreactor mate- rial (Fig. 2B) showed acceptable ampliﬁcation efﬁciency across 5 reactions (9.7 × 106 to 970 copies of the BirA target − estimated by spectrophotometry) from a total of 8 tenfold dilutions. The rea- sons for this difference are unclear but taken together these proﬁles serve to illustrate further that equal ampliﬁcation efﬁciency across a dilution series cannot be safely assumed. 2.6. Quantitative PCR assembly and data capture For the shake ﬂasks sample, when sonicated cells were used as template the number of acceptably efﬁcient reactions was unchanged from the puriﬁed DNA template, at 3 (Fig. 2A). The pres- ence of sonicated cells from the bioreactor sample reduced the number of acceptably efﬁcient reactions to 3, down from 5 with puriﬁed DNA (Fig. 2B). 2.9. Copies of target DNA determined by LRE qPCR 2.9. Copies of target DNA determined by LRE qPCR LRE qPCR, as described by Rutledge and Côté [26], was applied to estimate copy numbers. Lambda DNA for CAL1 calibration was purchased from New England BioLabs (Ipswich, MA, USA), prod- uct code N3011S. LRE analyser v. 0.97 [27] was used according to developer’s instructions. Puriﬁed lambda genome DNA samples of known DNA mass were used to calibrate the program and provide information on copy number in samples contaminated with cell debris. Three spectrophotometric estimations were plotted (Fig. 2C and D, grey circles) and extrapolated to provide a common benchmark for comparison of LRE qPCR and SC qPCR. For quantitation of tar- get DNA within disrupted cell samples from shake ﬂask growth, LRE qCPR data points matched more closely the trend of the spectropho- tometric data than did SC qPCR for the undiluted sample, containing 4.17 × 107 copies of the BirA targert, and across 5 further tenfold dilutions to 417 copies (Fig. 2C). For high cell density bioreactor material LRE qCPR also outperformed SC qPCR for measurement of 9.7 × 105 copies and across 4 tenfold dilutions to 97 copies (Fig. 2D). A caveat for bioreactor analysis is that both LRE qPCR and SC qPCR were unable to quantitate target DNA in undiluted samples and diverged signiﬁcantly from the spectrophotometric estimations for the ﬁrst two tenfold dilutions. 3.2. LRE and SC qPCR quantiﬁcation of a genomic target in cellular material E = 10 −1 slope Standardisation is a non-trivial goal in biology [21] and a range of statistical approaches exist for method comparison [10]. Here we assess the equivalence of SC qPCR and LRE qPCR by discussion of raw data, comparison with spectrophotometric data and direct, head-to-head statistical analysis. 2.4. PCR primers and proxy plasmid design The CAL1 primer pair (Fig. 1C, Table 1) directs ampliﬁca- tion of a speciﬁc 151 bp region of the Lambda bacteriophage genome [6]. Due to the ampliﬁcation performance of this 151 bp region, Rutledge and Stewart [28] designated the CAL1 reaction 26 A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 2.7. Determination of amplicon production efﬁciency Efﬁciency was calculated with the standard curve method, as set out by Rutledge and Côté [26], from iterative least-square ﬁtting of a linear function to various data points until a set of points within the desired limit of acceptance (R2 threshold of 0.99) was identiﬁed. Linear regression was then applied to calculate efﬁciency (E), with the equation: 2.8. Copies of target DNA determined by ‘Standard Curve’ qPCR 2.8. Copies of target DNA determined by ‘Standard Curve’ qPCR The standard curve generated as described above was used to estimate copies of target in samples contaminated by cell debris. Cq values of contaminated samples were plotted along the standard curve and converted into copy number using the equation: y Shokere et al. [31] used spectrophotometry to assess the accu- racy of qPCR with puriﬁed DNA as template. Unexpectedly, we observed that spectrophotometry could be used to measure DNA concentration in the presence of disrupted cells from samples of up to OD600 = 16 (Fig. 2D), even though we anticipated the pres- ence of such cell debris would distort the absorbance spectra. An R value (ratio of absorbance at 260 nm/280 nm) of 1.8 is ideal for accurate spectrophotometry. R values for sonicates of material from OD600 = 2.5 − OD600 = 16 cultures were typically close to 1.3, indi- cating the presence of high levels of protein, and also possibly RNA, contributed signiﬁcantly to the absorption at 260 nm. DNA concentrations as low as 2 ng/L were consistently measurable by spectrophotometry of sonicated cells. The low R value meant that we considered all spectrophotometric measurements in cell soni- cates strictly as crude estimations only, with their primary function to assist comparison of LRE and SC qPCR methods. target copy number = 10 Cq−b m target copy number = 10 Cq−b m Where b is the y-intercept and m is the slope of the standard curve. 3.3. LRE qPCR quantiﬁcation of a genomic target in absence and presence of cellular material We performed real time PCR with 5 ng of the naked pPROX1 plasmid (1.54 × 109 copies), encoding bacteriophage DNA sequence, as template and plotted Cq values as a function of ten- fold dilutions (Fig. 4A) to assess ampliﬁcation efﬁciency. For naked template DNA, efﬁciency within the range of 100 ± 10%, with a conﬁdence value r2 ≥ 0.99, was observed from the sample that had undergone one tenfold dilution (1.54 × 109 copies, 5 ng) to the eighth tenfold dilution (154 copies, 500ag). The lowest level of naked template DNA, 15.4 copies (50ag), in the ninth tenfold dilution, gave the same Cq value as 154 copies. SC qPCR is predicated on the use of a standard curve comprised of the same primers and target as those used in experimental reac- tions. Because puriﬁed DNA was used as standard curve for the SC qPCR experiment in Fig. 2C and D it cannot meaningfully be used to evaluate the accuracy of SC qPCR for puriﬁed DNA template. No such restriction applies to LRE qPCR and as such we plotted LRE qPCR data gathered using naked DNA and disrupted cell suspension as template alongside data points generated by spectrophotometry (Fig. 3). For disrupted cell samples derived from shake ﬂask cultiva- tion (Fig. 3A), there was close agreement between LRE qPCR and spectrophotometric estimation, for undiluted material (4.17 × 107 copies) and over ﬁve dilutions (to 417 copies). The copy numbers indicated by LRE qPCR plateaued for dilutions 6 and 7 (41.7 copies and 4.17 copies), suggesting either a false positive or that a cer- tain level of DNA remains permanently associated with cellular material. 3.4. qPCR ampliﬁcation efﬁciency for a bacteriophage target in cellular material 3.4. qPCR ampliﬁcation efﬁciency for a bacteriophage target in cellular material from shake ﬂask cultivation (Fig. 2E). The Y intercept of 0.16674 also suggested modest systematic bias. A Bland-Altman [3] plot of these data (Fig. 2G) indicated LRE qPCR had a slightly negative bias of SC qPCR data but that the methods were equivalent due to the fact that the mean bias range included zero difference [4]. XY plot com- parison of LRE qPCR and SC qPCR analysis of disrupted cells from bioreactor cultivation (Fig. 2F) showed less proportional bias (slope of 1.0074) than for shake ﬂask material but greater systemic bias (Y intercept of 0.2053). Bland-Altman plot (Fig. 2H) again indicated the methods were equivalent, as the mean bias range spanned the zero difference level [4]. from shake ﬂask cultivation (Fig. 2E). The Y intercept of 0.16674 also suggested modest systematic bias. A Bland-Altman [3] plot of these data (Fig. 2G) indicated LRE qPCR had a slightly negative bias of SC qPCR data but that the methods were equivalent due to the fact that the mean bias range included zero difference [4]. XY plot com- parison of LRE qPCR and SC qPCR analysis of disrupted cells from bioreactor cultivation (Fig. 2F) showed less proportional bias (slope of 1.0074) than for shake ﬂask material but greater systemic bias (Y intercept of 0.2053). Bland-Altman plot (Fig. 2H) again indicated the methods were equivalent, as the mean bias range spanned the zero difference level [4]. For quantiﬁcation of a genomic target (Figs. 2 and 3) cell son- icates were the only source of template and were diluted tenfold for each reaction. So while the amount of template decreased, the ratio of target to cellular material was maintained in each dilution. For industrial scale cultivation of E. coli, quantiﬁcation of a contam- inant organism such as bacteriophage is ideally effective at very low concentrations of contaminant. Consequently, for qPCR experi- ments with bacteriophage sequences we performed serial dilutions of the plasmid ﬁrst, and then added the same amount of cell son- icate material to every dilution, decreasing the relative amount of target versus cells for each dilution (Fig. 4). 3.1. qPCR ampliﬁcation efﬁciency for a genomic target in cellular material 3.1. qPCR ampliﬁcation efﬁciency for a genomic target in cellular material Efﬁciency of amplicon production, which can be deﬁned as the slope of Cq values when plotted as a function of reaction cycle, is key to many of the numerous statistical approaches to qPCR data analysis. Typically, efﬁciency within the range of 100 ± 10%, with a conﬁdence value (r2) of at least 0.99, is set as the limit for accurate quantitation. Many methods assume equal ampliﬁcation efﬁciency across all reactions in a dilution series [26]. Method comparison by XY plot [4] will result in a slope of 1.00 in the case of zero bias between methods. When compared to SC qPCR using an XY plot, LRE qPCR showed a small degree of proportional bias (slope of 1.1351) for quantitation of target in disrupted cells The genomic target sequence we used is present in the BirA gene, which is known to be present as a single copy in the E. coli genome. A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 27 Fig. 3. Inﬂuence of disrupted E. coli cells on LRE qPCR quantiﬁcation of an E. coli genomic target sequence. The LRE method was applied to real time PCR ﬂuorescence dat gathered using dilutions of shake ﬂask (graph A) and bioreactor material (graph B) and pure gDNA extracted from these materials. Grey data points indicate spectrophotometri data and dashed lines extrapolate these data to predict copy number at lower dilutions. The undiluted samples from shake ﬂask and bioreactor cultivation contain the sam cell and DNA content as detailed in Fig. 2. Fig. 3. Inﬂuence of disrupted E. coli cells on LRE qPCR quantiﬁcation of an E. coli genomic target sequence. The LRE method was applied to real time PCR ﬂuorescence data gathered using dilutions of shake ﬂask (graph A) and bioreactor material (graph B) and pure gDNA extracted from these materials. Grey data points indicate spectrophotometric data and dashed lines extrapolate these data to predict copy number at lower dilutions. The undiluted samples from shake ﬂask and bioreactor cultivation contain the same cell and DNA content as detailed in Fig. 2. 4.1. Quantifying the need for sample preparation A PCR-based assay that requires little or no sample preparation has the potential to help accelerate assay turnaround sufﬁciently to be used for at-line monitoring of bioindustrial processes [37] and the pressing challenges of scaling up synthetic biology solutions. For detection of a genomic target the presence of cellular mate- rial derived from bioreactor cultivation compromised quantitation by LRE qPCR more than when a comparable amount of shake ﬂask material was present (Fig. 3). Accurate quantitation of target in 215 ng DNA in the presence of OD600 = 2.5 shake ﬂask culture (Fig. 3A) was not matched for 50 ng in presence of OD600 = 1.6 bioreactor material (Fig. 3B, second tenfold dilution). The reason for the reduced LRE qPCR accuracy with bioreactor material is unknown, but the observation is consistent with the data from Fig. 2B that bioreactor material had a greater effect on ampliﬁca- tion efﬁciency than shake ﬂask material. This putative inhibitory effect of bioreactor material was not observed for detection of a bacteriophage target sequence present in a plasmid (Fig. 4C). An interaction between cellular material and DNA may be inﬂuenced by the provenance of the cells (shake ﬂask or bioreactor cultivation) and the nature of the target (plasmid or genomic DNA). However, any confounding effects arising from such factors can be readily surmounted by sample dilution. Efﬁciency of ampliﬁcation underlies many statistical approaches to analysis of real time PCR data and in some approaches is assumed to be equal across all reactions. Ampli- ﬁcation efﬁciency for a genomic target was not equal across all reactions, even for naked DNA (Fig. 2). Relative to ampliﬁcation efﬁciency for naked DNA, disrupted cells from bioreactor culti- vation had an inhibitory effect (Fig. 2B) that was not observed for shake ﬂask material (Fig. 2A). This implies that ampliﬁcation efﬁciency should be monitored for qPCR methods that assume equal ampliﬁcation efﬁciency across all reactions and that qPCR sample preparation is more critical for bioreactor-derived cellular material. Ampliﬁcation efﬁciency for 1.54 × 107 to 1540 copies (3–7 tenfold dilutions) of a bacteriophage target sequence was not inﬂu- enced by the presence of cells sonicates from cultures of up to OD600 = 160. Cell sonicates from OD600 = 2.5 culture permitted ade- quate ampliﬁcation efﬁciency for as few as 154 copies (8 tenfold dilutions) of the bacteriophage target sequence (Fig. 4A). 3.5. LRE and SC qPCR quantiﬁcation of bacteriophage target in cellular material We next used LRE qPCR and SC qPCR to derive absolute bacte- riophage DNA copy numbers in the presence of cellular material from shake ﬂask and bioreactor cultivation. For comparison, abso- lute copy numbers calculated by the two different methods were plotted alongside copy numbers derived from spectrophotometry of naked plasmid DNA. For samples derived from bioreactor cultivation the target num- bers indicated by LRE qPCR were depressed for the undiluted sample of OD600 = 160 (5 g, 9.71 × 108 copies of BirA target) and the next 2 tenfold dilutions (Fig. 3B). LRE qPCR closely matched spectrophotometric estimation of copy number over 3–8 tenfold dilutions for puriﬁed target DNA (7.57 × 105 to 7.57 copies) and 3–7 tenfold dilutions (9.7 × 105 to 97 copies) in the presence of disrupted cells (Fig. 3B). Copy numbers calculated using the LRE qPCR method were in agreement with spectrophotometric data in the range of 1.54 × 108–1.54 × 106 copies (500pg–5 pg pDNA) of target, despite the presence of bioreactor material of OD600 up to 160 in the undi- luted sample (Fig. 4C). LRE qPCR was less effective for quantiﬁcation of 1.5 × 104 or less copies of bacteriophage target sequence (Fig. 4C). A. Templar et al. / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 28 Fig. 4. Inﬂuence of disrupted E. coli on qPCR analysis of a bacteriophage target sequence. 5 ng (1.54 × 109 copies) of puriﬁed pPROX1 plasmid (encoding a bacteriophage target sequence) was used as template (in 1 L) and a series of tenfold dilutions made each with 4 L of dH O or cell sonicate added prior to ampliﬁcation A) Cq values Fig. 4. Inﬂuence of disrupted E. coli on qPCR analysis of a bacteriophage target sequence. 5 ng (1.54 × 109 copies) of puriﬁed pPROX1 plasmid (encoding a bacteriophage target sequence) was used as template (in 1 L) and a series of tenfold dilutions made, each with 4 L of dH2O or cell sonicate added prior to ampliﬁcation. A) Cq values derived from real time ﬂuorescence data were plotted as a function of tenfold dilutions of the plasmid. Copy numbers derived from B) SC qPCR and C) LRE qPCR methods were also plotted alongside copy number estimates extrapolated from spectrophotometric measurements of puriﬁed plasmid DNA (dashed lines). A. Templar et al. 4.2. Comparing SC qPCR and LRE qPCR SC qPCR and LRE qPCR were directly compared with respect to their agreement with spectrophotometric data for estimation of genomic target DNA concentration in samples from shake ﬂask (Fig. 2C) and bioreactor (Fig. 2D) cultivation. LRE qPCR data matched extrapolated spectrophotometric data for 6 out 8 data points for shake ﬂake samples (Fig. 2C) and 5 out 10 data points for biore- actor samples (Fig. 2D). This compares to at most 3 matching data points for SC qPCR. Further statistical analysis (Fig. 2E–H) suggested the methods could be regarded as equivalent. 5. Conclusions The accuracy proﬁle of LRE qPCR matched that of SC qPCR by the measures performed here using industrially relevant samples. In light of these observations, and previous validation of the proper- ties of LRE qPCR [25], we invite the synthetic biology community to use test CAL1 standard and LRE qPCR procedure for absolute qPCR. Accumulation of data and experience could lead to the establish- ment of CAL1 as a useful synthetic biology standard. 3.5. LRE and SC qPCR quantiﬁcation of bacteriophage target in cellular material / Biomolecular Detection and Quantiﬁcation 11 (2017) 21–30 29 For quantitation of a genomic target, shake ﬂask samples of OD600 = 2.5 still allowed quantitation of 4.17 × 107 copies of tar- get (215 ng DNA) in agreement with a spectrophotometric estimate (Fig. 3A). For bioreactor samples, dilution to OD600 = 0.16 was nec- essary for quantitation of 9.71 × 105 copies of target (5 ng DNA) in agreement with a spectrophotometric estimate (Fig. 3B). By contrast, quantitation in agreement with a spectrophotometric estimate was still possible in the presence of up to OD600 = 160 cel- lular material for 1.54 × 105 copies (0.5 ng pDNA) of a bacteriophage target sequence (Fig. 4C). These data suggest a simple processing step, with no DNA puriﬁcation, followed by 2–3 tenfold dilutions, may be sufﬁcient to render an industrial process stream sample amenable to qPCR analysis by the LRE qPCR method. SC qPCR data diverged from spectrophotometric estimations at the high, 1.54 × 109 copies (5 ng), and low, 150 copies (500ag), extremes of bacteriophage DNA concentration that were tested (Fig. 4B). SC qPCR had less range than LRE qPCR overall, but did show impressive accuracy extending to a copy number as low as 150 (Fig. 4B). 4.1. Quantifying the need for sample preparation This indi- cates that DNA puriﬁcation is unnecessary for quantiﬁcation of T7 bacteriophage DNA by qPCR and that instead a brief sonication step will enable accurate quantitation of as few as 1500 copies of target in a given sample. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bdq.2016.12.001. Acknowledgments This work was supported by the EPSRC (grant code EP/G034656/1) and BJS Biotechnologies Ltd. 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https://openalex.org/W2740182356	https://europepmc.org/articles/pmc5539428?pdf=render	English	null	Elongation Factor P Interactions with the Ribosome Are Independent of Pausing	MBio	2,017	cc-by	2,625	COMMENTARY crossm July/August 2017 Volume 8 Issue 4 e01056-17 ® mbio.asm.org 1 Elongation Factor P Interactions with the Ribosome Are Independent of Pausing Rodney Tollerson II,a,c Anne Witzky,b,c Michael Ibbaa,c Department of Microbiology, The Ohio State University, Columbus, Ohio, USAa; Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USAb; Center for RNA Biology, The Ohio State University, Columbus, Ohio, USAc Department of Microbiology, The Ohio State University, Columbus, Ohio, USAa; Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, USAb; Center for RNA Biology, The Ohio State University, Columbus, Ohio, USAc ABSTRACT Bacterial elongation factor P (EF-P) plays a pivotal role in the translation of polyproline motifs. To stimulate peptide bond formation, EF-P must enter the ri- bosome via an empty E-site. Using ﬂuorescence-based single-molecule tracking, Mohapatra et al. (S. Mohapatra, H. Choi, X. Ge, S. Sanyal, and J. C. Weisshaar, mBio 8:e00300-17, 2017, https://doi.org/10.1128/mBio.00300-17) monitored the cel- lular distribution of EF-P and quantiﬁed the frequency of association between EF-P and the ribosome under various conditions. Findings from the study showed that EF-P has a localization pattern that is strikingly similar to that of ribosomes. Intriguingly, EF-P was seen to bind ribosomes more frequently than the estimated number of pausing events, indicating that E-site vacancies occur even when ribosomes are not paused. The study provides new insights into the mechanism of EF-P-dependent peptide bond formation and the intricacies of translation elongation. Received 15 June 2017 Accepted 19 June 2017 Published 1 August 2017 Citation Tollerson R II, Witzky A, Ibba M. 2017. Elongation factor P interactions with the ribosome are independent of pausing. mBio 8:e01056-17. https://doi.org/10.1128/mBio .01056-17. Copyright © 2017 Tollerson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Michael Ibba, ibba.1@osu.edu. For the article discussed, see https://doi.org/10 .1128/mBio.00300-17. The views expressed in this Commentary do not necessarily reﬂect the views of this journal or of ASM. Received 15 June 2017 Accepted 19 June 2017 Published 1 August 2017 Citation Tollerson R II, Witzky A, Ibba M. 2017. Elongation factor P interactions with the ribosome are independent of pausing. mBio 8:e01056-17. https://doi.org/10.1128/mBio .01056-17. Copyright © 2017 Tollerson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Michael Ibba, ibba.1@osu.edu. For the article discussed, see https://doi.org/10 .1128/mBio.00300-17. The views expressed in this Commentary do not necessarily reﬂect the views of this journal or of ASM. Elongation Factor P Interactions with the Ribosome Are Independent of Pausing KEYWORDS elongation factor P, pausing, translation The views expressed in this Commentary do not necessarily reﬂect the views of this journal or of ASM. O f the proteogenic amino acids, proline plays a unique role, providing critical kink turns in proteins and increasing peptide stability (1). For example, in Mycobacte- rium tuberculosis, nearly 10% of the coding capacity is spent on translating proteins containing PE and PPE motifs (2). A less extreme but equally remarkable case is valyl-tRNA synthetase, which contains a universally conserved triprolyl motif required for substrate speciﬁcity (3). Due to its unique pyrrolidine ring structure, proline is both a poor peptide bond donor and acceptor, forming peptide bonds with puromycin from 10 to 1,000 times slower than other amino acids (4). Consequently, stretches of prolines can cause ribosome pausing and translational arrest. Unresolved ribosome pausing at polyproline motifs in bacteria can lead to loss of motility, virulence, and cell viability (4, 5). Bacteria use elongation factor P (EF-P) to alleviate ribosome pausing on polyproline motifs. Escherichia coli ribosome proﬁling data have shown that in the absence of EF-P, nearly half of PPX (where X is any third amino acid) motifs have a 10-fold-higher rate of ribosome occupancy than when EF-P is absent, which is indicative of strong pausing at these motifs (6). O EF-P must be posttranslationally modiﬁed at a conserved residue to perform its function. Genes required for modiﬁcation are highly pleiotropic, and knockout mutants mimic phenotypes displayed in efp deletion backgrounds. Though EF-P and its homo- logues are conserved throughout all domains of life, the structures of the posttransla- tional modiﬁcation (PTM) are not. For example, the E. coli and Bacillus subtilis EF-Ps are modiﬁed by the linear (R)--lysine and 5-aminopentanol, respectively, while the Pseu- domonas aeruginosa and Neisseria menengitidis EF-Ps are modiﬁed by a cyclic rhamnose moiety. The exact mechanism of EF-P-mediated ribosome rescue is currently unknown, but it is speculated that when modiﬁed, EF-P increases the stability of the P-site tRNA and proline within the ribosomal peptidyl transfer center (PTC), possibly changing the orientation of proline to be more amenable for peptide bond formation (7). Biochem- ical work suggests that the E. coli EF-P posttranslational modiﬁcation, (R)--lysine, ® mbio.asm.org 1 ® Commentary signiﬁcantly increases the association constant between EF-P and the ribosome, though the mechanism of this phenomenon is unknown (4). Elongation Factor P Interactions with the Ribosome Are Independent of Pausing Curiously, using the Thermus thermophilus crystal structure of EF-P bound to the 70S ribosome as a guide, rhamnose- modiﬁed EF-P seems to be unable to reach into the PTC in the same way that linear modiﬁcations would (8). This further confounds our understanding of the role of EF-P PTM, since structurally diverse modiﬁcations seem to play similar roles in facilitating peptide bond synthesis. To further investigate the role of EF-P and its PTM in peptide bond formation, Mohapatra et al. used ﬂuorescence microscopy to identify and quantify the interactions between EF-P and the ribosome through changes in spatial distribution and diffusion rates of the two factors (9). The authors of the study aimed to address a critical question in the ﬁeld: does EF-P selectively bind ribosomes paused at polyproline motifs via an empty E-site, or does this interaction occur randomly? Since EF-P accelerates peptide bond synthesis within paused ribosomes, one possibility is that ribosome pausing is a prerequisite for EF-P rescue (3). Previous studies have presented evidence that tRNA translocation during elongation is a modular event, making E-site vacancy rare outside spontaneous diffusion (10). In this case, only paused ribosomes would have empty E-sites, and EF-P entry via the E-site would be limited to the population of paused ribosomes. To determine the axial distribution and diffusion rate of EF-P and ribosomes in E. coli, Mohapatra et al. utilized mEos2-tagged EF-P and S2 ribosomal proteins. The utility of the mEos2 tag was the authors’ ability to speciﬁcally follow one to two individually tagged molecules at a time. By shifting the wavelength emitted through weak laser excitation, the investigators were able to accurately identify the location of speciﬁc molecules at 2-ms intervals. The data generated in their study provided evidence that EF-P and the ribosome share a similar distinctive three-peaked distribution, near the nucleoid and at the cell poles, within an E. coli cell under normal conditions. When the ribosomal spatial distribution is perturbed by the addition of antibiotics, EF-P distribu- tion is similarly affected. With addition of the translation-arresting agent chloramphen- icol, ribosomes and EF-P had a more nucleoid distribution. In contrast, treatment with a transcription inhibitor, rifampin, caused both factors to be more evenly distributed throughout the cell. Though distribution of EF-P very strikingly mimics that of the ribosome, the spatial organizations are not identical. July/August 2017 Volume 8 Issue 4 e01056-17 Elongation Factor P Interactions with the Ribosome Are Independent of Pausing EF-P binding to ribosomes with empty E-sites due to tRNA diffusion from actively translating ribosomes does not stimulate peptide bond formation. FIG 1 Constant interrogation of ribosomes by posttranslationally modiﬁed EF-P. (Top row) Independent of the pausing at an EF-P-dependent (PPX) motif (red circles), EF-P is unable to enter the ribosome via the E-site if the site is occupied by tRNA. (Bottom row) When the E-site is vacant, modiﬁed EF-P is able to bind to the ribosome. When the ribosome is paused at a PPX motif, the binding event stimulates peptide bond formation. EF-P binding to ribosomes with empty E-sites due to tRNA diffusion from actively translating ribosomes does not stimulate peptide bond formation. present per cell. Therefore, EF-P must “interrogate” ribosomes much more frequently than would be possible if paused ribosomes were the only target. Curiously, the percentage of total EF-P interacting with ribosomes increases from 30% to 45% when chloramphenicol is added to the cells, possibly due to an increased number of empty E-sites, and in this case even the “fast” EF-P molecules may be interacting transiently with ribosomes (inferred from a decrease in the “fast” mean diffusion coefﬁcient, from 4.3 m2/s to 1.2 m2/s). EF-PK34A also conformed to a two-state model, but both fast and slow diffusion coefﬁcients were strongly divergent from that of EF-P interacting with a ribosome, implying that EF-PK34A may never interact with translating ribosomes in a directed manner. Through investigation of the overall interaction cycle between EF-P and the ribo- some (about 23 ms), the calculated rate constant of EF-P binding to ribosomes was seen to be around 100 times lower than if binding was purely based on diffusion rates, highlighting the intricacies of EF-P interactions with the ribosome. Interestingly, the amount of time in which EF-P is bound to the ribosome is independent of whether translation elongation occurs; in effect, EF-P is randomly interacting with ribosomes independent of pausing. The time between binding events is shorter if translation is arrested, possibly due to the greater availability of ribosomes with empty E-sites. Mechanistically, EF-P function is intrinsically tied to the availability of vacant ribo- somal E-sites. The Mohapatra et al. study showed that not only does EF-P localize with ribosomes in the cell, but also that it is constantly interrogating ribosomes for available E-sites. When successful, the time EF-P spends in the E-site is independent of transla- tional pausing. Elongation Factor P Interactions with the Ribosome Are Independent of Pausing Axial distribution of EF-P is not as segregated as that of the ribosome, implying that they are not in constant interaction. As mentioned above, EF-P posttranslational modiﬁcation has been seen to dramat- ically increase association between EF-P and the ribosome. To determine the effect of EF-P modiﬁcation on its axial distribution, mEos2-tagged EF-PK34A, which cannot be modiﬁed, was expressed from a plasmid in E. coli harboring the chromosomal copy of wild-type EF-P to prevent defects associated with only nonfunctional EF-P. Axial distribution of EF-PK34A displayed a pattern similar to that of wild-type EF-P and ribosomes when E. coli cells were treated with rifampin; there was no obvious spatial organization, similar to that of a simulated homogeneous distribution. These ﬁndings correlate with the in vitro biochemical data, providing evidence that the K34A mutant has a major decrease in afﬁnity for the ribosome (4). However, it is difﬁcult to assess the impact that the continued presence of the wild-type copy of EF-P would have had in these experiments. To identify the fraction of EF-P that interacts with the ribosome, single-step diffusion dynamics were determined for EF-P under a variety of conditions. Quantiﬁcations of EF-P–ribosome interactions were made by comparing the rate of diffusion of EF-P to that of the ribosome and determining the best ﬁt in a two-state model. These states are “slow,” in which EF-P slows diffusion by interacting with another factor, in this case the ribosome, and “fast,” in which EF-P is not bound to ribosomes and is freely diffusing. By comparing the fraction of “slow” EF-P molecules to those which were “fast,” the Mohapatra group determined the fraction of ribosomes interacting with EF-P. Under normal conditions, the authors estimated that EF-P interacts with anywhere from 25% to 100% of translating ribosomes. This is in stark contrast with their ﬁndings that about 0.05% of the active E. coli translatome contains a PPX motif, with about 280 motifs July/August 2017 Volume 8 Issue 4 e01056-17 mbio.asm.org 2 ® Commentary FIG 1 Constant interrogation of ribosomes by posttranslationally modiﬁed EF-P. (Top row) Independent of the pausing at an EF-P-dependent (PPX) motif (red circles), EF-P is unable to enter the ribosome via the E-site if the site is occupied by tRNA. (Bottom row) When the E-site is vacant, modiﬁed EF-P is able to bind to the ribosome. When the ribosome is paused at a PPX motif, the binding event stimulates peptide bond formation. July/August 2017 Volume 8 Issue 4 e01056-17 Elongation Factor P Interactions with the Ribosome Are Independent of Pausing This paints a picture where EF-P is acting on any open E-site, but only ribosomes pausing at speciﬁc motifs beneﬁt from this event (Fig. 1). Lacking a speciﬁc sequence which recruits EF-P may be advantageous because of the expansive variety of EF-P-dependent pause motifs and the effect that distal sequences can have on pausing (11). This mechanism can only be viable if E-sites are empty at a much greater frequency than previously expected. The original mechanism of immediate occupation of the vacant E-site by P-site tRNA would not allow for EF-P interrogations occurring with anywhere from 25% to 100% of translating ribosomes. Frequent E-site vacancy also provides evidence that translation elongation is a dynamic process in which translocation of P-site tRNA is not required for E-site tRNA diffusion. The Mohapatra et al. study ﬁndings advance not only the ﬁeld of EF-P-dependent translation but also that of general bacterial translation, where a greater understanding of the elongation cycle is paramount July/August 2017 Volume 8 Issue 4 e01056-17 mbio.asm.org 3 ® Commentary ACKNOWLEDGMENTS We thank P. Kelly for helpful comments and suggestions. This work is supported by the National Institutes of Health (GM65183 to M.I.), NIH T32 (GM086252 to R.T. and A.W.), and an OSU Center for RNA Biology fellowship to A.W. This work is supported by the National Institutes of Health (GM65183 to M.I.), NIH T32 (GM086252 to R.T. and A.W.), and an OSU Center for RNA Biology fellowship to A.W. July/August 2017 Volume 8 Issue 4 e01056-17 REFERENCES 7. Blaha G, Stanley RE, Steitz TA. 2009. Formation of the ﬁrst peptide bond: the structure of EF-P bound to the 70S ribosome. Science 325:966–970. https://doi.org/10.1126/science.1175800. 1. 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https://openalex.org/W2999910794	https://europepmc.org/articles/pmc7017419?pdf=render	English	null	JNK Pathway Mediates Low Oxygen Level Induced Epithelial–Mesenchymal Transition and Stemness Maintenance in Colorectal Cancer Cells	Cancers	2,020	cc-by	12,850	Received: 23 December 2019; Accepted: 14 January 2020; Published: 16 January 2020 Abstract:(1)Background: Epithelial–mesenchymaltransition(EMT)andcancercellstemnessmaintenance (SM) are important factors for cancer metastasis. Although hypoxia has been considered as a possible factor for EMT induction and promotion of SM, studies in this area, apart from hypoxia-inducible factor (HIF) pathways and severe hypoxia, are scant. This study aimed to evaluate the effects of different oxygen levels on EMT induction and SM and elucidate the signaling pathways involved in colorectal cancer cells. (2) Methods: Cell morphological analysis, migration assay, immunofluorescence staining of cytoskeleton and Western blotting were performed on human colorectal cancer cells HT-29, DLD-1, and SW-480 cultured at 1%, 10%, and normal (21%) O2 levels. The role played by c-Jun N-terminal kinase (JNK) was evaluated through the use of the specific JNK inhibitor SP600125. (3) Results: This study evaluated 1% and 10% O2 are possible conditions for EMT induction and SM. This study also demonstrated the partial relieve of EMT induction and SM by SP600125, showing the importance of the JNK pathway in these processes. Furthermore, this study proposed a novel pathway on the regulation of Akt by JNK-c-Jun. (4) Conclusions: This study suggests 10% O2 as another possible condition for EMT induction, and SM and JNK pathways play important roles in these processes through multiple factors. Inhibition of JNK could be explored as treatment for inhibiting metastasis in colorectal cancer cells. Keywords: Akt; colorectal cancer; epithelial–mesenchymal transition; hypoxia; JNK; oxygen level; stemness maintenance cancers cancers cancers Cancers 2020, 12, 224; doi:10.3390/cancers12010224 Received: 23 December 2019; Accepted: 14 January 2020; Published: 16 January 2020 www.mdpi.com/journal/cancers Article Shing Yau Tam , Vincent W.C. Wu * and Helen K.W. Law * Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk * Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) Shing Yau Tam , Vincent W.C. Wu * and Helen K.W. Law * Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk * Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) Shing Yau Tam , Vincent W.C. Wu * and Helen K.W. Law * Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk * Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) 1. Introduction Metastasis is an important event in cancer progression and accounts for majority of cancer related deaths including colorectal cancer (CRC) [1]. Although much of the exact mechanism remains unknown, epithelial–mesenchymal transition (EMT) has been regarded as an important event for metastasis as it allows cancer cells to transform from the closely packed epithelial cell type to mesenchymal cell type with migratory and invasive properties [2]. The loss of adherent junctions between cells alters the cytoskeletal composition and cell polarity to form spindle-shaped cells. In cancer cells undergoing EMT, the actin cytoskeleton is reorganized from cortical thin bundles into thick contractile stress ﬁbers at the ventral cell surface [3]. The monomers of actin polymerize to form ﬁlamentous-actin (F-actin) and start the formation of various migratory protrusions including podosomes, invadopodia, ﬁlopodia, and lamellipodia. This process, known as dynamic actin reorganization, is a prerequisite for the migration and invasion of cancer cells [4,5]. Hypoxia induces EMT mainly by hypoxia-inducible factor (HIF) activation [6]. HIF-1α could bind directly to twist-related protein 1 (TWIST1), matrix metalloproteinase-9 (MMP-9), and histone deacetylase 3 (HDAC3), which eventually promote transcription of Snail [7]. Meanwhile, HDAC3 regulates the formation of histone methyltransferase complexes to induce vimentin and N-cadherin. For CRC, ubiquitin-specific www.mdpi.com/journal/cancers Cancers 2020, 12, 224 2 of 16 protease 47 (USP47) reduced E-cadherin expression by Snail regulation under hypoxic conditions [8], and the overexpression of TWIST1 and Snail is associated with poor prognosis [9]. protease 47 (USP47) reduced E-cadherin expression by Snail regulation under hypoxic conditions [8], and the overexpression of TWIST1 and Snail is associated with poor prognosis [9]. Apart from EMT of the primary tumor, cancer stem cells (CSCs) have been widely accepted as precursors of metastases [10]. CSCs are pluripotent for multilineage differentiation, capable for self-renewal, and have long longevity [11]. They vary in number among different types of cancer cell lines and human xenografts, affecting tumor differentiation and aggressiveness [12]. CSC proliferation and maintenance of stemness properties are controlled by similar pathways as EMT-inducing pathways such as Wnt, transforming growth factor-β (TGF-β), and Notch [13]. The pathways exert their influences on stemness maintenance (SM) by regulating pluripotency markers including octamer-binding transcription factor 4 (Oct-4), sex determining region Y-box 2 (SOX2), and Nanog. In CRC cells, these pluripotency markers are regarded as poor prognosis indicators of CRC. 1. Introduction In CRC stem cells, aberrant Wnt/β-catenin signaling is observed, and this is suggested to cause treatment resistance and disease relapse [13]. Although HIFs have been suggested to inﬂuence EMT and stemness though multiple pathways under hypoxic conditions, the inﬂuence of other hypoxia-related molecules such as c-Jun N-terminal kinase (JNK) has received little attention [6,13]. JNK is activated by various stresses and was initially regarded as an inducer of inﬂammation and apoptosis. However, JNK could also be activated by chronic hypoxia and inﬂuence autophagy [14]. JNK may inﬂuence EMT though TGF-β SMAD-independent pathway [6] and SM through Wnt/bone morphogenetic protein (BMP)/T cell factor (TCF)/Notch or interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathways [15]. Cancer cells constantly face diﬀerent oxygen level during development from hypoxia to about 10% oxygen level during metastasis in the bloodstream. In our previous autophagy study, we have reported that 10% oxygen level could exert prominent eﬀects on oxygen level driven regulators including HIFs and JNK [14]. Therefore, the impact of various oxygen levels on EMT and SM was further investigated in this study. 2. Results 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition From the photomicrographs taken after 48 h incubation, the three CRC cell lines (HT-29, DLD-1, and SW-480) showed different responses to different oxygen levels. For HT-29, which had more epithelial cell features among the three cell lines, cells became more loosely packed colonies under 1% O2 (Figure 1A), showing signs of EMT induction in the loss of cell–cell interactions [16]. Also, cell colonies incubated under 1% and 10% O2 became more irregularly shaped from the dominantly round shape in 21% O2. In the presence of JNK inhibitor, the cell colony shape in all three oxygen levels became rounder, similar to the control, when compared with those without the addition of JNK inhibitor. For DLD-1, which had intermediate epithelial and mesenchymal cell features among the three studied cell lines, there was no prominent changes of morphological features under diﬀerent oxygen levels (Figure 1B). Whereas, in the presence of JNK inhibitor, cell colonies of DLD-1 cultured in 10% O2 became more closely packed. SW-480 normally had the most mesenchymal cell features among the three cell lines. In the 1% and 10% O2 conditions, they acquired larger cell colonies of spindle-shaped cells when compared with cells in 21% O2, showing signs of EMT induction (Figure 1C). In the presence of JNK inhibitor, the EMT induction in 1% and 10% O2 were partially reversed by having more epithelial cell colonies and less spindled-shaped mesenchymal-like cells. 3 of 16 Cancers 2020, 12, 224 Cancers 2020, 12, x 3 of 16 Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under different oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample images from 5 independent experiments performed (200× magnification). 2.2. Cell Migration Was Reduced by JNK Inhibition Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29 Figure 1. showed an extremely slow migration rate, probab Figure S1); hence, they have been excluded in th 2.2. Cell Migration Was Reduced by JNK Inhibition ) yg conditions (C). Sample images from 5 independe g y y that there was no significant difference in migratory rate when cells were cultured in different oxygen levels (Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate among different oxygen levels. A significant reduction of migratory rate at 10% O2 in DLD-1 was found. Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29 showed an extremely slow migration rate, probably due to their epithelial properties (Supplementary Figure S1); hence, they have been excluded in the analysis. Results for DLD-1 and SW-480 showed that there was no signiﬁcant diﬀerence in migratory rate when cells were cultured in diﬀerent oxygen levels (Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate among diﬀerent oxygen levels. A signiﬁcant reduction of migratory rate at 10% O2 in DLD-1 was found. 2.2. Cell Migration Was Reduced by JNK Inhibition Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29 showed an extremely slow migration rate, probably due to their epithelial properties (Supplementary Figure S1); hence, they have been excluded in the analysis. Results for DLD-1 and SW-480 showed that there was no significant difference in migratory rate when cells were cultured in different oxygen levels (Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate among different oxygen levels A significant reduction of migratory rate at 10% O2 in DLD 1 was found Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there were no significant differences of migration rate between different oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there were no significant differences of migration rate between different oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally Figure 2. Wound healing assay under diﬀerent oxygen levels. Wound healing assay showed that there were no signiﬁcant diﬀerences of migration rate between diﬀerent oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally reduced the migration rate among diﬀerent oxygen levels in both cell lines, with 10% O2 having signiﬁcant reduction in DLD-1. 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition Cell morphological changes under diﬀerent oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under diﬀerent oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under diﬀerent oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample images from 5 independent experiments performed (200× magniﬁcation). Cancers 2020, 12, x 3 of 16 Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under different oxygen levels with SP600125 addition especially in 1% and 10% O2 Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under different oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample images from 5 independent experiments performed (200× magnification). 2.2. Cell Migration Was Reduced by JNK Inhibition Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29 Figure 1. 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition Cell morphological changes under diﬀerent oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under diﬀerent oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under diﬀerent oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample images from 5 independent experiments performed (200× magniﬁcation). Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under different oxygen levels with SP600125 addition especially in 1% and 10% O2 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation 4 (DLD-1). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation 4 (DLD-1). Immunoﬂuorescence staining of F-actin by phalloidin was conducted to evaluate the actin cytoskeleton. For HT-29, majority of cells had lamellipodia formation (Figure 3A). There was a trend of increased ﬁlopodia formation at lower oxygen levels. Ventral stress ﬁbers are described as thick and directed contractile ﬁbers [3]. There was a decreasing percentage of cells with ventral stress ﬁbers with lowering oxygen levels. JNK inhibition reduced the formation of lamellipodia, ﬁlopodia, and ventral stress ﬁbers in diﬀerent oxygen levels (Figure 3A). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation Immunofluorescence staining of F-actin by phalloidin was conducted to evaluate the actin cytoskeleton. For HT-29, majority of cells had lamellipodia formation (Figure 3A). There was a trend of increased filopodia formation at lower oxygen levels. Ventral stress fibers are described as thick and directed contractile fibers [3]. There was a decreasing percentage of cells with ventral stress fibers with lowering oxygen levels. JNK inhibition reduced the formation of lamellipodia, filopodia, and t l t fib i diff t l l (Fi 3A) Fi 3 I fl i i f F i d diff l l I fl Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows) and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD- 1 also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia formation among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and Figure 3. Immunoﬂuorescence staining of F-actin under diﬀerent oxygen levels. Immunoﬂuorescence staining of F-actin (red) and DAPI (blue) showed an increase in ﬁlopodia formation (yellow arrows) and a decrease in ventral stress ﬁber formation (green arrows) in 1% and 10% O2 conditions, while lamellipodia formation (white arrows) was similar among diﬀerent oxygen levels in HT-29 (A) DLD-1 also showed an increase in ﬁlopodia formation in 1% and 10% O2 with similar lamellipodia formation among diﬀerent oxygen levels (B) SW-480 showed similar amounts of lamellipodia and ﬁlopodia formation among diﬀerent oxygen levels, and ventral stress ﬁbers were not observed in SW-480. showed an extremely slow migration rate, probab Figure S1); hence, they have been excluded in th 2.2. Cell Migration Was Reduced by JNK Inhibition ) yg conditions (C). Sample images from 5 independe The data (means ± SEM) were expressed as the relative migration rate against the rate under 21% O2 + DMSO (C,D). p < 0.01, DMSO versus SP600125. N = 3 (SW-480) and 4 (DLD-1). Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there were no significant differences of migration rate between different oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there were no significant differences of migration rate between different oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally Figure 2. Wound healing assay under diﬀerent oxygen levels. Wound healing assay showed that there were no signiﬁcant diﬀerences of migration rate between diﬀerent oxygen level incubation in DLD-1 (A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally reduced the migration rate among diﬀerent oxygen levels in both cell lines, with 10% O2 having signiﬁcant reduction in DLD-1. The data (means ± SEM) were expressed as the relative migration rate against the rate under 21% O2 + DMSO (C,D). p < 0.01, DMSO versus SP600125. N = 3 (SW-480) and 4 (DLD-1). Cancers 2020, 12, 224 reduced t i ifi a 4 of 16 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). (C) The presence of JNK inhibitor SP600125 slightly reduced ﬁlopodia and lamellipodia formation among the three cell lines, while ventral stress ﬁber formation was promoted in DLD-1. Sample images were taken from 3 independent experiments. The counting results were shown as means ± SEM in percentage among all evaluated cells. At least 200 cells were evaluated for each sample. * p < 0.05 DMSO versus SP600125. N = 3. Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows) and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD- 1 also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia formation among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and Figure 3. Immunoﬂuorescence staining of F-actin under diﬀerent oxygen levels. Immunoﬂuorescence staining of F-actin (red) and DAPI (blue) showed an increase in ﬁlopodia formation (yellow arrows) and a decrease in ventral stress ﬁber formation (green arrows) in 1% and 10% O2 conditions, while lamellipodia formation (white arrows) was similar among diﬀerent oxygen levels in HT-29 (A) DLD-1 also showed an increase in ﬁlopodia formation in 1% and 10% O2 with similar lamellipodia formation among diﬀerent oxygen levels (B) SW-480 showed similar amounts of lamellipodia and ﬁlopodia formation among diﬀerent oxygen levels, and ventral stress ﬁbers were not observed in SW-480. (C) The presence of JNK inhibitor SP600125 slightly reduced ﬁlopodia and lamellipodia formation among the three cell lines, while ventral stress ﬁber formation was promoted in DLD-1. Sample images were taken from 3 independent experiments. The counting results were shown as means ± SEM in percentage among all evaluated cells. At least 200 cells were evaluated for each sample. * p < 0.05 DMSO versus SP600125. N = 3. Cancers 2020, 12, 224 5 of 16 For DLD-1, a smaller population of cells had lamellipodia formation when compared with HT-29, while a similar increase of ﬁlopodia formation in lower oxygen levels was observed (Figure 3B). JNK inhibition could slightly reduce lamellipodia and ﬁlopodia formation in 1% and 10% O2 but generally promoted ventral stress ﬁber formation in DLD-1. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). In SW-480, similar percentages of cells with lamellipodia and filopodia formations were recorded among different oxygen levels (Figure 3C). The presence of JNK inhibitor reduced formations of lamellipodia and filopodia. There was no observable ventral stress fiber formation in SW-480. 2.4. EMT Induced by Low Oxygen Levels via JNK Pathway Was Conﬁrmed by EMT Markers and Transcription Factors To further investigate the underlying signaling pathways in leading to these morphological and cytoskeleton changes and the role of JNK in EMT and stemness pathways, the expression of related proteins including EMT markers, EMT transcription factors, JNK pathway markers, other EMT related pathway markers, and SM markers were evaluated by Western blotting. The key EMT marker, E-cadherin, was signiﬁcantly down-regulated in 1% and 10% O2 in all three studied cell lines (Figure 4A). JNK inhibitor SP600125 (10 µM) could generally promote E-cadherin in diﬀerent oxygen levels, especially for 10% O2 in HT-29 (Figure 4B). The mesenchymal marker ﬁbronectin was generally promoted in 1% and 10% oxygen levels among the three cell lines with signiﬁcant promotion in 1% O2 on HT-29 (Figure 4C). The impact of SP600125 on ﬁbronectin had diverged responses among the three cell lines (Figure 4D). Down-regulation of ﬁbronectin in HT-29 was found in lower oxygen levels, whereas DLD-1 and SW-480 experienced up-regulation trends in 10% and 21% O2. Another mesenchymal marker, vimentin, was only detectable in SW-480. It was mildly promoted in 10% O2 but not in 1% O2 (Figure 4E). JNK inhibitor reduced vimentin expression under diﬀerent oxygen levels (Figure 4F). EMT transcription factors Snail and TWIST1 were also investigated in DLD-1 and SW-480 cells. Both Snail (Figure 4G) and TWIST1 (Figure 4I) were up-regulated in 1% and 10% O2 among both cell lines, with SW-480 having signiﬁcant promotions in both Snail and TWIST1 at lower oxygen levels, while DLD-1 could attain signiﬁcant increase in TWIST1. Both Snail (Figure 4H) and TWIST1 (Figure 4J) were generally inhibited by JNK inhibitor in DLD-1 and SW-480, especially for DLD-1 in lower oxygen levels. 6 of 16 Cancers 2020, 12, 224 Cancers 2020, 12, x 6 of 16 Ca ce s 0 0, , 6 o 6 Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under different oxygen levels. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under diﬀerent oxygen levels. EMT markers and transcription factors were evaluated by Western blotting in diﬀerent oxygen levels on HT-29, DLD-1, and SW-480 cell lines. The key epithelial EMT marker E-cadherin was signiﬁcantly down-regulated in all three studied cell lines in 1% and 10% O2 (A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in diﬀerent oxygen levels, especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker ﬁbronectin was generally up-regulated in lower oxygen levels with 1% O2 in HT-29 having signiﬁcant up-regulation (C) HT-29 experienced down-regulation of ﬁbronectin in the presence of SP600125, while DLD-1 and SW-480 had up-regulation of ﬁbronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E) SP600125 could substantially reduce vimentin expression under diﬀerent oxygen levels, especially for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in diﬀerent oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen levels were found with SW-480 having signiﬁcant increase in 1% and 10% O2 (G) Snail was generally suppressed by SP600125 with more prominent eﬀects in DLD-1 (H) TWIST1 also had signiﬁcant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression eﬀect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under different oxygen levels. EMT markers and transcription factors were evaluated by Western blotting in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). The key epithelial EMT marker E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2 (A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels, especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29 experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480 had up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E) SP600125 could substantially reduce vimentin expression under different oxygen levels, especially for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was generally suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had significant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD- 1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under diﬀerent oxygen levels. EMT markers and transcription factors were evaluated by Western blotting in diﬀerent oxygen levels on HT-29, DLD-1, and SW-480 cell lines. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). EMT markers and transcription factors were evaluated by Western bl i i diff l l HT 29 DLD 1 d SW 480 ll li Th k i h li l EMT Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under diﬀerent oxygen levels. EMT markers and transcription factors were evaluated by Western blotting Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under different oxygen levels. EMT markers and transcription factors were evaluated by Western blotting in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. The key epithelial EMT marker E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2 (A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels, especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29 experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480 had up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E) SP600125 could substantially reduce vimentin expression under different oxygen levels, especially for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was generally suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had significant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD- 1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Figure 4. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). The key epithelial EMT marker E-cadherin was signiﬁcantly down-regulated in all three studied cell lines in 1% and 10% O2 (A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in diﬀerent oxygen levels, especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker ﬁbronectin was generally up-regulated in lower oxygen levels with 1% O2 in HT-29 having signiﬁcant up-regulation (C) HT-29 experienced down-regulation of ﬁbronectin in the presence of SP600125, while DLD-1 and SW-480 had up-regulation of ﬁbronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E) SP600125 could substantially reduce vimentin expression under diﬀerent oxygen levels, especially for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in diﬀerent oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen levels were found with SW-480 having signiﬁcant increase in 1% and 10% O2 (G) Snail was generally suppressed by SP600125 with more prominent eﬀects in DLD-1 (H) TWIST1 also had signiﬁcant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression eﬀect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Cancers 2020, 12, 224 7 of 16 7 of 16 2.5. Conﬁrmation of JNK and Akt Pathway Activation in Low Oxygen Levels Cancers 2020, 12, x 2.5. Conﬁrmation of JNK and Akt Pathway Activation in Low Oxygen Levels Cancers 2020, 12, x 2.5. Conﬁrmation of JNK and Akt Pathway Activation in Low Oxygen Levels Cancers 2020, , JNK and Akt pathways are possible regulators of EMT transcription factors. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). It was conﬁrmed that JNK phosphorylation in CRC was detected in both 1% and 10% O2 conditions for the three studied cell lines, with signiﬁcant activations in HT-29 (1% and 10% O2) and DLD-1 (10% O2) (Figure 5A). JNK inhibitor did not directly aﬀect the phosphorylation status of JNK (Figure 5B). However, it suppressed the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in diﬀerent oxygen levels among all three studied cell lines. 2.5. Confirmation of JNK and Akt Pathway Activation in Low Oxygen Levels JNK and Akt pathways are possible regulators of EMT transcription factors. It was confirmed that JNK phosphorylation in CRC was detected in both 1% and 10% O2 conditions for the three studied cell lines, with significant activations in HT-29 (1% and 10% O2) and DLD-1 (10% O2) (Figure 5A). JNK inhibitor did not directly affect the phosphorylation status of JNK (Figure 5B). However, it suppressed the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in different oxygen levels among all three studied cell lines he direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in diﬀerent xygen levels among all three studied cell lines. JNK inhibitor did not directly affect the phosphorylation status of JNK (Figure 5B). However, it suppressed the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in different oxygen levels among all three studied cell lines. Figure 5. JNK and Akt pathway activation under different oxygen levels. The key markers of JNK pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT- 29, DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29 and DLD-1 (C) The indirect downstream effector of JNK pathway p62 showed mild up-regulation in 10% O and down regulation in 1% O (D) In the presence of SP600125 significant down regulations Figure 5. JNK and Akt pathway activation under diﬀerent oxygen levels. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). JNK was activated under lower oxygen levels in HT-29, DLD-1, and SW-480 with signiﬁcant promotion in HT-29 and DLD-1 (A) There were generally mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell lines, with greater eﬀects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely suppressed by SP600125 in lower oxygen levels, with signiﬁcant reductions found in HT-29 and DLD-1 8 of 16 Cancers 2020, 12, 224 (C) The indirect downstream eﬀector of JNK pathway p62 showed mild up-regulation in 10% O2 and down-regulation in 1% O2 (D) In the presence of SP600125, signiﬁcant down-regulations were found in diﬀerent oxygen levels among DLD-1 and SW-480 (E) However, for HT-29, signiﬁcant up-regulation of p62 was evaluated in 10% O2. Akt, which is another important EMT promoter, also demonstrated an increase in activation under lower oxygen levels in the three cell lines, with SW-480 having signiﬁcant activation (F) The 1% and 10% O2 activated Akt was abruptly inhibited by SP600125, particularly in HT-29 and SW-480 (G) The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29), 8–9 (DLD-1), or 10 (SW-480) (A,D,F). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 4–9 (HT-29), 5–9 (DLD-1), or 5–10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (B,C,E,G). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Cancers 2020, 12, x 8 of 16 were found in different oxygen levels among DLD-1 and SW-480 (E) However, for HT-29, significant up-regulation of p62 was evaluated in 10% O2. Akt, which is another important EMT promoter, also demonstrated an increase in activation under lower oxygen levels in the three cell lines, with SW-480 having significant activation (F) The 1% and 10% O2 activated Akt was abruptly inhibited by SP600125, particularly in HT-29 and SW-480 (G) The data (means ± SEM) were expressed as the For the indirect downstream eﬀector of JNK pathway, p62 had diverse changes under lower oxygen levels with general promotion at 10% O2 and general down-regulation at 1% O2 (Figure 5D). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). The key markers of JNK pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT-29, DLD-1, and SW-480 with signiﬁcant promotion in HT-29 and DLD-1 (A) There were generally mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell lines, with greater eﬀects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely suppressed by SP600125 in lower oxygen levels, with signiﬁcant reductions found in HT-29 and DLD-1 r different oxygen levels The key markers of JNK r different oxygen levels. The key markers of JNK K i d d l l l i HT r diﬀerent oxygen levels. The key markers of JNK Figure 5. JNK and Akt pathway activation under different oxygen levels. The key markers of JNK pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT- 29, DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29 and DLD-1 (C) The indirect downstream effector of JNK pathway p62 showed mild up-regulation in Figure 5. JNK and Akt pathway activation under diﬀerent oxygen levels. The key markers of JNK pathways were evaluated by Western blotting. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation g ( ) p 4 (DLD-1). JNK inhibitor suppressed p62 in DLD-1 and SW-480 and activated p62 in 10% O2 of HT-29 (Figure 5E). This evidence demonstrated the successful general JNK inhibition. relative expression compared with 21% O2 group, N = 9 (HT-29), 8–9 (DLD-1), or 10 (SW-480) (A,D,F). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 4–9 (HT-29), 5–9 (DLD-1), or 5–10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (B,C,E,G). * p < 0.05, p < 0.01, * p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). For the indirect downstream effector of JNK pathway, p62 had diverse changes under lower For Akt phosphorylation, all three cell lines showed general activation under lower oxygen levels with statistically signiﬁcant up-regulation in SW-480 (Figure 5F). For the parallel EMT-inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated Akt, with statistically signiﬁcant suppression found among HT-29 and SW-480 (Figure 5G). For the indirect downstream effector of JNK pathway, p62 had diverse changes under lower oxygen levels with general promotion at 10% O2 and general down-regulation at 1% O2 (Figure 5D). JNK inhibitor suppressed p62 in DLD-1 and SW-480 and activated p62 in 10% O2 of HT-29 (Figure 5E). This evidence demonstrated the successful general JNK inhibition. For Akt phosphorylation, all three cell lines showed general activation under lower oxygen levels with statistically significant up-regulation in SW-480 (Figure 5F) For the parallel EMT- 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated statistically significant suppression found among HT 29 and SW 480 (Figure 5G) 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A statistically significant suppression found among HT-29 and SW-480 (Figure 5G) Changes of SM markers of CSC, including Oct-4 and Nanog, in diﬀerent oxygen levels were also studied. Results revealed general promotion of both markers in lower oxygen levels with greater promotion of Oct-4, especially for HT-29 and DLD-1 (Figure 6A,B). Signiﬁcant up-regulations were found at Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the study of the eﬀect of JNK inhibition on Oct-4 and Nanog (Figure 6C), diverged results had been discovered in Oct-4 among the three cell lines. Oct-4 was generally suppressed by JNK inhibition among HT-29 and SW-480, while DLD-1 experienced slight up-regulation instead. In the meantime, Nanog was generally down-regulated by JNK inhibition among all three cell lines in 1% and 10% O2. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition Changes of SM markers of CSC, including Oct-4 and Nanog, in different oxygen levels were also studied. Results revealed general promotion of both markers in lower oxygen levels with greater promotion of Oct-4, especially for HT-29 and DLD-1 (Figure 6A,B). Significant up-regulations were found at Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the study of the effect of JNK inhibition on Oct-4 and Nanog (Figure 6C), diverged results had been discovered in Oct-4 among the three cell lines. Oct-4 was generally suppressed by JNK inhibition among HT-29 and SW-480, while DLD-1 experienced slight up-regulation instead. In the meantime, Nanog was generally down-regulated by JNK inhibition among all three cell lines in 1% and 10% O2 Figure 6. Cont. 9 of 16 Cancers 2020, 12, 224 Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight up-regulation in DLD-1 by SP600125 were found. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A statistically significant suppression found among HT-29 and SW-480 (Figure 5G) While for Nanog, general inhibition in 1% and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW- 480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO (0.1%), S: SP600125 (10 µM). 3. Discussion Figure 6. Oct-4 and Nanog expressions under diﬀerent oxygen levels. The key stemness maintenance markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower oxygen levels with signiﬁcant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1% and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO (0.1%), S: SP600125 (10 µM). Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). EMT h 3. Discussion the relatively stationary epithelial cell type to invasive mesenchymal cell type [2]. From the results of 48 h of incubation, lower oxygen levels (1% and 10% O2) induced morphological changes in CRC cells, and the cells became less organized as the cell–cell separation increased, especially for 1% O2. This demonstrated the loss of cell–cell contact, which is an initial step for EMT. In the presence of JNK inhibitor, dramatic changes of cell morphology were observed, and JNK inhibition could reverse the situation by inducing pro-epithelial cell morphology changes. Cancer cells undergoing EMT may have increased invasiveness and migratory power. The wound healing assay evaluated the impact of lower oxygen levels and JNK inhibition on the i t t f CRC ll B f th i t t f HT 29 l DLD 1 d SW 480 EMT has been regarded as an important event for metastasis as it transforms cancer cells from the relatively stationary epithelial cell type to invasive mesenchymal cell type [2]. From the results of 48 h of incubation, lower oxygen levels (1% and 10% O2) induced morphological changes in CRC cells, and the cells became less organized as the cell–cell separation increased, especially for 1% O2. This demonstrated the loss of cell–cell contact, which is an initial step for EMT. In the presence of JNK inhibitor, dramatic changes of cell morphology were observed, and JNK inhibition could reverse the situation by inducing pro-epithelial cell morphology changes. migratory rate of CRC cells. Because of the poor migratory rate of HT-29, only DLD-1 and SW-480 were analyzed. We did not detect a significant difference of relative migratory rate in different oxygen levels, probably due to the time limitation of this simple wound healing assay, which could only provide relatively accurate data within 24 h as cell division may affect the assay reliability, and EMT is a relatively slow process. Moreover, the high confluence of cell culture required in a wound healing Cancer cells undergoing EMT may have increased invasiveness and migratory power. The wound healing assay evaluated the impact of lower oxygen levels and JNK inhibition on the migratory rate of CRC cells. Because of the poor migratory rate of HT-29, only DLD-1 and SW-480 were analyzed. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A statistically significant suppression found among HT-29 and SW-480 (Figure 5G) For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1% and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW- 480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO (0.1%), S: SP600125 (10 µM). 3. Discussion Figure 6. Oct-4 and Nanog expressions under diﬀerent oxygen levels. The key stemness maintenance markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower oxygen levels with signiﬁcant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1% and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO (0.1%), S: SP600125 (10 µM). EMT h 3. Discussion We did not detect a signiﬁcant diﬀerence of relative migratory rate in diﬀerent oxygen levels, probably due to the time limitation of this simple wound healing assay, which could only provide relatively accurate data within 24 h as cell division may aﬀect the assay reliability, and EMT is a relatively slow process. Moreover, the high conﬂuence of cell culture required in a wound healing assay 10 of 16 Cancers 2020, 12, 224 could reduce the eﬀectiveness of EMT induction [17]. Nevertheless, the presence of JNK inhibitor reduced the migratory rate in diﬀerent oxygen levels, further supporting the notion that JNK promotes CRC migration. Apart from the macroscopic changes in cell morphology and migration, we also demonstrated EMT induction based on the cytoskeleton changes due to dynamic actin reorganization. Actin polymerization could occur by increasing F-actin formation to promote the formation of various migratory structures including lamellipodia, ﬁlopodia, and stress ﬁbers and the inhibition of migratory structure formation by JNK inhibitor. y Epithelial marker E-cadherin and mesenchymal markers ﬁbronectin and vimentin are important EMT markers. The loss of E-cadherin was conﬁrmed in all three CRC cell lines in 1% and 10% O2. Although hypoxia (1% O2) has been described in previous research as a factor of EMT induction, the signiﬁcant loss of E-cadherin in 10% O2 found in this study is a novel ﬁnding. E-cadherin expression was enhanced in the presence of JNK inhibitor with greater enhancement in 10% O2 and in HT-29. This suggests the general relieve of EMT induction by JNK inhibition. Moreover, the ineﬀective relieve in 1% O2 suggests the activation of other EMT-inducing pathways, such as the HIF pathway, while the predominant EMT-inducing pathway in 10% O2 is JNK-mediated. In line with the observation of EMT-related morphological changes, our results showed that ﬁbronectin was generally up-regulated under lower oxygen levels, especially in HT-29 and SW-480. While, for another mesenchymal marker, vimentin, its expression level was observable only in SW-480. This suggests that SW-480 had the highest mesenchymal status among the three cell lines studied. Results found that vimentin was generally up-regulated in 10% O2 but not for 1% O2. Vimentin is usually promoted by HIF-1α-HDAC3 in hypoxia [6], but this promotion was not seen in 1% O2 samples in this study. EMT h 3. Discussion It may be due to the short-lived nature of HIF-1α, while the promotion trend in 10% O2 may be due to another pathway that was activated at a low oxygen level. In the presence of JNK inhibitor, vimentin was signiﬁcantly suppressed. The trends of the results in mesenchymal markers coincided with E-cadherin results, as HT-29 achieved better relieve of EMT by JNK inhibitor. Moreover, the results suggested JNK mediation of vimentin. Various EMT transcription factors have been proposed among diﬀerent cancer types, including Snail, Slug, TWIST1/2, and zinc ﬁnger E-box-binding homeobox 1/2 (ZEB1/2), as they may aﬀect transcription of E-cadherin and other EMT markers [6]. Among the three cell lines studied, only Snail and TWIST1 could be detected in DLD-1 and SW-480 by Western blotting. This indicates the higher mesenchymal statuses of DLD-1 and SW-480 than that of HT-29. In HT-29, the low expression of Snail may be due to low expression of HDAC3 [18]. Snail binds to the promoter of CDH1 to repress E-cadherin transcription and therefore promote EMT [6]. The highest expression level of Snail in SW-480 among the three cell lines probably is due to the highest expression of HDAC3 [18,19], in which HDAC3 could promote Snail by HIF-1α-HDAC3-Snail pathway. From the results of this study, SW-480 had signiﬁcant up-regulation of Snail in both 1% and 10% O2, while DLD-1 only had substantial activation in 10% O2 but not for 1% O2. The diﬀerence of activation in 1% O2 mainly is due to the diﬀerence of activation between SW-480 and DLD-1 by HIF-1α-HDAC3-Snail pathway. Although HIF-1α-HDAC3-Snail may account for the activation of Snail in 1% O2, HIF-mediated EMT induction could not account for Snail up-regulation in 10% O2 and there should be another pathway leading to the up-regulation of Snail [20]. TWIST1 belongs to the basic helix-loop-helix (bHLH) transcription family, which is involved in cancer metastasis and flanks the CDH1 gene to repress E-cadherin [6]. TWIST1 is found to be a direct target of HIF-1α, rather than as an indirect target like Snail [6]. Our results demonstrated significant up-regulation of TWIST1 in both the HDAC3-deficient DLD-1 and HDAC3-activated SW-480. Similar to Snail, TWIST1 was also significantly up-regulated in 10% O2 for both DLD-1 and SW-480, demonstrating activation by another EMT-inducing pathway other than HIF-1α under low oxygen level conditions, as HIF-1α does not activate in 10% O2 [20]. EMT h 3. Discussion Both Snail and TWIST1 confirmed EMT induction in lower oxygen levels. Moreover, both Snail and TWIST1 were inhibited by JNK inhibition in both DLD-1 and SW-480. Cancers 2020, 12, 224 11 of 16 In this study, we have evaluated JNK pathway related proteins including JNK, its indirect down-stream regulator p62, and its possible up-stream regulator Akt. The JNK pathway is a possible delayed pathway that is activated in 10% O2 in light of our previous study [14]. Also, there was research showing that JNK signaling may contribute to Snail and TWIST1 activation by DNA methyltransferase 1 (DNMT1) or TGF-β1-induced EMT pathway by promoting ﬁbronectin and vimentin [21–27]. Our results demonstrated the general activation through hyperphosphorylation under low oxygen levels among all three cell lines, and this conﬁrmed the activation of JNK pathway among CRCs. p62 is an autophagy adaptor protein that may bind to various EMT regulators including TWIST1, mothers against decapentaplegic-4 (SMAD4), and vimentin [28–30]. Our results showed general down-regulation of p62 in 1% O2 while opposite results were found in 10% O2. Down-regulation of p62 in 1% O2 may be due to the promotion of autophagy under severe oxidative stress for cell survival [31], and this correlates with the lower expression of vimentin in SW-480 under 1% O2 versus 10% O2. While the up-regulation of p62 in 10% O2 corresponds to the activation of JNK-c-Jun-p62 and could contribute to the up-regulation of TWIST1 and vimentin in 10% O2. Consistent with the past study on the eﬀect of SP600125 on a variety of kinases and enzymes [32], SP600125 did not inhibit the phosphorylation of JNK but rather by inhibiting its downstream targets such as c-Jun. Results of p-c-Jun expression in the three cell lines demonstrated the eﬀective inhibition by SP600125, especially at low oxygen levels. The results of p-c-Jun and EMT transcription factors suggest the possible JNK-c-Jun-Snail/TWIST1 pathway for EMT induction under low oxygen levels. Moreover, ﬁbronectin and vimentin down-regulation by SP600125 found in HT-29 suggests possible activation of JNK-c-Jun-Fibronectin/vimentin pathway under low oxygen levels. While for the indirect JNK target p62, SP600125 could achieve signiﬁcant inhibitions for DLD-1 and SW-480 in diﬀerent oxygen levels. However, SP600125 could up-regulate p62 expression in HT-29 instead. The down-regulations of p62 and Snail/TWIST1 in DLD-1 and SW-480 suggest another possible JNK-mediated EMT-inducing pathway by JNK-c-Jun-p62-Snail/TWIST1 under low oxygen levels. EMT h 3. Discussion Akt, which is usually seen as an activator of EMT and an inhibitor of autophagy through phosphoinositide 3-kinase (PI3K)-Akt-Snail/Slug and PI3K-Akt-mechanistic target of rapamycin (mTOR) pathways, respectively, has also been regarded as a possible up-stream regulator of the JNK pathway in gastric cancer cells [33,34]. Our results showed that SW-480 had the most prominent up-regulation of Akt phosphorylation under lower oxygen levels, while HT-29 was less activated. This could be due to the diﬀerence in phosphatase and tensin homolog (PTEN) status among the three cell lines. PTEN is a tumor suppressor and can inhibit Akt activation [35]. As PTEN has the highest expression in HT-29 [36], this causes the weakened Akt activation in HT-29 as shown in the result of this study. SP600125 surprisingly caused hypophosphorylation of Akt, especially for HT-29 and SW-480, in which Akt was previously considered as a possible upstream regulator of JNK in previous research [33]. This suggests a novel feedback mechanism of JNK-c-Jun-Akt for further promotion of EMT as a positive feedback mechanism and inhibition of autophagy through Akt-mTOR pathway as negative feedback mechanism by preventing simultaneous stimulation of autophagy through Akt-mTOR and JNK pathways. There are numerous cancer stemness markers proposed in previous studies including pluripotency markers Oct-4, Nanog, and SOX2, which are considered as poor prognosis indicators of CRC [37,38]. Also, JNK may inﬂuence these markers through Wnt/BMP-TCF/Notch or IL-6-JAK-Stat3 pathways [15]. Oct-4 is the primary transcription factor required for stemness properties in CSCs. Overexpression of Oct-4, together or separately with other pluripotency markers such as Nanog, leads to tumor metastasis and recurrence in diﬀerent cancer types [39]. From the results, both Oct-4 and Nanog were generally stimulated under lower oxygen levels, with HT-29 and DLD-1 having more prominent up-regulation. This veriﬁed both 1% and 10% O2 as possible stimulants of SM of CRC cells. In the presence of JNK inhibitor, the low oxygen level driven general up-regulations of Oct-4 and Nanog among the three cells lines were partially inhibited, except for Oct-4 in DLD-1. This suggests the importance of JNK in SM under low-oxygen environments, which could sequentially aﬀect CSCs and the related tumorigenesis Cancers 2020, 12, 224 12 of 16 12 of 16 processes such as metastasis and radiosensitivity. The possible pathway could be Wnt/BMP-TCF/Notch or IL-6-JAK-Stat3 pathways as suggested in previous studies [15], which could be further conﬁrmed in future studies. EMT h 3. Discussion Combining the results of this study, we showed the hypoxia-driven EMT induction/SM and proposed 10% O2 as another possible condition for EMT induction and SM among CRC cells. The blood oxygen level is about 10% in general. This study suggests that the regulation of EMT/SM and its related tumor progression events such as metastasis could be triggered by the oxygen level in blood. Moreover, the partial relieve of EMT induction and SM promotion by JNK inhibitor was demonstrated, and this suggests the role of JNK pathway mediation of EMT under low oxygen levels. Additionally, as the majority of CRC cells usually have null SMAD4 expressions in microsatellite instability (MSI)-negative CRC cell lines, including HT-29 and SW-480, but not for MSI-positive cell lines such as DLD-1, and this depends on SMAD-independent pathways such as JNK pathway [40,41], the JNK pathway has unique importance in EMT induction of CRC. Furthermore, this study proposes a novel pathway on the regulation of Akt by JNK-c-Jun, which could further control EMT and autophagy in CRC cells under low oxygen conditions. Inhibition of JNK is a potentially rewarding strategy for inhibiting EMT progression and SM in CRC cells. Further investigations with in vivo and clinical studies are recommended to establish JNK inhibition as a possible strategy to limit metastasis in CRC. 4.3. Cell Morphological Analysis HT-29, DLD-1, or SW-480 cells (0.5 M) were seeded in T25 ﬂasks. After overnight settling and 24 h serum starvation, cells were incubated in diﬀerent O2 levels for 48 h with or without JNK inhibitor SP600125 (10 µM). The eﬀect of oxygen level in EMT induction was evaluated by photomicrographs taken by light microscopy with 200× magniﬁcation. The cell morphology was visually compared and analyzed in 5 independent experiments. 4.1. Cell Lines and Culture Conditions Human colorectal adenocarcinoma cell line HT-29 was purchased from PerkinElmer, Inc. (Waltham, MA, USA). Human colorectal adenocarcinoma cell lines DLD-1 and SW-480 were obtained from Professor Jun Yu, Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong (Hong Kong). All cells were cultured in DMEM medium with GlutaMAX supplement and HEPES and supplemented with 10% fetal bovine serum (FBS). 4.2. Hypoxic Condition and JNK Inhibition The hypoxic and blood oxygen condition was maintained by the SCI-tive Dual chamber hypoxia workstation (Baker Ruskinn, Sanford, ME, USA) at 1% and 10% O2 with 5% CO2, 37 ◦C, and 100% humidity respectively. Cells cultured in normal incubator at an atmospheric oxygen level of 21% were used as the control. For JNK inhibition, cells were treated for 48 h with 0.1% DMSO (control) or 10 µM of JNK inhibitor SP600125 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1% DMSO as titrated in a preliminary experiment [32]. 4.6. Western Blotting The cell density, incubation condition, and JNK inhibition were the same as that of the cell morphological analysis. Protein extraction and gel electrophoresis were performed as described in our previous paper [14]. The primary antibodies used are listed in Supplementary Table S1. The relative protein expression of each sample was evaluated by ImageJ software with β-Actin as loading controls. At least 4 independent sets were performed for each set of protein expression analyses. (All original western blot ﬁgures can be found in Supplementary Figures S2–S4) 4.5. Immunoﬂuorescence Staining Fifty thousand HT-29, DLD-1, or SW-480 cells were seeded in 24-well plates with coverslips inserted. The serum starvation and incubation under diﬀerent oxygen levels with or without JNK inhibitor SP600125 was the same as that of the cell morphological analysis. After incubation, cells were ﬁrst gently washed by phosphate-buﬀered saline (PBS) for three times, then ﬁxed with 4% paraformaldehyde (PFA) in PBS for 30 min. After washing by PBS for another three times, the cells were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. Alexa Fluor 594 Phalloidin (Thermo Fisher Scientiﬁc, Waltham, MA, USA) was added to the cells for staining F-actin in 1 h. Cells were mounted with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientiﬁc) after PBS washing. Cells and their cytoskeletons were visualized by confocal microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany). The eﬀect of oxygen level on cytoskeleton was quantiﬁed in terms of lamellipodia, ﬁlopodia, and ventral stress ﬁbers. At least 200 cells were scored in each of the three independent experiments, and the percentages of cells having the three cytoskeleton structures of diﬀerent conditions were plotted and compared. 4.4. Cell Migration Assay Wound healing assay was employed to assess cell migration properties. Thirty-five thousand HT-29, DLD-1, or SW-480 cells were seeded in each compartment of a Culture-Insert 2 Well in µ-Dish 35 mm (Ibidi LLC, Munich, Germany). After overnight settling and 24 h serum starvation, the wound gap was made by removing the silicone insert. After that, fresh culture medium was added with or without JNK inhibitor SP600125 (10 µM), and cells were allowed to migrate under different oxygen levels for 24 h. Photomicrographs were taken before and after incubation. The gap area was measured using the MRI Cancers 2020, 12, 224 13 of 16 13 of 16 Wound Healing Tool macro for ImageJ software (NIH) (http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/ Wound_Healing_Tool). The invasion rate was calculated as the relative gap area difference between 0 and 24 h against the control among at least 3 independent experiments. 4.7. Data Analysis Diﬀerent statistical tests have been used in analyzing the data. In general, values were plotted as mean ± SEM. Comparisons of means between independent groups were conducted by Student’s t test (2 groups) or Kruskal–Wallis one-way ANOVA (3 or more groups) with pairwise comparison. Comparison of mean between paired groups was conducted by paired t tests. Statistical analysis was conducted by Statistical Product and Service Solutions (SPSS) version 22 (IBM Corp, Armonk, NY, USA), and signiﬁcance level of p < 0.05 was considered as statically signiﬁcant. Author Contributions: Conceptualization, S.Y.T. and H.K.W.L.; Data curation, S.Y.T.; Formal analysis, S.Y.T.; Investigation, S.Y.T.; Methodology, S.Y.T. and H.K.W.L.; Resources, H.K.W.L.; Supervision, V.W.C.W. and H.K.W.L.; Validation, S.Y.T. and H.K.W.L.; Visualization, S.Y.T. and H.K.W.L.; Writing—original draft, S.Y.T.; Writing—review & editing, V.W.C.W. and H.K.W.L. All authors have read and agreed to the published version of the manuscript. 5. Conclusions It is clear that JNK, JNK pathway proteins, and transcription factors play important roles in EMT induction and SM of CRC cells under low-oxygen levels through multiple factors. Moreover, this study proposes a novel pathway of JNK-Akt serving as further control of EMT and other tumor progression processes such as autophagy. Thus, we suggest inhibition of JNK as a promising way for inhibiting EMT progression and SM in CRC cells. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/224/s1, Figure S1: Preliminary experiment of HT-29 wound healing assay; Figure S2: Original Western blots of Figure 4; Figure S3: Original Western blots of Figure 5; Figure S4: Original Western blots of Figure 6. Table S1: List of primary antibodies used in Western blotting. Author Contributions: Conceptualization, S.Y.T. and H.K.W.L.; Data curation, S.Y.T.; Formal analysis, S.Y.T.; Investigation, S.Y.T.; Methodology, S.Y.T. and H.K.W.L.; Resources, H.K.W.L.; Supervision, V.W.C.W. and H.K.W.L.; Validation, S.Y.T. and H.K.W.L.; Visualization, S.Y.T. and H.K.W.L.; Writing—original draft, S.Y.T.; Writing—review & editing, V.W.C.W. and H.K.W.L. All authors have read and agreed to the published version of the manuscript. 14 of 16 14 of 16 Cancers 2020, 12, 224 Funding: This research was supported by a Postgraduate Studentship to S.Y.T. and the Departmental Seeding Fund for H.K.W.L. from The Hong Kong Polytechnic University. Acknowledgments: We would like to acknowledge Jun Yu, Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong, for the provision of DLD-1 and SW-480 cell lines. We would also like to acknowledge the Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, for the provision of the hypoxia chamber. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References 1. Guglielmo, A.; Staropoli, N.; Giancotti, M.; Mauro, M. Personalized medicine in colorectal cancer diagnosis and treatment: A systematic review of health economic evaluations. Cost Eﬀ. Resour. Alloc. 2018, 16, 2. [CrossRef] [PubMed] 2. 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https://openalex.org/W2732219435	https://educationaltechnologyjournal.springeropen.com/track/pdf/10.1186/s41239-017-0067-9	English	null	A proficient and versatile online student-teacher collaboration platform for large classroom lectures	International journal of educational technology in higher education	2,017	cc-by	6,558	Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 DOI 10.1186/s41239-017-0067-9 RESEARCH ARTICLE Open Access Open Access A proficient and versatile online student-teacher collaboration platform for large classroom lectures ABM Tariqul Islam* , Jonas Flint, Philipp Jaecks and Clemens H. Cap *Correspondence: tariqul.islam.cse@gmail.com M.Sc in Computer Science, Universitat Rostock, Rostock, Germany Abstract The popularity of online collaboration on lecture content has been growing steadily over the last few decades because of its potential to enhance the overall learning experience. We propose a didactical approach of online collaboration where the students and the teachers can collaborate seamlessly on the lecture contents. The approach, which we call Multiscript (MS), offers two methods of online learning on one collaboration platform. In MS, we call one method the outside of class Multiscript (OMS) and another, the inside of class Multiscript (IMS). OMS is a form of distance online learning where the students can collaborate on the lecture contents while being outside of class, whereas IMS allows online collaboration among the students and the teacher during the lecture. In OMS, the teacher can share the slides along with audio annotations for each lecture slides and/or a single recorded audio for the whole lecture. The students can access the slides and discuss (via text and audio chat) with their fellow classmates about the slides and annotate them, post feedback about the slides and ask questions to the teacher directly via MS. In IMS, the students can create annotations for the slides and post feedback to the teacher about the slides. We design MS in such a way that it can be accessed by using just a web browser on any PC, tablet or mobile device. Keywords: Collaborative learning, Online learning, Open learning, E-learning, Remote collaboration, Distance education © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source provide a link to t Introduction Over the past few decades, the interest about online collaboration on classroom lecture content has been growing steadily among researchers (Er, Özden, & Arifo˘glu, 2009). This is happening primarily due to the prospect of online collaboration in enhancing the over- all learning experience. Collaboration on lecture content can take place mainly in two forms – face-to-face(F2F) collaboration where the students and the teacher collaborate by being in the same location while the lecture is going on, and online collaboration where the participants take part in remote collaboration on the lecture contents via online plat- forms. Online collaboration can be further categorized into two groups – synchronous and asynchronous collaboration. The learning activities and expectations of synchronous online collaboration are similar to those found in F2F collaboration. In synchronous col- laboration, typically the discussions and lectures on lecture contents occur at a certain time with the expectation of the participation of all the learners and the teacher. Whereas, Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 2 of 13 Page 2 of 13 in asynchronous online collaboration, the learners participate in the discussion with their peers on the lecture contents uploaded by the teacher, interact with their peers, deliver peer feedback, and reflect on the status of their own learning plans and outcomes (Er et al., 2009; Harris, Mishra, & Koehler, 2009; Simonson, Smaldino, Albright, & Zvacek, 2012). However, asynchronous online collaboration has grown to be a more popular learning type because of the the lower cost of the learning tools, requirement of minimal hard- ware, and it is used at the student’s pace (Meloni, 2010). In this paper, we refer to online collaboration as asynchronous online collaboration. While F2F collaboration typically has undeniable impact on learning experience, research, for example: (Swan, Shen, & Hiltz 2006; Strijbos, 2011), have found that the students consider online collaboration more autonomous than F2F collaboration due to the equal opportunity it presents to all the students for expressing opinions or asking questions about certain contents. Besides this, online collaboration provides the students with an opportunity to foster certain diligence and deliberation about their peer’s contri- bution (e.g. feedback or questions) about a lecture while they are developing their own comments (Richardson & Swan, 2003). Related work In this section, we firstly present an overview of the implications and importance of online collaboration on lecture contents and then, we present an overview of existing works on online collaborative learning. In an online classroom environment many of the learning activities and expectations are similar to those found in a traditional classroom (Harris et al., 2009; Hrastinski, 2008; Simonson et al., 2012). However, online learning provides some added benefits over traditional classroom learnings (Martnez-Caro, 2011). Online learning environ- ments provide the students and teachers with different approaches to interact, share, and offer them the ability to collaborate and ask questions in real-time through available learning technologies. Research, for example: (Díaz & Entonado, 2009; Er et al., 2009), indicate that the higher the students perceive the level of collaboration the more sat- isfied they become with the online learning environment. In both the traditional and online classroom environments, interaction and collaboration are identified as a major factor in successful learning outcomes (Bonk & Zhang, 2006; Martnez-Caro, 2011). Online learning environments provide flexibility and offer the students personalized learning opportunities which are achieved through feedback and interactive collaboration (Lorenzo & Ittelson, 2005). Here, the students can autonomously express their thoughts (Er et al., 2009). In case of both synchronous and asynchronous collaboration, research indicates that such collaboration enhances the overall student learning capability (Díaz & Entonado, 2009; Er et al., 2009). Through such collaboration, the students develop a sense of com- munity which is essential to sustain the educational experience over time (Garrison & Kanuka, 2004). In case of asynchronous collaborative learning, the students possess the opportunity to reflect on the learning objectives since they have the time to criti- cally synthesize their learning (Bonk & Zhang, 2004; Garrison & Kanuka, 2006). Besides this, through asynchronous learning technologies, a teacher can assess what students understand about the content and can adapt future course contents to facilitate a higher level and more in-depth understanding of the content (Bonk & Zhang, 2006; Hrastinski, 2008). The most significant tool which asynchronous collaboration provides is a learning space where learners can argue on debatable ideas in more objective and reflective ways (Garrison & Kanuka, 2004). Blended learning is the thoughtful integration of classroom face-to-face learning experi- ences with online learning experiences (Garrison & Kanuka, 2004). What makes blended learning particularly effective is its ability to facilitate a community of inquiry. Introduction International Journal of Educational Technology in Higher Education (2017) 14:29 Page 3 of 13 unified online collaboration environment. Therefore, MS accommodates the attributes of a blended learning approach (Poon, 2013) where a convergence of F2F meeting and dis- tance learning takes places via available technology. In addition to this, MS enhances the F2F meeting part of blended learning by incorporating the IMS functionalities into MS. We design MS in such a way that it can be accessed by using just a web browser on any available PC, tablet or mobile device. Introduction Research, for example: (Swan et al., 2006), about the impact of online collaboration stipulates that the aspect in which the students appre- hend the role of their colleagues, perceive their social presence and foster certain feelings for the collaborating community, seemingly influences the learning activity. Research, for example: (Richardson & Swan, 2003; Swan et al., 2006), also shows that online collabo- ration among the students and the teacher indeed plays an important role in stimulating the overall learning process by enrolling the students into a pleasant, autonomous and socially well-perceived medium. We can classify existing research on online collaborative learning into two principle types: distance online collaboration (DC) or remote collaboration, for example: (Brindley, Walti, & Blaschke, 2009) and in-class online collaboration (IC), for example: (Anderson et al., 2006). In case of DC, the participants usually stay at remote and geographically distinct places and create small groups to collaborate on certain tasks. On the contrary, IC normally occurs in class, between the students and the teachers, on lecture materials or on group projects. Normally, different types of devices such as PCs, Tablets or mobile phones are used to initiate DC or IC. To our knowledge, the existing online collaboration platforms on classroom lecture materials support either of these two types; we have not encountered any platform which completely possesses the functionalities of both types of collaboration in a single platform. Moreover, the existing platforms require various softwares and plugins to initiate and support the collaboration. In this paper, we propose a didactical approach of online collaboration, called Mul- tiscript (MS), which combines two online learning methods into one collaboration platform. We call our first approach outside-of-class-Multiscript (OMS) which is a form of distance online learning and the second approach inside-of-class-Multiscript (IMS) which occurs while the lecture is going on. OMS is suitable for the situations when the participants want to collaborate on the lecture materials after the lecture is over. It might happen that after attending the classroom lecture, the students still have some questions in mind which they want to clarify by collaborating with their peers, or they were absent from class due to a medical issue or traveling purpose. Whereas, IMS can be used by the students and the teacher to collaborate on the lecture content during the class. To our knowledge, MS is the first approach which supports both of these methods in one Islam et al. Related work Commu- nity furnishes the stabilizing and cohesive impact that balances the open communication and unlimited access to information on the web (Garrison & Kanuka, 2004). Our proposed MS accommodates the attributes of a blended learning approach where a convergence of F2F meeting and online learning takes places via available technology. Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 4 of 13 We mention in the previous section that existing works in this area can be classified into two main types – distance online collaboration (DC) and in-class online collaboration (IC). In case of DC, several research, for example: (Brindley et al., 2011; Strijbos, 2006; Swan et al., 2009), has been pursued which investigates the effect of collaborative learning, among a group of participants, on a specific project or homework. Furthermore, there are quite a few tools available which support DC for different purposes for different user groups. Groupboard (2016) is one such DC platform which allows participants to collaborate on a certain topic through text, audio and video chats. The collaboration in Groupboard doesn’t occur directly on lecture slides because the participants don’t have access to the slides; so they collaborate mainly on certain topics from the lecture delivered by the teacher. It also has an IC part with very limited functionality. Vyew (2016) is another DC tool which is used mostly for collaborating on group projects or homework. Vyew has the biggest similarity to our OMS approach in terms of functionality. In Vyew, teach- ers can upload lecture materials for real-time presentations and then students can meet online and collaborate together in real-time or separately over time. Here, users can create annotations on slides and post suggestions about a lecture content to the teacher via text chat. A few other notable DOC tools are Scribbler (2016), Twiddla (2016), RealtimeBoard (2016), GoToMeeting (2016) and WebEx (2016). Most of these platforms support a form of collaboration between a group of participants on specific projects such as artworks or text materials. There are also quite a few IC platforms which support collaboration among the stu- dents and the teacher during the class. The authors, in (Anderson et al., 2006), present Classroom Presenter which is an IC platform where the students submit their answers of the provided questions via wireless connectivity. Description of the approach We previously mention that we propose MS as a didactical approach which brings two online collaboration approaches (outside-of-class-Multiscript (OMS) and inside-of-class- Multiscript (IMS)) into a single platform. The basis of MS is the didactical aspect i.e. more own initiative of the students and more chances for student peer interaction, which even- tually motivates the design of our MS platform. We refer MS as a platform rather than an application because it combines the tasks of different applications (e.g. recording the audio lectures, maintaining student’s annotations and feedbacks on the slides, supporting audio and text chats on the lecture contents, fetching content feedback from a classroom response system called Tweedback (Vetterick et al., 2013) to deliver the final task. A lec- ture content typically contains a set of slides, optionally extended by private annotations of the student and public audio annotations of the teacher. The teacher can provide the audio annotations to explain a specific topic in detail. The teacher has the option to record the audio during the lecture presentation; the audio will be automatically attached to the relevant slide set. Besides this, if the teacher feels appropriate, s/he can add audio snippets and recording of the discussions of the students (in case they agree). Below, we present the main features which MS contains to support the online collabo- ration. There are two main categories of features – one is for the teachers and the other is for the students which we refer here as shared features. Features for teacher only • Upload and manage slide sets • Attach audio files to a single slide • Present the slides on Multiscript - Automatically sync the projected slides to the screens of the listeners - Different information, e.g. the number of listeners - Integration of the class room response system Tweedback (Vetterick et al., 2013) Related work Upon receiving the answers from the students, the teacher evaluates the responses, and randomly selects a few to annotate and display them simultaneously on a display medium. In Classroom Presenter, the students and the teacher use stationary Tablet PCs which are connected wirelessly. Therefore, it does not support web based connectivity and hence, it fails to reach a wide range of users to interact with the system. It also has the limitation of providing a reliable communica- tion platform. Besides this, Classroom Presenter might also suffer from the consequences of the loss of attention of the students and, the specific use of a single device limits the use of technology which could be used in various other ways. Furthermore, much research has been pursued in the field of Classroom Response Sys- tems (CRSs), for example: (Bruff 2009; Draper, Cargill, & Cutts, 2002; Kay & LeSage, 2009), which can be defined as a subset of IC platforms. Despite the fact that the CRSs are mostly not connected to the teacher’s slides, they provide wide possibilities to gather learners’ feedback. Over the years two main feedback types have been established: learn- ers can post messages to the audience and multiple choice questions answered by learners. Whereas both of these types are widely implemented into learning platforms, only a few platforms, for example: (Vetterick, Schwennigcke, Langfeld, Cap, & Sucharowski, 2014), preserve the feedback, gathered by these feedback types, for later use. Multiscript has the ability to collect IC feedback while presenting it later on in its OMS functionality. Another IC platform, namely Ubiquitous Classroom Response System, is presented by Caceffo and Da Rocha in (Caceffo & Da Rocha, 2011). This approach has the most similarity to our IMS approach. Its working procedure has certain resemblance to Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 5 of 13 the Classroom Presenter, however it manipulates the context information to devise a dynamic environment where students can utilize any available mobile device to respond to the teacher’s questions. In addition to this, it does not have the shortcomings of the Classroom Presenter since it works via web based connection for supporting the collaboration. Another similar approach to this system is presented by Oura et al. in (Oura, Kato, & Akahori, 2006). Shared features • Listen to audio files for more information • Annotate the slides with text and mark them with predefined tags to group them • Text chat (in every situation) • Annotate the slides with text and mark them with predefined tags to group them • Text chat (in every situation) • Audio chat (in outside of class) - Synchronize the slides between other audio chat users (like a small “in-class” situation among learners) Besides these, the following features are currently in development: Page 6 of 13 Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 • Consultation hour (see “Consultation hour” section) • Notes directly on the slides • Audio stream of a live session • Public annotations for a slide to group similar questions/notes, that can also be answered/approved by the teacher • Record a live session (in class) and automatically assign the recording to the presented slides (with a possible replay of the complete session) Here, we respectively describe the idea behind devising OMS and IMS and the functional requirements to support their working process. In OMS, the students are able to seamlessly collaborate, via web connection, with their peers on the lecture slides uploaded by the teacher. Here, a teacher, after delivering a lecture, can share the lecture slides by uploading them to the MS server. The students can access the lecture slides on any available PCs, tablets, notebooks or mobile devices at any time just by using a web browser. While sharing the lecture slides, a teacher can attach audio annotations for each of the slides and/or attach a single audio for the whole lecture. So, the students can listen to the audio annotations for each slide while studying the slides and/or go through all the slides while listening to the entire lecture audio in the background. Figure 1 shows an illustration of OMS mode. In OMS mode, the students are offered four different kinds of features, they can: • Perform text chats with their peers • Participate in audio chats with their peers • Annotate the slide for their own purpose • Post feedback about the slides and ask questions to the teacher For the last case, the feedback and the questions are sent to the teacher as a ‘newslet- ter’ and the teacher, along with providing appropriate response, can also rate the feedback/questions. Shared features The teacher is also able to provide reason for his/her voting. In IMS, the students can collaborate on the lecture content while being in the classroom. Here, the students can create annotations on the slides for themselves and post feedback Fig. 1 Illustration of two collaboration platforms (OMS and IMS) of proposed Multiscript (MS) platform – the left and the right part illustrate the OMS and IMS approaches respectively Fig. 1 Illustration of two collaboration platforms (OMS and IMS) of proposed Multiscript (MS) platform – the left and the right part illustrate the OMS and IMS approaches respectively Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 7 of 13 about the slides which is read by the teacher later. The teacher can respond to the feedback and rate it similar to OMS mode. This mode is illustrated in Fig. 1. While the teacher delivers a lecture in IMS, the slides are shown in three different modes: • via the projector in the classroom • on the browser of a student’s device • on his own computer’s screen In the second case, the slide displayed on a student’s device is synchronized with the projector’s current slide. In the third case, the teacher’s screen shows a few additional fea- tures along with the slides. S/he can see the currently selected slide, a preview of the other slides and some notes for herself. The teacher can also control, from his/her machine, the number of collaborating options which will be available on the students’ devices. Apart from the functional features described above, Multiscript (MS) has a few other features which are designed for providing the best possible user experience. One of such features is the fast interaction experience of the users with MS. We develop a robust com- munication platform within MS so that the interaction with MS is fast and responsive enough to meet user’s satisfaction. Besides, since the users consider a communication platform as a comfortable one only when the user-interface is easy to use and has a min- imalist design, we preserve these qualities within MS. We also consider the fact that the interface of a robust communication platform like MS should have a flexible interface which fits on different screen sizes of different devices (e.g., PCs, Tablets, mobile phones). Shared features We develop the MS platform in such a way that it can be displayed intuitively on various screen sizes on a range of devices. Besides this, while designing the MS platform, we have considered the age of the users. We kept the design simple enough so that people with minimum knowledge about Inter- net can familiarize themselves with MS with minimum effort. Moreover, MS has the capability to handle quite a large number of users at a time. In future, it might be used by a large number of users, so we have considered the access peak of the system when many users start to use MS at once. The scalability of MS allows large number of connec- tions simultaneously. Besides this, we keep the identity of the students anonymous in MS, so that they can avoid the account creation process and collaborate autonomously with their peers. We have designed MS to be modular so that, in future, new features can be integrated easily into it. Form of collaboration in Multiscript We have designed MS to be a collaboration platform for the learners and the teachers. Here, we explain which type of collaboration we consider while designing the MS plat- form. We consider the following three different situations about how the collaboration can occur. Feedback from the participants As stated in the related work section, IC feedback can be gathered in two ways: learners post messages to the audience and multiple choice questions answered by learners. While the first one is covered by the previous chapter, the second one adds a huge benefit to the teachers’ use of Multiscript. As used in Classroom Response Systems, Multiscript (MS) also offers a function for the teachers to ask multiple choice questions (“quiz”). Teachers are able to spontaneously create a quiz whenever they intend to get a picture of the overall understanding level of the students. Within MS, teachers can create this “bigger picture” by asking a set of questions with predefined answers to the students and then, judging the responses of those questions upon receiving from the students; here, the students have to map their knowledge and understanding into the given answers. Multiscript, after collecting all the students’ answers, summarizes and present them to the teacher. Furthermore, Multiscript preserves these summaries (quiz results), so the teachers, on the one hand, can revise their teaching material and methods afterwards to improve their teaching. On the other hand, students can revise their knowledge, understanding and pitfalls afterwards to prepare better for the exam. Consultation hour Here, the teacher provides a consultation hour for the students. Now, we can think of two scenarios – either the teacher provides a fixed time (e.g. 1 p.m. to 3 p.m.) or a more spontaneous approach is taken. The first scenario allows for a remote version of the usual consultation, but allows all participants to operate from their current location, removing the need for traveling. In the second scenario, the teacher is working on MS and notices that some students are learning on a specific slideset. Due to the text chat or some other form of notification, the teacher notices that there is an interesting discussion going on. Therefore, the teacher decides to join the discussion and invites the student to an audio session to discuss the problem in detail. Additionally, to the existing features like the slides and the text/audio chat, the teacher will get the possibility to draw on a blackboard-like element to write down formula, explanations etc. in an easy way. Spontaneous meeting among students Here, we consider a spontaneous learning situation. It might happen that some students are learning on MS and one has a question regarding a difficult topic. So, the students can take advantage of MS and resolve their respective queries. Planned meeting among students This situation can be described by a group of students, that made an actual appoint- ment on MS. Using other communication services (email, messengers etc) all students made an appointment, e.g. at 3 p.m. to meet on MS Slide Set “Lecture01” by Prof. X. The students don’t have to find each other because they already have a specific meeting point. Page 8 of 13 Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Architecture of the Multiscript platform Since we plan to support a wide range of devices (with varying screen size and oper- ating system), rather than developing individual app for each cases, we develop a web application, which serves HTML, for the MS platform. Since most of the devices (sta- tionary or mobile) possess the capability to render HTML5, Javascript and CSS (Kamboj & Gupta, 2012), we develop one browser-based client interface which intuitively fits on these devices. This removes the necessity to maintain different repositories for different versions (mobile phone, desktop, iOS, Android etc.) of the proposed MS plat- form. Moreover, we also consider an easy and minimal design for the user interface. Hence, to achieve a unique development platform and user friendly interface design, Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 9 of 13 we choose to use established frameworks for the web-based user interface. We use Express and SocketIO framework, Node.js, Jade and AngularJS for building the web application of MS. While designing the user interface of MS platform, we decide to subdivide the interface into different views in such a way that every feature has a separate view of its own. The teacher’s view on the MS interface contains a number of control- ling options by means of which s/he can control which feature the students are able to see on their own machines. Figure 2 outlines the technology stack of MS. In MS, we allow anonymous participation of the students so that the peers and the teacher cannot trace back to the user. To achieve this, we remove the necessity to register an individual with a university account, rather we allow everyone. Neverthe- less, we define certain thresholds to prevent any misuse of the MS platform with an anonymous account. Performance, scalability and responsiveness are the factors which matter most for a good web application, and yet, these are the requirements which are quite difficult to achieve. To develop a stable and fast web application for MS, we looked into numerous solutions for asynchronous web servers which would work with two-way communica- tion channel in tandem. We decide to use the Node.js as our web server. It uses V8, the Google Chromes JavaScript engine. It is event-driven, stable and has the capability to perform thousands of concurrent connections. Architecture of the Multiscript platform For MS, a two-way communication chan- nel is necessary, because both, the server and the client, send messages to each other. In MS, the client is the browser of both the students and the teacher. We use the SocketIO library as the two-way communication layer which is well implemented for Javascript and for the Node.js web server. To support audio chat in MS, we use WebRTC. We choose to use MongoDB, a non-relational database which possesses flexible scheme to store all permanent data. Data layer MongoDB Browser layer Angular JS SocketIO jQuery Business layer Node.js SocketIO Mongoose Express Req. Jade Websocket:realtime HTTP:static Fig. 2 Technology stack diagram of Multiscript platform Req. Fig. 2 Technology stack diagram of Multiscript platform Fig. 2 Technology stack diagram of Multiscript platform Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 10 of 13 Page 10 of 13 Fig. 3 Data flow within the Multiscript platform Fig. 3 Data flow within the Multiscript platform Data flow In MS, the users are assigned hash values as UserID and a lecture content is assigned a unique LessonID. The data flow in MS are initiated – either by the teacher or by the students. Figure 3 shows the data flow diagram within MS platform. When the teacher uploads lecture content (called a Slideset) with audio annotations, an upload command is executed within MS. As a result, a corresponding LessonID is generated for the Slideset, and subsequently, the Slideset with audio annotations and LessonID are sent to the MS server to be stored. When a student selects a Slideset from the list of displayed Slidesets, a request is sent to the MS server to fetch the Slideset. Upon getting the request, the MS server fetch the audio annotations by the teacher content of the selected slide text and audio chat area annotation area for the students currently selected slide slide no. user settings Fig. 4 OMS collaboration window Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 11 of 13 start presentation on projector audio annotation area for teacher Fig. 5 IMS collaboration window Slideset and associated annotations (if already existed) for that particular student and send them to the students browser. When the students create new annotations and/or feedback for a particular slide, those are sent (along with UserID and LessonID) to the MS server to be stored. Slideset and associated annotations (if already existed) for that particular student and send them to the students browser. When the students create new annotations and/or feedback for a particular slide, those are sent (along with UserID and LessonID) to the MS server to be stored. Slideset and associated annotations (if already existed) for that particular student and send them to the students browser. When the students create new annotations and/or feedback for a particular slide, those are sent (along with UserID and LessonID) to the MS server to be stored. Competing interests Th th d l th t Competing interests The author declares that they have no competing interests. Development status Currently, as of April 2017, we have completed the development of most of the features for both OMS and IMS mode, except the feedback feature (currently under development) for the students. The current version of MS is accessible at https://ms.twbk.de/. An overview of the OMS and the IMS modes, and the outlook of teacher’s window when the lecture is being presented via the projector is depicted in Figs. 4, 5 and 6 respectively. Figure 4 depicts that the students can look into the slide contents while listening to the audio anno- tations, collaborate anonymously with their peers via audio and text chat, and annotate presentation is running on projector number of users joined on this lecture running time for the slide Fig. 6 IMS window – teacher’s window while the presentation is running on projector Fig. 6 IMS window – teacher’s window while the presentation is running on projector Islam et al. International Journal of Educational Technology in Higher Education (2017) 14:29 Page 12 of 13 the slides for themselves. Figure 5 shows that the teacher can start the presentation on the projector from his/her device while his/her screen shows different options (e.g., pre- view of current and other slides, audio annotation area). S/he can write notes about the slides or the lecture for him while s/he is presenting the slide to the students. Figure 6 depicts the teacher’s window when s/he has started the lecture and the slides are being displayed on the projector. It shows different real-time statistics such as how many users have joined the lecture via MS, running time of the lecture slides etc. Publisher’s Note Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 5 December 2016 Accepted: 26 June 2017 References Anderson, R., Chung, O., Davis, K. M., Davis, P., Prince, C., Razmov V, ...Simon, B. (2006). Classroom presenter – a classroom interaction system for active and collaborative learning. In Proc. Workshop Impact of Pen-Based Technology on Education (WIPTE). Bonk, C. J., & Zhang, K. (2006). 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(2002). Electronically enhanced classroom interaction. Australian journal of educational technology, 18(1), 13–23. Er, E., Özden, M. Y., & Arifo˘glu, A. (2009). A blended e-learning environment: A model proposition for integration of asynchronous and synchronous e-learning. International Journal Of Learning, 16(2), 449–460. Er, E., Özden, M. Y., & Arifo˘glu, A. (2009). A blended e-learning environment: A model proposition for integration of asynchronous and synchronous e-learning. International Journal Of Learning, 16(2), 449–460. Conclusion Online collaboration on lecture content has gained much popularity due to its potential in enhancing the learning process. We propose a didactical approach of online collabo- ration, called Multiscript (MS). MS offers a unique collaboration platform in which the participants can collaborate, using just a web browser, both while being inside of class and outside of class. MS is optimized to be used on different devices with varying screen size. We design the overall MS collaboration platform to be fast, responsive and scalable, and keep the user interface easy and simple to meet user demand and satisfaction. We allow anonymous access for the students to persuade an autonomous participation during col- laborating with peers or the teacher. We have already started testing the usability of the MS platform for the computer science lecturers at our university. MS can also be used in every other study area. Authors’ contributions All authors read and approved the final manuscript. Authors’ contributions All authors read and approved the final manuscript. References Garrison, D., & Kanuka, H. (2004). Blended learning: Uncovering its transformative potential in higher education. The Internet and Higher Education, 7(2), 95–105. G i (2016) R i d f h // i / , , , ( ) g g p g Internet and Higher Education, 7(2), 95–105. Gotomeeting (2016). Retrieved from https://www.gotomeeting.com/. G b d (2016) R i d f h // b d / Gotomeeting (2016). 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Making classroom response systems more social In Csedu (pp 153–161) Vetterick, J., Garbe, M., & Cap, C. H. (2013). Tweedback: A live feedback system for large audiences. In Csedu (pp. 194–198). Vetterick, J., Schwennigcke, B., Langfeld, A., Cap, C. H., & Sucharowski, W. (2014). Making classroom response systems more social. In Csedu (pp. 153–161). Webex (2016). Retrieved from https://www.webex.com/.
https://openalex.org/W2116091236	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Table_1_from_Targeting_the_Phosphoinositide_3-Kinase_p110-_Isoform_Impairs_Cell_Proliferation_Survival_and_Tumor_Growth_in_Small_Cell_Lung_Cancer/22449030/1/files/39900090.pdf	English	null	Targeting the Phosphoinositide 3-Kinase p110-α Isoform Impairs Cell Proliferation, Survival, and Tumor Growth in Small Cell Lung Cancer	Clinical cancer research	2,013	cc-by	1,126	Suppl. Table 1. Biostatistical analysis of the transcriptional networks impaired by p110α modification process (37.4%; 1.420e-13), ma (38.4%; 1.604e-13) p-Value zScore gScore p-Value zScore gScore p-Value zScore gScore Suppl. Table 1. Biostatistical analysis of the transcriptional networks impaired by p110α Suppl. Table 1. Suppl. Table 1. Biostatistical analysis of the transcriptional networks impaired by p110α Biostatistical analysis of the transcriptional networks impaired by p110α inhibition No Network GO Processes p-Value zScore gScore 1 SP1 cellular process (92.5%; 1.476e-20), developmental process (49.8%; 2.302e-20), anatomical structure development (44.4%; 5.950e-19), metabolic process (73.0%; 5.470e-18), cellular component organization (42.3%; 6.564e-17) 0.000E+00 100.52 100.52 2 HNF4-alpha cellular metabolic process (68.4%; 1.211e-20), primary metabolic process (66.7%; 2.208e-18), metabolic process (72.6%; 4.695e-17), cellular process (89.3%; 7.217e-15), cellular macromolecule metabolic process (50.0%; 8.455e-14) 0.000E+00 99.89 99.89 3 c-Myc cellular metabolic process (71.1%; 1.934e-23), primary metabolic process (70.2%; 4.226e-22), cellular nitrogen compound metabolic process (50.7%; 1.487e-20), metabolic process (75.6%; 5.741e-20), nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (47.1%; 9.153e-20) 0.000E+00 97.11 97.11 4 ESR1 (nuclear) developmental process (48.2%; 2.340e-11), positive regulation of cellular process (37.6%; 3.012e-11), positive regulation of metabolic process (27.0%; 3.968e-11), response to estrogen stimulus (11.3%; 4.872e-11), positive regulation of macromolecule metabolic process (25.5%; 5.365e-11) 8.740E-237 77.36 77.36 5 p53 regulation of metabolic process (56.6%; 1.172e-13), regulation of primary metabolic process (51.9%; 1.489e-13), regulation of macromolecule metabolic process (49.6%; 2.922e-13), regulation of cellular metabolic process (51.2%; 7.187e-13), cellular macromolecule metabolic process (54.3%; 7.250e-11) 4.920E-215 73.75 73.75 6 CREB1 regulation of metabolic process (57.8%; 1.726e-14), regulation of primary metabolic process (52.3%; 9.134e-14), regulation of macromolecule metabolic process (49.2%; 7.076e-13), regulation of cellular metabolic process (50.8%; 1.666e-12), regulation of cellular process (75.0%; 2.980e-12) 2.300E-213 73.47 73.47 7 EGR1 regulation of macromolecule metabolic process (55.4%; 8.872e-16), cellular macromolecule metabolic process (62.5%; 5.353e-15), macromolecule metabolic process (65.2%; 2.335e-14), cellular metabolic process (73.2%; 7.536e-14), regulation of primary metabolic process (54.5%; 1.119e-13) 2.330E-183 68.14 68.14 8 HIF1A cellular macromolecule metabolic process (69.0%; 3.345e-18), macromolecule metabolic process (70.0%; 2.755e-16), metabolic process (85.0%; 3.678e-16), cellular metabolic process (78.0%; 7.322e-16), protein modification process (40.0%; 9.638e-16) 4.410E-165 64.67 64.67 9 Androgen receptor primary metabolic process (77.0%; 4.894e-15), macromolecule metabolic process (67.0%; 3.589e-14), protein modification by small protein conjugation (18.0%; 4.469e-14), cellular macromolecule metabolic process (63.0%; 7.110e-14), metabolic process (82.0%; 7.895e-14) 2.010E-163 64.34 64.34 10 NF-kB protein modification by small protein conjugation (19.2%; 2.513e-15), protein ubiquitination (18.2%; 1.318e-14), protein modification by small protein conjugation or removal (19.2%; 4.673e-14), protein modification process (37.4%; 1.420e-13), macromolecule modification (38.4%; 1.604e-13) 9.170E-162 64.02 64.02 pp y p No Network GO Processes 1 SP1 cellular process (92.5%; 1.476e-20), develo 2.302e-20), anatomical structure developm metabolic process (73.0%; 5.470e-18) organization (42.3%; 6.564e-17) 2 HNF4-alpha cellular metabolic process (68.4%; 1.211e process (66.7%; 2.208e-18), metabolic proc cellular process (89.3%; 7.217e-15), cellular process (50.0%; 8.455e-14) 3 c-Myc cellular metabolic process (71.1%; 1.934e process (70.2%; 4.226e-22), cellular nitrog process (50.7%; 1.487e-20), metabolic proc nucleobase, nucleoside, nucleotide and nucle (47.1%; 9.153e-20) 4 ESR1 (nuclear) developmental process (48.2%; 2.340e-11) cellular process (37.6%; 3.012e-11), positive process (27.0%; 3.968e-11), response to es 4.872e-11), positive regulation of macromo (25.5%; 5.365e-11) 5 p53 regulation of metabolic process (56.6%; 1 primary metabolic process (51.9%; 1.4 macromolecule metabolic process (49.6%; 2 cellular metabolic process (51.2%; 7.187e-13 metabolic process (54.3%; 7.250e-11) 6 CREB1 regulation of metabolic process (57.8%; 1 primary metabolic process (52.3%; 9.1 macromolecule metabolic process (49.2%; 7 cellular metabolic process (50.8%; 1.666e-1 process (75.0%; 2.980e-12) 7 EGR1 regulation of macromolecule metabolic pro cellular macromolecule metabolic proces macromolecule metabolic process (65.2% metabolic process (73.2%; 7.536e-14), regula process (54.5%; 1.119e-13) 8 HIF1A cellular macromolecule metabolic proces macromolecule metabolic process (70.0% process (85.0%; 3.678e-16), cellular met 7.322e-16), protein modification process (40.0 9 Androgen receptor primary metabolic process (77.0%; 4.8 metabolic process (67.0%; 3.589e-14), prote protein conjugation (18.0%; 4.469e-14), metabolic process (63.0%; 7.110e-14), me 7.895e-14) 10 NF-kB protein modification by small protein conjug protein ubiquitination (18.2%; 1.318e-14), small protein conjugation or removal (19. GO Processes GO Processes cellular process (92.5%; 1.476e-20), developmental process (49.8%; 2.302e-20), anatomical structure development (44.4%; 5.950e-19), metabolic process (73.0%; 5.470e-18), cellular component organization (42.3%; 6.564e-17) 0.000E+00 100.52 100.52 cellular process (92.5%; 1.476e-20), developmental process (49.8%; 2.302e-20), anatomical structure development (44.4%; 5.950e-19), metabolic process (73.0%; 5.470e-18), cellular component organization (42.3%; 6.564e-17) 0.000E+00 100.52 100.52 cellular metabolic process (68.4%; 1.211e-20), primary metabolic process (66.7%; 2.208e-18), metabolic process (72.6%; 4.695e-17), cellular process (89.3%; 7.217e-15), cellular macromolecule metabolic process (50.0%; 8.455e-14) 0.000E+00 99.89 99.89 cellular metabolic process (68.4%; 1.211e-20), primary metabolic process (66.7%; 2.208e-18), metabolic process (72.6%; 4.695e-17), cellular process (89.3%; 7.217e-15), cellular macromolecule metabolic process (50.0%; 8.455e-14) 0.000E+00 99.89 99.89 cellular metabolic process (71.1%; 1.934e-23), primary metabolic process (70.2%; 4.226e-22), cellular nitrogen compound metabolic process (50.7%; 1.487e-20), metabolic process (75.6%; 5.741e-20), nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (47.1%; 9.153e-20) 0.000E+00 97.11 97.11 developmental process (48.2%; 2.340e-11), positive regulation of cellular process (37.6%; 3.012e-11), positive regulation of metabolic process (27.0%; 3.968e-11), response to estrogen stimulus (11.3%; 4.872e-11), positive regulation of macromolecule metabolic process (25.5%; 5.365e-11) 8.740E-237 77.36 77.36 developmental process (48.2%; 2.340e-11), positive regulation of cellular process (37.6%; 3.012e-11), positive regulation of metabolic process (27.0%; 3.968e-11), response to estrogen stimulus (11.3%; 4.872e-11), positive regulation of macromolecule metabolic process (25.5%; 5.365e-11) 8.740E-237 77.36 77.36 regulation of metabolic process (56.6%; 1.172e-13), regulation of primary metabolic process (51.9%; 1.489e-13), regulation of macromolecule metabolic process (49.6%; 2.922e-13), regulation of cellular metabolic process (51.2%; 7.187e-13), cellular macromolecule metabolic process (54.3%; 7.250e-11) 4.920E-215 73.75 73.75 regulation of metabolic process (56.6%; 1.172e-13), regulation of primary metabolic process (51.9%; 1.489e-13), regulation of macromolecule metabolic process (49.6%; 2.922e-13), regulation of cellular metabolic process (51.2%; 7.187e-13), cellular macromolecule metabolic process (54.3%; 7.250e-11) 4.920E-215 73.75 73.75 regulation of metabolic process (57.8%; 1.726e-14), regulation of primary metabolic process (52.3%; 9.134e-14), regulation of macromolecule metabolic process (49.2%; 7.076e-13), regulation of cellular metabolic process (50.8%; 1.666e-12), regulation of cellular process (75.0%; 2.980e-12) 2.300E-213 73.47 73.47 regulation of metabolic process (57.8%; 1.726e-14), regulation of primary metabolic process (52.3%; 9.134e-14), regulation of macromolecule metabolic process (49.2%; 7.076e-13), regulation of cellular metabolic process (50.8%; 1.666e-12), regulation of cellular process (75.0%; 2.980e-12) 2.300E-213 73.47 73.47 regulation of macromolecule metabolic process (55.4%; 8.872e-16), cellular macromolecule metabolic process (62.5%; 5.353e-15), macromolecule metabolic process (65.2%; 2.335e-14), cellular metabolic process (73.2%; 7.536e-14), regulation of primary metabolic process (54.5%; 1.119e-13) 2.330E-183 68.14 68.14 cellular macromolecule metabolic process (69.0%; 3.345e-18), macromolecule metabolic process (70.0%; 2.755e-16), metabolic process (85.0%; 3.678e-16), cellular metabolic process (78.0%; 7.322e-16), protein modification process (40.0%; 9.638e-16) 4.410E-165 64.67 64.67 primary metabolic process (77.0%; 4.894e-15), macromolecule metabolic process (67.0%; 3.589e-14), protein modification by small protein conjugation (18.0%; 4.469e-14), cellular macromolecule metabolic process (63.0%; 7.110e-14), metabolic process (82.0%; 7.895e-14) 2.010E-163 64.34 64.34 protein modification by small protein conjugation (19.2%; 2.513e-15), protein ubiquitination (18.2%; 1.318e-14), protein modification by small protein conjugation or removal (19.2%; 4.673e-14), protein modification process (37.4%; 1.420e-13), macromolecule modification (38.4%; 1.604e-13) 9.170E-162 64.02 64.02
https://openalex.org/W1990335170	https://www.repository.cam.ac.uk/bitstream/1810/246186/1/Geophys.%20J.%20Int.-2014-Nunn-1724-41.pdf	English	null	Imaging the lithosphere beneath NE Tibet: teleseismic P and S body wave tomography incorporating surface wave starting models	Geophysical journal international	2,013	cc-by	14,513	Ceri Nunn,1 Steven W. Roecker,2 Frederik J. Tilmann,3,4 Keith F. Priestley,1 Ross Heyburn,5 Eric A. Sandvol,6 James F. Ni,7 Yongshun John Chen,8 Wenjin Zhao9 and the INDEPTH IV Team 1Department of Earth Sciences, University of Cambridge, Cambridge, UK. E-mail: cn277@cam.ac.uk 2Department of Earth and Environmental Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 3Helmholtz-Centre Potsdam, GFZ German Research Centre for Geosciences, Potsdam, Germany 4Department of Earth Sciences, Freie Universit¨at Berlin, Berlin, Germany 5AWE Blacknest, Reading, UK g 6University of Missouri, Columbia, MO 65211, USA 7New Mexico State University, Las Cruces, NM 88003, USA 8Peking University, Beijing, China 9Chinese Academy of Geological Sciences, Beijing, China g 6University of Missouri, Columbia, MO 65211, USA 7New Mexico State University, Las Cruces, NM 88003, USA 8Peking University, Beijing, China 9Chinese Academy of Geological Sciences, Beijing, China Accepted 2013 November 21. Received 2013 November 19; in original form 2013 July 31 C⃝The Authors 2013. Published by Oxford University Press on behalf of The Royal Astronomical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.1093/gji/ggt476 doi: 10.1093/gji/ggt476 Geophysical Journal International Geophys. J. Int. (2014) 196, 1724–1741 doi: 10.1093/gji/ggt476 Advance Access publication 2013 December 24 S U M M A R Y The northeastern margin of the Tibetan Plateau, which includes the Qiangtang and Songpan- Ganzi terranes as well as the Kunlun Shan and the Qaidam Basin, continues to deform in response to the ongoing India–Eurasia collision. To test competing hypotheses concerning the mechanisms for this deformation, we assembled a high-quality data set of approximately 14 000 P- and 4000 S-wave arrival times from earthquakes at teleseismic distances from the International Deep Proﬁling of Tibet and the Himalaya, Phase IV broad-band seismometer deployments. We analyse these arrival times to determine tomographic images of P- and S-wave velocities in the upper mantle beneath this part of the plateau. To account for the effects of major heterogeneity in crustal and uppermost mantle wave velocities in Tibet, we use recent surface wave models to construct a starting model for our teleseismic body wave inversion. We compare the results from our model with those from simpler starting models, and ﬁnd that while the reduction in residuals and results for deep structure are similar between models, the results for shallow structure are different. Checkerboard tests indicate that features of ∼125 km length scale are reliably imaged throughout the study region. Using synthetic tests, we show that the best recovery is below ∼300 km, and that broad variations in shallow structure can also be recovered. We also ﬁnd that signiﬁcant smearing can occur, especially at the edges of the model. We observe a shallow dipping seismically fast structure at depths of ∼140– 240 km, which dies out gradually between 33◦N and 35◦N. Based on the lateral continuity of this structure (from the surface waves) we interpret it as Indian lithosphere. Alternatively, the entire area could be thickened by pure shear, or the northern part could be an underthrust Lhasa Terrane lithospheric slab with only the southern part from India. We see a deep fast wave velocity anomaly (below 300 km), that is consistent with receiver function observations of a thickened transition zone and could be a fragment of oceanic lithosphere. In NE Tibet, it appears to be disconnected from faster wave velocities above (i.e. it is not downwelling or subducting here). Our models corroborate results of previous work which imaged a relatively slow wave velocity region below the Kunlun Shan and northern Songpan-Ganzi Terrane, which is difﬁcult to reconcile with the hypothesis of southward-directed continental subduction at the GJI Seismology C⃝The Authors 2013. Geophysical Journal International Geophys. J. Int. (2014) 196, 1724–1741 Advance Access publication 2013 December 24 doi: 10.1093/gji/ggt476 1 I N T RO D U C T I O N The high (∼5 km) elevation of the Tibetan Plateau is a consequence of the collision between the Indian and Eurasian plates during the closure of the Tethys Ocean. Based on a change in plate motion, most researchers suggest collision between India and Eurasia prob- ably began 50–55 Ma (e.g. Molnar & Tapponnier 1975; Patriat & Achache 1984; Copley et al. 2010), although Yin & Harrison (2000) suggest it may have begun earlier and Aitchison et al. (2007) prefer a later collision time of ∼34 Ma. The International Deep Proﬁling of Tibet and the Himalaya, Phase IV (INDEPTH IV) (Fig. 1) was set up to image the northeastern margin of the Tibetan collision zone and explore the mechanisms for the continued growth of the plateau. In this paper, we describe the results of an arrival time tomography study of the upper mantle beneath northeast Tibet using recordings of P and S waves from earthquakes at teleseismic distances from the INDEPTH IV array. The evolution of the Tibetan Plateau (Fig. 1) involved numerous collisions, with terranes accreted to Eurasia during the closure of the Tethys Ocean. In eastern Tibet, the Lhasa and Qiangtang terranes are separated by the Bangong–Nujiang Suture (BNS), which began as a rift in the Early Ordovician or Carboniferous, and later became a convergent margin. The BNS closed during the late Jurassic (Dewey et al. 1988). The Qiangtang and the Songpan-Ganzi terranes are sep- arated by the Jinsha Suture (JS), which was formed by the closure of the Songpan-Ganzi Ocean (Yin & Harrison 2000) in the late Triassic or earliest Jurassic (Dewey et al. 1988). The northern edge of the Songpan-Ganzi is marked by the major left-lateral strike- slip Kunlun Fault (KF). The KF has major branches on the eastern side of Tibet: the South Kunlun thrust fault (SKF) and the North Kunlun strike-slip fault (NKF). The crust is approximately 75 km thick beneath the Lhasa Terrane and becomes thinner (63–74 km) north of the BNS (Kind et al. 2002; Yue et al. 2012). The thick- ness of the lithosphere is less well established. One way to estimate the thickness is to look for the lithosphere–asthenosphere bound- ary (LAB) using negative converters in receiver function studies. Mechie & Kind (2013) note that three studies show the LAB to be at 150–200 km depth underneath northern Lhasa and the Qiangtang (Kumar et al. 2006; Zhao et al. 1 I N T RO D U C T I O N 2011; Yue et al. 2012). High Pn (i.e. uppermost mantle) velocities are found beneath the southern plateau and major basins, including the Tarim and Qaidam (Hearn et al. 2004; Liang & Song 2006). A belt of low Pn veloc- ities stretches across the Songpan-Ganzi and Qiangtang terranes (McNamara et al. 1997), suggesting that the uppermost mantle be- neath the Songpan-Ganzi and Qiangtang terranes is warm and may be weak while that beneath the basins and southern Tibet is strong and cold. In this study, we use P and S body wave teleseismic data, along with a starting model based on surface wave analysis. Teleseismic regional tomography is sensitive to lateral velocity contrasts, and provides resolution to depths comparable to the aperture of the array. However, because it uses relative arrival times, it is insensitive to absolute wave velocities. It also is prone to smearing images in ar- eas lacking crossing rays, particularly in shallow regions such as the crust (e.g. Aki et al. 1977; Priestley & Tilmann 2009). In the absence of any prior information about the distribution of wave velocities in the crust, investigators generally use either station corrections de- rived from average residuals (e.g. Frederiksen et al. 1998; Graeber et al. 2002) or force as much of the misﬁt as possible into the crust (e.g. Abers & Roecker 1991) to isolate signal due exclusively to structure in the mantle. However, this approach can underestimate variations of mantle velocities (Waldhauser et al. 2002; Priestley & Tilmann 2009). Results from previous investigations in northeast- ern Tibet (e.g. Yue et al. 2012) suggest large variations in Moho depth, as well as considerable lateral heterogeneity in crustal wave velocities. The Kunlun Shan lies to the north of the KF, where it is marked by a steep drop in topography, and bounds the Qaidam Basin in a series of buried thrusts called the North Kunlun Thrust System (NKTS). The 3 km high Qaidam Basin is a desert, with 8 km of sediment, and a total crustal thickness of 50 km (Karplus et al. 2011; Yue et al. 2012). The Qaidam is bound to the west by the Qimen Tagh Thrust Belt, to the northwest by the left-lateral strike-slip Altyn Tagh Fault and to the northeast by the mainly north-dipping high-angle North Qaidam Thrust System (NQTS). S U M M A R Y Published by Oxford University Press on behalf of The Royal Astronomical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1724 Imaging the lithosphere beneath NE Tibet 1725 northern margin. Wave velocities in the shallow mantle beneath the Qaidam Basin are faster than normal, and more so in the east than the west. Key words: Tomography; Mantle processes; Body waves; Seismic tomography; Continental margins: convergent; Asia. Key words: Tomography; Mantle processes; Body waves; Seismic tomography; Continental margins: convergent; Asia. et al. 2012; Zhang et al. 2012), joint body wave and surface wave tomography (Obrebski et al. 2012), receiver functions (Kind et al. 2002; N´abˇelek et al. 2009; Zhao et al. 2010; Yue et al. 2012), reﬂection seismology (Hauck et al. 1998) and gravity modelling (Jim´enez-Munt et al. 2008) has led to the general acceptance that the Indian lithosphere has subducted underneath at least the southern part of the plateau. Considerable controversy remains concerning the northern edge of the plateau. In particular, Tapponnier et al. (2001) have posited the existence of southward-directed subduction of Asian lithosphere below northern Tibet, and Kind et al. (2002) point to a prominent south-dipping interface in P-receiver functions to corroborate this hypothesis. Subduction of Asian lithosphere is also supported by the identiﬁcation of two interfaces that generate negative polarity S-wave mode conversions at ∼100 and ∼170 km depth below northern Tibet that were interpreted by Zhao et al. (2011) as the lower boundaries of stacked mantle lithospheres of Tibetan and Asian provenance. These interfaces appear connected to a shallow LAB at 120 km below the Qaidam Basin. At the same time, the receiver function study of Yue et al. (2012) found no candidate interfaces associated with subduction of Asian lithosphere, and the Rayleigh wave tomography study of Ceylan et al. (2012) found no evidence of continuous higher velocity zones that would be consistent with subducting lithosphere from the north. 1 I N T RO D U C T I O N The Qilian Shan chain lies to the north of the basin, ﬂanked by the south-dipping Qilian Shan Thrust Belt (Yin et al. 2008). In contrast to body wave tomography, surface wave tomography generally exhibits good depth resolution in the upper 250–300 km depth, but tends to smear short-wavelength lateral anomalies. We use an approach previously used by Rawlinson & Fishwick (2012), The role of the Indian lithosphere in the evolution of Tibet remains a subject of debate, but evidence from surface wave tomography (Shapiro & Ritzwoller 2002; Priestley et al. 2006; Lebedev & van der Hilst 2008), body wave tomography (Li et al. 2008; Liang 1726 C. Nunn et al. To provide an estimate of measurement error, for the purpose of weighting the inversion, uncertainties (σ d) in the relative P-wave ar- rival times were estimated by ﬁrst assigning events into one of three quality classes. We then searched for pairs of observations where events within 20 km of each other were observed at the same station. There were over 100 event-pairs with a minimum of 11 stations and a maximum of 65 stations common to both events (resulting in an average of 989 paired observations per event-pair). Using the statis- tics calculated from the pairs, we assigned an uncertainty to each of the quality classes. 2107 picks were assigned an uncertainty of 0.20 s, 11 126 picks assigned 0.28 s and 590 picks assigned 0.47 s. This compares with initial traveltime residuals of 0.51–0.53 s for our P models. which allows the surface waves to account for the shallow hetero- geneity and to provide absolute velocities and the long-wavelength background structure (down to ∼400 km, albeit at lower resolution below ∼200 km depth). We use a surface wave model (based on Acton et al. 2010; Priestley et al. 2006) to construct a starting model for our body wave inversion. This model incorporates the major differences in crustal wave velocities in this region, and represents the long-wavelength background structure in the shallow mantle. We compare our results to those starting with a simple 1-D model, and one where we explic- itly account for expected differences in Moho depth. Our approach allows us to make a correction for shallow heterogeneity, but is also able to sample lateral heterogeneity within the mantle. There were insufﬁcient data to allow a similarly quantitative es- timate of the S-wave arrival time uncertainties. 2 DATA We use data from the INDEPTH IV array (2007–2009, Fig. 1) which comprises 113 stations, 15 of which form part of the dense (∼5 km spacing) Kunlun 1-D proﬁle (Zhao et al. 2008). Relative arrival times for both P and S waves were determined using the multichannel cross-correlation (MCCC) method devel- oped by VanDecar & Crosson (1990). P arrivals were correlated on the vertical component after bandpass ﬁltering between 0.4 and 1.8 Hz; S arrivals were correlated on the transverse component after bandpass ﬁltering between 0.03 and 0.2 Hz. The analysis windows for P-wave correlation were assigned manually for each event, with a mean length of 2.8 s. S-wave correlation was less straightfor- ward as cycle-skipping was common. The analysis window for S waves had a mean length of 14.9 s. Each S-wave correlation was checked manually for cycle-skipping or for later phases inﬂuencing the correlation, and seismograms exhibiting poor correlation were removed before recorrelating. At least three picks were made for each of the included events. We include example correlations with the Supporting Information. We examined 937 P-wave teleseismic events with an epicentral distance ranging from 30◦to 90◦and moment magnitudes (MW) be- tween 5.0 and 6.9. 498 events were excluded due to low waveform quality, and a further 152 to avoid events from the east and south- east dominating the signal. Our assembled data set contains 13 823 P-wave arrivals recorded from 287 events (Fig. 2, red circles). Figure 1. A map of the study region showing terranes and major faults (after Yin et al. 2008): barbed lines are thrusts, continuous lines are strike-slip and dashed lines are sutures. Red triangles are INDEPTH IV stations, and lines L1–L5 are the location of our proﬁles. The location of the study area is shown on the right. Abbreviations on the map are: MFT, Main Frontal Thrust; LT, Lhasa Terrane; QT, Qiangtang Terrane; SG, Songpan-Ganzi Terrane; KS, Kunlun Shan; QS, Qilian Shan; IYS, Indus-Yalu Suture; BNS, Bangong-Nujiang Suture; JS, Jinsha Suture; JRF, Jinsha River Fault; KF, Kunlun Fault; SKF, South Kunlun Fault; NKF, North Kunlun Fault; NKTS, North Kunlun Thrust System; QTFS, Qimen Tagh Fault System; QSTB, Qilian Shan Thrust Belt; NQTS, North Qaidam Thrust System; AT, Altyn Tagh; AB, Alxa Block. Note that there is some dispute about the nature of the SKF and the vergence of the NKTS. Figure 1. 1 I N T RO D U C T I O N To at least provide a working estimate for purposes of weighting, we calculated the uncertainty for six event-pairs within 20 km of each other. For all events, we then estimated the uncertainty manually by considering how well the waveforms correlated across the network for each event. Finally, the manually estimated uncertainties were scaled us- ing a scaling factor of 0.7, based on a comparison between the statistics for the six event-pairs and the manual estimates for the same events. The mean assigned uncertainty for the 3957 S arrivals used in this study is 0.7 s, the lowest uncertainty 0.3 s and the maximum uncertainty 1.7 s. This compares with an initial travel- time residual rms of 1.06–1.13 s for our S models. While these estimates do not appear unreasonable, we emphasize that they are themselves quite uncertain, and, as discussed below, are likely to be too conservative. 3 M E T H O D We analyse the P and S arrival times using a method described in detail in Roecker et al. (2004, 2006) and Li et al. (2009). Traveltimes are calculated by adding the time from the base of the regional model to a given station to that from the hypocentre to the base of the model. Times within the regional model are computed using the ﬁnite difference eikonal equation solver of Hole & Zelt (1995), adapted for teleseisms and spherical coordinates by Li et al. (2009). Times from the hypocentre to the base of the model are taken from traveltime tables for a standard earth model (IASP91: Kennett & Engdahl 1991). By Fermat’s principle, the true traveltime is the minimum of these sums. Ray paths are then determined by following the steepest traveltime gradient, and are updated with each iteration. The S-wave version of starting model SW (Fig. 6a) is based on shear wave velocities obtained from prior surface wave analysis. At depths less than 100 km we use the results of Acton et al. (2010), based on the inversion of Rayleigh group velocities at periods of 10–70 s, for the crust and uppermost mantle. For depths larger than 150 km up to the base of our model grid at 600 km depth we use an updated model based on a fundamental and higher mode Rayleigh wave inversion between 50 and 160 s described in Priest- ley et al. (2006), but which utilizes 11 times as many waveforms. Below 325 km, this model shows only small (<±2 per cent) varia- tion from PREM (Dziewonski & Anderson 1981). Between 100 and 150 km depth, we grade the Acton and Priestley models into each other. The expected depth resolution is ∼20 km above 100 km depth and ∼30–50 km at greater depths. The expected lateral resolution is ∼100–200 km above 100 km depth and ∼200–400 km at greater depths. Two model grids are deﬁned: a ﬁne spaced grid for the ﬁnite dif- ference computation of the traveltimes and ray paths, and a coarser grid for the inversion. We choose a ﬁne grid spacing that is 5 km in depth and 0.0625◦(∼7 km) laterally, and a coarse grid which is 25 km in depth and 0.25◦(∼27 km) laterally. The depth range is from 10 km above sea level to 600 km depth. 2 DATA A map of the study region showing terranes and major faults (after Yin et al. 2008): barbed lines are thrusts, continuous lines are strike-slip and dashed lines are sutures. Red triangles are INDEPTH IV stations, and lines L1–L5 are the location of our proﬁles. The location of the study area is shown on the right. Abbreviations on the map are: MFT, Main Frontal Thrust; LT, Lhasa Terrane; QT, Qiangtang Terrane; SG, Songpan-Ganzi Terrane; KS, Kunlun Shan; QS, Qilian Shan; IYS, Indus-Yalu Suture; BNS, Bangong-Nujiang Suture; JS, Jinsha Suture; JRF, Jinsha River Fault; KF, Kunlun Fault; SKF, South Kunlun Fault; NKF, North Kunlun Fault; NKTS, North Kunlun Thrust System; QTFS, Qimen Tagh Fault System; QSTB, Qilian Shan Thrust Belt; NQTS, North Qaidam Thrust System; AT, Altyn Tagh; AB, Alxa Block. Note that there is some dispute about the nature of the SKF and the vergence of the NKTS. Imaging the lithosphere beneath NE Tibet 1727 Figure 2. Data from 287 teleseismic events provided 13 823 P-wave arrivals (red circles). 78 teleseismic events provided 3957 S-wave arrivals (blue diamonds). tions is weighted to reﬂect the uncertainties in the observations. Perturbations are regulated in two ways: ﬁrst, the least-squares so- lution is damped to prevent overstepping at any single iteration, and, second, short-wavelength perturbations are smoothed using a moving average window prior to modifying the existing model (e.g. Li et al. 2009). The values one chooses for these regulators (size of damper and length of moving window) are somewhat arbitrary and we experimented with a range of values until we found a combi- nation that led to stable convergence while ﬁtting the observations reasonably well without requiring a too complicated model. Based on our trade-off curves (Fig. 4) we chose a damper of 200 s2 km−2 and smooth over ﬁve nodes in each cardinal direction (the target node and two nodes either side), corresponding to 1.25◦laterally and 125 km in depth. 3.1 Starting models at University of Cambridge on October 17, http://gji.oxfordjournals.org/ Downloaded from The results presented in this paper use recent surface wave models as a starting model for our teleseismic body wave inversion. This is to mitigate the effects of a highly heterogeneous crust and uppermost mantle. In this section, we describe the preparation of this model (SW), and compare it with two other models: a simple 1-D model (AK) and a model where we explicitly account for Moho depth variations (MO). Figure 2. Data from 287 teleseismic events provided 13 823 P-wave arrivals (red circles). 78 teleseismic events provided 3957 S-wave arrivals (blue diamonds). S-wave arrival times were derived from recordings of 78 events with epicentral distances ranging from 25◦to 80◦and moment magni- tudes (MW) between 5.2–6.9 (Fig. 2, blue diamonds). To balance the azimuthal distribution of data for the S tomography, we restricted selection of events from the east and southeast to those with MW > 5.9. After reviewing waveform quality, we retained 3957 S arrivals from an original set of 4846 (Fig. 2). The ray coverage for both P and S is shown in Fig. 3. 3.1.1 Preparation of different starting models The starting models are compared in Figs 5 and 6. Starting model AK is a simple 1-D model that uses global reference model ak135 (Kennett et al. 1995). Starting model MO was generated using the Moho depth model of Yue et al. (2012), which is based on stacking direct and multiple Ps converted phases using the slant-stack method of Zhu & Kanamori (2000). Below the Moho, we used the average at the corresponding depth from model SW (see below). Above the Moho, we assumed a P velocity of 6.4 km s−1 and an S velocity of 3.6 km s−1. We adjust the nodes either side of the Moho, such that the integrated traveltime approximates the traveltime across the Moho discontinuity. 3 M E T H O D In order to mitigate the effects of variation in structure outside the model, we remove the mean and work with only relative traveltimes. Because P- and S-wave traveltimes can be inﬂuenced in different ways by structure outside the model, we solve for variations in P and S wave velocities independently. The inverse problems are linearized and the partial derivatives for each observation are cal- culated along the ray paths. The resulting series of linear equations are solved iteratively using the Least-Squares (LSQR) algorithm of Paige & Saunders (1982). Each row of the system of linear equa- The average P-wave velocity for each depth within starting model SW (Fig. 5a) was estimated from the surface wave models by assum- ing the same Vp/Vs ratio at a particular depth as ak135. Variations in mantle velocities normally reﬂect changes in temperature rather than composition; we expect that compositional changes will re- sult in velocity anomalies <1 per cent. In addition, temperature has a greater effect on S velocities: a 100◦C increase in temperature 1728 C. Nunn et al. Figure 3. P-wave (left-hand column) and S-wave (right-hand column) ray coverage along proﬁles L1, L2, L3, L4 and L5. Rays are shown within 60 km of each proﬁle. The topography and major faults and sutures are projected onto the proﬁle, along with stations within 50 and 150 km of the proﬁle (blue and grey riangles, respectively). Proﬁle locations in Fig. 1. owers Vs by 0.7–4.5 per cent and Vp by 0.5–2 per cent (Goes et al. 2000). Hence, the variation in mantle P velocities should be less than those in S, and we set the P-velocity anomalies to be only half the magnitude of those in the S-wave model. 3.1.2 Comparing results using different starting models The ﬁnal results from the three different models show broadly the same features (Fig. 5a: fourth row). In order to facilitate the Figure 3. P-wave (left-hand column) and S-wave (right-hand column) ray coverage along proﬁles L1, L2, L3, L4 and L5. Rays are shown within 60 km of each proﬁle. The topography and major faults and sutures are projected onto the proﬁle, along with stations within 50 and 150 km of the proﬁle (blue and grey triangles, respectively). Proﬁle locations in Fig. 1. 3.1.2 Comparing results using different starting models lowers Vs by 0.7–4.5 per cent and Vp by 0.5–2 per cent (Goes et al. 2000). Hence, the variation in mantle P velocities should be less than those in S, and we set the P-velocity anomalies to be only half the magnitude of those in the S-wave model. The ﬁnal results from the three different models show broadly the same features (Fig. 5a: fourth row). In order to facilitate the 1729 Imaging the lithosphere beneath NE Tibet Imaging the lithosphere beneath NE Tibet 1729 Figure 4. The trade-off curves between the roughness of the imposed model perturbations and rms residual. (a) P-wave inversions and (b) S-wave inversions after four inversions for model SW. Dashed lines connect the same damping parameter, and solid shapes show the smoothing parameter. We chose damping of 200 s2 km−2 and smoothing across ﬁve nodes for both P and S (circled in red). The star shows the initial residual (the initial roughness is zero because we consider model perturbations). Figure 4. The trade-off curves between the roughness of the imposed model perturbations and rms residual. (a) P-wave inversions and (b) S-wave inversions after four inversions for model SW. Dashed lines connect the same damping parameter, and solid shapes show the smoothing parameter. We chose damping of 200 s2 km−2 and smoothing across ﬁve nodes for both P and S (circled in red). The star shows the initial residual (the initial roughness is zero because we consider model perturbations). discussion of the models, we add labels to the larger features for ease of reference. The body waves cannot retrieve the shallowest structure, because there is little crossing ray coverage immediately below the stations, and so the largest differences between the mod- els are within the shallow structure. In model AK, anomaly A is strongly smeared vertically (see Fig. 5a for labels). By comparison, in model SW, the depth range is more constrained, and consequently the amplitude of the anomaly is higher. Below 300 km, the models are very similar because of improved crossing ray coverage at these depths, and also because there is less structure in starting model SW. calculated traveltimes are absorbed in relatively minor changes in model perturbations at all depths (Figs 5 and 6, third row). 3.1.3 Reduction of the residuals All three sets of models show a large reduction in the residuals (Fig. 7). For P, there are small differences for initial ﬁt, with model AK the best, and model SW the worst (Fig. 7a). For S, there are slightly larger differences in the initial ﬁt, with model SW the best, and model MO the worst (Fig. 7b). Surprisingly, in both cases, the models eventually reach nearly the same ﬁt to the residuals. 3.1.2 Comparing results using different starting models We ﬁnd that our images are robust, with only minor differences between the models below 300 km, despite major differences in the shallow structure that reﬂect the differences in starting models. We note that for P, the residuals converge to a value close to our estimate of the picking error (Fig. 7). As discussed earlier (Section 2), we were not able to determine a reliable quantitative estimate of S uncertainty from the correlated waveforms, and the ﬁnal residuals suggest that our original estimate of 0.7 s was too conservative. The ﬁnal rms of the ﬁt is 0.52 s, which is still nearly twice that for P, and so would seem to be a reasonable estimate of actual S uncertainty. The map views of the results (Fig. 5b) also show broadly similar features, especially C, D and E. However, the boundary of A (shown by white dashed lines) is 50–100 km further north in model SW. Recovery is affected by the station distribution: there are holes where the coverage is sparser. The residuals do not indicate that one model is statistically better than the others. However, the main differences between models AK, MO and SW are above 200 km, in the region where surface waves have good resolution. As an additional test on our conﬁdence in model SW, we calculated surface wave group traveltimes through starting model SW, and compared them with the traveltimes for all three ﬁnal models. We found that including the body waves into model SW introduced only small variations to the surface wave traveltimes, and that SW provided a signiﬁcantly better ﬁt to the surface waves than the other two models (Supporting Information Fig. S9). Therefore, we prefer to use model SW, since it is compatible with two independent data sets (the body and surface waves). We use it to provide constraints for the shallow structure and the long- wavelength deeper structure. Features A–G and I are also recovered in the S-wave models (Fig. 6a). Neither Model AK nor MO retrieve H, although it remains in model SW with reduced amplitude in comparison to starting model SW. Feature A does not reach as far north in model AK and MO as in SW (Figs 6a and b), probably because it is obscured by vertical smearing of feature B. 3.2 Synthetic tests Panels in (a) show proﬁles along L2 for Model AK, MO an We show the starting model (top and second rows), the difference between starting and ﬁnal model (third row) and the ﬁnal model (fourth row, with majo ures labelled). The starting models in the top row are plotted as absolute seismic velocities (in km s−1). As the body waves have no sensitivity for lay rages, the remainder of the proﬁles are plotted as percentage anomalies relative to the average at any particular depth for that particular model. This ha effect of balancing positive and negative anomalies at each depth, and we plot all our results in this way. Topography and stations are plotted as in Fig. els in (b) show map views for the starting model (top row), difference (middle row) and ﬁnal model (bottom row) at 175 km depth. For comparison, dashe te lines indicate the northern edge of A for each model. The map views are also plotted relative to the layer average. Stations are shown as grey triangle extent of the body wave ray coverage is indicated by the black line on both the map views and the proﬁles. Figure 5. A comparison of P-wave velocity models resulting from three different starting models. Panels in (a) show proﬁles along L2 for Model AK, MO and SW. We show the starting model (top and second rows), the difference between starting and ﬁnal model (third row) and the ﬁnal model (fourth row, with major features labelled). The starting models in the top row are plotted as absolute seismic velocities (in km s−1). As the body waves have no sensitivity for layer averages, the remainder of the proﬁles are plotted as percentage anomalies relative to the average at any particular depth for that particular model. This has the effect of balancing positive and negative anomalies at each depth, and we plot all our results in this way. Topography and stations are plotted as in Fig. 3. Panels in (b) show map views for the starting model (top row), difference (middle row) and ﬁnal model (bottom row) at 175 km depth. For comparison, dashed white lines indicate the northern edge of A for each model. The map views are also plotted relative to the layer average. Stations are shown as grey triangles. 3.2 Synthetic tests We conducted a series of tests to evaluate the resolution and ro- bustness of our results. Using the same method as our forward calculation (Section 3), we calculated traveltimes using an eikonal equation solver within the test model and traveltimes from a stan- dard earth model outside. The data sets we use for these synthetic tests mimic the real data as closely as possible. We use the same source–receiver pairs as in the real distribution, and add random The different starting models can generate differences in the relative arrival times of up to 1.4 s for S, though typical differences are on the order of one or two-tenths of a second, substantially less than the estimated picking errors. Changing the starting models (which affect only the shallow structure in this case) results in almost no change to the ﬁt of the residuals. What differences there are in 1730 730 C. Nunn et al. C. Nunn et al. 730 C. Nunn et al. igure 5. A comparison of P-wave velocity models resulting from three different starting models. Panels in (a) show proﬁles along L2 for Model AK, MO and W. We show the starting model (top and second rows), the difference between starting and ﬁnal model (third row) and the ﬁnal model (fourth row, with major eatures labelled). The starting models in the top row are plotted as absolute seismic velocities (in km s−1). As the body waves have no sensitivity for layer verages, the remainder of the proﬁles are plotted as percentage anomalies relative to the average at any particular depth for that particular model. This has he effect of balancing positive and negative anomalies at each depth, and we plot all our results in this way. Topography and stations are plotted as in Fig. 3. anels in (b) show map views for the starting model (top row), difference (middle row) and ﬁnal model (bottom row) at 175 km depth. For comparison, dashed white lines indicate the northern edge of A for each model. The map views are also plotted relative to the layer average. Stations are shown as grey triangles. he extent of the body wave ray coverage is indicated by the black line on both the map views and the proﬁles. ure 5. A comparison of P-wave velocity models resulting from three different starting models. 3.2.1 Synthetic test: ‘complex’ model 3.2 Synthetic tests Figure 7. The reduction in the residual for (a) P-wave models and (b) S-wave models. Model AK in green, MO in blue and SW in red. The dashed grey line shows the residual, we might expect given our a priori estimation of the measurement errors. Note that we believe that our estimate of the measurement errors b t ti f S Figure 8. Synthetic test to understand how well we can retrieve a known, complicated model. (a) The input is Model SW (after 30 iterations). (b) Sy-AK: the inversion is carried out using Model AK as the initial model. (c) Sy-CO: using the same synthetics, but with Model CO as the initial model. This has simple structure below 125 km (marked by the dashed white line) but replicates the complicated structure above. Shown along proﬁle L2 (Fig. 11). the broadest features above 125 km. Note that A, which is nearly horizontal in the input model, becomes close to vertical, especially on Sy-AK (Fig. 8b), and in both cases the strong fast wave velocities 3.2.2 Checkerboard tests In order to understand the resolution we can achieve, as well as ex- ploring how well models can retrieve an image below a complicated crust and uppermost mantle, we carried out a variation on the stan- dard ‘checkerboard’ test (Fig. 9). Below 125 km we created a model consisting of alternating positive and negative velocity anomalies of 5 per cent on a background of ak135. The crust and uppermost mantle structure of model SW was used for the upper part of the input model. The checker dimensions were 1.25◦width in latitude and longitude, and 75 or 100 km depth, separated by neutral regions of 0.75◦in latitude and longitude and 50 km in depth in order to be able to better judge smearing. The P checker pattern (including the crust and upper mantle struc- ture) is alternately inverted using an initial model which replicates the upper 125 km, with ak135 below (PCh-CO, Fig. 9a), or a model which only has the simple ak135 structure (PCh-AK, Fig. 9b). The checker patterns are well retrieved in both cases, although smearing occurs along the ray paths at the edge of the model, where there is no crossing of the rays. The checkers have marginally improved amplitudes for PCh-CO, and marginally less smearing into the crust (Fig. 9a). PCh-AK reﬂects the major scale shallow structure, but misses the detail. The S checker pattern is a little more smeared towards the edges, and amplitude recovery a little lower, but the overall pattern is similar (SCh-CO, Fig. 9c). 3.2 Synthetic tests The extent of the body wave ray coverage is indicated by the black line on both the map views and the proﬁles. Imaging the lithosphere beneath NE Tibet 1731 Imaging the lithosphere beneath NE Tibet 1731 gure 6. A comparison of S-wave velocity models resulting from three different starting models. Figure format as in Fig. 5. aussian errors with the same standard deviation as the picking ror (0.26 s for P and 0.57 s for S). (Fig. 8a). We compare the results when the upper part of the model is provided as part of the starting model versus a much simpler starting model. For one starting model, we used Model AK (Sy- AK, Fig. 8b). For the second starting model we used a combined ure 6. A comparison of S-wave velocity models resulting from three different starting models. Figure format as in Fig. 5. Figure 6. A comparison of S-wave velocity models resulting from three different starting models. Figure format as in Fig. 5 Gaussian errors with the same standard deviation as the picking error (0.26 s for P and 0.57 s for S). Gaussian errors with the same standard deviation as the picking error (0.26 s for P and 0.57 s for S). (Fig. 8a). We compare the results when the upper part of the model is provided as part of the starting model versus a much simpler starting model. For one starting model, we used Model AK (Sy- AK, Fig. 8b). For the second starting model we used a combined model which replicated the upper 125 km exactly and used ak135 below that depth (Sy-CO, Fig. 8c). 3.2.1 Synthetic test: ‘complex’ model 3.2.1 Synthetic test: ‘complex’ model 3.2.1 Synthetic test: ‘complex’ model Below 125 km the features are generally well recovered, and the output models are quite similar. As expected, Sy-AK recovers only We were interested in how well a complex (but known) model can be retrieved. Model SW (after 30 iterations) was used as the input 1732 C. Nunn et al. C. Nunn et al. Figure 7. The reduction in the residual for (a) P-wave models and (b) S-wave models. Model AK in green, MO in blue and SW in red. The dashed grey line shows the residual, we might expect given our a priori estimation of the measurement errors. Note that we believe that our estimate of the measurement errors may be too conservative for S. 3.3 Synthetic test for probable structures 9a for S). The proﬁle is along 95.5◦(indicated on map views). The map views are at 225 and 550 km. The initial perturbation was ±5 per cent, d the location of each checker is outlined. Figure 9. We created a checkerboard model consisting of a complicated upper 125 km above a checkerboard pattern imposed on a 1-D model. Coloured rectangles show 5 per cent positive and negative anomalies in the input model; areas in between have 0 per cent anomaly in the input model. (a) PCh-CO is the P-wave checkerboard retrieved using a starting model which replicates the upper 125 km (above dashed white line). (b) PCh-AK is the P-wave checkerboard retrieved using Model AK as the starting model. (c) SCh-CO is the S-wave checkerboard retrieved using a starting model which replicates the upper 125 km (the equivalent of Fig. 9a for S). The proﬁle is along 95.5◦(indicated on map views). The map views are at 225 and 550 km. The initial perturbation was ±5 per cent, and the location of each checker is outlined. absent from the input model. This is due to a combination of smear- ing and our imaging of relative anomalies across boundaries. Feature H is not particularly well retrieved, and G becomes connected to A, despite being separated by a 200 km gap in the input model. At 100 km, A has smeared upwards. The southwest of the model is slower than the southeast, consistent with the location of B, but the slow velocities of B are only observed north of the area of A. ﬁnd that there is considerable similarity between the main features for P and S, except that the S model shows less short wavelength structure. Also, we ﬁnd that our P-wave results move further from the starting point. This behaviour may be due to the greater number of P-wave observations, but could also be a result of the P-wave starting model being less accurate than the S-wave version, because we have to make assumptions on Vp/Vs when translating the ve- locities from S to P. Note that the body waves do not sample the regions outside the area bounded by the black lines in these ﬁgures, and so the anomalies outside these regions come exclusively from the surface wave starting model. 3.3 Synthetic test for probable structures In order to evaluate how different types of features may be recovered or generate artefacts, we created a model based on some simple shapes designed to represent structures that we may be able to image. We are interested in determining how various features might smear, the effect of uneven coverage, and how dealing with relative anomalies might affect the ﬁnal picture. Figure 8. Synthetic test to understand how well we can retrieve a known, complicated model. (a) The input is Model SW (after 30 iterations). (b) Sy-AK: the inversion is carried out using Model AK as the initial model. (c) Sy-CO: using the same synthetics, but with Model CO as the initial model. This has simple structure below 125 km (marked by the dashed white line) but replicates the complicated structure above. Shown along proﬁle L2 (Fig. 11). The input model contains a number of rectangular regions, la- belled A, B, C, G, H and L (Fig. 10, left-hand column), which represent structures that we ﬁnd in our models (Figs 11 and 12). Note that the model is plotted as relative percentage anomalies, and that only the labelled structures are included in the input models. the broadest features above 125 km. Note that A, which is nearly horizontal in the input model, becomes close to vertical, especially on Sy-AK (Fig. 8b), and in both cases the strong fast wave velocities at A and G in the input model smear into each other. The P results (Fig. 10, centre column) show that features A and C are well retrieved at 175 km, as are G and L at 500 km. However, E and K, and to a lesser extent D, F and I, all appear despite being 1733 Imaging the lithosphere beneath NE Tibet gure 9. We created a checkerboard model consisting of a complicated upper 125 km above a checkerboard pattern imposed on a 1-D model. Coloured ctangles show 5 per cent positive and negative anomalies in the input model; areas in between have 0 per cent anomaly in the input model. (a) PCh-CO is the wave checkerboard retrieved using a starting model which replicates the upper 125 km (above dashed white line). (b) PCh-AK is the P-wave checkerboard trieved using Model AK as the starting model. (c) SCh-CO is the S-wave checkerboard retrieved using a starting model which replicates the upper 125 km (the uivalent of Fig. 3.3 Synthetic test for probable structures As discussed in Section 3.1.2, the shallow structure is controlled mostly by starting model SW, and the structure below ∼300 km is controlled by the body waves. Between ∼100 and 300 km, both are exerting an inﬂuence. The recovery using S waves is similar (Figs 10 m–r), with slightly more vertical smearing (e.g. C in Fig. 10o), and poorer recovery for H (Fig. 10o). Note that we are adding noise based on measurement errors which may be too conservative for the S waves (Sections 2 and 3.1.2), and thus the results may also be too conservative. We ﬁnd that the range of percentage perturbations imposed by the body waves is higher in P than in S. For example, at 175 km, the total percentage range is 9.3 and 6.6 per cent for P and S, respectively. This is likely to be due to the larger P data set, and possibly also due to different choices of damping parameters. However, the combined inﬂuence of the body waves and the starting models results in ﬁnal models which have a greater range in S than in P (since we set the 4 R E S U LT S As discussed earlier, we prefer model SW and base our discussion on it. The images of Vp and Vs we obtain from modelling P and S-wave arrival times (Figs 11 and 12, equivalent ﬁgures for MO and AK are shown in Figs S5–S8) reveal considerable heterogeneity be- neath northeastern Tibet both laterally and as a function of depth. We 1734 1734 C. Nunn et al. C. Nunn et al. Figure 10. Input to a synthetic test for probable structures (left-hand column), P results (cental coulmn) and S results (right-hand column). Results shown along L2 and L4, and at 175, 500, 100 and 300 km depth. Note that perturbations were only applied to the labelled features in the input model (white labels). As before, the model is plotted as relative percentage anomalies, and so the background colours change in some cases, most obviously in the 150–250 km depth range. Black text labels either indicate smearing or features not originally in the input model. Figure 10. Input to a synthetic test for probable structures (left-hand column), P results (cental coulmn) and S results (right-hand column). Results shown along L2 and L4, and at 175, 500, 100 and 300 km depth. Note that perturbations were only applied to the labelled features in the input model (white labels). As before, the model is plotted as relative percentage anomalies, and so the background colours change in some cases, most obviously in the 150–250 km depth range. Black text labels either indicate smearing or features not originally in the input model. P percentage anomalies to be only half the percentage anomalies of S in the starting models). P percentage anomalies to be only half the percentage anomalies of S in the starting models). A by the slow anomaly H. In L1, at the very edge of our coverage (Fig. 11c), there is a connection through a fast structure J, although this is probably due to smearing (Section 3.3). I is another deep fast anomaly that lies beneath the Kunlun Mountains and reaches to the northern edge of the model (L2, Fig. 11a). There are also fast structures to the east and west (L1 and L3), although the shape is less well deﬁned where the coverage is poorer. Along L3, K is a slow anomaly from the IYS to the JS. 4.1 P-wave results Feature A is a shallow dipping, fast anomaly that extends from ∼140–240 km depth (Fig. 11a), and across the southern edge of the model at 175 km (Fig. 11b). The amplitude reduces gradually between the two dashed white lines. B is a slow wave velocity region on the western side of the region, extending from the Kunlun Shan, through the Songpan-Ganzi into the Qiangtang Terrane, up to the dashed white line (Fig. 11g). B extends from mid-crustal depths to ∼120 km on sections L1 and L2 (Figs 11c and a) 4 R E S U LT S In the south, it lies close to the base of the model, but is ∼100 km shallower further north (Figs 11d and i). L is a deep linear feature beneath L5 concentrated below 400 km depth (Figs 11e, f and i). 4.2 S-wave results At 175 km, the eastern side of the Qaidam Basin is faster than the west (C and D in Fig. 11b). E is a slow anomaly extending across the Songpan-Ganzi Terrane and the Kunlun Shan, terminating sharply near the NKTS (Figs 11a, b and d). Its southern boundary lies close to the JS, up to the boundary with A. E is between the fast regions A and C, and so potentially may only be slow in comparison to these areas, rather than exhibiting particularly slow wave velocities. In L2 (Fig. 11a), E blends in the west into the deeper slow anomaly F, which lies beneath the Songpan-Ganzi, Kunlun Shan and Qaidam Basin at depths of ∼250–475 km. The S-wave results (Fig. 12) are generally similar to the P-wave results (Fig. 11). All of the features described in the P-wave model appear in the S-wave model as well, but with some varia- tion in shape and amplitude. We focus on the important differences here. Feature A extends 50–100 km further north (Fig. 12b). There is an area on the eastern side which extends ∼200 km north of the NKF, although it is arguable whether or not this forms a continuous structure (it is interrupted by slower velocities on L4, Fig. 12e). C is shallower, and the boundaries are less steep (Fig. 12a). In L2 (Fig. 12a), there is some connection between A and G, but it is low G is a deep fast anomaly beneath the Qiangtang and Lhasa ter- ranes west of 92◦E (Fig. 11i). On L2 (Fig. 11a), it is separated from Imaging the lithosphere beneath NE Tibet 1735 Figure 11. Results: P-wave velocity anomalies along L1, L2, L3, L4 and L5 (c, a, d, e, f) and at 125, 175, 300 and 550 km depth (g, b, h, i). The model uses teleseismic body wave data and an initial starting model provided by surface waves. The extent of the body wave ray coverage is indicated by the black line on both the map views and the proﬁles, and so anomalies outside this region come purely from the starting model. Major features are labelled. The amplitude of A reduces gradually between the two dashed white lines on the map view at 175 km. White lines indicate the extent of B at 125 km and G and L at 550 km. 4.2 S-wave results As before, both proﬁles and map views are plotted as relative percentage anomalies (see Fig. 5 for fuller explanation) and topography and stations are plotted as in Fig. 3. Imaging the lithosphere beneath NE Tibet 1735 at University of Cambridge on Octobe http://gji.oxfordjournals.org/ Downloaded from Figure 11. Results: P-wave velocity anomalies along L1, L2, L3, L4 and L5 (c, a, d, e, f) and at 125, 175, 300 and 550 km depth (g, b, h, i). The model uses teleseismic body wave data and an initial starting model provided by surface waves. The extent of the body wave ray coverage is indicated by the black line on both the map views and the proﬁles, and so anomalies outside this region come purely from the starting model. Major features are labelled. The amplitude of A reduces gradually between the two dashed white lines on the map view at 175 km. White lines indicate the extent of B at 125 km and G and L at 550 km. As before, both proﬁles and map views are plotted as relative percentage anomalies (see Fig. 5 for fuller explanation) and topography and stations are plotted as in Fig. 3. 1736 C. Nunn et al. Figure 12. Results: S-wave velocity anomalies along L1, L2, L3, L4 and L5 (c, a, d, e, f) and at 125, 175, 300 and 550 km depth (g, b, h, i). The same features are labelled as in the P-wave models (Fig. 11), although the location may be shifted. The amplitude of A in the S-wave model reduces gradually between the wo dashed blue lines on the map view at 175 km, and the white lines show the equivalent for the P-wave results (Fig. 11). White lines indicate the extent of B at 125 km and G and L at 550 km. amplitude, and they continue to appear as separate structures. On L1 and L3 (Figs 12c and d) A and G are disconnected. There is a slow anomaly along the bottom of L2 (Fig. 12a), which is fast in the P waves (although also slower than G and I). Finally, the strongly deﬁned linear shape of L in the P waves is more diffuse in the S waves (Figs 11i and 12i). A and H are stronger features. It is likely that H remains from the starting model, rather than being clearly retrieved by the body 1736 C. 5.1 The lithosphere underneath Tibet Beneath the Lhasa and Qiangtang terranes, our images show a shallow dipping, fast feature at depths of ∼120–220 km, below a high-amplitude slow anomaly from ∼30–110 km (A and B , respec- tively, in Figs 11 and 12; interpretative cartoon Fig. 13). The overall shape of A comes from starting model SW, but the body waves have sharpened the change from fast to slow, and moved the boundary further south. Although P- and S-wave velocities behave differently in cases of temperature and compositional variations, we ﬁnd that the shape of the structure is similar in our P and S models. How- ever, as upper-mantle seismic velocities are much more sensitive to temperature than to compositional variations (e.g. Goes et al. 2000; Schutt & Lesher 2006), the fast velocities are likely to be due to colder than average temperatures irrespective of any compositional anomaly which might additionally exist. A third explanation for A is that pure shear thickening of the entire collision zone has forced some lithospheric material deeper, which will make it cooler than normal material at the same depth, until it can return to thermal equilibrium (McKenzie & Priestley 2008). Several other studies have imaged a continuous high velocity body stretching from the Himalaya northwards (e.g. Shapiro & Ritzwoller 2002; Lebedev & van der Hilst 2008; Li et al. 2008; Priestley et al. 2008; Hung et al. 2010; Zhang et al. 2012) although A popular model for plateau uplift involves delamination or re- moval of the bottom part of the lithosphere due to Rayleigh–Taylor Figure 13. Interpretation of the crust and upper mantle architecture of the central Tibetan Plateau (92◦–93◦E), modiﬁed from Yue et al. (2012). The central box indicates where our coverage is best. We observe a fast region underneath the plateau (dark green), which we interpret as underthrusting Indian lithosphere. Alternatively, it could represent a region which has been thickened by pure shear or the northern part could represent an underthrust Lhasa Terrane lithospheric slab. Radiogenic heating (McKenzie & Priestley 2008) of the thickened crust has heated the lower crust and upper mantle beneath the Qiangang and Songpan- Ganzi terranes, and the top of the underthrusting lithosphere (Craig et al. 2012). Qaidam lithosphere is faster than normal lithospheric mantle. Between the Qaidam and the underthrusting Indian Plate is an area of relatively warm material, but it is not hot enough to cause melt. 4.2 S-wave results Nunn et al. C. Nunn et al. 1736 C. Nunn et al. Figure 12. Results: S-wave velocity anomalies along L1, L2, L3, L4 and L5 (c, a, d, e, f) and at 125, 175, 300 and 550 km depth (g, b, h, i). The same features are labelled as in the P-wave models (Fig. 11), although the location may be shifted. The amplitude of A in the S-wave model reduces gradually between the two dashed blue lines on the map view at 175 km, and the white lines show the equivalent for the P-wave results (Fig. 11). White lines indicate the extent of B at 125 km and G and L at 550 km. deﬁned linear shape of L in the P waves is more diffuse in the S waves (Figs 11i and 12i). amplitude, and they continue to appear as separate structures. On L1 and L3 (Figs 12c and d) A and G are disconnected. There is a slow anomaly along the bottom of L2 (Fig. 12a), which is fast in the P waves (although also slower than G and I). Finally, the strongly A and H are stronger features. It is likely that H remains from the starting model, rather than being clearly retrieved by the body Imaging the lithosphere beneath NE Tibet 1737 waves (Figs 12a, c, d and h). However, A is also well retrieved by all the S-wave models (Fig 6). the apparent northward extent of this anomaly differs by several 100 km between different methodologies, and also along the strike of the Himalayan Arc. Hence, feature A is commonly interpreted as underthrusting Indian lithosphere (e.g. Kind et al. 2002; Li et al. 2008; Chen et al. 2010). 5 D I S C U S S I O N Alternatively, A could represent lithospheres of two different ori- gins. The southern part could originally come from India, and the northern part could represent an underthrust Lhasa Terrane litho- spheric slab. This has been proposed by Yue et al. (2012), based on a shallow dipping (6◦–10◦) interface that generates mode con- versions in receiver functions and dips from ∼80 km below the BNS to ∼120 km below the Kunlun Mountains. A belt of Eocene– Oligocene potassic volcanism beneath the Qiangtang suggests that there may have been earlier continental subduction (Roger et al. 2000; Ding et al. 2003), although it is unclear whether it would have been from the north or south. Our results suggest that there is no present subduction, because there is no connection between fast structures at 140 km and the surface. 5.2 Slow velocities beneath the Songpan-Ganzi and Kunlun Shan An explanation for the shallow low velocities (B) was put for- ward by McKenzie & Priestley (2008) who argue that radiogenic heat production in the thickened crust of Tibet is sufﬁcient to heat the mid-crust, which eventually heats the lower crust and the upper man- tle. This causes a temperature inversion, with temperatures highest in the mid-crust, and a negative temperature gradient beneath this region. This heating could result in an appreciable reduction in the shear wave velocities in the thickened mid-crust after only 10 Ma (McKenzie & Priestley 2008). It also could heat the lithosphere un- derneath (either the underthrusting Indian lithosphere or remnant Qiangtang lithosphere) to ∼120 km depth (Craig et al. 2012). The relatively narrow slow wave velocity region E beneath the Songpan-Ganzi Terrane and the Kunlun Shan at 175 km (Fig. 11b) is also seen in other tomographic studies (e.g. Li et al. 2008; Ceylan et al. 2012; Liang et al. 2012; Obrebski et al. 2012; Zhang et al. 2012). Zhang et al. (2012) suggested this area may be a mantle diapir upwelling between Indian and Asian lithospheres, and Ceylan et al. (2012) suggested that it may be shear heating along the KF. However, there is no recent volcanism in the Songpan-Ganzi, east of ∼92◦E (Searle et al. 2011), so this area is presumably not hot enough to produce melt. It is also likely that the lithosphere is 150–200 km thick beneath the Songpan-Ganzi and Kunlun Shan (Kumar et al. 2006; Zhao et al. 2011; Yue et al. 2012). Therefore, we think that E is slow only in a relative sense. It is sandwiched between two particularly fast areas A and C at the same depth. A number of studies have seen large differences in Pn and shear wave splitting between northern and southern Tibet. High Pn (i.e. uppermost mantle) velocities are found beneath most of the plateau south of the BNS, contrasting with a belt of low Pn velocities that stretches across the Songpan-Ganzi and Qiangtang terranes (McNamara et al. 1997; Hearn et al. 2004; Liang & Song 2006). The signiﬁcant shear wave splitting observed in northern Tibet con- trasts with the isotropic or weakly anisotropic fabric of southern Tibet and the Indian Shield (Chen & ¨Ozalaybey 1998; Huang et al. 2000; Chen et al. 2010). 5.1 The lithosphere underneath Tibet Below 350 km, we see an area of slower mantle, which coincides with a deepening of the 660 discontinuity (Yue et al. 2012), which could potentially be the remains of a disconnected oceanic slab which has passed through the 410. A depression in the 410 km discontinuity coincides with a slower area of mantle in our results, and may indicate small-scale upwelling. Figure 13. Interpretation of the crust and upper mantle architecture of the central Tibetan Plateau (92◦–93◦E), modiﬁed from Yue et al. (2012). The central box indicates where our coverage is best. We observe a fast region underneath the plateau (dark green), which we interpret as underthrusting Indian lithosphere. Alternatively, it could represent a region which has been thickened by pure shear or the northern part could represent an underthrust Lhasa Terrane lithospheric slab. Radiogenic heating (McKenzie & Priestley 2008) of the thickened crust has heated the lower crust and upper mantle beneath the Qiangang and Songpan- Ganzi terranes, and the top of the underthrusting lithosphere (Craig et al. 2012). Qaidam lithosphere is faster than normal lithospheric mantle. Between the Qaidam and the underthrusting Indian Plate is an area of relatively warm material, but it is not hot enough to cause melt. Below 350 km, we see an area of slower mantle, which coincides with a deepening of the 660 discontinuity (Yue et al. 2012), which could potentially be the remains of a disconnected oceanic slab which has passed through the 410. A depression in the 410 km discontinuity coincides with a slower area of mantle in our results, and may indicate small-scale upwelling. 1738 C. Nunn et al. instabilities (e.g. Molnar 1988; Molnar et al. 1993). We image fast velocities underneath the southern plateau, suggesting that the litho- sphere is thickened rather than delaminated. at least 100–350 km depth. We see a fast anomaly below 400 km, on the southwestern side of our region (G in Figs 11a and i), but it is separated from A by a region of slower velocities. In our area, we observe no well-resolved steeply dipping features, although in areas of poorer coverage, we do see a connection between A and G. However, this is likely to be due to smearing. Our synthetic test (Fig. 10) shows that two anomalies (one shallow and one deep) can easily become connected. 5.2 Slow velocities beneath the Songpan-Ganzi and Kunlun Shan These changes to the shear wave splitting and Pn have been interpreted as marking the edge of the advancing Indian lithosphere. Instead, we suggest that these changes mark the edge of the region where signiﬁcant heating occurs (the onset of B). Heating of the uppermost mantle would lower Pn and Sn. Similarly, Craig et al. (2012) suggest that the transition from weak anisotropy or isotropy to signiﬁcant anisotropy may reﬂect where the material becomes hot and weak enough to deform signiﬁcantly and develop an anisotropic fabric. However, it is also possible that B represents partially asthenospheric material or remnant weak Tibetan litho- sphere that has been underthrust by Indian mantle lithosphere. 5.1 The lithosphere underneath Tibet Assuming a collision age of ∼50 Ma, plate reconstructions from marine magnetic anomalies and fracture zone reconstructions show that there may be up to ∼3200 km of India-Asian conver- gence accommodated on the eastern side of Tibet, much more than the 450–900 km of documented Himalayan shortening (van Hinsbergen et al. 2011). There is thus sufﬁcient Indian mantle litho- spheric material available to account for anomaly A, but seismic methods cannot uniquely identify the provenance of the material in the collision zone. 5.3 No evidence of southward subduction of Asian lithosphere As discussed in Section 1, a number of investigators have proposed the southward subduction of Asian lithosphere beneath northern Tibet (Tapponnier et al. 2001; Kind et al. 2002; Zhao et al. 2011). If this were the case, we would expect to see a connection between the Qaidam lithosphere and structures in the plateau, and potentially see fast structures underthrusting or dipping into the mantle. Like Liang et al. (2012), Obrebski et al. (2012) and Li et al. (2008), we image relatively slower material here, which is difﬁcult to recon- cile with continuous southward subduction. Similarly, the Rayleigh wave tomography of Ceylan et al. (2012) and Lebedev & van der Hilst (2008) and the receiver function images of Yue et al. (2012) and Karplus et al. (in preparation) found no evidence of subduction in this region. p y p An E–W contrast, similar to our shallow feature B, also appears in the S-wave tomographic model of Liang et al. (2012) and is also consistent with the Pn study of Liang & Song (2006), which found low uppermost mantle velocities in the Songpan-Ganzi Terrane and the northern and western Qiangtang terrane, with particularly low wave velocities corresponding to B. The E–W contrast is also consis- tent with an area of Sn blockage seen by Barron & Priestley (2009). The propagation of the Sn phase is reduced in areas where there is a negative shear wave velocity gradient. At high frequencies, Sn is blocked across the entire plateau. However, in the mid-frequencies, Sn propagates with reduced efﬁciency in areas west of 93◦E in our area (corresponding to B). As described earlier, a negative shear wave velocity gradient can occur when the mid-crust is heated. At low frequency, Sn waves are able to propagate efﬁciently across the whole of Tibet, suggesting that the low velocities below the Qiang- tang are limited to the uppermost few tens of kilometres of the mantle. R E F E R E N C E S Abers, G.A. & Roecker, S.W., 1991. Deep structure of an arc-continent collision: earthquake relocation and inversion for upper mantle P and S wave velocities beneath Papua New Guinea, J. geophys. Res., 96(B4), 6379–6401. Acton, C.E., Priestley, K., Gaur, V.K. & Rai, S.S., 2010. Group velocity to- mography of the Indo-Eurasian collision zone, J. geophys. Res., 115(B12), B12335, doi:10.1029/2009JB007021. Aitchison, J.C., Ali, J.R. & Davis, A.M., 2007. When and where did India and Asia collide? J. geophys. Res.: Solid Earth, 112(B5), 2156–2202. Aki, K., Christofferson, A. & Husebye, E., 1977. Determination of the three-dimensional seismic structure of the lithosphere, J. geophys. Res., 82, 277–296. Barron, J. & Priestley, K., 2009. Observations of frequency-dependent Sn propagation in northern Tibet, Geophys. J. Int., 179(1), 475–488. AC K N OW L E D G E M E N T S We thank all members of the scientiﬁc teams of the INDEPTH IV/ASCENT seismic arrays. INDEPTH IV Fieldwork funding was provided by Deutsche Forschungsgemeinschaft, the Deutsches GeoForschungsZentrum-GFZ and by NSFEAR-CD-0409939 and CGS-1212010511809 in China. ASCENT funding was provided by Missouri University of Science & Technology. The INDEPTH IV Instruments were provided by SEISUK (NERC) (Brisbourne 2012), GIPP (GFZ) and ASCENT instruments by PASSCAL (IRIS). CN is supported by a Natural Environment Research Council studentship (grant NE/H52449X/1), with CASE funding from AWE Blacknest. Figures were prepared with Generic Mapping Tools (GMT) software (Wessel & Smith 1998). We thank James Mechie, Dan McKenzie, Timothy Craig, Alex Copley and Tuna Eken for their comments on the draft manuscript. The manuscript has been improved by com- ments from anonymous reviewers and the editor. at University of Cambridge on October 1 http://gji.oxfordjournals.org/ Downloaded from Finally, E, F and H (Fig. 11a) combined correspond to slow wave velocities imaged by Wittlinger et al. (1996). Also, Yue et al. (2012) see a thinning of the transition zone (probably due to deepening at the 410 discontinuity) in a location approximately corresponding to F. However, in our synthetic test (Fig. 10), the contrast between A and the rest of the region, as well as smearing from shallow anomaly B, was sufﬁcient to retrieve at least some of the amplitude of slow anomaly at F. Wittlinger et al. (1996) suggested that E, F and H may be a mantle plume of hot upwelling material. However, they note no melt from the plume can have reached the surface, since the Neogene volcanism in the Qiantang is further west than their proﬁle (Yin & Harrison 2000). We think it is unlikely that there is a major plume in the area. 5.5 Mantle structure beneath 300 km We see a deep, fast anomaly in the southwest (G in Figs 11i, a and c) and another in the northwest (I in Figs 11i and a), separated by a region which is slower (in a relative sense) at the same depths. G corresponds to a depression in the 660 km discontinuity (Yue et al. 2012). A reduction of the velocity contrast at the 660 had also been inferred from modelling the triplication of seismic waveforms with bounce points further west, in central Tibet (Chen & Tseng 2007). Following earlier suggestions by the works just cited and others, we therefore interpret D as a fragment of lithosphere that has detached and sunk into the mantle. This lithospheric fragment could either be a part of the detached Tethyan oceanic slab, or could be related to subduction or delamination of lithosphere after the onset of the continent–continent collision. We also see deep slow structures K and L. Both may represent small upwelling structures (with K possibly related to the emplacement of G). Alternatively, our synthetic tests suggest K may be an artefact of G since a low am- plitude slow structure is recovered next to G (Figs 10h and n). Deep slow wave velocities have been previously observed in southeastern Tibet, where we see K and L (Koulakov 2011). 5.4 Qaidam Basin At depths of ∼125 km, the Qaidam Basin is underlain by a fast region, particularly on the eastern side. Similarly, Hearn et al. (2004) and Liang & Song (2006) ﬁnd high Pn (i.e. uppermost mantle) velocities beneath the Qaidam. The LAB is observed at depths of 100–150 km with S-receiver functions (Zhao et al. 2011) and Yue et al. (2012) also infer a shallower Qaidam LAB on their western proﬁle than on their eastern proﬁle. The Qaidam has been suggested by many workers to act in a similar way to the larger Tarim Basin (e.g. Braitenberg et al. 2000). Both basins are thought to be cratonic in origin, and overlain by deep sediments. Interpretation of the seismicity and GPS observa- tions has shown that the Tarim rotates like a rigid block (Braitenberg et al. 2000; Reigber et al. 2001). In the Qaidam, GPS observa- tions show that it is currently taking up more shortening than the Further west (at ∼90◦E), between the IYS and BNS, Tilmann et al. (2003) imaged a fast, steeply dipping body that extends from Imaging the lithosphere beneath NE Tibet 1739 Songpan-Ganzi or Qiangtang terranes, albeit considerably less than the mountains of the Qilian Shan (Gan et al. 2007). Yin et al. (2008) show that Cenozoic upper crustal shortening decreases eastwards across the basin from >48 per cent in the west to <1 per cent in the east. Our observation that the east is faster (and presumably colder and more rigid) than the west may explain why there has been less shortening in the east. western side of our model. This coincides with an area of reduced propagation of the Pn phase (Barron & Priestley 2009), and is likely to be due to warming of the uppermost mantle due to radiogenic heating in the thickened crust (McKenzie & Priestley 2008). We ﬁnd that the Qaidam Basin is faster than normal mantle wave velocities to depths of around ∼180 km, with the east faster than the west. The fast region coincides with an area which has seen only small amounts of shortening over the Cenozoic (Yin et al. 2008). We ﬁnd no connection between structures beneath the Qaidam and further south in the plateau. From this we conclude that there is no evidence for southward subduction of Eurasian lithosphere. 6 C O N C LU S I O N S Tomographic image of the crust and upper mantle beneath the western Tien Shan from the MANAS broadband deployment: possible evidence for lithospheric delamination, Tectonophysics, 477(1–2), 49–57. Craig, T.J., Copley, A. & Jackson, J., 2012. 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Traveltimes for global earthquake location and phase identiﬁcation, Geophys. J. Int., 105, 429–465. Kennett, B.L.N., Engdahl, E.R. & Buland, R., 1995. Constraints on seismic velocities in the Earth from traveltimes, Geophys. J. Int., 122, 108–124. Roecker, S., Thurber, C. & McPhee, D., 2004. Joint inversion of gravity and arrival time data from Parkﬁeld: new constraints on structure and hypocenter locations near the SAFOD drill site, Geophys. Res. Lett., 31(12), L12S04, doi:10.1029/2003GL019396. Kind, R. et al., 2002. Seismic images of crust and upper mantle beneath Tibet: evidence for Eurasian Plate subduction, Science, 298, 1219–1221. Roecker, S., Thurber, C., Roberts, K. & Powell, L., 2006. Reﬁning the image of the San Andreas Fault near Parkﬁeld, California using a ﬁnite difference travel time computation technique, Tectonophysics, 426(1–2), 189–205. Koulakov, I., 2011. High-frequency P and S velocity anomalies in the up- per mantle beneath Asia from inversion of worldwide traveltime data, J. geophys. Res., 116(B4), doi:10.1029/2010JB007938. Kumar, P., Yuan, X., Kind, R. & Ni, J., 2006. Imaging the colliding Indian and Asian lithospheric plates beneath Tibet, J. geophys. Res.: Solid Earth, 111(B10), B06308, doi:10.1029/2005JB003930. Roger, F., Tapponnier, P., Arnaud, N., Sch¨arer, U., Brunel, M., Zhiqin, X. & Jingsui, Y., 2000. An Eocene magmatic belt across central Tibet: mantle subduction triggered by the Indian collision?, TerraNova, 12(3), 102–108. Lebedev, S. & van der Hilst, R.D., 2008. Global upper-mantle tomography with the automated multimode inversion of surface and S-wave forms, Geophys. J. Int., 173, 1–14. Schutt, D.L. & Lesher, C.E., 2006. Effects of melt depletion on the density and seismic velocity of garnet and spinel lherzolite, J. geophys. Res., 111(B5), B05401, doi:10.1029/2003JB002950. Imaging the lithosphere beneath NE Tibet 1741 Searle, M.P., Elliott, J.R., Phillips, R.J. 6 C O N C LU S I O N S & Chung, S.-L., 2011. Crustal- lithospheric structure and continental extrusion of Tibet, J. geol. Soc., 168(3), 633–672. Zhao, W. et al., 2011. Tibetan plate overriding the Asian plate in central and northern Tibet, Nature Geosci., 4(12), 870–873. Zhao, W. et al., 2011. Tibetan plate overriding the Asian plate in central and northern Tibet, Nature Geosci., 4(12), 870–873. Zhu, L. & Kanamori, H., 2000. Moho depth variation in southern California from teleseismic receiver functions, J. geophys. Res., 105(B2), 2969– 2980. Shapiro, N.M. & Ritzwoller, M.H., 2002. Monte-Carlo inversion for a global shear-velocity model of the crust and upper mantle, Geophys. J. Int., 151, 88–105. Tapponnier, P., Zhiqin, X., Roger, F., Meyer, B., Arnaud, N., Wittlinger, G. & Jingsui, Y., 2001. Oblique stepwise rise and growth of the Tibet Plateau, Science, 294, 1671–1677. S U P P O RT I N G I N F O R M AT I O N Additional Supporting Information may be found in the online ver- sion of this article: Additional Supporting Information may be found in the online ver- sion of this article: Tilmann, F., Ni, J. & the INDEPTH III Seismic Team, 2003. Seismic imaging of the downwelling Indian lithosphere beneath central Tibet, Science, 300, 1424–1427. Figure S1. Histograms showing initial and ﬁnal residuals for P and S models AK, MO and SW. VanDecar, J.C. & Crosson, R., 1990. Determination of teleseismic rela- tive phase arrival times using multi-channel cross correlation and least squares, Bull. seism. Soc. Am., 80, 150–169. Figure S2. An additional 5939 P-wave arrivals from 152 events (green squares) were used to test the ﬁt to the various models (Fig. S1). Waldhauser, F., Lippitsch, R., Kissling, E. & Ansorge, J., 2002. High- resolution teleseismic tomography of upper-mantle structure using an a priori three-dimensional crustal model, Geophys. J. Int., 150, 403–414. Figure S3. Example of a waveform for a typical P-wave event Figure S4. Example of a waveform for a typical S-wave event; traces are aligned according to relative delays determined by cross- correlation. Wessel, P. & Smith, W.H.F., 1998. New, improved version of generic mapping tools released, EOS, Trans. Am. geophys. Un., 79(47), 579. Wittlinger, G. et al., 1996. Seismic tomography of northern Tibet and Kun- lun: evidence for crustal blocks and mantle velocity contrasts, Earth planet. Sci. Lett., 139, 263–279. Wittlinger, G. et al., 1996. Seismic tomography of northern Tibet and Kun- lun: evidence for crustal blocks and mantle velocity contrasts, Earth planet. Sci. Lett., 139, 263–279. Yin, A. & Harrison, T.M., 2000. Geologic evolution of the Himalayan- Tibetan Orogen Annu Rev Earth planet Sci 28(1) 211–280 Figure S5. Model AK (P waves). Figure S5. Model AK (P waves). Figure S6. Model MO (P waves). Figure S6. Model MO (P waves). p Yin, A. & Harrison, T.M., 2000. Geologic evolution of the Himalayan- Tibetan Orogen, Annu. Rev. Earth planet Sci., 28(1), 211–280. Figure S7. Model AK (S waves). Figure S8. Model MO (S waves). Yin, A., Dang, Y.-Q., Zhang, M., Chen, X.-H. & McRivette, M.W., 2008. Cenozoic tectonic evolution of the Qaidam basin and its surrounding regions (part 3): structural geology, sedimentation, and regional tectonic reconstruction, Bull. geol. Soc. Am., 120(7–8), 847–876. Figure S9. A comparison between predicted group traveltimes from ﬁnal model AK, MO and SW with starting model SW, for a group of surface wave paths. Figure S10. S U P P O RT I N G I N F O R M AT I O N We generate noise-free synthetic P-wave traveltimes through starting model MO (a) and SW (c), and recover these traveltimes against starting model AK (http://gji.oxfordjournals. org/lookup/suppl/doi:10.1093/gji/ggt476/-/DC1). Figure S10. We generate noise-free synthetic P-wave traveltimes through starting model MO (a) and SW (c), and recover these traveltimes against starting model AK (http://gji.oxfordjournals. org/lookup/suppl/doi:10.1093/gji/ggt476/-/DC1). Yue, H. et al., 2012. Lithospheric and upper mantle structure of the northeastern Tibetan Plateau, J. geophys. Res., 117(B5), B05307, doi:10.1029/2011JB008545. Zhang, H., Zhao, D., Zhao, J. & Xu, Q., 2012. Convergence of the Indian and Eurasian plates under eastern Tibet revealed by seismic tomography, Geochem. Geophys. Geosyst., 13(6), doi:10.1029/2012GC004031. Geochem. Geophys. Geosyst., 13(6), doi:10.1029/2012GC0040 Please note: Oxford University Press are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Zhao, J. et al., 2010. The boundary between the Indian and Asian tectonic plates below Tibet, Proc. Natl. Acad. Sci. USA, 107(25), 11 229–11 233. Zhao, W. et al., 2008. Seismology across the northeastern edge of the Tibetan Plateau, EOS, Trans. Am. geophys. Un., 89(48), 487.
https://openalex.org/W4366425758	https://www.ijhpm.com/article_4433_843e2d928fb9a23be7fdcc6dd28916cf.pdf	English	null	National Neurotrauma Registry Data in Low- and Middle-Income Countries - Current Status and Future Requirements; Comment on "Neurotrauma Surveillance in National Registries of Low- and Middle-Income Countries: A Scoping Review and Comparative Analysis of Data Dictionaries"	International journal of health policy and management	2,023	cc-by	2,001	Abstract Since 1990 National Trauma Registries, — taking the form of “not for profit” small and medium enterprises — have been integral to improvementsin major injury case fatality in high-income settings. This is laudable but unsatisfactory as globally most years of life lost to injury occur in low- and middle-income countries (LMICs). International Journal of Health Policy and Management, recently published a scoping review of neurotrauma registries in LMICs by Barthelemy et al; from this the commentary reflects on the state of the art and how these LMIC registries could be taken to “the next level” as meaningful tools for improving major injury patient care. Keywords: Registry, Neurotrauma, LMIC Copyright: © 2023 The Author(s); Published by Kerman University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/ licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Lecky F. National neurotrauma registry data in low- and middle-income countries – current status and future requirements: Comment on “Neurotrauma surveillance in national registries of low- and middle-income countries: a scoping review and comparative analysis of data dictionaries.” Int J Health Policy Manag. 2023;12:7935. doi:10.34172/ijhpm.2023.7935 Correspondence to: Fiona Lecky Email: f.e.lecky@sheffield.ac.uk T T he global burden of injury falls disproportionately on young people living in low- and middle-income countries (LMICs)1 and the majority of injury deaths involve neurotrauma (injury to the brain, and or spinal cord).2 The recent World Health Assembly resolution illustrates a critical need to understand how healthcare and trauma care systems in LMICs can minimise mortality and disability from RTCs and other major injury vectors — mitigating the unacceptable human cost of “development” (Global Emergency and Trauma Care Initiative; https://www.who.int/ news/item/27-05-2019-72nd-world-health-assembly-adopts- resolution-on-emergency-and-trauma-care). this might be given the “state of the art.” Barthélemy and colleagues conducted a scoping review by searching the literature since 1991 for reports of national trauma registries in LMICs where the data dictionaries may be accessible, they also randomly but not comprehensively searched LMIC ministries of health. CURE, School of Health and Related Research, University of Sheffield, Sheffield, UK. https://ijhpm.com Int J Health Policy Manag 2023;12:7935 doi 10.34172/ijhpm.2023.7935 Article History: Received: 11 January 2023 Accepted: 3 April 2023 ePublished: 2 May 2023 Correspondence to: Fiona Lecky Email: f.e.lecky@sheffield.ac.uk Commentary National Neurotrauma Registry Data in Low- and Middle- Income Countries – Current Status and Future Requirements Comment on “Neurotrauma Surveillance in National Registries of Low- and Middle-Income Countries: A Scoping Review and Comparative Analysis of Data Dictionaries” Fiona Lecky* ID Abstract In total 15 LMICs were identified as having national trauma registries active at some point over the study period with 16 different registries, however only one registry had all the “minimum neurotrauma data” elements of the international registry for trauma and emergency care (IRTEC).5 Although the study had limitations particularly around searching it is impossible not to conclude from this review that currently LMIC trauma registries have limited capacity to support neurotrauma care improvement at national and international level — both in terms of their breadth and depth. In recent years hospital case fatality from traumatic brain injury and multisystem injury has been shown to halve in high-income countries (HICs) — associated with improved access to skilled resuscitation and specialist neuroscience care within designated trauma care systems.3,4 It has only been possible to demonstrate and publish this evidence with the data acquired, analysed, published and maintained by national trauma registries. Clinicians and Ministries of Health in LMICs — supported by the World Health Organization (WHO) Acute and Trauma Care Programme are keen to see this approach replicated in low resource settings: The current publication by Barthélemy et al “Neurotrauma Surveillance in National Registries of Low- and Middle-Income Countries: A Scoping Review and Comparative Analysis of Data Dictionaries”5 provides important insights into how feasible Disease or patient registries are collections of secondary data related to patients with a specific diagnosis, condition, or procedure. Secondary data is extracted from the patient care record rather than requiring new patient contact making them efficient resources for healthcare quality improvement (QI), assurance and comparative effectiveness research. National and international registries are usually anonimised making data analysis ethical when used for governance, service improvement or research. In order to understand why trauma and neurotrauma registries are not ubiquitous in LMICs one must understand their development in HICs over Lecky the last 30 years. provided through a variety of funding arrangements from road traffic collision insurance companies, central ministry or surgical college funding or subscriptions from individual hospitals.6-8 Trauma registry co-ordination centres are usually situated within higher education institutions rather than as independent entities, and it is worth noting the efficiencies that emanate from an “all major injury” inclusion criterion rather than solely neurotrauma. The rigour of the approach taken by Barthélemy and colleagues suggest the GEMCARN question should be given priority by research funders. References 1. Haagsma JA, Graetz N, Bolliger I, et al. The global burden of injury: incidence, mortality, disability-adjusted life years and time trends from the Global Burden of Disease study 2013. Inj Prev. 2016;22(1):3-18. doi:10.1136/injuryprev-2015-041616 1. Haagsma JA, Graetz N, Bolliger I, et al. The global burden of injury: incidence, mortality, disability-adjusted life years and time trends from the Global Burden of Disease study 2013. Inj Prev. 2016;22(1):3-18. doi:10.1136/injuryprev-2015-041616 2. Lecky F, Bouamra O, Woodford M. Changing epidemiology of polytrauma. In: Pape HC, Peitzman AB, Rotondo MF, Giannoudis PV, eds. Damage Control Management in the Polytrauma Patient. Cham: Springer; 2017. p. 27-32. doi:10.1007/978-3-319-52429-0_3 3. Fuller G, Bouamra O, Woodford M, et al. Temporal trends in head injury outcomes from 2003 to 2009 in England and Wales. Br J Neurosurg. 2011;25(3):414-421. doi:10.3109/02688697.2011.570882 4. Moran CG, Lecky F, Bouamra O, et al. Changing the system-major trauma patients and their outcomes in the NHS (England) 2008-17. EClinicalMedicine. 2018;2-3:13-21. doi:10.1016/j.eclinm.2018.07.001 5. Barthélemy EJ, Hackenberg AEC, Lepard J, et al. Neurotrauma surveillance in national registries of low- and middle-income countries: a scoping review and comparative analysis of data dictionaries. Int J Health Policy Manag. 2021;11(11):2373-2380. doi:10.34172/ijhpm.2021.167 4. Moran CG, Lecky F, Bouamra O, et al. Changing the system-major trauma patients and their outcomes in the NHS (England) 2008-17. EClinicalMedicine. 2018;2-3:13-21. doi:10.1016/j.eclinm.2018.07.001 The Global Emergency Care research network (GEMCARN) prioritised 7 key questions for improving trauma and emergency care systems. The third highest priority was given to the question “What are the obstacles to implementing emergency care/trauma registry-based systems in LMICs?”14 Given the history and requirement for a successful neurotrauma registry in resource rich settings one can guess that the barrier is not just a database and/or clinical record which IRTEC has addressed, but the ability to prioritise initiating and maintaining registry based QI and governance in resource limited settings when the actual provision of resuscitation, lifesaving treatment and training clinicians understandably take priority. However, to suggest that “it’s resources” is glib — probably oversimplifying a complex issue. Studies of why data in mass casualty incidents is so limited also suggest it is a cultural issue as much as limited resources.15 As with many small and medium enterprises success depends on leadership by key individuals, appetite for change, motivation to succeed — but resources and training in consistent reproducible data collation, reporting and interpretation for QI are also needed. International Journal of Health Policy and Management, 2023;12:7935 2 Abstract y Trauma and neurotrauma registries have been very much a “bottom up” small or medium enterprise and not for profit development in HICs. They are the equivalent of small business “start-ups” which have taken off. The entrepreneurial spirit was born in groups of clinicians, hospital managers, data scientists and patients in Europe, North America, and Australasia. The motivation was the need for data — rather than dictat and dogma — as the primary driver informing QI, governance, research, and national guidelines6-9 to improve survival and reduce disability in major injury victims. Investment in Trauma Registry reach and depth has been supported by ministries and healthcare commissioners; witnessing registry potential from published studies in single hospitals or geographical regions. Core neurotrauma data items are relatively sparse 8 in IRTEC and 40 from the Utstein template, recently replicated by the Collaborative European Neurotrauma Effectiveness Research in Traumatic Brain Injury.10-12 However, for trauma and neurotrauma registries to “live and breathe” data collection and reporting needs to be continuous and updated. Trauma receiving hospitals need a designated staff member paid to enter data and a clinician supporting this and receiving the registry reports which should benchmark the performance of each hospital against its peers — international and national norms — in a recent data set. The trauma or neurotrauma lead for the registry needs to be responsible for feeding back the reports at hospital trauma audit meetings for learning, governance and QI to occur. A credible mortality risk adjustment model is also key so that hospitals can estimate whether their acute care survival is better or worse than expected, this should be possible with the IRTEC data fields to risk adjust using the Kampala Trauma Score.13 Without these feedback loops for QI, governance and learning the registry can wither on the vine and become historical and less relevant like any ageing research dataset. Ethical issues Not applicable. Ethical issues Not applicable. Competing interests Author declares that she has no competing interests. Competing interests Competing interests Author declares that she has no competing interests. p g Author declares that she has no competing interests. Funding Global Emergency Care Research Network (GEMCARN)/Lead applicant/ GCRF-QR/£50,000/2019-20. Acknowledgements This is an invited commentary after providing review on the International Journal of Health Policy and Management scoping review from Barthélemy et al. Ethical issues Not applicable. Competing interests Author declares that she has no competing interests. Ethical issues Not applicable. comparison of the Kampala Trauma Score (KTS) with the Revised Trauma Score (RTS), Injury Severity Score (ISS) and the TRISS method in a Ugandan trauma registry. Eur J Trauma. 2003;29(6):392-398. doi:10.1007/s00068-003-1277-5 countries (LMICs): results of research prioritisation setting exercise. BMC Emerg Med. 2020;20(1):68. doi:10.1186/s12873-020-00362-7 15. Jafar AJN, Fletcher RJ, Lecky F, Redmond AD. A pilot of a UK emergency medical team (EMT) medical record during a deployment training course. Prehosp Disaster Med. 2018;33(4):441-447. doi:10.1017/ s1049023x18000468 14. Lecky FE, Reynolds T, Otesile O, et al. Harnessing inter-disciplinary collaboration to improve emergency care in low- and middle-income International Journal of Health Policy and Management, 2023;12:7935 3 References In HICs resources for dedicated registry staff in the co-ordination centre are 5. Barthélemy EJ, Hackenberg AEC, Lepard J, et al. Neurotrauma surveillance in national registries of low- and middle-income countries: a scoping review and comparative analysis of data dictionaries. Int J Health Policy Manag. 2021;11(11):2373-2380. doi:10.34172/ijhpm.2021.167 6. Champion HR, Copes WS, Sacco WJ, et al. The Major Trauma Outcome Study: establishing national norms for trauma care. J Trauma. 1990; 30(11):1356-1365. doi:10.1097/00005373-199011000-00008 7. Yates DW, Woodford M, Hollis S. Preliminary analysis of the care of injured patients in 33 British hospitals: first report of the United Kingdom major trauma outcome study. BMJ. 1992;305(6856):737-740. doi:10.1136/ bmj.305.6856.737 8. Cameron PA, Finch CF, Gabbe BJ, Collins LJ, Smith KL, McNeil JJ. Developing Australia’s first statewide trauma registry: what are the lessons? ANZ J Surg. 2004;74(6):424-428. doi:10.1111/j.1445- 1433.2004.03029.x 9. National Institute for Health and Care Excellence (NICE). Head Injury: Assessment and Early Management. NICE; 2019. 10. World Health Organization (WHO). WHO International Registry for Trauma and Emergency Care. WHO; 2020. 11. Ringdal KG, Coats TJ, Lefering R, et al. The Utstein template for uniform reporting of data following major trauma: a joint revision by SCANTEM, TARN, DGU-TR and RITG. Scand J Trauma Resusc Emerg Med. 2008;16:7. doi:10.1186/1757-7241-16-7 12. Lecky FE, Otesile O, Marincowitz C, et al. The burden of traumatic brain injury from low-energy falls among patients from 18 countries in the CENTER-TBI Registry: a comparative cohort study. PLoS Med. 2021; 18(9):e1003761. doi:10.1371/journal.pmed.1003761 ( ) j p 13. MacLeod JBA, Kobusingye O, Frost C, Lett R, Kirya F, Shulman C. A 13. MacLeod JBA, Kobusingye O, Frost C, Lett R, Kirya F, Shulman C. A 13. International Journal of Health Policy and Management, 2023;12:7935 2 Lecky Lecky
https://openalex.org/W54445393	https://hal.inria.fr/hal-01451742/document	English	null	Mass Customization in Supply Chain Level: Development of a Conceptual Framework to Manage and Assess Performance	IFIP advances in information and communication technology	2,013	cc-by	4,389	To cite this version: Mahnoosh Zebardast, Silvia Malpezi, Marco Taisch. Mass Customization in Supply Chain Level: Development of a Conceptual Framework to Manage and Assess Performance. 20th Advances in Production Management Systems (APMS), Sep 2013, State College, PA, United States. pp.81-90, 10.1007/978-3-642-41263-9_11. hal-01451742 Mass Customization in Supply Chain Level: Development of a Conceptual Framework to Manage and Assess Performance Mahnoosh Zebardast, Silvia Malpezi, Marco Taisch Mahnoosh Zebardast, Silvia Malpezi, Marco Taisch Distributed under a Creative Commons Attribution 4.0 International License Mass Customization in Supply Chain Level: Development of a Conceptual Framework to Manage and Assess Performance Mahnoosh Zebardast1, Silvia Malpezi1, Marco Taisch1 1Politecnico di Milano, Department of Management, Economics and Industrial Engineering Via Lambruschini 4/b 20156, Milano, Italy {mahnoosh.zebardast and silvia.malpezi}@mail.polimi.it, marco.taisch@polimi.it Abstract. Recent market interest on customized offers and intensive competition on attracting market globally, lead companies to implement supply chain management to improve performance and gain competi- tive advantage. To this aim, Supply chain management in customer- oriented environment is pursuing the transition from traditional supply chain into concurrent flexible and efficient one. This paper aims to un- derstand specifically how supply chain within this environment needs to be configured and managed in order to enable efficient customization for mass market. To reach this goal, a conceptual framework and list of indicators to support the framework have been developed and tested. Keywords: Mass customization, Supply chain management, Perfor- mance measurement. HAL Id: hal-01451742 https://inria.hal.science/hal-01451742v1 Submitted on 1 Feb 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction and Research Objectives There is a growing recognition among scholars and practitioners that individual businesses no longer compete as stand-alone entities, but rather as supply chains [3]. Recently, we are now entering the area of “network compe- tition,” where the awards will go to those organizations who can better struc- ture, coordinate, and manage the relationships with partners in a network which is committed for a better, closer, and more agile relationships with final customers [3]. It can be argued that in today’s challenging global markets, the route to sustainable advantage lies in being able to leverage the respective strengths and competencies of network partners to achieve greater respon- siveness to market needs [3]. Evidently market interest on more customized offers better aligned to individual’s needs, brings further challenges in terms of complexity and uncertainty. Being competitive in this environment requires concurrent efficiency and flexibility [8]; accordingly enables the ability to provide higher variety at lower cost, enabling strategies of mass customization to be pursued [3]. Mass customization is defined differently by many scholars time after time [5], [27], [29], [41] but the most well-known definition used by this study, is given by Piller as “Customer co-design process of products and services, which meet the needs of each individual customer with regard to certain product features. All operations are performed within a fixed solution space, characterized by stable but still flexible and responsive processes”[28]. p y p p Inspired by extensive literature review, we recognized that the literature on mass customization in supply chain level has been growing recently and there are still some areas that need further research. In particular we believe that more understanding is needed on how to configure and manage a supply chain in such environment. Therefore, the purpose of this paper is to analyze the configuration and management of supply chain while implementing mass customization. More specifically, the research objectives of this paper are (1) understanding factors necessary to be considered in order to configure and manage the supply chain while implementing mass customization; and (2) recognizing how these factors can be measured. To reach these goals, we first did the literature review and tried to cluster it into research areas to construct our understandings. Four research areas relevant to our objectives were identi- fied. Afterwards relevant factors and indicators related to each research areas were identified and structured in a consistent and understandable framework. 1 Introduction and Research Objectives In this framework, factors were structured from literature while indicators were developed by this study. As last step we tried to validate the framework by three case studies that employed mass customization at the time of data collection in diverse industries. We shaped this paper as follows: first, we delivered a literature review, next we explained the methodology employed. Then, we presented the framework and finally, we explained validation phase and made the conclusion. 2 Literature review With the aim of mass customization, customer needs to be involved in- to value creation processes. This involvement can happen in different stages, in relation to different actors within supply chain The degree of customer involvement in literature is known as customization level and has been dis- cussed extremely by scholars [6], [18], [30], [36, 37]. Moreover, the capabil- ity of supply chain in implementing it is known as postponement. Graman defines postponement as the capability of supply chain in delaying the activi- ties associated to differentiation of product_ customization processes_ closer to the time that demand is known [8]. Literature positions postponement dif- ferently. Some recognize it only in manufacturing operations [2], [11], [32, 33] while some others take a broad view and distinguish it in supply chain level specially emphasize on differentiation in distribution point [39]. Within those who consider postponement in supply chain level, many discuss about issues such as the conflict between product variety and quick response time [16], or product growth and cost control at certain point [32, 33]. Generally literature discusses about postponement by either focusing on types of post- ponement (time, form and place),their evaluation and comparison [11], [17], [39, 40]; or targeting management of inventory to set optimal level of invento- ry [2], [8], [24], [32, 33]. In both of these groups modularization has been recognized as an enabling method for efficient customization. Based on our literature review, this study analyses modularization to the aim of a better understanding about this method by focusing on its characteristics and ad- vantages [4], [19], [35], [44]. It specifically discusses about the need for a more intimate relationship among supply chain partners to produce, supply and manage the inventory of modules for customization [13], [20], [22, 23]. Literature has put more attention on relationships among partners in customer oriented environments where a more flexible and efficient supply chain is requested. The relationship is interpreted as integration and cooperation be- tween supplier-manufacturer, manufacturer-customer; and among internal divisions of manufacturer [15], [22], [25], [34], [45, 46]. Literature rarely differentiates cooperation and integration and draws the line between them. Some studies, like Pan and Holland [4], defines cooperation as a beneficial relationship between actors namely customer, manufacturer and other partners such as supplier and distributers. 2 Literature review It is believed that this relationship aims to improve outcomes like customer satisfaction, time to market or resource us- age by setting common objectives and reducing duplicated activities for in- creasing value added activities [46]. Integration is a more rigorous concept which aims to integrate the actors in both ends (downstream and upstream) to achieve an optimal output. It includes integration of processes, activities, loca- tions and etc. to optimize the performance of all actors as a whole [15], [20, 21]. Moreover, it decreases uncertainties and increases flexibility and respon- siveness [20, 21]. 3 Methodology In the first step of this research “supply chain” and “mass customiza- tion” were searched in keywords and abstract sections without any restriction or preference over journals. In total 71 articles from 39 different journals were selected to be reviewed. Papers were analyzed in a more detailed level and their main focuses in supply chain were recognized and subsequently clus- tered (e.g. postponement, modularity and etc.). The output of this step was twelve clusters covering different strategies and methods, called research are- as. These research areas have been the starting point for the construction of our framework. Since the conceptual framework was based on literature, this study validated it empirically by three case studies in different industries. Unit 3 of analysis was manufacturer supply chain (only including first layer suppli- ers), research domain was manufacturing industry/sector implementing mass customization at the time of data collection, regardless of the size or the level of customization; and expected respondents were operation manager and owner. Validation phase was done by online questionnaire. In particular, the questionnaire consisted of four parts representing framework subjects and aimed to test associated factors and indicators. Questions were multiple choice (without restriction on number of choices) and open-end. Multiple choice questions were used for validation of factors and indicators while open-end questions aimed to initiate respondents to add missing impacting factors and/or indicators. 4 Conceptual Framework A conceptual framework was developed to support configuration and man- agement of supply chain. It identifies four main decision areas that are about relationship management, postponement, level of customization, and modular- ity level. To reach our objectives, by conceptual framework, we tried to un- derstand relevant elements to be considered for each decision area. For in- stance in order to manage relationship along a supply chain, this study expects that an industry needs to consider elements such as customer integration level and supplier selection criteria. By following, we structure the work in four sections associated to the framework decision areas. In each section, first we briefly define the decision area and associated elements. Afterwards, we spec- ify relevant impacting factors and indicators for each element (see Table 1). Relationship Management Internal integration level Customer integration level Cooperation level with partners Supplier selection criteria Customization Level Product characteristics Market characteristics Partners characteristics Modularity Level Product characteristics Production system characteristics Partners characteristics Postponement Production system characteristics Partners characteristics Mc in SC level Mc in SC level Figure 1_ Conceptual Framework 4.1 Relationship management The first decision area is related to different relations that a company (manu- facturer) should have with players in supply chain. The aim is to primarily understand who is considered as key partner and is necessary to build close relationship; and how to manage these different kinds of relationships while practicing mass customization. To reach this goal, four types of elements are necessary to be considered. These elements are briefly defined by following: Internal integration level: This element refers to relations inside the manu- facturer and points out the importance of internal capabilities in satisfying customer needs responsively. In particular, this element brings out level of interaction between internal departments and employees. y y y g Internal integration level: This element refers to relations inside the manu- facturer and points out the importance of internal capabilities in satisfying customer needs responsively. In particular, this element brings out level of interaction between internal departments and employees. Customer integration level: This element highlights the importance of cus- tomer and their value-adding contribution inside the supply chain processes. Specifically this element deals with the extension of customer contribution and management of the transferred knowledge. Cooperation level with partners: This element focuses on how to define the extent of relation and then how to manage it with different actors such as sup- pliers and distributors. Partners’ selection criteria: This element emphasizes on the basics which needs to be considered in order to select some partners over the others. 4.4 Postponement: Last subject is related to the postponement strategy known as capability of a supply chain to perform customization in a way to delay differentiation or customization closer to the time that demand for the product is known [8]. The aim of this group is to understand appropriate position of customer inte- gration point. To reach this goal, we identify two elements necessary to be considered. These elements are briefly defined by following: Partners’ characteristics: This element refers to partners’ capability in either carrying out the customization in their location (e.g. distributors’ ability in customizing products) or being collaborative and responsive to support core company postponement strategy. Production system characteristics: This element refers to production capa- bilities inside of the manufacturing which operationally support customer intervention. 4.2 Customization level The second area relates to the marginal value that customization brings to the end customer. Definition of this value impacts on the way supply chain oper- ates and creates the customization marginal value. Mass customization levels can be driven from tailored customization (customization in fabrication), cus- tomized standardization (customization in assembly); and segmented stand- ardization (customization in package and distribution) [18]. In order to identi- fy the customization level, a company needs to consider three elements. These elements are briefly defined by following: Product characteristics: This element refers to product features that support decisions related to customization. Partners’ characteristics: This element refers to capabilities, characteristics and relationships of actors inside the supply chain. 5 Market characteristics: This element refers to extent of customization in relation to market need. Market characteristics: This element refers to extent of customization in relation to market need. 4.3 Modularity level: Third decision area is related to a method known as modularization that ena- bles a company to efficiently customize products. This study considers only the set of elements, related to the production process and supply chain charac- teristics, that impacts on product modularization, hence excluding elements related to other types of modularization (such as organizational modularity). The impacting elements are briefly defined by following: The impacting elements are briefly defined by following: Product characteristics: This element refers to product features that support decisions related to customization. Partners’ characteristics: This element refers to capabilities of actors inside the supply chain which operationally support modularization. Production system characteristics: This element refers to production capa- bilities inside of the manufacturing which operationally support modulariza- tion. 5 Validation and conclusion As a final step this study conducted three case studies to validate its findings. In particular by case studies, we tried to understand if impacting factors are practically considered in configuration and management of supply chain. Moreover we tried to test indicators relevance by asking if they are already applied in practice or can be considered beneficial. Based on our case studies, all impacting factors were validated but certain indicators were not. Customer willingness in participation was never measured and its measure- ment was believed challenging. Moreover, although all three cases had modu- lar product, but believed that proposed indicator for product architecture was not representative; instead they assert that ration of customizable modules cost on production cost was suggested to be substitute. As a result of this study we create a better understanding of how different factors impact on configuration and management of supply chain in customer-oriented environment. Moreo- ver, we suggest a list of indicators that can support decisions with a better indication on circumstances. Acknowledgements. This work was partly done by Merve Murathanoglu as her master thesis in Politecnico di Milano University. Moreover, this paper has been partly funded by the European Commission through S-MC-S (Sus- tainable Mass Customization – Mass Customization for Sustainability) Project (Grant Agreement No: FoF.NMP.2010-2 260090 - S-MC-S) and project called “New business models for manufacturing and services”. 7 7 Table 1_List of Indicators dedicated to conceptual framework Elements Impacting factors Factor Description Indicator Internal integra- tion level Capability in exchanging in- formation [20], [21], [25], [45] This factor impacts on ability of a company to exchange information internally and be responsive to final customers Number of times that information has been exchanged between departments on a time base (e.g. 5 Validation and conclusion weekly/ monthly) Organizational readiness [25] This factor refers to employees’ skill in understanding the end customer needs and their ability in translat- ing it into know-hows Order response time Customer inte- gration level Customer willingness in partic- ipation [15] This factor refers to customer willingness in sharing actual demand and in providing feedbacks to issues such as product development Percentage of customers participating in data collection phase via employed methods/mechanisms/tools Information exchange level [15], [25], [26], [45] This factor focuses on the actual level of information exchanged which has a direct impact on effective implementation of mass customization Number of methods/mechanisms/tools employed to gather data from customers Cooperation level with partners Joint profits [46] This factor refers to decisions made jointly to bring out better results in set of aspects such as profit Percentage of partners with joint profit agreement Type of shared information [34] This factor refers to set of information that its sharing can bring advantages to partners. Sharing infor- mation such as delivery due date, cost of delay, total time of manufacturing; and distribution with partners, are some examples which beside profit, brings improvements in service level, quality and etc. Percentage of partners with shared information with core manufacturer Supplier selec- tion criteria Agility of supplier [45] This factor refers to dynamic characteristics of a customer-oriented environment. Consequently, concur- rent flexible and responsive suppliers are required Percentage of orders committed to be fulfilled on time Capacity of supplier [19] This factor refers to power of manufacturer on supplier. In particular it focuses on portion of capacity allocated to the manufacturer Percentage of capacity allocated to manufacturer Information exchange level [20], [21], [22], [25], [26], [45] This factor refers to internal capability of supplier in sharing information Expected time interval in sharing information between supplier and manufacturer (e.g. weekly/ monthly) Product characteristics Product architecture [16], [38] This factor refers to architecture of the product which can impact on its customizability in different levels Percentage of customizable modules/parts Partners characteristics Supply chain information exchange level [38] This factor refers to level of information integration among partners Expected time interval in sharing information between partners(e.g. weekly/ monthly) Cost of customization [16] This factor refers to the trade-off between cost and value of customization. References 1. 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https://openalex.org/W2599986470	https://scholarcommons.sc.edu/cgi/viewcontent.cgi?article=1111&context=sph_environmental_health_sciences_facpub	English	null	Altered gut microbiome in a mouse model of Gulf War Illness causes neuroinflammation and intestinal injury via leaky gut and TLR4 activation	PloS one	2,017	cc-by	12,623	Faculty Publications Environmental Health Sciences Faculty Publications University of South Carolina University of South Carolina Scholar Commons Scholar Commons Faculty Publications Environmental Health Sciences 1-1-2017 Altered Gut Microbiome in a Mouse Model of Gulf War Illness Altered Gut Microbiome in a Mouse Model of Gulf War Illness Causes Neuroinflammation and Intestinal Injury via Leaky Gut and Causes Neuroinflammation and Intestinal Injury via Leaky Gut and TLR4 Activation TLR4 Activation Firas Alhasson Suvarthi Das Ratanesh K. Seth University of South Carolina, sethr@mailbox.sc.edu Diptadip Dattaroy Varun Chandrashekaran See next page for additional authors Follow this and additional works at: https://scholarcommons.sc.edu/ sph_environmental_health_sciences_facpub Part of the Environmental Health Commons Publication Info Publication Info PloS One, Volume 12, Issue 3, 2017. This Article is brought to you by the Environmental Health Sciences at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact digres@mailbox.sc.edu. University of South Carolina University of South Carolina Scholar Commons Scholar Commons Author(s) Author(s) Firas Alhasson, Suvarthi Das, Ratanesh K. Seth, Diptadip Dattaroy, Varun Chandrashekaran, Caitlin N. Ryan, Luisa S. Chan, Traci Testerman, James Burch, Lorne J. Hofseth, Ronnie Horner, Mitzi Nagarkatti, Prakash Nagarkatti, Stephen M. Lasley, and Saurabh Chatterjee This article is available at Scholar Commons: https://scholarcommons.sc.edu/ sph_environmental_health_sciences_facpub/108 Altered Gut Microbiome in a Mouse Model of Gulf War Illness Altered Gut Microbiome in a Mouse Model of Gulf War Illness Causes Neuroinflammation and Intestinal Injury via Leaky Gut and Causes Neuroinflammation and Intestinal Injury via Leaky Gut and TLR4 Activation TLR4 Activation Publication Info Publication Info PloS One, Volume 12, Issue 3, 2017. This Article is brought to you by the Environmental Health Sciences at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact digres@mailbox.sc.edu. Author(s) Author(s) RESEARCH ARTICLE OPEN ACCESS Citation: Alhasson F, Das S, Seth R, Dattaroy D, Chandrashekaran V, Ryan CN, et al. (2017) Altered gut microbiome in a mouse model of Gulf War Illness causes neuroinflammation and intestinal injury via leaky gut and TLR4 activation. PLoS ONE 12(3): e0172914. doi:10.1371/journal. pone.0172914 ☯These authors contributed equally to this work. ¤ Current address: Department of Medicine (Gastroenterology), University of Massachusetts Medical School, Worcester, Massachusetts, United States of America * schatt@mailbox.sc.edu ☯These authors contributed equally to this work. ¤ Current address: Department of Medicine (Gastroenterology), University of Massachusetts Medical School, Worcester, Massachusetts, United States of America * schatt@mailbox.sc.edu ☯These authors contributed equally to this work. ¤ Current address: Department of Medicine (Gastroenterology), University of Massachusetts Medical School, Worcester, Massachusetts, United States of America * schatt@mailbox.sc.edu Editor: Partha Mukhopadhyay, National Institutes of Health, UNITED STATES Altered gut microbiome in a mouse model of Gulf War Illness causes neuroinflammation and intestinal injury via leaky gut and TLR4 activation Firas Alhasson1☯, Suvarthi Das1☯¤, Ratanesh Seth1☯, Diptadip Dattaroy1, Varun Chandrashekaran1, Caitlin N. Ryan2, Luisa S. Chan2, Traci Testerman3, James Burch4, Lorne J. Hofseth5, Ronnie Horner6, Mitzi Nagarkatti3, Prakash Nagarkatt Stephen M. Lasley7, Saurabh Chatterjee1* Firas Alhasson1☯, Suvarthi Das1☯¤, Ratanesh Seth1☯, Diptadip Dattaroy1, Varun Chandrashekaran1, Caitlin N. Ryan2, Luisa S. Chan2, Traci Testerman3, James Burch4, Lorne J. Hofseth5, Ronnie Horner6, Mitzi Nagarkatti3, Prakash Nagarkatti3, Stephen M. Lasley7, Saurabh Chatterjee1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America, 2 Second Genome Inc., San Francisco, California, United States of America, 3 Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina, United States of America, 4 Department of Epidemiology and Biostatistics, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, United States of America, 5 Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina, United States of America, 6 Department of Health Services, Policy and Management, Arnold School of Public Health, University of South Carolina. Columbia, South Carolina, United States of America, 7 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, Illinois, United States of America Introduction Prior research shows that, shortly after the first Persian gulf war (GW), a series of unique symptoms plagued many of the deployed veterans [1, 2]. These symptoms included chronic fatigue, gastrointestinal (GI) disturbances, and irritable bowel syndrome (IBS), in addition to post traumatic stress disorder and neurological abnormalities[1]. Many of these symptoms are difficult to categorize as they have no known cause, no objective findings on clinical examina- tion, no diagnostic biomarkers, no known tissue pathology, and no curative therapy[3]. Competing interests: The inclusion of researchers from Second Genome Inc. CNR and LSC does not alter our adherence to PLOS ONE policies on sharing data and materials [http://www.PLOSone. org/static/editorial.action#competing]. Well-conducted clinical studies and nationwide federal surveys provide documented evi- dence that exposure to GW agents such as pesticides and insect repellents, along with the con- sumption of nerve agent pre-treatment sets containing Pyridostigmine Bromide (PB), are among the major causative factors implicated in the etiopathogenesis of Gulf War Illness (GWI) [1, 4–8]. Likewise, epidemiological studies provide critical support for a significant bio- logical gradient between the concentration and frequent use of PB pills and the increased severity of the illness [9]. Rodent studies in a GWI model showed corticosterone primed neu- roinflammation [10]. Interestingly, exposure to PB, Permethrin and corticosterone has been found to cause oxidative stress in liver and neuronal cells [11–15]. Recent studies show an association between mood and cognitive dysfunction and hippocampal pathology is epito- mized by decreased neurogenesis, partial loss of principal neurons, and mild inflammation in a model of GWI[16]. Further, results with an increase in soluble cytokine receptors and decrease in IL6 indicate that specific inflammatory proteins may be associated with brain structure and function as indexed by hippocampal volume and PTSD symptoms[17]. GWI networks had more abundant connections but were less organized. A detailed analysis indi- cated that IL-1alpha and CD2+/CD26+ nodes strongly influenced this characteristic modula- tion of B and T cell network motifs. This potentially heightened lymphocyte and HPA axis responsiveness to IL-1 stimulation in the context of a mixed Th1:Th2 immune signature sup- ports an autoimmune component in GWI etiology[18]. Dysbiosis and leaky gut contributes to Gulf War Illness decision to publish, or preparation of the manuscript. decision to publish, or preparation of the manuscript. Abstract Received: November 29, 2016 Accepted: February 10, 2017 Published: March 22, 2017 Copyright: © 2017 Alhasson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Many of the symptoms of Gulf War Illness (GWI) that include neurological abnormalities, neuroinflammation, chronic fatigue and gastrointestinal disturbances have been traced to Gulf War chemical exposure. Though the association and subsequent evidences are strong, the mechanisms that connect exposure to intestinal and neurological abnormalities remain unclear. Using an established rodent model of Gulf War Illness, we show that chemical exposure caused significant dysbiosis in the gut that included increased abundance of phy- lum Firmicutes and Tenericutes, and decreased abundance of Bacteroidetes. Several gram negative bacterial genera were enriched in the GWI-model that included Allobaculum sp. Altered microbiome caused significant decrease in tight junction protein Occludin with a con- comitant increase in Claudin-2, a signature of a leaky gut. Resultant leaching of gut caused portal endotoxemia that led to upregulation of toll like receptor 4 (TLR4) activation in the small intestine and the brain. TLR4 knock out mice and mice that had gut decontamination showed significant decrease in tyrosine nitration and inflammatory mediators IL1β and MCP-1 in both the small intestine and frontal cortex. These events signified that gut dysbio- sis with simultaneous leaky gut and systemic endotoxemia-induced TLR4 activation contrib- utes to GW chemical-induced neuroinflammation and gastrointestinal disturbances. Received: November 29, 2016 Accepted: February 10, 2017 Published: March 22, 2017 Copyright: © 2017 Alhasson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data are contained in the figures, tables, supplementary information, and the NCBI/EBI database under accession number PRJEB19474. Funding: This work has been supported by NIH Pathway to Independence Award, R00ES019875 to Saurabh Chatterjee. Second Genome Inc provided support in the form of salaries for authors [CNR and LSC], but did not have any additional role in the study design, data collection and analysis, 1 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 ONE \| DOI:10.1371/journal.pone.0172914 March 22, 20 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Introduction With evidence of the exposure of these chemicals in the war theater through oral, nasal, and dermal routes, it is reasonable to predict that the above-listed exposures, either in unison or individually, can cause severe organ-spe- cific or systemic oxidative stress and inflammation, effects that may depend on the route of exposure. Chemical exposures through an oral or systemic route have been shown to cause significant inflammation and intestinal oxidative stress[19–21]. GI disturbance in GWI closely resembles inflammatory bowel disease/syndrome[2]. Although there is no significant evidence of oxida- tive stress-mediated changes in the gut microbiome, intestinal inflammation associated with IBS, alcoholic steatohepatitis, obesity, nonalcoholic fatty liver disease and metabolic syndrome, they have been strongly linked to alterations in the gut microbiome[22–29]. There are at least 3000 species of bacteria residing in the human gut, and there is a unique prototype of bacterial diversity in every individual[30]. The last decade saw a tremendous increase in our under- standing of how gut bacteria participate in health and disease. Significant areas in which gut bacteria have been found to play a role are obesity, metabolic syndrome and inflammatory bowel syndrome[30]. There is increasing evidence that the gut microbiome plays a role in modulating the neuronal responses[31]. Studies have shown that IBS patients are characterized by a reduction of butyrate-producing bacteria, known to improve intestinal barrier function, as well as a reduction of methane-producing microorganisms. This is a major mechanism of hydrogen disposal in the human colon, which could explain the excess of abdominal gas in IBS [32]. Significant data from studies of obese, metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) patients have identified an increase in phylum Firmicutes over Bacteriodetes, which is otherwise a dominating phylum in normal, healthy individuals[22, 23]. On a family 2 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness level, increases in Bacteriodaceae, Porphyomonadaceae, Prevotellaceae, Clostridiaceae and Enterobacteriaceae have been associated with adverse outcomes, while a decrease of Lactobaci- laceae was seen in patients with IBS and NAFLD[24, 26, 33–35]. GWI is also characterized by systemic rise of pro-inflammatory cytokines, including TNF-α and IL1β[18, 36–38]. Interest- ingly, IBS, Obesity, NAFLD and nonalcoholic steatohepatitis have a significant pro-inflamma- tory component that includes macrophage activation, and triggering of toll-like receptor pathway. Materials Neomycin trisulfate salt (Neomycin), Enrofloxacin, Pyridostigmine bromide (PB), Permithrin, lipopolysaccharides (LPS) and Corticosterone were purchased from Sigma- Aldrich (St. Louis, MO). Anti- claudin-2, anti- Occludin, anti-TLR4, anti-flotillin, anti 3-nitrotyrosine (3NT), anti-IL1β, and anti-MCP-1 primary antibodies were purchased from Abcam (Cambridge, MA). Species specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) was purchased from Vector Laboratories (Burlingame, CA). Fluo- rescence conjugated (alexa fluor) secondary antibodies, ProLong Gold antifade mounting media with DAPI and Pierce LAL chromogenic endotoxin quantitation kit was bought from Thermo Fisher Scientific (Waltham, MA). All other chemicals which were used in this project purchased from sigma only if otherwise specified. Paraffin-embedding of tissue sections on slides were done by AML laboratories (Baltimore, MD). Microbiome analysis was done by Sec- ond Genome, the microbiome company (San Francisco, CA). Introduction Several research reports also found strong evidence of a decrease in expression of gut junction proteins leading to portal endotoxemia[39–42]. With sufficient evidence from GW veteran studies about the existence of chronic fatigue and GI disturbances coupled with neuro- nal inflammation, it is all the more important that we explore newer and novel mechanistic pathways, especially the role of the gut microbiome in modulating adverse outcomes in GWI such as neuroinflammation and intestinal injury[2]. level, increases in Bacteriodaceae, Porphyomonadaceae, Prevotellaceae, Clostridiaceae and Enterobacteriaceae have been associated with adverse outcomes, while a decrease of Lactobaci- laceae was seen in patients with IBS and NAFLD[24, 26, 33–35]. GWI is also characterized by systemic rise of pro-inflammatory cytokines, including TNF-α and IL1β[18, 36–38]. Interest- ingly, IBS, Obesity, NAFLD and nonalcoholic steatohepatitis have a significant pro-inflamma- tory component that includes macrophage activation, and triggering of toll-like receptor pathway. Several research reports also found strong evidence of a decrease in expression of gut junction proteins leading to portal endotoxemia[39–42]. With sufficient evidence from GW veteran studies about the existence of chronic fatigue and GI disturbances coupled with neuro- nal inflammation, it is all the more important that we explore newer and novel mechanistic pathways, especially the role of the gut microbiome in modulating adverse outcomes in GWI such as neuroinflammation and intestinal injury[2]. The present study examines the effect of exposure of gulf war chemicals PB and Permethrin and their resultant alteration of the gut microbiome as a key pathway to neuroinflammation and GI disturbances via systemic endotoxemia and toll like receptor pathway activation. This research that uses an established GWI rodent model, antibiotic treatment regimen (to nullify the effect of gut bacteria) and transgenic TLR4 knockout mice show a distinct role of the altered gut microbiome in causing inflammatory intestinal injury and neuronal inflammation. PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Animals Adult wild type male (C57BL/6J mice) and adult mice that contained the disrupted TLR4 gene (TLR4 KO)(B6.B10ScN-Tlr4lpsdel/JthJ) were purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were implemented in accordance with NIH guideline for human care and use of laboratory animals and local IACUC standards. All procedures were approved by The University of South Carolina at Columbia, SC. Mice were housed individually and fed with chow diet at 22–24˚C with a 12-h light/ 12-h dark cycle. All mice were sacrificed after animal experiments had been completed. Right after anesthesia, blood from the mice was drawn using cardiac puncture, in order to preserve serum for the experiments. The mice brain was removed immediately after terminal surgery. Olfactory bulbs (OB) and Frontal cortex (FC) were dis- sected out and were fixed by using Bouin’s fixative solution. Fecal pellets and luminal contents were collected from the animals, followed by dissection of small intestine and colon. The tis- sues were fixed using 10% neutral buffered formalin. Distal segments of small intestines were used for the staining and visualizations. 3 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness Microbiome analysis Fecal pellets and luminal contents were collected from the animals of each group after sacrifice, and then sent to Second Genome, for microbiome analysis. Second Genome performed nucleic acid isolation with the MoBio PowerMag1 Microbiome kit (Carlsbad, CA) according to manufacturer’s guidelines and optimized for high-throughput processing. For detailed methods used at Second Genome, refer to supplementary methods. Chemical exposure and rodent model of Gulf War Illness Mice were exposed to gulf war chemicals based on established rodent models of Gulf War Illness with some modifications[7, 10]. The treated mice group (GW-T) and TLR4 KO (GW-TLR4 KO) mice group were dosed tri-weekly for one week with PB (2mg/kg) and Permethrin.(200 mg/kg) via oral route. After completion of PB/Permethrin dosages, mice were administered corticosterone intraperitoneally with a dose of 100μg/mice/day for 5 days of the week for one week. The dose of corticosterone was selected from the study which exposed mice to 200mg/L of corticosterone through drinking water. The i.p dose of corticosterone had similar immunosuppression as examined by low splenic T cell prolifer- ation (data not shown).). Similarly, another group of mice (GW-Ab) were exposed to PB/ Permethrin and corticosterone as above mentioned dosages along with antibiotics (Neo- mycin 45 mg/kg and Enrofloxacin 1mg/kg) thrice per week for two weeks for intestinal decontamination. The control group (GW-Con) of mice was received saline injections and vehicle for oral gavage in the same paradigm. Lipopolysaccharides (LPS) exposure In order to generate TLR4 activated positive control, wild type male (C57BL/6J) mice were exposed to LPS as published earlier[43]. Briefly, mice received single bolus infusion of LPS (12 mg/kg) and mice were euthanized after 24 h for tissue and serum. A control group was also included, where normal mice received saline in place of LPS. LPS was dissolved in pyrogen- free saline and administered through the intraperitoneal (i.p.) route. PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Laboratory methods Immunohistochemistry. The brain and small intestine were collected from mice and fixed in Bouin’s solution and 10% neutral buffered formalin respectively. The fixed tissues were embedded in paraffin and cut in 5 μM thick section. These sections were subjected to deparaffi- nization using standard protocol [44]. Epitope retrieval solution and steamer (IHC-Word, Woodstock, MD) were used for epitope retrieval for deparaffinized sections. 3% H2O2 was used for the recommended time to block the endogenous peroxidase. After serum blocking, the pri- mary antibodies such as IL1β and MCP-1 were used in recommended concentrations. Species- specific biotinylated conjugated secondary antibodies and streptavidin conjugated with HRP were used to implement antigen specific immunohistochemistry. 3,3’-Diaminobenzidine (DAB) (Sigma Aldrich, St Louis, MD) was used as a chromogenic substrate. Mayer’s Hematoxylin solu- tion (Sigma Aldrich) was used as a counter stain. Sections were washed between steps using phosphate buffered saline 1X. Finally, stained sections were mounted in Simpo-mount (GBI lab- oratories, Mukilteo, WA). Tissue sections were observed using Olympus BX51 microscope (Olympus, America). Cellsens software from Olympus America (Center Valley, PA) was used for morphometric analysis of images. Immunofluorescence staining for microscopy. Paraffin embedded sections were depar- affinized using standard protocol. Epitope retrieval solution and steamer were used for epitope 4 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness retrieval of sections. Primary antibodies such as Claudin-2, Occludin, 3NT, TLR4, and Flotillin were used at recommended dilutions. Species specific secondary antibodies conjugated with Alexa Fluor (633-red and 488-green) were used at advised dilution. In the end, the stained sec- tions were mounted using prolong gold antifade reagent with DAPI. Sections were observed under–Olympus florescence microscope using 20X, 40X objective lens. - LAL assays. Serum from mice were diluted 1:50 and used for quantifying the portal endo- toxin concentration, using the Pierce LAL chromogenic endotoxin quantitation kit (following manufacturer’s protocol) Western blot. 25 mg of tissue from each small intestinal sample was immediately homog- enized in 250 μl of RIPA buffer with protease and phosphatase inhibitors cocktail (Pierce, Rockford, IL) using slow speed (to avoid frothing) mechanical homogenizer. The homogenate was centrifuged and the supernatant was collected and saved for experimental use. 30 μg of denatured protein from each sample was loaded per well of novex 4–12% bis-tris gradient gel (Life technologies, Carlsbad, CA) and subjected for standard SDS-PAGE. Laboratory methods Separated protein bands were transferred to nitrocellulose membrane using precut nitrocellulose/filter paper sandwiches (Bio-Rad, Hercules, CA) and Trans–Blot Turbo transfer system (Bio-Rad) using 30 minutes transfer protocol. Further, blots were blocked with 5% non-fat milk solution pre- pared in Tris-buffered saline with 0.05% tween-20 (TBS-T). Primary antibodies were used at recommended dilutions in blocking buffer and incubated 2h at room temperature or over- night at 4˚C. Species-specific (source of primary antibody) anti-IgG secondary antibody con- jugated with HRP were used at recommended dilutions in blocking buffer and incubated 2h at room temperature. Pierce ECL Western Blotting substrate (Thermo Fisher Scientific Inc., Rockford, IL) was used in dark to develop the blot. Finally, the blot was imaged using G: Box Chemi XX6 (Syngene imaging systems) and subjected to densitometry analysis using Image J software. Statistical analysis Prior to initiation of the study, we conducted calculations for each experimental condition with appropriate preliminary data to confirm that the sample number is sufficient to achieve a minimum statistical power of 0.80 at an alpha of 0.05. Student’s t-test was used to compare means between two groups at the termination of treatment. One way analysis of variance (ANOVA) was used with post-hoc comparisons among different exposure conditions or treat- ments (e.g., least significant differences or LSD statistic) to compare means among multiple groups at the end of the 2-week treatment period. A one-way ANOVA- was applied as needed, to evaluate differences among treatment groups. A Neuman-Keuls post-hoc test was carried out following ANOVA application. We used standard procedures for evaluating immunohis- tochemistry and relative mRNA expression data, as described in our previous publications. Gulf war chemical exposure and chronic corticosterone administration causes gut dysbiosis GW-T had significant increase in OTU-richness compared to GW-Con (KW p-value: 0.01, Fig 2A left panel, Table C in S2 File) There was a significant increase in Shannon diversity of GW-T over GW-Con (KW p-value: 0.01, Fig 2A right panel, Table C in S2 File). There were significant differences in microbiome beta diversity between the two groups (PERMANOVA p-value of 0.005, Fig 2B). There were 109 significantly different Operational Taxonomic Units (OTUs) out of a total 577 OTUs between GW-T and GW-Con. only 18 OTUs that were able to be classified at the genus level were shown in the plot (Fig C, in S1 File). Certain OTUs representing the genera Allo- baculum, Coprococcus, Dorea, Turibacter and Ruminococcus were abundant in GW-T, com- pared to GW-Con, the latter showing either fewer or no OTUs classifiable (attributable) to the same genera (Fig 3). Gulf war chemical exposure and chronic corticosterone administration causes gut dysbiosis GW chemical exposed group (GW-T) showed significantly higher percent relative Phylum- level abundance of Firmicutes -and Tenericutes over the Bacteroidetes (Kruskal-Wallis, KW signed rank p-value = 0.03, Table A in S1 File) as compared to the unexposed group (GW-Con, Fig 1A). GW-T also showed a significant increase in Ruminococcaceae and two other unclassified/unnamed families from the order Clostridiales (KW p-value = 0.01, Table B in S2 File). There was a reported decrease in abundance of family-level OTU S24-7. (KW p-value = 0.03,.Table B in S2 File) (Fig 1B), Gulf war chemical exposure altered bacte- rial alpha diversity in the intestinal lumen. GW-T had significant increase in OTU-richness compared to GW-Con (KW p-value: 0.01, Fig 2A left panel, Table C in S2 File) There was a significant increase in Shannon diversity of GW-T over GW-Con (KW p-value: 0.01, Fig 2A right panel, Table C in S2 File). There were significant differences in microbiome beta diversity between the two groups (PERMANOVA p-value of 0.005, Fig 2B). There were 109 significantly different Operational Taxonomic Units (OTUs) out of a total 577 OTUs between GW-T and GW-Con. only 18 OTUs that were able to be classified at the genus level were shown in the plot (Fig C, in S1 File). Certain OTUs representing the genera Allo- baculum, Coprococcus, Dorea, Turibacter and Ruminococcus were abundant in GW-T, com- pared to GW-Con, the latter showing either fewer or no OTUs classifiable (attributable) to the same genera (Fig 3). differences in the composition of gut bacteria both at the phylum and family levels (Fig 1). GW chemical exposed group (GW-T) showed significantly higher percent relative Phylum- level abundance of Firmicutes -and Tenericutes over the Bacteroidetes (Kruskal-Wallis, KW signed rank p-value = 0.03, Table A in S1 File) as compared to the unexposed group (GW-Con, Fig 1A). GW-T also showed a significant increase in Ruminococcaceae and two other unclassified/unnamed families from the order Clostridiales (KW p-value = 0.01, Table B in S2 File). There was a reported decrease in abundance of family-level OTU S24-7. (KW p-value = 0.03,.Table B in S2 File) (Fig 1B), Gulf war chemical exposure altered bacte- rial alpha diversity in the intestinal lumen. Gulf war chemical exposure and chronic corticosterone administration causes gut dysbiosis Chemical exposures and chronic drug use cause significant oxidative stress in the liver, intes- tine and the kidneys signifying that the drugs and their intermediates might also have a cyto- toxic effect on the normal gut microbial flora [44–47]. To study whether exposure of gulf war chemicals and the resultant chronic stress could alter the gut microbiome in the GWI-mice model, intestinal contents (illeal and cecal segments) and fecal pellets were analyzed for gut microbiome diversity by 16s V4 sequencing. Results showed that there was a significant 5 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness :10 1371/journal pone 0172914 March 22 2017 6 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 6 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 1. Gulf war chemical exposure alters gut microbiome. A. Proportional abundance of phyla: Graphical representation of the most abundant taxa of bacteria at the phylum level. Groups compared are gulf war chemical exposed group (GW-T) and control group fed with vehicle (GW-Con). -. Groups include individual samples numbered at the time of V4 16S rRNA sequencing. GW-T show marked higher percent relative Phylum-level abundance of Firmicutes, and Tenericutes over Bacteroidetes (KW p-value: 0.03) compared to GW-Con. B. Proportional abundance of families: Graphical representation of most abundant taxa at the family level in GW-T compared to GW-Con groups. GW-T show a significant increase in Ruminococcaceae and 2 other unclassified/unnamed Operational taxonomic units (OTUs) (KW p-value: 0.01) All profiles are inter-compared in a pair-wise fashion to determine a dissimilarity score and store it in a distance dissimilarity matrix. Distance functions produce low dissimilarity scores when comparing similar samples. Abundance-weighted sample pair-wise differences were calculated using the Bray-Curtis dissimilarity. Bray-Curtis dissimilarity is calculated by the ratio of the summed absolute differences in counts to the sum of abundances in the two samples (Bray and Curtis 1957). The binary dissimilarity values were calculated with the Jaccard index. This metric compares the number of mismatches (OTUs present in one but absent in the other) in two samples relative to the number of OTUs present in at least one of the samples (Jaccard 1912). Kruskal-Wallis rank sum test on top 8 most abundant phyla. Percent relative abundance means are provided. doi:10.1371/journal.pone.0172914.g001 doi:10.1371/journal.pone.0172914.g001 differences in the composition of gut bacteria both at the phylum and family levels (Fig 1). PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Altered gut microbiome modulates tight junction protein levels of Claudin-2 and Occludin Several studies have shown that intestinal inflammation is accompanied by leaching of the gut resulting in flow of intestinal content including gut bacterial components in the portal circula- tion[48–52]. The “leaky gut” is often as a result of a significant decrease in tight junction pro- teins Claudin-1 and Occludin with an increase in Claudin-2[49]. Results showed that there was a significant increase in Claudin-2 and decrease in Occludin levels in the lower small intes- tinal segments in the GW-T group when compared to GW-Con groups (Fig 4A–4G). Admin- istration of an antibiotic regimen of enrofloxacin and neomycin to achieve gut sterility and decontamination significantly decreased the Claudin-2 and increased Occludin protein levels as shown by immunofluorescence microscopy, western blot and subsequent morphometry of the images (Fig 4A–4G). The results suggested that altered gut microbiome in GW-T group with marked increases in Firmicutes (Phylum) and several genus such as Allobaculum, Copro- coccus, Dorea, Turibacter and Ruminococcus that are known to be associated with inflamma- tion, might play a significant role in expression of tight junction proteins Claudin-2 and Occludin. 7 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness I:10.1371/journal.pone.0172914 March 22, 2017 8 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 8 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 2. Alpha diversity estimates: Gulf war chemical exposure alters bacterial diversity in the intestinal lumen while there is no change among the individual groups themselves A. Left panel: OTU richness. Graphical representation of the number of OTUs present in each sample. Chao1 calculates the estimated sample richness (number of OTUs) based on sequencing depth and taking into account rare taxa that may be present in a sample GW-T has significant increase in OTU-richness compared to GW-Con (KW p-value: 0.01). Right panel: Graphical representation of Shannon diversity differences between GW-T and GW-Con. Shannon diversity utilizes the richness of a sample along with the relative abundance of the present OTUs to calculate a diversity index. There is an observed significant increase in Shannon diversity of GW-T over GW-Con (KW p-value: 0.01) B. Weighted ordination of GW-Con and GW-T groups. Dimensional reduction of the Bray-Curtis distance between microbiome samples, using the PCoA ordination method. Samples were separated according to groups along Axis 2 (PERMANOVA p-value = 0.005). doi:10.1371/journal.pone.0172914.g002 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 4. GW chemical-induced altered gut microbiome modulates gap junction protein levels in the small intestine. A. Tissue levels of Claudin- 2 in control (GW-Con), treated (GW-T) and antibiotic treated (GW-Ab) samples as observed by immunofluorescent microscopy after labelling the protein with red fluorescent secondary antibody. Nuclear staining is shown by DAPI (blue) stain. B. Tissue levels of Occludin in control (GW-Con), treated (GW-T) and antibiotic treated (GW-Ab) samples as observed by immunofluorescent microscopy after labelling the protein with red fluorescent secondary antibody. Nuclear staining is shown by DAPI (blue) stain. C. Morphometry of fluorescence intensity as observed in the region of interest (ROI) Claudin-2 immunoreactivity and D. occludin immunoreactivity. E. Western blot analysis of Claudin-2 and Occludin in small intestine tissue homogenate. F-G. morphometric analysis of western blot. (p<0.05). Data is represented as Mean+/- SE. Fig 4. GW chemical-induced altered gut microbiome modulates gap junction protein levels in the small intestine. A. Tissue levels of Claudin- 2 in control (GW-Con), treated (GW-T) and antibiotic treated (GW-Ab) samples as observed by immunofluorescent microscopy after labelling the protein with red fluorescent secondary antibody. Nuclear staining is shown by DAPI (blue) stain. B. Tissue levels of Occludin in control (GW-Con), treated (GW-T) and antibiotic treated (GW-Ab) samples as observed by immunofluorescent microscopy after labelling the protein with red fluorescent secondary antibody. Nuclear staining is shown by DAPI (blue) stain. C. Morphometry of fluorescence intensity as observed in the region of interest (ROI) Claudin-2 immunoreactivity and D. occludin immunoreactivity. E. Western blot analysis of Claudin-2 and Occludin in small intestine tissue homogenate. F-G. morphometric analysis of western blot. (p<0.05). Data is represented as Mean+/- SE. doi:10.1371/journal.pone.0172914.g004 doi:10.1371/journal.pone.0172914.g004 significantly decreased the endotoxin concentration in the blood (Fig 5B)(P<0.05). The results suggested that altered bacterial diversity and increase in certain bacterial genus might have resulted in the translocation of bacterial products into the systemic circulation. The transloca- tion is possible via the intestinal tight junctions that have been rendered leaky following a sig- nificant decrease in Occludin and increase in Claudin-2 (Fig 4). Altered gut microbial diversity in gulf war chemical exposed mice cause gut leaching and portal endotoxemia The role of gut microbiota in various chronic inflammatory diseases and cancer has recently been established by extensive studies[53–55]. Several studies show an increase in endotoxin levels in experimental models upon changes in gut microbiome and enhanced intestinal per- meability to endotoxins appears to be the primary cause of such systemic inflammation[56– 61]. To study the role of gut microbiome changes-induced portal and systemic endotoxemia in gulf war chemical-exposed mice, blood endotoxin levels were analyzed by Limulus Amebocyte lysis (LAL) assay. Results showed that there was a significant increase (almost 2.0 fold) in sys- temic endotoxin levels in GW-T group as compared to GW-Con mice (Fig 5A)(P<0.05) while decontamination of the gastrointestinal tract with antibiotics in mice treated with antibiotics Fig 3. Differentially abundant features in GW-T vs. GW-Con: Each point represents an OTU belonging to each Genus. Features were considered significant if their FDR-corrected p-value was less than or equal to 0.05, and the absolute value of the Log-2 fold change was greater than or equal to 1. There were 109 significantly different features detected out of 577 tests. Only 18 features that were able to be classified at the genus level are shown in the plot. doi:10 1371/journal pone 0172914 g003 Fig 3. Differentially abundant features in GW-T vs. GW-Con: Each point represents an OTU belonging to each Genus. Features were considered significant if their FDR-corrected p-value was less than or equal to 0.05, and the absolute value of the Log-2 fold change was greater than or equal to 1. There were 109 significantly different features detected out of 577 tests. Only 18 features that were able to be classified at the genus level are shown in the plot. Fig 3. Differentially abundant features in GW-T vs. GW-Con: Each point represents an OTU belonging to each Genus. Features were considered significant if their FDR-corrected p-value was less than or equal to 0.05, and the absolute value of the Log-2 fold change was greater than or equal to 1. There were 109 significantly different features detected out of 577 tests. Only 18 features that were able to be classified at the genus level are shown in the plot. doi:10.1371/journal.pone.0172914.g003 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 9 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness S ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 10 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 10 / 26 Gut microbial changes, altered diversity in gulf war chemical exposure causes toll like receptor activation To study the role of altered gut bacterial composition, resultant leaky gut and systemic endo- toxemia in causing neuroinflammation and intestinal injury, studies were conducted to analy- ses the activation of TLR4 in frontal cortex, olfactory bulb and the ileum. TLR4 activation was analyzed by localizing the receptor in lipid rafts of the tissue plasma membrane. Results showed that the TLR4 co-localization in lipid rafts (trafficking) as shown by the increased number of colocalization events was significantly increased in olfactory bulb regions of the brain in the GW-T group when compared to GW-Con group (Figs 6A and 7A)(P<0.05). Decontamination of the intestinal tract by using an antibiotic regimen significantly decreased the TLR4 trafficking into the lipid rafts (Figs 6A and 7A). Similar significant increases were found in the intestinal segment of GW-T mice when compared with GW-Con group while Fig 5. Portal endotoxemia following gulf war chemical exposure-induced altered gut microbiome. A. Endotoxin concentration as measured by Limulus amebocyte lysate (LAL) assay from serum of mice exposed to GW-chemicals. B. serum endotoxin levels in mice that were administered antibiotics (Neomycin and Enrofloxacin) to eliminate gut bacteria. (p<0.05). Data is represented as Mean +/- SE. Fig 5. Portal endotoxemia following gulf war chemical exposure-induced altered gut microbiome. A. Endotoxin concentration as measured by Limulus amebocyte lysate (LAL) assay from serum of mice exposed to GW-chemicals. B. serum endotoxin levels in mice that were administered antibiotics (Neomycin and Enrofloxacin) to eliminate gut bacteria. (p<0.05). Data is represented as Mean +/- SE. doi:10.1371/journal.pone.0172914.g005 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 11 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 12 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 12 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 6. TLR4 activation following gulf war chemical exposure-induced altered gut microbiome. A. Immunofluorescence microscopy of olfactory bulb showing (TLR4 (red) trafficking to the lipid rafts (green), an essential process for TLR4 activation causing a co-localization of TLR4 in flotillin-rich rafts (yellow). B. Representative images of TLR4-flotillin co-localization in the small intestine shown by white arrow heads pointing to the yellow spots created by an overlay of red (TLR4) and green (Flotillin). C. Higher magnification (60x oil) representative image of the co-localization (TLR4 and Flotillin) in the small intestine from GW-treated samples. Gut microbial changes, altered diversity in gulf war chemical exposure causes toll like receptor activation doi:10.1371/journal.pone.0172914.g006 doi:10.1371/journal.pone.0172914.g006 treatment with antibiotics caused a significant decrease in the TLR4 trafficking when com- pared with the GW-T group (Figs 6B and 7B)(p<0.05). TLR4-Flotillin (lipid raft protein) colo- calization was found in both the luminal side and the submucosal interface as shown by the higher magnification image (Fig 6C). The results suggested that bacterial content lipopolysac- charide might be the prime component in causing TLR4 activation in both brain and intestinal tissues. These observations further confirmed with LPS treated mice groups. The LPS exposed mice showed significant increase in TLR4 trafficking into the lipid rafts in both small intestine and olfactory bulb (Fig H in S1 File). This raises the possibility that altered gut microbiota, leaky gut and systemic endotoxemia might activate the TLR4 signaling pathway. TLR4 activation following gulf war chemical exposure-induced gut microbial changes cause neuronal and intestinal tyrosine nitration, a marker for both nitrative and oxidative stress TLR4 activation following gulf war chemical exposure-induced gut microbial changes cause neuronal and intestinal tyrosine nitration, a marker for both nitrative and oxidative stress Oxidative and nitrative stress has been shown to be an integral cause of neuronal and intestinal inflammation[62–65]. Most often oxidative stress can result from an active TLR4 signaling pathway that causes NADPH oxidase activation and release of superoxide radicals[66, 67]. Fig 7. A. Graphical analysis of the number of co-localizations represented by yellow in individual groups of tissues (olfactory bulb) per 200 cells counted from randomly chosen microscopic fields. B. Graphical analysis of the number of co-localizations represented by yellow in individual groups of tissues (Small intestine) per 200 cells counted from randomly chosen microscopic fields (p<0.05). Data is represented as Mean+/- SE. doi:10.1371/journal.pone.0172914.g007 Fig 7. A. Graphical analysis of the number of co-localizations represented by yellow in individual groups of tissues (olfactory bulb) per 200 cells counted from randomly chosen microscopic fields. B. Graphical analysis of the number of co-localizations represented by yellow in individual groups of tissues (Small intestine) per 200 cells counted from randomly chosen microscopic fields (p<0.05). Data is represented as Mean+/- SE. doi:10.1371/journal.pone.0172914.g007 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 13 / 26 NE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness NE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 14 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 14 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 8. Oxidative stress as evidenced by tyrosine nitration of brain and intestinal tissues following GW chemical exposure and subsequent changes in the gut microbiome and TLR4 activation. A. Frontal cortex tissue slices showing immunoreactivity to 3-nitrotyrosine (red) followed by olfactory bulb (B) and small intestine(C). D, E and F shows the quantitative analysis of fluorescence intensities in the region of interest (ROI) in frontal cortex (D), Olfactory Bulb (E) and small intestine (F). (p<0.05). Fig 8. Oxidative stress as evidenced by tyrosine nitration of brain and intestinal tissues following GW chemical exposure and subsequent changes in the gut microbiome and TLR4 activation. A. Frontal cortex tissue slices showing immunoreactivity to 3-nitrotyrosine (red) followed by olfactory bulb (B) and small intestine(C). D, E and F shows the quantitative analysis of fluorescence intensities in the region of interest (ROI) in frontal cortex (D), Olfactory Bulb (E) and small intestine (F). (p<0.05). doi:10.1371/journal.pone.0172914.g008 doi:10.1371/journal.pone.0172914.g008 Nitric oxide is also released as a consequence of TLR4 activation and can react with superoxide in diffusion controlled rates to generate peroxynititre which in turn causes tyrosine nitration [66]. Results from immunofluorescence microscopy of brain and intestinal tissue slices probed with anti-3-nitrotyrosine antibodies showed that immunoreactivity of 3-nitrotyrosine in frontal cortex, olfactory bulb and small intestine significantly increased in GW-T group when com- pared to GW-Con group (Fig 8A–8F)(p<0.05) while use of TLR4 knockout mice or use of intestinal decontamination (data not shown) significantly decreased the formation of tyrosine nitration in these tissues (Fig 8A–8F)(p<0.05). The results suggested that the altered gut micro- biome and the leaky gut caused increased oxidative stress through a TLR4 dependent pathway. TLR4 activation following gulf war chemical exposure-induced gut microbial changes cause neuronal and intestinal inflammation TLR4 activation following gulf war chemical exposure-induced gut microbial changes cause neuronal and intestinal inflammation Gulf war chemical exposure and rodent models of gulf war illness have shown increased levels of proinflammatory cytokines in the brain[10, 16, 68]. Interestingly inflammatory bowel disease also exhibits a strong inflammatory response primarily from high levels of IL1β and other che- mokines[69]. To show that altered gut microbiome, a leaky gut and systemic endotoxemia result in local intestinal injury and an ectopic neural inflammation, tissue slices from the frontal cor- tex, and small intestine were probed for immunoreactivities against IL1β and monocyte che- moattractant protein-1 (MCP-1). Results showed that IL1β protein levels were significantly higher in the frontal cortex and small intestine segments of the GW-T group when compared to GW-Con group (Figs 9A, 9C, 10A and 10C)(p<0.05) while use of antibiotics for intestinal decontamination or TLR4 knockout mice showed a significant decrease in the immunoreactivi- ties/protein levels of IL1β. Similar results were obtained for the protein levels of MCP-1 where the GW-T group had significantly higher levels of this chemokine as compared to GW-Con GW-Ab and TLR4 knockout mice groups. Other regions of the brain like cerebellum, hippo- campus and hypothalamus were not examined. The results suggested that gulf war chemical exposure and the resultant changes in the gut microbial content had a key role to play in neuro- nal and intestinal inflammation primarily through a TLR4 dependent process. PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Discussion Our results show that regular consumption of pyridostigimine bromide (PB) and concomitant use of insecticide permethrin (as modelled in mice, Fig 11) in chronic stress alters the gut microbiome. The altered microbiome causes leaky gut and systemic endotoxemia leading to neuronal and intestinal inflammation. Recent studies in chronic inflammatory diseases like osteoarthritis, chronic kidney disease, inflammatory bowel syndrome report of altered micro- bial diversity[53–55, 70–72]. Obesity and nonalcoholic fatty liver disease that have significant low-lying inflammation have been associated with gut-microbial changes[53, 55].Systemic endotoxemia is known to activate Toll like receptor activation[73]. Often gram negative bacte- ria and its cell wall component lipopolysaccharide activate both TLR2 and TLR4 receptors in different organ systems. TLR4 activation is the most common occurrence with concomitant release of proinflammatory cytokines that are quintessential for inflammation[73, 74]. The present study is a novel report of a parallel mechanism (the other being the direct effects of this drug on the neuronal functions) of inflammation and tissue injury that can have far PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 15 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Dysbiosis and leaky gut contributes to Gulf War Illness Fig 9. Gulf war chemical exposure-induced changes in gut microbiome modulates neuroinflammation and intestinal cytokine release. A. Frontal cortex tissue slices were probed for IL-1β immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. Specific immunoreactivity to IL-1β as evident by dark brown spots are indicated by black arrows and areas of interest are outlined by red circles. B. Frontal cortex tissue slices were probed for MCP-1 immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. Specific immunoreactivity to MCP-1 as evident by dark brown stain/spots are indicated by black arrows and areas of interest are outlined by red circles. C. Small intestine tissue slices were probed for IL-1β immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. D. Small intestine tissue slices were probed for MCP-1 immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. Fig 9. Gulf war chemical exposure-induced changes in gut microbiome modulates neuroinflammation and intestinal cytokine release. A. Frontal cortex tissue slices were probed for IL-1β immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. Specific immunoreactivity to IL-1β as evident by dark brown spots are indicated by black arrows and areas of interest are outlined by red circles. B. Frontal cortex tissue slices were probed for MCP-1 immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. Specific immunoreactivity to MCP-1 as evident by dark brown stain/spots are indicated by black arrows and areas of interest are outlined by red circles. C. Small intestine tissue slices were probed for IL-1β immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. D. Small intestine tissue slices were probed for MCP-1 immunoreactivity in control (GW-Con, treated (GW-T), TLR4 knockout mice treated with GW Chemical exposure (GW-TLR4KO) and antibiotic treated (GW-Ab) groups. doi:10.1371/journal.pone.0172914.g009 reaching effects on the health of gulf war veterans. The above results that provide a mechanistic input and a strong link to gut dysbiosis and inflammation and advance our understanding in other diseases like IBS, NAFLD and metabolic syndrome that altered microbiome can cause chronic inflammation to persist. The present study also throws light on possible microbiome- brain axis or vice versa for disease development in war veterans. The present study showed a direct relationship of gulf war chemical exposure and altered gut microbiota diversity with changes in the Phylum and family levels. It is important to note that the phylum Firmicutes that has been found to be enriched in GW-T group is associated with obesity, metabolic syndrome, chronic fatigue and importantly inflammatory bowel disease, a common occurrence among gulf war veterans[57, 61, 75]. Our data also show that several bacterial family and genera were enriched in the GW-T group. They include Ruminoc- coccaceae that has been associated with fibrosis, neuroinflammation and memory [76]. The present study also reports genus specific effects of bacteria in GW-T group that is associated in both small intestine and neuroinflammation[77]. Allobaculum sp. has been shown to be posi- tively correlated with intestinal inflammation and a leaky gut (decreased tight junction pro- teins) [59]. Coprococcus and Turicibacter have been shown to be negatively correlated with disease severity and inflammation but these reports are contrary to what we found in our pres- ent study [69]. GW-T was highly correlated with neuroinflammation and gut leaching that had abundant Coproccoccus and Turicibacter (Fig 3). The apparent difference may be due to the exposure paradigm and disease type. The role pf PB in in causing neuroinflammation is unclear. Though GW-chemical exposure that included PB caused astrocytic activation and ICAM-1 increase a firm mechanistic argument in favor of the neuroinflammatory pathology is lacking[7]. [10, 68]. On the other hand PB did not show a significant increase in neuroinflam- mation[10]. Since stress alone can cause changes in the gut microbiome, the lack of stress-only group is a limitation in our studies. Though GWI is a combination of all the factors modelled, stress related altered microbial phenomenon can have tremendous implications. The present study identifies a specific role of gut microbiome in neuroinflammation and intestinal leaki- ness though it does not shed light on the blood brain barrier leakiness which is important to allow proinflammatory cytokines to target neuronal tissues. Dysbiosis and leaky gut contributes to Gulf War Illness PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 16 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 16 / 26 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 To address this specific question the present study hypothesized that it may not be the proinflammatory cytokines but high endotoxin concentration that bind to pattern recognition receptors in organ systems, in this case the brain and the small intestine, and cause a localized pro-inflammatory effect. Support for this hypothesis comes from the results that show an enrichment of gram negative bacteria in the GW-T group followed by a decrease in tight junction protein Occludin (Fig 4 and Fig 5). Interestingly, decreased levels of tight junction protein Occludin and increased Claudin-2 overwhelmingly show gut leakiness more often associated with IBS, NAFLD and alcoholic steatohepatitis[49, 52]. It is likely that in the present study, enriched gram negative bacteria and its components leaked into the systemic circulation and could access different organ sys- tems including the brain. There are reports that brain uptake of circulating LPS is so low that most effects of peripherally administered LPS are likely mediated through LPS receptors PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 17 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 10. A-D. Morphometry of the immunoreactivities in tissue slices using the stained regions of interest. Data normalized against control and plotted in Microcal-Origin software (p<0.05). Data is represented as Mean+/- SE. doi:10.1371/journal.pone.0172914.g010 Fig 10. A-D. Morphometry of the immunoreactivities in tissue slices using the stained regions of interest. Data normalized against control and plotted in Microcal-Origin software (p<0.05). Data is represented as Mean+/- SE. doi:10 1371/journal pone 0172914 g010 Fig 10. A-D. Morphometry of the immunoreactivities in tissue slices using the stained regions of interest. Data normalized against control and plotted in Microcal-Origin software (p<0.05). Data is represented as Mean+/- SE. doi:10 1371/journal pone 0172914 g010 Fig 10. A-D. Morphometry of the immunoreactivities in tissue slices using the stained regions of interest. Data normalized against control and plotted in Microcal-Origin software (p<0.05). Data is represented as Mean+/- SE. Fig 10. A-D. Morphometry of the immunoreactivities in tissue slices using the stained regions of interest. Data normalized against control and plotted in Microcal-Origin software *(p<0.05). Data is represented as Mean+/- SE. doi:10.1371/journal.pone.0172914.g010 doi:10.1371/journal.pone.0172914.g010 doi:10.1371/journal.pone.0172914.g010 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 18 / 26 NE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 Dysbiosis and leaky gut contributes to Gulf War Illness Fig 11. Schematic representation of dosage pattern: A. PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 dosage pattern of control group (GW-Con) n = 4, C57BL/6J mice were exposed to vehicle/ solvent (Veh) of the Gulf war chemicals and antibiotics. B. C57BL/6J (n = 6) and TLR4 KO (n = 6) mice were exposed to gulf war chemicals Pyridostigmine bromide (Pb), Permethrin (Per) and Corticosterone (Cort). C. C57BL/6J (n = 6) mice were co-exposed to gulf war chemicals (Pb, Per and Cort) and Antibiotics (Ab). Fi 11 S h ti t ti f d tt A d tt f t l (GW C ) 4 C57BL/6J i d t hi l / Fig 11. Schematic representation of dosage pattern: A. dosage pattern of control group (GW-Con) n = 4, C57BL/6J mice were exposed to vehicle/ solvent (Veh) of the Gulf war chemicals and antibiotics. B. C57BL/6J (n = 6) and TLR4 KO (n = 6) mice were exposed to gulf war chemicals Pyridostigmine bromide (Pb), Permethrin (Per) and Corticosterone (Cort). C. C57BL/6J (n = 6) mice were co-exposed to gulf war chemicals (Pb, Per and Cort) and Antibiotics (Ab). doi:10.1371/journal.pone.0172914.g011 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 PLOS ONE \| DOI:10.1371/journal.pone.0172914 March 22, 2017 19 / 26 Dysbiosis and leaky gut contributes to Gulf War Illness located outside the blood brain barrier further confirming our argument that TLR4 activation is likely in this scenario [78]. A recent study also showed that the endothelial cells and perivas- cular macrophages at the blood brain barrier play a significant role in allowing immune signals enter the brain [79]. Further, neuroinflammation may be trigerred from other systemic inflam- matory signals from the intestinal tract [80]. Evidence found in our study showed that toll like receptor 4, a pattern recognition receptor for the gram negative bacterial cell wall is activated in the small intestine, and the olfactory bulb (Fig 6A–6F). Though we did not study the immune signal flow at the blood brain barrier, an activation of the TLR4 via its trafficking to the lipid rafts is a strong indication that proinflammatory signals have reached the brain causing a signifi- cant increase in cytokines and chemokines. Our results further showed that IL1β and MCP-1 were significantly elevated in frontal cortex and small intestine along with tyrosine nitration (Figs 8 and 9 and 10). GWI is associated with chronic immune activation with higher levels of IL-1α, TNF-α, sTNFRII and IL-1β [10, 16]. S1 File. Supporting information. (DOCX) S2 File. Supporting tables. (DOCX) S2 File. Supporting tables. (DOCX) Acknowledgments The authors gratefully acknowledge the technical services of Benny Davidson at the IRF, Uni- versity of South Carolina School of Medicine. We also thank the Instrumentation resource facility (IRF) at the University of South Carolina for equipment usage and consulting services. Our results of increased protein levels of IL1β and MCP-1 in the frontal cortex and the small intestine show a progressive inflammatory pattern since the chemokine action of MCP-1 will further allow infiltration of leukocytes in the affected tissue[81]. This may be true to the intestine and is highly likely in the brain since activated microglia and resident cells expressing cytokines exist in that organ [82]. These cells can infiltrate brain tissue and can amplify the pro-inflammatory signal cascade initiated by the activation of TLR4 (Fig 6). However more studies with respect to neuroinflammation and the leaky gut in GWI will be required to definitively conclude the direct role of gut leaching in GWI. Taken together, we present substantial evidence that might indicate an existence of an alter- native immune network that arises from the ability of gulf war chemicals to alter the micro- biome and enrich several gram-negative bacterial population. Further the altered microbiome- induced gut leakiness and endotoxemia might contribute to neuroinflammation and intestinal injury that are TLR4 dependent. The present study also is likely to support the notion that anti- bacterials like minocycline and probiotics might be a good therapeutic regimen in Gulf war ill- ness and other associated disorders. The studies also might have tremendous translational impact since studies show that Ninety-nine percent of mouse genes are shared with humans at the host genetic level, and they share key similarities with the human gut microbiome at the phylum through family levels making them a powerful model system for evaluating host– microbiota interactions applicable to human biology [83]. However subtle differences in the gastrointestinal tract anatomy and corresponding bacterial species though small may influence the translational approach in future and requires cautious optimism. Author Contributions Conceptualization: SC. References 1. Gulf War and Health: Volume 8: Update of Health Effects of Serving in the Gulf War. Washington DC: 2010 by the National Academy of Sciences; 2010. 2. Chronic Multisymptom Illness in Gulf War Veterans: Case Definitions Reexamined. Washington DC: 2014 by the National Academy of Sciences; 2014. 3. Iversen A, Chalder T, Wessely S. 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https://openalex.org/W2544125281	https://jurnal.ar-raniry.ac.id/index.php/didaktika/article/download/458/369	Indonesian	null	REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis	Jurnal Ilmiah Didaktika/Jurnal Ilmiah Didaktika : Media Ilmiah Pendidikan dan Pengajaran	2,012	cc-by	5,976	Jurnal Ilmiah DIDAKTIKA Februari 2012 VOL. XII NO. 2, 348-367 Jurnal Ilmiah DIDAKTIKA Februari 2012 VOL. XII NO. 2, 348-367 Abstract Teaching learning process is a two-sided process which occures significantly. It will run well if teachers could give positive influence to their students based on their level of competency. In general, the influence covers cognitive, affective, and psychomotoric aspects. To have an effective process of teaching, teachers should be skillful in using appropriate method, media, and evaluation as well as all the supporting elements to make the intercative process could occur in the classroom. In teaching learning process, teacher should play active roles, so does students. In the classroom, teacher often face many problems, among others, there are still students who are lack of understanding to the material taught. Related to that, teacher should do remedial to the students aiming at diagnosing the students’ problem in mastering the concept. By doing so, they would know whether the instructional tools, or method, media, and evaluation technique are not appropriate to their students. From the literature review done , it is found that teachers should do the remedial teaching to their students. Besides, in order to set an active, creative, innovative, and fun teaching learning situation, teachers should have some competencies. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis Masbur Dosen tetap pada Fakultas Tarbiyah IAIN Ar-Raniry Abstrak Proses pembelajaran adalah dua sisi proses yang muncul secara signifikan. Proses pembelajaran akan berjalan dengan lancar jika guru dapat memberikan pengaruh yang positif terhadap siswa-siswa mereka berdasarkan level kompetensi mereka. Pada umumnya, pengaruh meliputi aspek kognitif, afektif dan psikomotor. Untuk mencapai proses pembelajaran yang efektif, pengajar harus memiliki keterampilan dalam menggunakan cara yang sesuai, media, dan juga evaluasi dan juga seluruh unsur yang mendukung untuk menjadikan proses pembelajaran yang interaktif di dalam kelas. Dalam pembelajaran, pendidik harus berperan aktif, karena masih ada pelajar yang belum memahami materi ajar. Terkait dengan hal itu, pendidik harus melakukan remedial pada siswa yang bertujuan untuk mendiagnosis permasalah pelajar dalam memahami konsep. Dengan hal itu, mereka akan mengetahui tentang materi ajar, metode, media, dan tehnik evaluasi yang tidak sesuai dengan mereka. Dari beberapa kajian teori yang dilakukan, ditemukan bahwa pendidik harus melakukan remedial untuk menentukan keadaan yang aktif, kreatif, inovatif dan menyenangkan, pendidik harus memiliki beberapa kompetensi. Kata Kunci: teaching, remedial, solusi Masbur 2 Safwan Amin, Pengantar Psikologi Pendidikan, Banda Aceh: Yayasan Pena, 2003, hal.50. 1 Made Pidarta, Landasan Kependidikan, Jakarta: Rineka cipta, 1997, hal. 11. 1 Made Pidarta, Landasan Kependidikan, Jakarta: Rineka cipta, 1997, hal. 11. 2 Safwan Amin, Pengantar Psikologi Pendidikan, Banda Aceh: Yayasan Pena, 2003, hal.50. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis kadang dapat cepat menangkap apa yang dipelajari, kadang-kadang terasa amat sulit. Setiap individu memang tidak ada yang sama. Perbedaan individual ini pulalah yang menyebabkan perbedaan tingkah laku belajar di kalangan anak didik. Dalam keadaan di mana siswa tidak dapat belajar sebagaimana mestinya, itulah yang disebut dengan “kesulitan belajar”.3 Adapun persoalan yang dihadapi siswa sehingga mempengaruhi hasil belajarnya yang mengakibatkan prestasinya rendah, hal ini karena dipengaruhi oleh beberapa faktor, baik yang bersifat internal maupun eksternal yang dialami oleh para siswa. Persoalan instern misalnya minat, bakat, tingkat intelegensi, pengetahuan, sikap. Persoalan ekstern misalnya meliputi lingkungan keluarga, misalnya kondisi ekonomi yang rendah, keluarga yang tidak harmonis, lingkungan sosial tempat tinggal, misalnya teman permainan yang nakal dan lingkungan sekolah, seperti letak sekolah yang jauh dengan tempat tinggal, fasilitas sekolah yang minim dan sebagainya. 3 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar, Jakarta: Rineka Cipta, 2004, hal. 77. 4 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 152. 5Abd. Rachmat Abror, Psikologi Pendidikan,Yoyakarta: Tiara Wacana Yogya, 1993, hal. 185. PENDAHULUAN Pendidikan menurut pandangan Islam merupakan suatu upaya membimbing, membina peserta didik yang dilakukan secara sadar dan terencana, agar terbentuknya suatu kepribadian yang utama sesuai dengan nilai-nilai agama Islam. Adapun Tujuan pendidikan menurut GBHN Tahun 1993 dijelaskan bahwa; “kebijaksanaan pembangunan sektor pendidikan ditujukan untuk meningkatkan kualitas manusia Indonesia, yaitu manusia yang beriman dan bertakwa kepada Tuhan Yang Maha Esa, berbudi pekerti luhur, berkepribadian, mandiri, maju, tangguh, cerdas, kreatif, terampil, berdisiplin, beretos kerja, propesional, bertanggung jawab, produktif, dan sehat jasmani- rohani”.1 “kebijaksanaan pembangunan sektor pendidikan ditujukan untuk meningkatkan kualitas manusia Indonesia, yaitu manusia yang beriman dan bertakwa kepada Tuhan Yang Maha Esa, berbudi pekerti luhur, berkepribadian, mandiri, maju, tangguh, cerdas, kreatif, terampil, berdisiplin, beretos kerja, propesional, bertanggung jawab, produktif, dan sehat jasmani- rohani”.1 Belajar adalah key term (kata kunci) yang sangat urgen dalam usaha pendidikan, sehingga tanpa belajar sesungguhnya tidak ada pendidikan.2 Pada dasarnya belajar adalah suatu proses usaha yang melibatkan aktivitas mental yang terjadi dalam diri manusia, sebagai akibat dari proses interaksi aktif dengan lingkungannya untuk memperoleh suatu perubahan dalam bentuk pengetahuan, pemahaman, tingkah laku. Dalam rangka membantu peserta didik mencapai standar isi dan standar kompetensi lulusan, pelaksanaan atau proses pembelajaran perlu diusahakan agar interaktif, inspiratif, menyenangkan, menantang, memotivasi peserta didik untuk berpartisipasi aktif, serta memberikan kesempatan yang cukup bagi prakarsa, kreativitas, dan kemandirian sesuai dengan bakat, minat, dan perkembangan fisik serta psikologis peserta didik. Untuk mencapai tujuan dan prinsip-prinsip pembelajaran tersebut pasti dijumpai adanya peserta didik yang mengalami kesulitan atau masalah belajar. Oleh karena itu, setiap satuan pendidikan perlu menyelenggarakan program pembelajaran remedial atau perbaikan. Dalam keseluruhan proses pendidikan di sekolah, kegiatan belajar merupakan kegiatan yang paling pokok. Ini berhasil tidaknya pencapaian tujuan pendidikan banyak tergantung kepada bagaimana proses belajar yang dialami oleh siswa sebagai anak didik. Aktivitas belajar bagi setiap individu, tidak selamanya berlangsung secara wajar. Kadang-kadang lancar, kadang-kadang tidak, kadang- Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 3 3 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar, Jakarta: Rineka Cipta, 2004, hal. 77. 4 Abu Ahmadi dan Widodo Supriono Psikologi Belajar hal 152 Hakikat Remedial Teaching Pengajaran remedial (remedial teaching) secara etimologis berasal dari kata remedy (Inggris) yang artinya menyembuhkan, membetulkan, perbaikan, pengulangan. Sedangkan teaching adalah mengajar, cara mengajar atau mengajarkan.4 Pengajaran remedial secara terminologis adalah suatu kegiatan belajar mengajar yang bersifat menyembuhkan atau perbaikan ke arah pencapaian hasil yang diharapkan. Pengajaran remedial menurut Abd. Rachmat Abror adalah bentuk pengajaran perbaikan yang diberikan kepada seseorang siswa untuk membantu memecahkan kesulitan belajar yang dihadapinya.5 Menurut Abin Syamsuddin, pengajaran remedial adalah sebagai upaya guru untuk menciptakan suatu situasi yang memungkinkan individu atau kelompok siswa (dengan kerakter) tertentu lebih mampu meningkatkan prestasi seoptimal mungkin sehingga dapat 350 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur memenuhi kriteria keberhasilan minimal yang diharapkan.6 Menurut Abu Ahmadi dan Widodo Supriono, pengajaran remedial adalah suatu bentuk khusus pengajaran yang bersifat menyembuhkan, membetulkan atau membuat menjadi baik.7 Berdasarkan pengertian di atas dapat disimpulkan bahwa, pengajaran remedial adalah suatu layanan pendidikan atau suatu bentuk program pembelajaran yang dilaksanakan dengan perlakuan khusus yang diberikan guru pada siswa yang mengalami kesulitan dan hambatan dalam kegiatan belajar mengajar, sehingga siswa tersebut mencapai standar kompetensi yang telah ditentukan. 6Abin Syamsuddin Makmun, Psikologi Kependidikan Perangkat Sistem Pengajaran Modul, Bandung: Remaja Rosdakarya, 2005, hal. 343. Tujuan dan Fungsi Pelaksanaan Remedial di Sekolah Dalam pengajaran remedial adanya proses yang terjadi antara guru (subjek) dan siswa (objek), yaitu terjadinya proses interaksi dan pentransferan ilmu pengetahuan untuk tujuan tersebut, guru harus menguasai kompetensi peserta didik. Tujuan pengajaran remedial menurut Abu Ahmadi dan Widodo Supriono secara umum tidak berbeda dengan pengajaran dalam rangka mencapai tujuan belajar yang telah ditetapkan. Secara khusus pengajaran perbaikan bertujuan agar siswa yang mengalami kesulitan belajar dapat mencapai prestasi belajar yang diharapkan melalui proses perbaikan.8 Tujuan pembelajaran remedial adalah untuk membantu siswa yang mengalami kesulitan belajar dengan memperbaiki prestasi belajarnya. Adapun fungsi pengajaran remedial antara lain: 9 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 155. 7Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 153. 8 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 154. 6Abin Syamsuddin Makmun, Psikologi Kependidikan Perangkat Sistem Pengajaran Modul, Bandung: Remaja Rosdakarya, 2005, hal. 343. 7Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 153. 8 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 154. 9 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 155. 1. Fungsi korektif Fungsi korektif adalah dapat dilakukan pembetulan atau perbaikan terhadap hal-hal yang dipandang belum memenuhi apa yang diharapkan dalam proses pembelajaran.9 Sebelum proses belajar mengajar dimulai, guru membuat perencanaan pembelajaran agar memperoleh hasil yang diharapkan. Dengan Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis demikian, guru dapat mengetahui perbedaan individual siswa dan kesulitan belajar siswa tersebut. 11 Abin Syamsuddin Makmun, Psikologi kependidikan…, hal. 355. 2. Fungsi pemahaman Fungsi pemahaman yaitu memungkinkan guru, siswa dan pihak lain dapat memperoleh pemahaman yang lebih baik terhadap pribadi siswa.10 Kepribadian siswa sangat mempengaruhi hasil belajarnya. Oleh karena itu, guru atau pihak lain dapat memahami kepribadian pada diri siswa atau perbedaan pada masing-masing siswa. 3. Fungsi penyesuaian 3. Fungsi penyesuaian Fungsi penyesuaian yaitu pengajaran remedial dapat membentuk siswa untuk bisa beradaptasi atau menyesuaikan diri dengan lingkungannya. Siswa dapat belajar sesuai dengan kemampuannya sehingga peluang untuk mencapai hasil lebih baik lebih besar. Tuntutan disesuaikan dengan jenis, sifat, dan latar belakang kesulitan sehingga termotivasi untuk belajar. Adapun pelaksanaan program ini dapat dilakukan secara relevan dengan tingkat yang dimiliki siswa dikarenakan faktor individual siswa dalam memahami suatu bidang studi. Maka fungsi penyesuaian ini memungkinkan individual siswa dengan karakter tertentu dapat termotivasi untuk belajar. 11 Abin Syamsuddin Makmun, Psikologi kependidikan…, hal. 355. 10 Ischak S. W. dan Warji R, Program Remedial Dalam Proses Belajar Mengajar, Yogyakarta: Liberti, 1987, hal. 87. 4. Fungsi pengayaan Fungsi pengayaan yaitu dapat memperkaya proses belajar mengajar. Pengayaan dapat melalui atau terletak dalam segi metode yang dipergunakan dalam pengajaran remedial sehingga hasil yang diperoleh lebih banyak, lebih dalam atau dengan singkat prestasi belajarnya lebih kaya. Adanya daya dukung fasilitas teknis, serta sarana penunjang yang diperlukan. Sasaran pokok fungsi ini ialah agar hasil remedial itu lebih sempurna dengan diadakannya pengayaan.11 Semakin banyak hasil belajar yang diperoleh dan semakin dalam ilmu yang didapat, maka prestasi belajarnya pun semakin meningkat. 5. Fungsi terapetik 5. Fungsi terapetik Fungsi terapetik yaitu secara langsung ataupun tidak, pengajaran perbaikan dapat memperbaiki atau menyembuhkan kondisi kepribadian yang menyimpang. 352 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur Penyembuhan ini dapat menunjang penyampaian prestasi belajar dan pencapaian prestasi yang baik dapat mempengaruhi pribadi. Prosedur Pelaksanaan Remedial yang Efektif Untuk memperlancar pengajaran remedial dengan sempurna sehingga hasil yang diinginkan tercapai lebih baik, maka pelaksanaan harus melalui langkah- langkah yang tepat dan sistematis.12 Adapun prosedur pengajaran remedial yaitu: 1. Meneliti kembali kasus 13 Muhammad Nashiruddin Al Abani, Shahih Sunan Ibnu Majah, Kamp. Melayu: Pustaka Azzam, 2005, hal. 55. 12 Abin syamsuddin Makmun, Psikologi kependidikan…, hal. 343. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis saja, tetapi juga dari ilmu pengetahuan. Sedangkan orang yang kuat baik dari fisik maupun ilmu pengetahuannya, ia mampu mengatasi faktor-faktor penyebab kesulitan belajar. Untuk mengatasi hal tersebut salah satu solusi yang tepat untuk meningkatkan hasil belajar adalah membutuhkan pengajaran remedial yang mampu memberikan potensi yang lebih baik. 14 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar..., hal. 186. 1. Meneliti kembali kasus Meneliti kembali kasus adalah mendiagnosis kasus kesulitan belajar dengan kriteria di bawah minimal yang dicapai dari hasil belajarnya. Meneliti kembali kasus dengan permasalahannya merupakan tahapan paling fundamental dalam pengajaran remedial karena merupakan landasan titik tolak langkah-langkah berikutnya. Adapun tujuan penelitian kembali kasus ini adalah agar memperoleh gambaran yang jelas mengenai kasus tersebut, serta cara dan kemungkinan pemecahannya. Berdasarkan atas penelitian kasus akan dapat ditentukan siswa- siswa yang perlu mendapatkan pengajaran remedial. Kemudian ditentukan besarnya kelemahan yang dialami dan dalam bidang studi apa saja mengalami kelemahan. Dalam hadist Rasulullah Ṣallallāhu ‘alayhi Wasallam mengatakan: ﻋﻦ أﺑﻰ ھﺮﯾﺮة رﺿﻰ ﷲ ﻋﻨﮫ ﻗﺎل: ﻗﺎل رﺳﻮل P ﺻﻠﻰ ﷲ ﻋﻠﯿﮫ وﺳﻠﻢ: اﻟﻤﺆﻣﻦ اﻟﻘﻮي ﺧﯿﺮ وأﺣﺐ إﻟﻰ ﷲ ﻣﻦ (اﻟﻤﺆﻣﻦ اﻟﻀﻌﯿﻒ.)رواه اﺑﻦ ﻣﺎﺟﺔ ﻋﻦ أﺑﻰ ھﺮﯾﺮة رﺿﻰ ﷲ ﻋﻨﮫ ﻗﺎل: ﻗﺎل رﺳﻮل P ﺻﻠﻰ ﷲ ﻋﻠﯿﮫ وﺳﻠﻢ: اﻟﻤﺆﻣﻦ اﻟﻘﻮي ﺧﯿﺮ وأﺣﺐ إﻟﻰ ﷲ ﻣﻦ (اﻟﻤﺆﻣﻦ اﻟﻀﻌﯿﻒ.)رواه اﺑﻦ ﻣﺎﺟﺔ Artinya:“Dari Abu Hurairah Raḍiyallāhu ‘anhu, ia berkata: “Telah bersabda Rasulullah Ṣallallāhu ‘alayhi Wasallam: “Seorang mukmin yang kuat itu lebih baik dan lebih dicintai oleh Allah dari pada orang mukmin yang lemah ”. (H. R. Ibnu Majah).13 Artinya:“Dari Abu Hurairah Raḍiyallāhu ‘anhu, ia berkata: “Telah bersabda Rasulullah Ṣallallāhu ‘alayhi Wasallam: “Seorang mukmin yang kuat itu lebih baik dan lebih dicintai oleh Allah dari pada orang mukmin yang lemah ”. (H. R. Ibnu Majah).13 Artinya:“Dari Abu Hurairah Raḍiyallāhu ‘anhu, ia berkata: “Telah bersabda Rasulullah Ṣallallāhu ‘alayhi Wasallam: “Seorang mukmin yang kuat itu lebih baik dan lebih dicintai oleh Allah dari pada orang mukmin yang lemah ”. (H. R. Ibnu Majah).13 Berdasarkan hadist di atas dapat dipahami bahwa orang yang lemah akan menjadi beban bagi orang lain, orang lemah bukan hanya dilihat dari segi fisiknya Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| \| 353 354 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 2. Menentukan tindakan yang harus dilakukan Menentukan tindakan yang harus dilakukan yaitu menentukan alternatif pilihan yang relevan dengan karakteristik kasus yang ditangani.14 Langkah ini merupakan lanjutan dari langkah pertama. Dari hasil penelaah dan penelitian kembali kasus yang dilakukan pada langkah pertama itu akan diperoleh karakteristik kasus yang ditangani tersebut, yaitu dapat diklasifikasikan ke dalam tiga golongan yaitu berat, cukup, dan ringan. Dikatakan kasus berat jika siswa belum memiliki cara belajar yang baik, juga memiliki hambatan emosional. Kasus yang cukup adalah jika siswa telah mampu menemukan pola belajar tetapi belum dapat berhasil karena ada hambatan psikologis. Sedangkan pada kasus ringan jika siswa belum menemukan cara belajar yang baik. Setelah karakteristik harus ditentukan, maka tindakan pemecahan perlu dipikirkan, yaitu sebagai berikut: a. Kalau kasusnya ringan, tindakan yang ditentukan adalah memberikan pengajaran remedial. a. Kalau kasusnya ringan, tindakan yang ditentukan adalah memberikan pengajaran remedial. b. Kalau kasusnya cukup dan berat, maka sebelum diberikan pengajaran remedial harus diberi layanan konseling lebih dahulu, yaitu untuk mengatasi hambatan- hambatan emosional yang mempengaruhi cara belajarnya. b. Kalau kasusnya cukup dan berat, maka sebelum diberikan pengajaran remedial harus diberi layanan konseling lebih dahulu, yaitu untuk mengatasi hambatan- hambatan emosional yang mempengaruhi cara belajarnya. 3. Pemberian layanan bimbingan dan konseling 3. Pemberian layanan bimbingan dan konseling Layanan bimbingan dan konseling adalah proses bantuan atau pertolongan yang diberikan oleh guru/konselor kepada siswa melalui pertemuan tatap muka atau hubungan timbal balik antara keduanya, agar siswa memiliki kemampuan atau kecakapan dalam melihat dan menemukan masalahnya serta mampu menyelasaikan masalahnya sendiri. Memberikan arahan atau interaksi antara guru 354 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur dan siswa dalam memecahkan suatu masalah yang menjadi hambatan mental emosional dalam menghadapi kegiatan belajar. Pelayanan bimbingan dan konseling yaitu untuk memberikan jasa, manfaat atau kegunaan, ataupun keuntungan-keuntungan tertentu kepada individu-individu yang menggunakan pelayanan tersebut.15 Tujuan dari layanan ini adalah mengusahakan terciptanya kesehatan agar siswa yang menjadi kasus itu terbebas dari hambatan mental emosional dan ketegangan batinnya, kemudian siap sedia kembali menghadapi kegiatan belajar secara wajar dan realistis. 4. Pelaksanaan pembelajaran remedial 15 Prayitno dan Erman Amti, Dasar-dasar dan Bimbingan Konseling, Jakarta: Rineka Cipta, 2004, hal. 225. 16 Abin syamsuddin Makmun, Psikologi Kependidikan…, hal. 343. 16 Abin syamsuddin Makmun, Psikologi Kependidikan…, hal. 343. 4. Pelaksanaan pembelajaran remedial Pelaksanaan pembelajaran remedial merupakan suatu program yang diberikan guru untuk memperbaiki prestasi belajar siswa yang dibawah kriteria ketuntasan minimal. Program ini sebagai upaya guru untuk menciptakan suatu situasi yang memungkinkan individu atau kelompok siswa (dengan karakter) tertentu lebih mampu meningkatkan prestasi seoptimal mungkin sehingga dapat memenuhi kriteria keberhasilan minimal yang diharapkan.16 Sasaran pokok pada langkah ini adalah peningkatan prestasi maupun kemampuan menyesuaikan diri sesuai dengan ketentuan keberhasilan yang telah ditetapkan. 17 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…..., hal. 180. 6. Melakukan re-evaluasi dan re-diagnostik Melakukan re-evaluasi dan re-diagnostik adalah menafsirkan dengan membandingkan kriteria seperti pada proses belajar mengajar yang sesungguhnya. Adapun dari hasil penafsiran itu dapat terjadi 3 kemungkinan dan rekomendasi yang dapat diberikan yaitu: a. Kasus menunjukkan peningkatan prestasi yang dihasilkan sesuai dengan kriteria yang diharapkan, maka selanjutnya diteruskan ke program berikutnya. a. Kasus menunjukkan peningkatan prestasi yang dihasilkan sesuai dengan kriteria yang diharapkan, maka selanjutnya diteruskan ke program berikutnya. b. Kasus menunjukkan peningkatkan prestasi, namun belum memenuhi kriteria yang diharapkan, maka diserahkan pada pembimbing untuk diadakan pengayaan. c. Kasus belum menunjukkan perubahan yang berarti dalam hal prestasi, maka perlu didiagnosis lagi untuk mengetahui letak kelemahan pengajaran remedial untuk selanjutnya diadakan ulangan dengan alternatif yang sama. 5. Melakukan pengukuran kembali terhadap prestasi belajar 5. Melakukan pengukuran kembali terhadap prestasi belajar Melakukan pengukuran kembali terhadap prestasi adalah dengan mengadakan tes terhadap perubahan pribadi siswa untuk mengetahui proses pengajaran remedial secara menyeluruh. Langkah ini adalah melakukan pengukuran terhadap perubahan pada diri siswa yang diberikan pengajaran remedial. Apakah ia sudah mencapai apa yang direncanakan pada kegiatan pelaksanaan remedial atau belum. Maka untuk mengetahui hal itu perlu dilakukan pengukuran terhadap prestasinya kembali dengan alat post-tes atau tes sumatif yang seperti dipergunakan pada proses belajar mengajar yang sesungguhnya. Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 355 REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis 6. Melakukan re-evaluasi dan re-diagnostik 6. Melakukan re-evaluasi dan re-diagnostik 6. Melakukan re-evaluasi dan re-diagnostik 356 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 1. Pendekatan individual Pendekatan individual merupakan interaksi antara guru-siswa secara individual dalam proses belajar mengajar.19 Pendekatan individual adalah suatu upaya untuk memberikan kesempatan kepada siswa agar dapat belajar sesuai dengan kebutuhan, kecepatan, dan caranya.20 Jadi pendekatan individual adalah pendekatan bersifat perorang, yaitu dikarenakan perbedaan individual siswa atau mempunyai karakteristik tersendiri yang berbeda dari satu siswa dengan siswa lain baik dari cara mengemukakan pendapat, daya serap maupun tingkat kecerdasan dan sebagainya. Persoalan kesulitan belajar lebih mudah dipecahkan dengan menggunakan pendekatan individual, walaupun suatu saat pendekatan kelompok dibutuhkan. Pendekatan dalam Pelaksanaan Remedial di Sekolah Ada beberapa pendekatan belajar dalam pelaksanaan remedial dengan harapan dapat membantu siswa dalam memecahkan berbagai masalah. Menurut Saiful Bahri Djamarah adalah baik pendekatan yang bersifat umum maupun pendekatan yang bersifat keagamaan (khusus).18 Antara lain yaitu: 7. Pengayaan (Tugas Tambahan) 7. Pengayaan (Tugas Tambahan) Pengayaan adalah memperkaya ilmu pengetahuan atau memperluas ilmu pengetahuan siswa dengan memberi tugas tambahan, baik tugas yang dikerjakan di rumah maupun tugas yang dikerjakan di kelas.17 Langkah ini sama dengan langkah ketiga dan bersifat pilihan (optimal) yang kondisional. Sasaran pokok langkah ini ialah agar hasil remedial itu lebih sempurna dengan tindakan pengayaan. Adapun prosedur pelaksanaan remedial menurut Muhammad Entang adalah identifikasi kasus dan faktor-faktor yang mempengaruhi timbulnya kesulitan belajar tidak akan bermanfaat apabila tidak diikuti dengan tindakan- tindakan yang dapat membantu para siswa yang mengalami kesulitan belajar. Sebelum mengambil tindakan-tindakan tersebut seorang guru perlu merencanakan cara yang menurut pertimbangannya akan dapat membantu siswa. Rencana yang disusun hendaknya didasarkan pada hasil identifikasi faktor-faktor yang mempengaruhi timbulnya kesulitan belajar. Berdasarkan uraian di atas, dapat disimpulkan bahwa melaksanakan pembelajaran remedial berdasarkan prosedur-prosedur yang telah ditentukan agar 356 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur proses pembelajaran tersebut berjalan dengan lancar sehingga menemukan letak kesulitan belajar pada diri siswa dan melaksanakan pembelajaran remedial. 18 Syaiful Bahri Djamarah dan Aswan Zain, Strategi Belajar Mengajar, Jakarta: Rineka Cipt 2002, hal. 61. 22 Oemar Hamalik, Kurikulum dan Pembelajaran, Jakarta, Bumi Aksara, 2008, hal. 111. 21 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar..., hal. 183. 19 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 184. 18 Syaiful Bahri Djamarah dan Aswan Zain, Strategi Belajar Mengajar, Jakarta: Rineka Cipta, 2002, hal. 61. 19 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar…, hal. 184. 20 Baharuddin, Pendidikan dan Psikologi Perkembangan, Jogjakarta: Ar-Ruzz Media, 2009, hal. 180. 21 Abu Ahmadi dan Widodo Supriono, Psikologi Belajar..., hal. 183. 22 Oemar Hamalik, Kurikulum dan Pembelajaran, Jakarta, Bumi Aksara, 2008, hal. 111. 20 Baharuddin, Pendidikan dan Psikologi Perkembangan, Jogjakarta: Ar-Ruzz Media, 2009 hal. 180. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis menumbuhkan sikap sosial anak didik, hal ini disadari bahwa anak didik adalah sejenis makhluk homo socius, yakni makhluk yang cenderung hidup bersama.23 Siswa dibiasakan hidup bersama, bekerja sama dalam kelompok, akan menyadari bahwa dirinya ada kekurangan dan kelebihan. Yang mempunyai kelebihan mau membantu mereka yang mempunyai kekuranga. 3. Pendekatan bervariasi Pendekatan bervariasi adalah bermacam-macam pendekatan yang dilakukan dalam kegiatan belajar agar terlaksananya proses belajar mengajar yang efektif. Pendekatan ini terjadi karena siswa mempunyai tingkat motivasi yang berbeda, pada satu sisi siswa memiliki motivasi yang rendah, tetapi pada sisi yang lain mempunyai motivasi yang tinggi. Maka pendekatan bervariasi ini sebagai alat yang dapat guru gunakan untuk kepentingan pengajaran. 4. Pendekatan edukatif Edukatif adalah sesuatu yang bersifat mendidik dan segala hal yang berkenaan dengan pendidikan.24 Pendekatan edukatif yaitu pendekatan yang dilakukan oleh guru, baik dari setiap tindakan, sikap, dan perbuatan yang guru lakukan harus bernilai pendidikan, dengan tujuan untuk mendidik siswa agar menghargai norma hukum, norma susila, norma moral, norma sosial dan norma agama. Adapun yang penting untuk diingat adalah bahwa pendekatan individual, pendekatan kelompok, dan pendekatan bervariasi harus berdampingan dengan pendekatan edukatif, dengan tujuan untuk mendidik siswa. 5. Pendekatan pengalaman 23 Muhammad . AR, “Strategi Pembelajaran Al-Qur’an Hadist di MTsN Meureudu”, Skripsi, Banda Aceh: IAIN, 2010, hal. 35. 24 Djalinur Syah Dkk, Kamus Pelajar Kata Serapan Bahasa Indonesia, Cet 1, Jakarta: Rineka Cipta, 1993, hal. 50. 2. Pendekatan kelompok Pendekatan kelompok adalah adanya interaksi diantara anggota kelompok dengan harapan terjadi perbaikan pada diri siswa yang mengalami kesulitan belajar.21 Dalam kegiatan belajar-mengajar di kelas adanya guru membentuk kelompok kecil. Kelompok tersebut umumnya terdiri dari 3-8 orang siswa. Dalam pembelajaran kelompok kecil, guru memberikan bantuan atau bimbingan kepada tiap kelompok lebih intensif.22 Pendekatan kelompok bertujuan membina dan Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 357 REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis 6. Pendekatan pembiasaan Pembiasaan adalah suatu proses pembentukan kebiasaan-kebiasaan baru atau perbaikan kebiasaan-kebiasaan yang telah ada. Tujuannya agar siswa memperoleh sikap-sikap dan kebiasaan-kebiasaan perbuatan baru yang lebih tepat dan relatif menetap. Pendekatan dengan proses pembentukan kebiasaan-kebiasaan baru atau perbaikan kebiasaan-kebiasaan yang telah ada. Tujuannya agar siswa memperoleh sikap-sikap dan kebiasaan-kebiasaan perbuatan baru yang lebih tepat dan positif.26 Pendidikan agama Islam sangat penting dalam hal ini, karena dengan pendidikan pembiasaan itulah diharapkan siswa senantiasa mengamalkan ajaran agamanya, yaitu dengan memberikan kesempatan pada siswa untuk senantiasa mengamalkan ajaran agamanya dalam kehidupan sehari-hari. Untuk itu maka metode mangajar yang perlu dipertimbangkan, antara lain adalah metode latihan (drill), pelaksanaan tugas, demonstrasi dan pengalaman langsung di lapangan. 5. Pendekatan pengalaman Pengalaman merupakan suatu kejadian atau perbuatan yang pernah terjadi pada masa dahulu dan mempunyai nilai atau manfaat untuk masa depan. Pendekatan pengalaman yaitu suatu pendekatan yang pembelajarannya harus dilandaskan pada pengalaman siswa sebelumnya, karna siswa juga sudah memiliki 358 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur pengalaman tentang al-Qur’an Hadist di rumah dan masyarakat.25 Belajar dari pengalaman adalah lebih baik daripada sekadar bicara, dan tidak pernah berbuat sama sekali. Untuk pendidikan agama Islam, pendekatan pengalaman yaitu suatu pendekatan yang memberikan pengalaman keagamaan pada siswa dalam rangka penanaman nilai keagamaan. 25 Ramli Maha, Perencanaan pembelajaran Sistem PAI, Banda Aceh: Selamat Sejahtera, 2000, hal. 48. 26 Tohiri, Psikologi Pembelajaran Pendidikan Agama Islam, Jakarta: Raja Grafindo Persada, 2008, hal. 103. 27 Nana Syaodih Sukmadinata, Landasan Psikologi Proses Pendidikan, Cet. 3, Bandung: Remaja Rosdakarya, 2005, hal. 78. 27 Nana Syaodih Sukmadinata, Landasan Psikologi Proses Pendidikan, Cet. 3, Bandun Remaja Rosdakarya, 2005, hal. 78. 26 Tohiri, Psikologi Pembelajaran Pendidikan Agama Islam, Jakarta: Raja Grafindo Persad 2008, hal. 103. 25 Ramli Maha, Perencanaan pembelajaran Sistem PAI, Banda Aceh: Selamat Sejahter 2000, hal. 48. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis rangsangan (stimulus) dari luar diri seseorang. Rangsangan itu misalnya ceramah, sindiran, pujian, ejekan, anjuran, perintah, sikap dan perbuatan. Emosi mempunyai peranan yang penting dalam pembentukan kepribadian seseorang. Pendekatan emosional dimaksud di sini adalah suatu usaha untuk menggugah perasaan dan emosi siswa dalam meyakini, memahami, dan menghayati ajaran agamanya. Maka metode mengajar yang perlu dipertimbangkan adalah metode ceramah, bercerita, sosiodrama. 8. Pendekatan rasional Pendekatan rasional ialah pembelajaran yang berpotensi untuk menumbuhkan daya pikir sendiri pada siswa guna memahami, mengamalkan, dan meyakini konsep-konsep dalam pembelajaran remedial al-Qur’an Hadist.28 Pendekatan rasional yaitu kemampuan memecahkan masalah dengan menggunakan pertimbangan dan strategi akal sehat, logis dan sistematis. Pendekatan dengan menggunakan kemampuan berfikir secara logis dan sistematis. Dengan kekuatan akalnya manusia dapat membedakan mana perbuatan yang baik dan mana perbuatan yang buruk. Untuk mendukung pemakaian pendekatan ini, maka metode mengajar yang perlu diberikan adalah metode ceramah, tanya jawab, diskusi, kerja kelompok, latihan dan pemberian tugas. 28 Khairuman, “Strategi Pembelajaran Bidang Studi Al-Qur’an Hadist Pada MAN Sawang Aceh Selatan”, Skripsi, Banda Aceh: IAIN, 2010, hal. 40. 7. Pendekatan emosional Emosi adalah gejala kejiwaan yang ada di dalam diri seseorang. Emosi berhubungan dengan masalah perasaan. Emosi atau perasaan adalah sesuatu yang peka. Emosi seperti halnya juga perasaan merupakan suatu suasana hati yang membentuk suatu kontinum atau garis. Kontinum ini bergerak dari ujung yang paling positif yaitu sangat senang sampai dengan ujung yang paling negatif yaitu sangat tiadak senang.27 Emosi akan memberi tanggapan (respons) bila ada Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 359 REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis 9. Pendekatan fungsional Pendekatan fungsional adalah suatu pendekatan atau suatu ilmu pengetahuan yang dipelajari bukan hanya untuk mengisi kekosongan intelektual, tetapi diharapkan berguna untuk diimplementasikan ke dalam kehidupan sehari- hari. Pendekatan ini bahwa ia relatif menetap dan setiap saat apabila dibutuhkan, perubahan tersebut dapat direduksi dan dimanfaatkan. Perubahan fungsional dapat diharapkan memberi manfaat yang luas, misalnya ketika siswa menempuh ujian dan menyesuaikan diri dengan lingkungan dalam mempertahankan kelangsungan hidup. Dalam hal ini diperlukan penggunaan metode mengajar, antara lain metode latihan, pemberian tugas, ceramah, tanya jawab dan demonstrasi. 360 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur 11. Pendekatan kebermaknaan Pendekatan kebermaknaan adalah suatu pendekatan dalam pembelajaran yang mempunyai arti atau dapat lebih berarti bagi siswa. Bahan pelajaran dan kegiatan pembelajaran, menjadi lebih bermakna bagi siswa jika berhubungan dengan kebutuhan siswa yang berkaitan dengan pengalaman, minat, tata nilai dan masa depan yang harus dijadikan pertimbangan dalam mengambil keputusan pengajaran dan pembelajaran untuk membuat pelajaran lebih bermakna bagi siswa. Menurut Oemar Hamalik, pendekatan pembelajaran dalam pelaksaan remedial adalah pendekatan sistem pembelajaran, yang mana pendekatan sistem sebagai suatu pandangan tertentu mengenai proses pembelajaran di mana berlangsung kegiatan belajar mengajar, terjadinya interaksi antara siswa dan guru, memberikan kemudahan bagi siswa untuk belajar secara efektif dan menggunakan metodologi untuk merancang sistem pembelajaran. Metode ini akan menghasilkan suatu sistem pembelajaran efektif dan efisien.29 Berdasarkan pendapat di atas, bahwa dengan berbagai pendekatan- pendekatan yang diberikan maka akan terjadi interaksi antara guru dan siswa dalam kegiatan belajar mengajar. Dengan demikian, keberhasilan siswa juga sangat berpengaruh melalui pendekatan-pendekatan dalam belajar sehingga menghasilkan potensi yang lebih baik. 29 Oemar Hamalik, Kurikulum dan Pembelajaran, Jakarta: Bumi Aksara, 2008, hal. 111. 10. Pendekatan keagamaan Pendekatan keagamaan adalah suatu pendekatan yang dilakukan dalam setiap bidang studi atau mata pelajaran umum dapat menyatu dengan nilai-nilai agama. Hal ini dimaksud agar nilai budaya ilmu tidak sekuler, seperti mata pelajaran biologi dapat dihubungkan dengan masalah agama dalam surat Yasin ayat 34, bahwa pelajaran biologi tidak dapat dipisahkan dari ajaran agama. 31 Muhibbin Syah, Psikologi Pendidikan Dengan Pendekatan Baru, Edisi Revisi, Bandung: Remaja Rosdakarya, 2003, hal. 174. 30 Mulyono Abdurrahman, Pendidikan Bagi Anak Berkesulitan Belajar, Jakarta: Rineka Cipta, 2003, hal. 20. 32 Abdul Majid, Perencanaan Pembelajaran, Bandung: Remaja Rosdakarya, 2005, hal. 227. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis memberikan pengajaran remedial, guru lebih dahulu menegakkan diagnosis, yaitu menentukan jenis dan penyebab kesulitan serta alternatif strategi pengajaran remedial yang efektif dan efisien.30 Menurut Muhibbin Syah, langkah-langkah diagnostik yang ditempuh guru antara lain: 1. Melakukan observasi kelas untuk melihat prilaku menyimpang siswa ketika mengikuti pelajaran. 2. Memeriksa penglihatan dan pendengaran siswa khususnya yang diduga mengalami kesulitan belajar. 3. Mewawancarai orang tua atau wali siswa untuk mengetahui hal ihwal keluarga yang mungkin menimbulkan kesulitan belajar. 4. Memberikan tes diagnostik bidang kecakapan tertentu untuk mengetahui hakikat kesulitan belajar yang dialami siswa. 5. Memberikan tes kemampuan intelegensi (IQ) khususnya kepada siswa yang diduga mengalami kesulitan belajar.31 Diagnosis menurut Saiful Bahri Djamarah, dapat berupa hal-hal sebagai berikut: Diagnosis menurut Saiful Bahri Djamarah, dapat berupa hal-hal sebagai berikut: 1. Keputusan mengenai jenis kesulitan belajar anak didik yaitu berat dan ringannya tingkat kesulitan yang dirasakan anak didik. 2. Keputusan mengenai faktor-faktor yang ikut menjadi suber penyebab kesulitan belajar anak didik. 3. Keputusan mengenai faktor utama yang menjadi sumber penyebab kesulitan belajar anak didik. Sedangkan menurut Mulyono Abdurrahman, ada tujuh langkah yang hendaknya diikuti oleh guru dalam menegakkan diagnosis tersebut, antara lain yaitu: Sedangkan menurut Mulyono Abdurrahman, ada tujuh langkah yang hendaknya diikuti oleh guru dalam menegakkan diagnosis tersebut, antara lain yaitu: 1. Identifikasi Mendiagnosis Pelaksanaan Remedial. Siswa berkesulitan belajar memerlukan program pelayanan remedial. Program remedial hendaknya dilaksanakan oleh guru khusus yang memiliki keahlian dalam bidang pendidikan bagi siswa berkesulitan belajar. Sebelum Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 361 1. Identifikasi Identifikasi adalah menentukan siswa mana yang mengalami masalah dalam belajar.32 Menentukan potensi siswa yang memerlukan pelayanan remedial 30 Mulyono Abdurrahman, Pendidikan Bagi Anak Berkesulitan Belajar, Jakarta: Rineka Cipta, 2003, hal. 20. 362 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur dengan memperhatikan laporan guru melalui hasil tes inteligensi atau melalui instrumen informal. Sekolah yang ingin menyelenggarakan program pengajaran remedial yang sistematis hendaknya melakukan identifikasi untuk menentukan siswa-siswa yang memerlukan atau berpotensi memerlukan pelayanan pengajaran remedial. Pelaksanaan identifikasi dapat dilakukan dengan memperhatikan laporan guru kelas atau sekolah sebelumnya, hasil tes inteligensi yang dilakukan secara masal atau individual, atau melalui insrtumen informal, misalnya dalam bentuk lembar observasi guru atau orang tua. 2. Menentukan Prioritas Prioritas adalah diutamakan atau didahulukan pada yang lain.33 Menentukan prioritas yaitu mengutamakan atau mendahulukan siswa-siswa yang mengalami kesulitan belajar yang tergolong berat untuk memperoleh layanan remedial yang sistematis dari guru khusus remedial. Oleh karena itu, sekolah perlu menentukan prioritas siswa mana yang diperkirakan dapat diberi pelayanan pengajaran remedial oleh guru kelas atau guru bidang studi, dan siswa mana perlu dilayani oleh guru khusus. Siswa-siswa berkesulitan belajar yang tergolong berat mungkin perlu memperoleh prioritas utama untuk memperoleh pelayanan pengajaran remedial yang sistematis dari guru khusus remedial. 33 Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia,Edisi 3, Jakarta: Balai Pustaka, 2002, hal. 896. 3. Menentukan Potensi Potensi adalah kemampuan yang mempunyai kemungkinan untuk dikembangkan. Menentukan potensi merupakan menentukan tingkat kemampuan siswa setelah identifikasi dilakukan dengan dilaksanakan tes inteligensi. Potensi siswa biasanya berdasarkan atas skor tes inteligensi. Oleh karena itu, setelah identifikasi siswa berkesulitan belajar ditentukan, maka untuk menentukan potensi siswa diperlukan tes inteligensi. REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis berkesulitan belajar, sedangkan kalau prestasinya seimbang dengan kapasitas intelegensinya maka tidak dapat dikelompokkan sebagai siswa berkesulitan belajar. 34 Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia,Edisi 3, Jakarta: Balai Pustaka, 2002, hal. 942. 6. Analisis Berbagai Faktor yang Terkait Menganalisis berbagai faktor yang terkait yaitu melakukan pemeriksaan yang dilakukan oleh para ahli-ahli psikolog kemudian dikaitkan dengan observasi yang dilakukan oleh guru agar mengetahui diagnosis dan dapat menentukan strategi pengajaran remedial yang efektif dan efisien. Pada langkah ini, guru remedial perlu melakukan analisis terhadap hasil- hasil pemeriksaan ahli-ahli lain seperti psikolog, dokter dan konselor. Berdasarkan hasil analisis terhadap hasil pemeriksaan berbagai bidang keahlian dan mengaitkan mereka dengan hasil observasi yang dilakukan sendiri, guru remedial dapat menegakkan suatu diagnosis yang diharapkan dapat digunakan sebagai landasan dalam menentukan strategi pengajaran remedial yang efektif dan efisien. 7. Menyusun rekomendasi untuk pengajaran remedial 5. Menentukan gejala kesulitan Menentukan gejala kesulitan ialah penentuan jenis penyakit atau jenis kesulitan belajar dengan meneliti (memeriksa) gejala-gejalanya atau proses pemeriksaan terhadap hal yang dipandang tidak beres, dengan melakukan observasi dan analisis kasus kesulitan belajar. Pada langkah ini guru remedial perlu melakukan observasi dan analisis cara siswa belajar. Cara siswa mempelajari suatu bidang studi sering dapat memberikan informasi diagnostik tentang sumber penyebab yang orisinil dari suatu kesulitan. Gejala kesulitan tersebut dapat digunakan sebagai landasan dalam menentukan diagnosis, yang selanjutnya dapat digunakan sebagai landasan dalam menentukan strategi pembelajaran yang sesuai. 4. Menentukan Penguasaan Bidang Studi yang Perlu Diremediasi Menentukan penguasaan bidang studi adalah dengan memperhatikan data prestasi belajar siswa. Guru remedial perlu memiliki data tentang prestasi belajar tersebut dengan taraf inteligensinya. Kalau prestasi belajar siswa menyimpang jauh di bawah kapasitas inteligensinya maka dapat dikelompokkan sebagai siswa 33 Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia,Edisi 3, Jakarta: Balai Pustaka, 2002, hal. 896. Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 363 363 7. Menyusun rekomendasi untuk pengajaran remedial Rekomendasi yaitu dengan persetujuan dari pihak-pihak tertentu.34 Menyusun Rekomendasi adalah menyusun suatu penyelenggaraan program pengajaran remedial dalam bentuk suatu program pendidikan yang diindividualkan (individuallized education program), yang pelaksanaannya perlu dievaluasi lebih dahulu oleh suatu tim yang disebut Tim Penilai Program Pendidikan Individual, 364 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur yang terdiri dari guru khusus remedial, guru reguler, kepala sekolah, konselor, dokter, psikolog, orang tua, dan kalau mungkin juga siswa yang bersangkutan.35 Mendiagnosis pelaksanaan remedial yang diuraikan oleh para ahli di atas dapat penulis simpulkan bahwa sebelum menetapkan alternatif pemecahan masalah kesulitan belajar siswa, guru sangat dianjurkan untuk terlebih dahulu melakukan identifikasi dan observasi (upaya mengenali gejala dengan cermat) terhadap fenomena yang menunjukkan kemungkinan adanya kesulitan belajar pada siswa tersebut. Upaya seperti ini disebut diagnosis yang bertujuan menetapkan jenis penyakit yakni jenis kesulitan belajar siswa. Dalam melakukan diagnosis diperlukan adanya prosedur yang terdiri atas langkah-langkah tertentu yang diorientasikan pada ditemukannya kesulitan belajar jenis tertentu yang dialami siswa. SIMPULAN Remedial teaching adalah suatu upaya guru untuk menciptakan situasi yang memungkinkan individu atau kelompok siswa (dengan karakter) tertentu agar lebih mampu meningkatkan prestasi seoptimal mungkin sehingga dapat memenuhi kriteria keberhasilan minimal yang diharapkan. Program remedial hendaknya dilaksanakan oleh guru khusus yang memiliki keahlian dalam bidang pendidikan bagi siswa berkesulitan belajar. Sebelum memberikan pengajaran remedial, guru lebih dahulu melakukan diagnosis, yaitu menentukan jenis dan penyebab kesulitan serta alternatif strategi pengajaran remedial yang efektif dan efisien. Rekomendasi adalah menyusun suatu penyelenggaraan program pengajaran remedial dalam bentuk suatu program pendidikan yang diindividualkan (individuallized education program), yang pelaksanaannya perlu dievaluasi lebih dahulu oleh suatu tim yang disebut Tim Penilai Program Pendidikan Individual, yang terdiri dari guru khusus remedial, guru reguler, kepala sekolah, konselor, dokter, psikolog, orang tua, dan kalau mungkin juga siswa yang bersangkutan. Mendiagnosis pelaksanaan remedial dapat disimpulkan bahwa, sebelum menetapkan alternatif pemecahan masalah kesulitan belajar siswa, guru sangat dianjurkan untuk terlebih dahulu melakukan identifikasi dan observasi (upaya mengenali gejala dengan cermat) terhadap fenomena yang menunjukkan kemungkinan adanya kesulitan belajar pada siswa Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 365 Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 365 366 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 REMEDIAL TEACHING SEBAGAI SUATU SOLUSI: Suatu Analisis Teoritis tersebut. Upaya seperti ini disebut diagnosis yang bertujuan menetapkan jenis penyakit yakni jenis kesulitan belajar siswa. tersebut. Upaya seperti ini disebut diagnosis yang bertujuan menetapkan jenis penyakit yakni jenis kesulitan belajar siswa. 366 \| Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 Masbur DAFTAR PUSTAKA Amin, Safwan, Pengantar Spikologi Pendidikan, Banda Aceh: YayasanPena, 2003. Ahmadi, Abu dan Widodo Supriono, Psikologi Belajar, Jakarta: Rineka Cipta, 2004. Bahri, Syaiful Djamarah dan Aswan Zain, Strategi Belajar Mengajar, Jakarta: Rineka Cipta, 2002. AR, Muhammad, “Strategi Pembelajaran Al-Qur’an Hadist di MTsN Meureudu”, Skripsi, Banda Aceh: IAIN, 2010. Abdurrahman, Mulyono, Pendidikan Bagi Anak Berkesulitan Belajar, Jakarta: Rineka Cipta, 2003. Baharuddin, Pendidikan dan Psikologi Perkembangan, Jogjakarta: Ar-Ruzz Media, 2009. Departemen Pendidikan Nasional, Kamus Besar Bahasa Indonesia, Edisi 3, Jakarta: Balai Pustaka, 2002. Hamalik, Oemar, Kurikulum dan Pembelajaran, Jakarta: Bumi Aksara, 2008. Khairuman, “Strategi Pembelajaran Bidang Studi Al-Qur’an Hadist Pada MAN Sawang Aceh Selatan”, Skripsi, Banda Aceh: IAIN, 2010. Majid, Abdul, Perencanaan Pembelajaran, Bandung: Remaja Rosdakarya, 2005. Maha Ramli, Perencanaan pembelajaran Sistem PAI, Banda Aceh: Selamat Sejahtera, 2000. Nashiruddin, Muhammad Al Abani, Shahih Sunan Ibnu Majah, Kamp. Melayu: Pustaka Azzam, 2005. Prayitno dan Erman Amti, Dasar-dasar dan Bimbingan Konseling, Jakarta: Rineka Cipta, 2004. Pidarta, Made, Landasan Kependidikan, Jakarta: Rineka Cipta, 1997. Rachmat, Abd. Abror, Psikologi Pendidikan,Yoyakarta: Tiara Wacana Yogya, 1993. Syamsuddin, Abin Makmun, Psikologi Kependidikan Perangkat Sistem Pengajaran Modul, Bandung: Remaja Rosdakarya, 2005. S. W. Ischak, dan Warji R, Program Remedial Dalam Proses Belajar Mengajar, Yogyakarta: Liberti, 1987. Syah, Djalinur, Dkk, Kamus Pelajar Kata Serapan Bahasa Indonesia, Cet 1, Jakarta: Rineka Cipta, 1993. Syaodih, Nana Sukmadinata, Landasan Psikologi Proses Pendidikan,Cet. 3, Bandung: Remaja Rosdakarya, 2005. Syah, Muhibbin, Psikologi Pendidikan Dengan Pendekatan Baru, Edisi Revisi, Bandung: Remaja Rosdakarya, 2003. Tohiri, Psikologi Pembelajaran Pendidikan Agama Islam, Jakarta: Raja Grafindo Persada, 2008. Jurnal Ilmiah Didaktika Vol. XII, No. 2, Februari 2012 \| 367
https://openalex.org/W4234085643	https://ro.uow.edu.au/cgi/viewcontent.cgi?article=1102&context=asshpapers	English	null	Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years	Research Square (Research Square)	2,020	cc-by	8,198	University of Wollongong University of Wollongong Research Online Research Online Faculty of Arts, Social Sciences and Humanities - Papers Faculty of Arts, Social Sciences & Humanities 2020 Proximal and distal predictors of self-regulatory change in children aged 4 Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years to 7 years Kate Williams Steven J. Howard University of Wollongong, stevenh@uow.edu.au University of Wollongong University of Wollongong Research Online Research Online Faculty of Arts, Social Sciences and Humanities - Papers Faculty of Arts, Social Sciences & Humanities 2020 Proximal and distal predictors of self-regulatory change in children aged 4 Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years to 7 years Kate Williams Steven J. Howard University of Wollongong, stevenh@uow.edu.au University of Wollongong University of Wollongong Research Online Research Online Faculty of Arts, Social Sciences and Humanities - Papers Faculty of Arts, Social Sciences & Humanities 2020 Proximal and distal predictors of self-regulatory change in children aged 4 Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years to 7 years Kate Williams Steven J. Howard University of Wollongong, stevenh@uow.edu.au ocial Sciences and Humanities Faculty of Arts, Social Sciences & Humanities Faculty of Arts, Social Sciences and Humanities - Papers Faculty of Arts, Social Sciences & Humanities Proximal and distal predictors of self-regulatory change in children aged 4 Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years to 7 years Follow this and additional works at: https://ro.uow.edu.au/asshpapers Follow this and additional works at: https://ro.uow.edu.au/asshpapers Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Williams, K. & Howard, S. (2020). Proximal and distal predictors of self-regulatory change in children aged 4 to 7 years. BMC Pediatrics, 20 (1), 226-1-226-9. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Abstract Background: Background: Growth in early self-regulation skills has been linked to positive health, wellbeing, and achievement trajectories across the lifespan. While individual studies have documented specific influences on self-regulation competencies in early childhood, few have modelled a comprehensive range of predictors of self-regulation change across health, development, and environment simultaneously. This study aimed to examine the concurrent associations among a range of proximal and distal influences on change in children's self-regulation skills over 2 years from age 4-5 years. Methods: Methods: Data from the Longitudinal Study of Australian Children (N = 4983) were used in a structural equation model, predicting a multi-source composite measure of self-regulation at each of 4-5 years and 6-7 years. By controlling for earlier self-regulation and covariates, the model examined the relative contributions of a comprehensive range of variables to self-regulation change including health, development, educational, home environment, time-use, and neighbourhood characteristics. Results: Results: The significant predictors of children's self-regulation growth across 4 to 7 years were fewer behavioural sleep problems, higher gross motor and pre-academic skills, lower levels of maternal and paternal angry parenting, and lower levels of financial hardship. There were also marginal effects for high-quality home learning environments and child-educator relationships. Conclusion: Conclusion: Findings suggest that if we are to successfully foster children's self-regulation skills, interventionists would do well to operate not only on children's current capacities but also key aspects of their surrounding context. This journal article is available at Research Online: https://ro.uow.edu.au/asshpapers/96 Williams and Howard BMC Pediatrics (2020) 20:226 https://doi.org/10.1186/s12887-020-02133-6 Open Access Abstract Background: Growth in early self-regulation skills has been linked to positive health, wellbeing, and achievement trajectories across the lifespan. While individual studies have documented specific influences on self-regulation competencies in early childhood, few have modelled a comprehensive range of predictors of self-regulation change across health, development, and environment simultaneously. This study aimed to examine the concurrent associations among a range of proximal and distal influences on change in children’s self-regulation skills over 2 years from age 4–5 years. Methods: Data from the Longitudinal Study of Australian Children (N = 4983) were used in a structural equation model, predicting a multi-source composite measure of self-regulation at each of 4–5 years and 6–7 years. By controlling for earlier self-regulation and covariates, the model examined the relative contributions of a comprehensive range of variables to self-regulation change including health, development, educational, home environment, time-use, and neighbourhood characteristics. Results: The significant predictors of children’s self-regulation growth across 4 to 7 years were fewer behavioural sleep problems, higher gross motor and pre-academic skills, lower levels of maternal and paternal angry parenting, and lower levels of financial hardship. There were also marginal effects for high-quality home learning environments and child-educator relationships. Conclusion: Findings suggest that if we are to successfully foster children’s self-regulation skills, interventionists would do well to operate not only on children’s current capacities but also key aspects of their surrounding context. Keywords: Early childhood, Self-regulation, Self-control, Predictive model with strong early childhood self-regulation skills linked with a wide range of health and achievement outcomes across the lifespan, including positive mental and physical health, and educational attainment [1–3]. In contrast, poorer self- regulation in early childhood has been linked with school adjustment difficulties [4], behaviour problems [5], adoles- cent risk-taking [2], and adult disordered behaviour [6]. Proximal and distal predictors of self- regulatory change in children aged 4 to 7 years Kate E. Williams1* and Steven J. Howard2 * Correspondence: k15.williams@qut.edu.au 1School of Early Childhood & Inclusive Education, Faculty of Education, Queensland University of Technology, QUT, Level 4 E Block, Victoria Park Road, Kelvin Grove, QLD 4059, Australia Full list of author information is available at the end of the article Background Self-regulation refers to the ability to exert control over our cognition, emotion, and behaviour in ways that are adaptive to functioning. These skills develop across the lifespan, but most rapidly in early childhood alongside cortical maturation processes. In terms of self-regulation development, early improvements appear to be better, Early childhood is a period in which growth in self- regulation is not only particularly desirable, but also demonstrably possible. In fact, growth in self-regulation skills in the early years of life (controlling for early self- * Correspondence: k15.williams@qut.edu.au 1School of Early Childhood & Inclusive Education, Faculty of Education, Queensland University of Technology, QUT, Level 4 E Block, Victoria Park Road, Kelvin Grove, QLD 4059, Australia Full list of author information is available at the end of the article Williams and Howard BMC Pediatrics (2020) 20:226 Page 2 of 9 Table 1 Sample characteristics Study sample characteristic Percentage Boys 51% English as main home language 86% Aboriginal or Torres Strait Islander 3.8% Mothers with incomplete high school education 22% Mothers with university education 28% Attending preschool program at 4–5 years 95% M (SD) Child age at 4–5 year data collection 56.9 months (2.65) Child age at 6–7 year data collection 81.9 months (2.96) Household income per week $1661.93AUD ($1294.05) regulatory levels and family environment) has been found to reduce risk of childhood behaviour problems [5], ado- lescent crime, self-harm, and mental health problems [2], as well as enhance academic learning trajectories [7]. Given limited understanding of antecedents of early self- regulation change that can shift trajectories and outcomes more broadly, intervention approaches remain incongru- ous. For instance, approaches to self-regulation interven- tion include computerized executive function training, specialized preschool curricula, physical activities, arts and music, motor skill development, and so forth [8–10]. While it is indeed likely that multiple approaches will be effective, and ideally suited to different contexts, needs and children, the design of interventions would neverthe- less be improved through a more comprehensive and holistic understanding of early childhood factors and ex- periences that support self-regulation development. teacher-, and observer-report ratings of self-regulation were standardized and then averaged to create a single composite score (M = 0, SD = 1). Constituent items of this factor index the extent to which children can control and sustain their attention, and control their behaviour and emotions (see Table 2). Internal consistency was high (alpha = 0.84 at 4–5 years, 0.86 at 6–7 years). The individual and environmental conditions that sup- port optimal development in self-regulation across early childhood remain relatively unclear. Various lines of inquiry have identified longitudinal predictors associated with better point-in-time self-regulation in early child- hood including rich home learning environments [11], positive parenting approaches [12], stronger motor [13] and language development [14], and well-adjusted sleep behaviours [15]. However, very few studies have exam- ined the extent to which these, and other plausible prox- imal and distal factors predict change in self-regulation over time. The aim of this study is to investigate the concurrent associations among a range of proximal and distal influences on change in children’s self-regulation skills over 2 years beginning at 4–5 years of age. Approach to analysis and missing data A structural equation model was tested in Mplus version 7.11. Figure 1 depicts the model, showing self-regulation at 6–7 years predicted by the full range of variables described above, while controlling for self-regulation measured two years earlier. This approach to modelling means the esti- mates for the predictors represent their impact on residua- lized change in self-regulation from 4 to 7 years of age, because the effect of the earlier measure of self-regulation has already been accounted for. Additionally, effects of stable covariates present from birth on earlier self- Participants This study used data from the population-representative Kindergarten (K) cohort of the Longitudinal Study of Australian Children (LSAC), with full study design de- tails described elsewhere [16]. In brief, for the K cohort, 4983 children aged 4–5-years old were recruited in 2004 with biennial data collection occurring since then. Data collection involves parent and teacher questionnaires, computer assisted interviews with parents and children, and direct assessments with children. The current study uses data collected for the K cohort across two waves (when children were 4- to 5-years old and 6- to 7-years old). Table 1 describes the characteristics of the sample. Predictors of self-regulation change were selected from the domains of health, development, home environment, education, time use, and neighbourhood measured when children were 4–5-years old. Details of each of these are provided in Table 3. Where parent-report is indicated, this was provided by Parent 1 (defined by LSAC as the parent who knows the study child best, which in 97% of cases was the mother). Control variables included in the analyses were gender, age of assessment (in months) at baseline, birth weight percentile, whether or not the child had ever been breastfed, Aboriginal and Torres Strait Islander status, Non-English speaking home background, maternal edu- cation level (on a 6-point scale from incomplete high school to postgraduate degree), and household income bracket. We used data from the age 4–5 years data col- lection for these variables, providing the most complete data possible (before attrition in the longitudinal study). Measures Self-regulation was assessed at 4–5 and 6–7 years of age using a factor score we have previously established as a re- liable indicator of children’s self-regulatory capacity with good predictive validity of broad later-life outcomes into adolescence [2]. A total of 20 survey items from parent-, Page 3 of 9 Williams and Howard BMC Pediatrics (2020) 20:226 Table 2 Items included in the self-regulation measure at 4–5 years and 6–7 years Construct Respondent Item Impulsive Aggression Parent and teacher Often has temper tantrums/hot tempers Parent and teacher Often fights with other children or bullies them Parent and teacher Often argumentative with adults Hyperactivity Parent and teacher Restless, overactive, cannot stay still for long Parent and teacher Constantly fidgeting or squirming Parent and teacher If this child is upset, it is hard to comfort him/her Lack of Persistence & Inattention Parent and teacher The child likes to complete one task or activity before going on to the next (reversed) Parent and teacher Sees takes through to the end, good attention span (reversed) Parent and teacher The child stays with an activity (e.g., puzzle, construction, kit, reading) for a long time (reversed) Parent, teacher, and observer Easily distracted, concentration wanders Impulsivity Parent and teacher Can stop and think things out before acting (reversed) Parent and teacher Shares readily with other children (reversed) Observer Degree of negative mood (withdrawn, uncooperative, sulky, seeming upset, angry) to interview Table 2 Items included in the self-regulation measure at 4–5 years and 6–7 years Construct Respondent stronger self-regulatory skills at 6–7 years including be- ing a female, having a higher birthweight percentile, identifying as non-Aboriginal and Torres Strait Islander, having a mother with a higher level of education, and a higher household income. regulation were controlled for. Correlations among all pre- dictor variables were included in the model, with the stron- gest significant correlation as r = .45 for the correlation among teacher-reported gross motor and fine motor skills. Due to the large sample size, we use a conservative p value of < .01 to indicate a significant effect and < .02 for a mar- ginally significant effect. Discussion The amount of missing data varied across waves and variables, ranging from no missing data for socio- demographic characteristics at 4–5 years to 45% missing data for the self-regulation scores at 4–5 and 6–7 years due primarily to item-level missing data from teacher non-report. The data were considered missing at ran- dom (MAR) because it was unlikely that the presence of a missing value was related to the response that would have been given [31]. We used full information max- imum likelihood with a robust estimator to address missing data, allowing us to retain 98% of the sample in the statistical models. We used the sampling weights provided for LSAC [32] to account for sampling error. This is the first paper to model a comprehensive and con- current set of predictors across health, development, and environment in relation to self-regulatory development of young children, across a two-year period beginning from age 4–5 years. Controlling for a range of background fac- tors, significant predictors of self-regulatory growth in- cluded: fewer behavioural sleep problems; higher gross motor and pre-academic skills; lower levels of maternal and paternal angry parenting; lower levels of financial hardship; and marginal effects for home learning environ- ment and child-educator relationships. As predictors were modelled simultaneously, significant findings provide a (likely conservative) estimate of the associations between each variable and self-regulation change, over and above the combined associations of all other variables in the model. While previous studies have provided insight into the transactional mechanisms between some factors known to influence self-regulation (e.g. parenting and sleep), this model better reflects the complexity of chil- dren’s lives and the combined impact of a range of factors on self-regulatory change. Thus the study makes an im- portant contribution toward prevention and intervention efforts by identifying the most salient and high-potential factors to target for self-regulation interventionists taking Results α = .72 Five items modelled as a latent variable as per prior studies [18]. E.g. child has problems on 4 or more nights a week with waking during the night (yes/no); this child’s sleep is a small/moderate/ large problem. Peabody Picture Vocabulary Test [19] of receptive vocabulary in which children listen to a spoken word and are asked to point to the matching picture given a set of four pictures. Higher scores represent higher receptive vocabulary skills. On a 4-point scale from ‘much less competent than peers’ to ‘more competent than peers’ On a 4-point scale from ‘much less competent than peers’ to ‘more competent than peers’ Who Am I test [20]. Children write their names, copy shapes, write words and numbers; scored according to skill level. α = .89 [21] Composite measure (weighted mean score) as per LSAC technical advice [22] using four adapted items from the National Longitudinal Study of Children & Youth [23]. Each item rated on 5-point scale from ‘never or almost never’ to ‘almost always’. E.g. how often are you angry when you punish this child? H = .72. Composite measure as per LSAC technical advice [22] using five items from the National Longitudinal Survey of Children and Youth [23]. Each item rates on a 10-point scale from ‘not at all’ to ‘all of the time’. E.g. how often does this child get away with things that you feel should have been punished? H = .80 for father; .82 for mothers. Kessler K6 screening scale [24] of six items (summed and averaged) about respondents’ feelings over the past four-week period. Rates on 5-point scale from ‘all of the time’ to ‘none of the time’. E.g. in the past 4 weeks how often have you felt hopeless? α = .84 for mothers, .82 for fathers. Single item book reading; plus latent variable with five indicators of other home learning activities including music, art, and play as used in other LSAC studies [25]. Each rated on 4-point scale of frequency of adult-child engagement for each activity in the last week from ‘not in the past week’ to ‘6–7 days in the week’. 7-item count index ranging from 0 to 7, based on summing Yes = 1, No = 0 responses to 7 items including couldn’t pay bills, gone without meals as used in prior LSAC research [26]. Results Paternal parenting consistency Father Maternal mental health Mother Kessler K6 screening scale [24] of six items (summed and averaged) abou over the past four-week period. Rates on 5-point scale from ‘all of the ti E.g. in the past 4 weeks how often have you felt hopeless? α = .84 for m Paternal mental health Father Home learning environment Parent Single item book reading; plus latent variable with five indicators of othe including music, art, and play as used in other LSAC studies [25]. Each ra frequency of adult-child engagement for each activity in the last week to ‘6–7 days in the week’. Financial hardship Parent 7-item count index ranging from 0 to 7, based on summing Yes = 1, No including couldn’t pay bills, gone without meals as used in prior LSAC re Argumentative parental relationships Parent Composite of 5 items (summed and averaged) rated on a 5-point scale my partner and I argue; disagree over child-rearing etc. α = .80 Stressful life events Parent 13-item count index ranging from 0 to 13 based on summing Yes = 1, N exposure to adverse life events over the past year including marital brea per prior LSAC research [27]. Education Teacher-child relationship Teacher 8-item composite drawn from the Student Teacher Relationship Scale [28 factor modelling [29]. Each item rated on 5-point scale from ‘definitely d applies’. E.g. share affectionate relationships, easy to be in tune with feel Time use Extra-curricular sport Parent Sum of 3 items indicating participation (yes / no) in extra-curricular swim sport Extra-curricular music / dance Parent Sum of 2 items indicating participation (yes / no) in extra-curricular mus Weekday TV hours Parent Number of hours watching TV on a typical weekday Weekday computer hours Parent Number of hours using a computer on a typical weekday Physical activity Parent Parent-rated child enjoyment of physical activity on a 5-point scale from physical activities’ to ‘very much likes physical activity’ Neighbourhood Liveability Parent Composite (sum) of 8 items each rated on 4 point scale from ‘strongly d Table 3 Predictors of self-regulation growth in the model Construct Data source Measure Physical Health Summary score from the Pediatric Quality of Life Inventory (PedsQL) [17]. Summed and average score of 8 items each rated on 5-point scale, tapping a child’s level of functioning in daily activities that rely on good physical health. E.g. problems with running. Results The model was a good fit for the data and accounted for 42% of variance in self-regulation at 6–7 years, with all estimates shown in Table 4. Self-regulation skills at 6–7 years, after controlling for self-regulation skills at 4–5 years, were predicted by fewer behavioural sleep prob- lems, higher gross motor and pre-academic skills, lower levels of maternal and paternal angry parenting, and lower levels of financial hardship. There were also mar- ginal effects for the home learning environment and child-educator relationships. Covariates associated with Williams and Howard BMC Pediatrics (2020) 20:226 Page 4 of 9 Table 3 Predictors of self-regulation growth in the model Construct Data source Measure Health & Health Behaviours Physical health Parent Physical Health Summary score from the Pediatric Quality of Life Inventory and average score of 8 items each rated on 5-point scale, tapping a chil daily activities that rely on good physical health. E.g. problems with runn Diet quality Parent Units of high sugar drinks consumed in the last week Behavioural sleep problems Parent Five items modelled as a latent variable as per prior studies [18]. E.g. chi more nights a week with waking during the night (yes/no); this child’s s large problem. Development Receptive vocabulary Assessed Peabody Picture Vocabulary Test [19] of receptive vocabulary in which ch word and are asked to point to the matching picture given a set of four represent higher receptive vocabulary skills. Gross motor development Teacher On a 4-point scale from ‘much less competent than peers’ to ‘more com Fine motor development Teacher On a 4-point scale from ‘much less competent than peers’ to ‘more com Pre-academic skills Assessed Who Am I test [20]. Children write their names, copy shapes, write word according to skill level. α = .89 [21] Home environment Maternal parenting anger Mother Composite measure (weighted mean score) as per LSAC technical advic items from the National Longitudinal Study of Children & Youth [23]. Eac scale from ‘never or almost never’ to ‘almost always’. E.g. how often are punish this child? H = .72. Paternal parenting anger Father Maternal parenting consistency Mother Composite measure as per LSAC technical advice [22] using five items fr Longitudinal Survey of Children and Youth [23]. Each item rates on a 10 to ‘all of the time’. E.g. how often does this child get away with things t been punished? H = .80 for father; .82 for mothers. Results Composite of 5 items (summed and averaged) rated on a 5-point scale from ‘never’ to ‘always’. E.g. my partner and I argue; disagree over child-rearing etc. α = .80 13-item count index ranging from 0 to 13 based on summing Yes = 1, No = 0 responses about exposure to adverse life events over the past year including marital breakdown, death of friend, as per prior LSAC research [27]. 8-item composite drawn from the Student Teacher Relationship Scale [28] following prior LSAC factor modelling [29]. Each item rated on 5-point scale from ‘definitely does not apply’ to ‘definitely applies’. E.g. share affectionate relationships, easy to be in tune with feelings α = .81 Composite (sum) of 8 items each rated on 4-point scale from ‘strongly disagree’ to ‘strongly agree’. E.g. this is a safe neighbourhood, this neighbourhood has good parks. α = .76 Composite of 31 variables (e.g. income, unemployment, occupation and education) computed by the Australian Bureau of Statistics [30]. Williams and Howard BMC Pediatrics (2020) 20:226 Page 5 of 9 Fig. 1 Conceptual model tested through structural equation model analyses Fig. 1 Conceptual model tested through structural equation model analyses a holistic approach to supporting self-regulatory growth in young children. reciprocal associations across time. A second and related explanation is that the pre-academic assessment used here may have tapped children’s visual-motor skills given it was a pencil and paper task requiring the writing of letters. While there was no visual-motor data available for chil- dren in this dataset, scores on the pre-academic test did correlate (r = .40) with the fine motor variable in our model (single item of teacher report of fine motor compe- tence). Recent research has suggested that visual-motor skills and cognitive self-regulation, as enabled by executive functions, co-develop in a bidirectional manner [35] and it may be that our findings are reflecting a small portion of this transactional process at this period of development. That is, children who scored more highly on the pre- academic score may have done so due to higher visual- motor skills, which may themselves co-develop with and support self-regulatory growth. Substantial research and theory supports both acute and persistent associations of self-regulation with learning and academic skills [33] with self-regulation typically posi- tioned as a predictor of academic skills. Results Indeed, tasks that are motor-demanding for young children, such as navigating uneven surfaces and/or obsta- cles, are more cognitively demanding and lead to more Williams and Howard BMC Pediatrics (2020) 20:226 Page 6 of 9 Page 6 of 9 Page 6 of 9 Table 4 Standardized coefficients for the predictors of self- regulation at 6–7 years controlling for prior self-regulation and covariates β 95% CI Covariate associations with self-regulation at 4–5 years Female .50 .44–.57 Age .01 .04–.11 Birthweight percentile .07 .03–.11 Breastfed −.15 −.29 - -.01 Aboriginal Torres Strait Islander −.53 −.81 - -.26 Non-English home language .01 −.10–.11 Maternal education level .13 .09–.17 Household income .13 .08–.17 Stability of self-regulation 4–5 years to 6–7 years .54 .49–.59 Predictors of self-regulation at 6–7 years controlling for above Health Physical health status .02 −.03–.05 High sugar drink intake .02 −.01–.06 Sleep problems −.08 −.13 - -.04 Development Vocabulary .01 −.03–.06 Gross motor .06 .02–.10 Fine motor −.05 −.10–.00 Pre-academic skills .12 .09–.16 Home environment Maternal angry parenting −.10 −.15–.06 Paternal angry parenting −.12** −.16 - -.07 Maternal consistent parenting −.01 −.04–.05 Paternal consistent parenting .02 −.02–.07 Maternal mental health −.01 −.06–.04 Paternal mental health .02 −.03–.06 Shared book reading frequency .03 −.01–.07 Home learning activities .06* .01–.10 Financial hardship −.07** −.12 - -.02 Argumentative parental relationships −.03 −.07–.02 Stressful life events −.00 −.04–.04 Education Educator-child relationship .06* .01–.11 Time use Extra-curricular sport −.02 −.05–.02 Extra-curricular music/dance .02 −.01–.05 Weekday TV hours .04 .00–.08 Weekday computer hours .01 −.03–.05 Physical activity −.03 −.06–.00 N i hb h d cognitive errors than less cognitively demanding motor tasks [39]. As such, one possibility is that this finding is in- dicative of the concomitance between self-regulatory and motor skills. However, that gross motor skills were associ- ated with change in self-regulation may additionally suggest that the acquisition of motor proficiency creates new learn- ing opportunities [40] such as experiences that serve to fos- ter self-regulation (e.g., increased mobility causing children to encounter rules associated with access, involvement in physically active shared play providing opportunities for im- pulse control and turn-taking, etc.) As such, gross motor skills may open a gateway to important self-regulation- promoting experiences and activities, whereas low levels of gross motor skills might consume much of the cognitive re- source that otherwise could be directed toward these same activities. Results In a related find- ing, but with self-regulation as the outcome, in our model pre-academic skills were one of the strongest predictors of self-regulation growth. It is clear why self-regulation would predict learning and academic skills: the ability to direct and sustain attention, tackle new challenges, resist maladaptive impulses, and work collaboratively and pro- socially with others – all hallmarks of high self-regulation – serve to support on-task behaviour, effort and persist- ence during learning. However, there is comparatively less research focused on the possible reciprocal effects with pre-academic skills predicting self-regulation growth. A number of explanations are feasible. First, it is likely that self-regulation and early literacy and numeracy skills, as represented by our pre-academic skill assessment, develop in a bidirectional manner across early childhood [34, 35]. For example, time spent in focussed literacy and numer- acy learning activities provides the opportunity to extend and enhance self-regulatory capacities, particularly in at- tentional and cognitive control aspects. It is likely that had we had an earlier and multiple measures of both self- regulation and early concept comprehension, literacy, and numeracy, we would have established birdirectional and Pre-school gross motor abilities were also significantly, al- beit modestly, associated with children’s self-regulation growth. This is consistent with suggestions of common mechanisms (i.e., executive functions) that are implicated in both self-regulation and motor learning [36–38], such that both show common areas of neural activation, are impaired after damage to neural regions for the other, and are often both impaired in cognitive disorders, such as ADHD and dyslexia. Results Another factor that was modestly but significantly and uniquely related to self-regulation growth was sleep prob- lems. This aligns with a large body of existing research that identifies sleep problems as a key contributor to day- time self-regulatory problems in young children both in the short [41] and long term [18, 42]. It is possible that be- havioural sleep problems in young children reflect an underlying phenotype associated with regulatory problems [43, 44], and/or that early behavioural sleep problems ini- tiate a developmental cascade that disrupts emotional and attentional development over time [15]. Either way, brief sleep interventions are known to be safe and effective in improving both sleep behaviours and daytime self- regulatory functioning in young children in both typically- developing [45–47] and clinical populations [48, 49]. p g p p Our finding that angry parenting was associated with less growth in self-regulation for children echoes a range of prior studies that have linked aggressive, controlling parenting with poor self-regulation in children [50–54]. However, this study extends that work by including not only mothers’ but also fathers’ parenting, a rare inclu- sion. We suggest that angry parenting as measured here is indicative of dysregulated parenting, and potentially of overall emotional regulation skills of parents. Mecha- nisms through which this might limit self-regulatory growth in children include heritability pathways in terms of self-regulation capabilities [55], and socialisation path- ways in which children learn about self-regulatory behaviours through modelling their parents’ behaviours. It is also important to note that child-driven effects are possible, as reflected in prior studies that show dysregu- lation in young children is associated with increased parenting stress and more-negative parenting ap- proaches [56, 57]. These bidirectional relationships be- tween parenting and children’s self-regulation, which are likely to establish mutual promotion/exacerbation pro- cesses over time, were not modelled in this study and should be the focus of future longitudinal work. Williams and Howard BMC Pediatrics (2020) 20:226 Page 7 of 9 Page 7 of 9 Williams and Howard BMC Pediatrics (2020) 20:226 A number of socioeconomic variables were associ- ated with enhanced self-regulatory growth including higher household incomes, higher maternal educa- tion levels and living in households with lower levels of financial hardship. Availability of data and materials The dataset analysed for the current study is available from the Australian Data Archive https://dataverse.ada.edu.au/dataset.xhtml?persistentId=doi:1 0.26193/JOZW2U Funding There was no funding attached to this study. Conclusion h l k While we know that self-regulation is important for a broad range of longitudinal achievement and wellbeing outcomes, and that early childhood is a key window for self-regulatory growth, we have not yet been overly ef- fective in intervention efforts. One reason for this might be that we need more holistic and evidence-informed theories and approaches to self-regulatory development, rather than a focus on single factors that appear predict- ive in isolation. We need more complex modelling of the interactions between these various factors and their association with self-regulation change (not just predic- tion at one time point). The findings of this study suggest a starting point for further detailed research that aims to achieve this. Marginal effects were also found for the association be- tween educator-child relationships and the home learning environment, with self-regulatory change. The finding re- garding importance of educator-child relationship in terms of children’s early self-regulation development reflects other similar findings in both Australia [61] and Europe [62]. Positive student-teacher relationships likely matter because they set the context within which teachers can en- act strategies particularly important for acquiring self- regulation during the preschool developmental period [63] including co-regulation, modelling and coaching [64]. Our findings regarding the home learning environment align with a prior American longitudinal study linking parental involvement in home learning activities with children’s self-regulatory development [65]. Results The experience of significant financial hardships such as those tapped here is likely associated with stressful home environments, which impact on children’s physiology and neurode- velopment in ways that limit their capacity for self- regulation development [58, 59]. Indeed, early self- regulation has been identified as one of the foremost mechanisms through which early stressors and socioeconomic disadvantage can lead to poorer aca- demic and wellbeing outcomes [60]. For these rea- sons, much of the prevention and intervention focus to date has been on children from disadvantaged backgrounds in an effort to address socio-economic gradients in achievement likely mediated through early self-regulatory capacity. Our findings suggest this focus is well-placed. even large-scale studies such as these are missing key ingredients related to self-regulatory growth. Our under- standings could be enhanced through studies which capture potential variables that are not often measured, including chronic stress (e.g. cortisol), psychophysio- logical arousal and regulation, sensory processing, and more detailed understandings of the nature of home learning and early education and care activities. Finally, it is important to note that participants in this study were recruited in 2004. While it is anticipated that there has been limited change in most lifestyle factors investi- gated (e.g., parenting), new cohort studies are required to better understand the influence of more prominent societal change such as increased access and use of digital devices. Authors’ contributions KW co-conceptualised the study, undertook all final analyses and was a major contributor to the writing of the paper. SH created the key outcome composite variables for self-regulation, co-conceptualised the study and was a major contributor to the writing of the paper. All authors have read and approved the manuscript. Limitations Although this study included a comprehensive array of predictors of self-regulation growth across a specific period in early childhood, there are a number of limita- tions related primarily to measurement. Most measures were broad and blunt instruments of their constructs. This reflects the nature of the population dataset, in which a broad spectrum of measures capturing child de- velopment and the environment were desired, rather than an in-depth measurement of any particular con- structs. In addition, our self-regulation composite was only available at two time points in this dataset, meaning that more sophisticated growth curve modelling, which requires a minimum of three time points, could not be undertaken. It is also important to note that although we included a wide array of predictors, nearly 60% of the variance in our self-regulation composite at 6–7 years was still unexplained by the model. This suggests that Ethics approval and consent to participate f Ethics approval for participants in the Longitudinal Study of Australia Children was approved by the Human Research Ethics Committee of the Australian Institute of Family Studies. Ethics approval for participants in the Longitudinal Study of Australia Children was approved by the Human Research Ethics Committee of the Australian Institute of Family Studies. K cohort: Kindergarten cohort; LSAC: Longitudinal Study of Australian Children Abbreviations K cohort: Kindergarten cohort; LSAC: Longitudinal Study of Australian Children References 25. Hayes N, Berthelsen DC, Nicholson JM, Walker S. Trajectories of parental involvement in home learning activities across the early years: associations with socio-demographic characteristics and children’s learning outcomes. Early Child Dev Care. 2018;188(10):1405–18. 1. McClelland MM, Acock AC, Piccinin A, Rhea SA, Stallings MC. Relations between preschool attention span-persistence and age 25 educational outcomes. Early Child Res Q. 2013;28:314–24. 2. Howard SJ, Williams KE. Early self-regulation, early self-regulatory change, and their longitudinal relation sto adolescents' academic, health, and mental well-being outcomes. 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Author details 1 h l f l Author details 1School of Early Childhood & Inclusive Education, Faculty of Education, Queensland University of Technology, QUT, Level 4 E Block, Victoria Park Road, Kelvin Grove, QLD 4059, Australia. 2Early Start, School of Education, Faculty of Social Sciences, University of Wollongong, Wollongong, Australia Road, Kelvin Grove, QLD 4059, Australia. 2Early Start, School of Education, Faculty of Social Sciences, University of Wollongong, Wollongong, Australia 23. Statistics Canada. National Longitudinal Survey of Children & Youth Cycle 3 Survey Instruments 1998–99. Book 1 - Parent & Child; 1999. Received: 5 March 2020 Accepted: 7 May 2020 Received: 5 March 2020 Accepted: 7 May 2020 24. Furukawa TA, Kessler RC, Slade T, Andrews G. The performance of the K6 and K10 screening scales for psychological distress in the Australian National Survey of mental health and well-being. Psychol Med. 2003;33(2): 357–62. Consent for publication Not applicable. 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https://openalex.org/W4388934481	https://bmcpulmmed.biomedcentral.com/counter/pdf/10.1186/s12890-023-02726-8	English	null	Assessing the impact of socioeconomic status on incidental lung nodules at an urban safety net hospital	BMC pulmonary medicine	2,023	cc-by	8,803	Abstract Introduction Lower socioeconomic status has been identified as an emerging risk factor for health disparities, including lung cancer outcomes. Most research investigating these outcomes includes patients from formal lung cancer screening programs. There is a paucity of studies assessing the relationship between socioeconomic sta- tus and incidental lung nodules. This study aimed to investigate the association between socioeconomic status and the size of incidental lung nodules on initial presentation at an urban safety net hospital, which did not have a formal lung cancer screening program or incidental lung nodule program. Methods A retrospective chart review was conducted on patients with incidental lung nodules on CT chest imaging who were referred from primary care to a pulmonology clinic at a safety net hospital. Patients with incomplete nodule characteristics information were excluded. Data on demographics, comorbidities, smoking history, insurance type, immigration status, and geographical factors were collected. Less commonly studied determinants such as crime index, cost of living, and air quality index were also assessed. Logistic regression analysis was performed to assess relationships between nodule size and socioeconomic determinants. Results Out of 3,490 patients with chest CT scans, 268 patients with ILNs were included in the study. 84.7% of patients represented racial or ethnic minorities, and most patients (67.8%) had federal insurance. Patients with non- commercial insurance were more likely to have larger, inherently higher-risk nodules (> 8 mm) compared to those with commercial insurance (OR 2.18, p 0.01). Patients from areas with higher unemployment rates were also less likely (OR 0.75, p 0.04) to have smaller nodules (< 6 mm). Patients representing racial or ethnic minorities were also more likely to have nodules > 8 mm (OR 1.6, p 0.24), and less likely to have nodules < 6 mm (OR 0.6, p 0.32), however, these relationships were not statistically significant. Conclusion This study found that lower socioeconomic status, indicated by having non-commercial insurance, was associated with larger incidental lung nodule size on initial presentation. While it is established that socioeconomic status is associated with disparities in lung cancer screening, these findings suggest that inequalities may also be present in those with incidental lung nodules. Further research is needed to understand the underlying mechanisms and develop interventions to address these disparities in incidental lung nodule evaluation and improve outcomes. Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 https://doi.org/10.1186/s12890-023-02726-8 Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 https://doi.org/10.1186/s12890-023-02726-8 BMC Pulmonary Medicine Open Access Assessing the impact of socioeconomic status on incidental lung nodules at an urban safety net hospital Mateus Fernandes1, Cristian Milla2,3, Ahmed Gubran2, Sandra Barrazueta2, Brian Altonen2,4, Anthony DiVittis2, Woodhull Resident Research Team1 and Stephen Kuperberg2,5* © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Keywords Lung nodule, Lung cancer, Inequity, Disparity, Socioeconomic, Disadvantage, Deprivation *Correspondence: Stephen Kuperberg kuperbes2@nychhc.org Full list of author information is available at the end of the article Introduction primary study location. NYCHHC is the largest munici- pal provider of safety-net care in the United States, serv- ing more than one million city residents who either utilize federal insurance, are uninsured or undocumented [13]. In addition, Woodhull is situated in a designated health- care professional shortage area in Brooklyn, NY, where a significant proportion of the population is uninsured or underinsured. The center had no formal LCS or ILN sur- veillance program during the study period, and individu- als were not enrolled in external LCS or ILN programs. Despite global collaborative efforts in prevention and man- agement, lung cancer continues to be the leading cause of cancer-related deaths worldwide [1]. On the basis of strong evidence [2–7], a key intervention now widely employed to attenuate the impact of lung cancer is lung cancer screen- ing (LCS), which serves as a public health measure aiming to identify high-risk individuals at an early stage and thus confer favorable potential for surgical intervention and sur- vival [2, 4]. Indeed, multiple randomized trials have con- firmed the value of lung cancer screening in reducing lung cancer mortality in high-risk individuals via early detection [5–7]. However, an increasing number of lung nodules are detected incidentally. As computed tomography (CT) chest imaging has become more common, there has been a par- allel increase in the detection of incidental lung nodules (ILNs) [8]. ILNs are now a common finding with a preva- lence of 10% to 30% on chest CT scans [9]. Individuals with ILNs have different characteristics and smoking behavior compared to those eligible for LCS, with less than half meet- ing the updated USPSTF screening criteria [10]. Therefore, LCS and ILN surveillance programs seem to reach different but complementary at-risk populations [9, 10]. We performed a retrospective chart review of individu- als ≥ 18 years of age who were referred from primary care services to the pulmonology clinic from February 2019 to February 2022. Of these individuals, we included all refer- rals with newly discovered, incidental lung nodules seen on CT chest, since a formal lung cancer screening program was not in place at our facility during this timeframe. Individu- als with nodules identified on chest radiographs only, and those with incomplete description or missing nodule meas- urements were excluded. For example, if the nodules were described as “small”, or no size was reported, then the nod- ule as excluded. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 2 of 10 Introduction A lung nodule was classified as an inciden- tal nodule if there was no record of a lung mass or nodule in the patient’s medical history, including previous imaging reports and clinical notes. Pulmonary nodules were classi- fied as any lesion that measured less than 30 mm located in the pulmonary parenchyma. The size of nodules was recorded in millimeters of greatest diameter, measured lin- early on two-dimensional imaging. When multiple nodules were present, the largest nodule diameter was recorded for analysis. The nodule size recorded for analysis was deter- mined by the radiologist report of the CT chest scan. y In both LCS and ILN surveillance, guideline concordant evaluation and follow-up rates are low [11]. Unfortunately, significant disparities have been uncovered based on race, income, and geographic location in LCS programs. How- ever, there is a paucity of studies evaluating how social determinants of health are related to ILNs [11]. Studies have shown that individuals with ILNs are more likely to be African American, nonsmokers or have stopped smok- ing [9, 12]. Other authors have demonstrated that individ- uals from ILN programs who were diagnosed with cancer were more likely to be uninsured or have federal insur- ance [10], providing further evidence of SES inequalities. For this reason, diverse efforts are being made to prioritize these socioeconomically driven challenges and overcome barriers to optimal lung cancer evaluation [11]. The objec- tive of this study was to assess the impact of poverty and SES on incidental lung nodule size in an urban, socioeco- nomically deprived population of high-risk individuals. Approval by the New York University School of Medi- cine Institutional Review Board was obtained prior to the initiation of the study. Nodule sizes were grouped into three tiers of risk per Fleischner Society Guidelines for evaluation of the soli- tary pulmonary nodule [14]. Recommendations for fol- low-up were based on nodule size being less than 6 mm, between 6 and 8 mm, and greater than 8 mm [14]. We allocated nodules to each of these categories for analysis. Demographics a Current/former smoker, 2 individuals missing data Demographics Age (years) Median 25th to 75th percentile 65 58 to 74 Sex Male 145 (54.1%) Female 123 (45.9%) Race/Ethnicity African American 71 (26.5%) Asian 6 (2.2%) Nonwhite Hispanic 150 (56.0%) White 41 (15.3%) Comorbidities Hypertension 180 (67.2%) Diabetes 86 (32.1%) Asthma 73 (27.2%) COPD 93 (34.7%) Current/former smokera 200 (74.5%) History of COVID-19 54 (20.1%) Nodules/Mass Characteristics Nodule Size (mm) Median 25 th to 75 th percentile 9 5 to 16 Nodule < 6 mm Nodule 6–8 mm 56 (20.9%) Nodule > 8 mm 109 (40.7%) Mass (> 30 mm) 29 (10.8%) a Current/former smoker, 2 individuals missing data Demographics Age (years) Median 25th to 75th percentile 65 58 to 74 Sex Male 145 (54.1%) Female 123 (45.9%) Race/Ethnicity African American 71 (26.5%) Asian 6 (2.2%) Nonwhite Hispanic 150 (56.0%) White 41 (15.3%) Comorbidities Hypertension 180 (67.2%) Diabetes 86 (32.1%) Asthma 73 (27.2%) COPD 93 (34.7%) Current/former smokera 200 (74.5%) History of COVID-19 54 (20.1%) Nodules/Mass Characteristics Nodule Size (mm) Median 25 th to 75 th percentile 9 5 to 16 Nodule < 6 mm Nodule 6–8 mm 56 (20.9%) Nodule > 8 mm 109 (40.7%) Mass (> 30 mm) 29 (10.8%) Statistical analysis S l l Statistical analysis was performed using JASP (version 0.16.4), which is a computer-based statistics program. Descriptive statistics on frequencies and medians were obtained to describe our study sample and nodule charac- teristics. Logistic regression was used to obtain adjusted odds ratios assessing the relationship between nodule size (dependent variable) and SES determinants (covari- ates) for individual and geographical markers of SES. A logistic regression model was used for analysis of each of the three nodule size categories. For each model, patients belonging to the same nodule category were coded as ‘yes’, while other patients were coded as ‘no’. For example, for the < 6 mm model, patients with ILNs less than 6 mm were coded as ‘yes’ while patients from the other two cat- egories (6 mm to 8 mm, and greater than 8 mm) were coded as ‘no’. Independent variables consisted of race/ ethnicity, insurance type, immigration status, and geo- graphical factors including median income, poverty level, cost of living index, air quality index, violent crime index, educational attainment, and unemployment rate. Insur- ance type was defined by one composite group labeled ‘noncommercial insurance’, which included individuals without commercial insurance and uninsured individuals. Race was defined by one composite group labeled ‘non- white race’, which included all individuals of races other than white. Odds ratios derived from the logistic regres- sion models were adjusted for covariates including age in years, sex (male as reference), smoking status (current or former smoker), and history of COPD. A p value of < 0.05 was determined to be statistically significant, and the effect size was determined by odds ratios. a Current/former smoker, 2 individuals missing data tables (Tables 3 and 4), grouped by relevant variables (individual and geographical determinants). Resultsh There were 3490 individuals with chest CT scans between February 2019 and February 2022 who were referred to the pulmonology clinic. After exclusion cri- teria were applied, 3222 individuals were excluded due to various criteria, most common being the absence of nodules or missing nodule data. A total of 268 individuals were included in the final study (Fig. 1). i Individuals were mostly within the age group 58–74 years with a median of 65 years. Most individuals were from a racial or ethnic minority group (84%), with the largest group being nonwhite Hispanic individuals (56%), followed by African American, White, and Asian individu- als. Comorbidities were prevalent, most commonly hyper- tension followed by COPD. There was a high prevalence of tobacco use at 74.5%. There was a high prevalence of clini- cally significant lesions (defined as nodules with size 6 mm or greater) at 72.4%, most of which were 8 mm or larger. Data collection Data were collected using electronic medical record review. The data were initially collected by members of the research team and then reviewed for accuracy by the primary author. Individual patient charts were reviewed to obtain relevant information, including demograph- ics (age, gender, ethnicity, race, zip code), comorbidities, smoking history, and determinants of socioeconomic sta- tus. Insurance type and immigration status were obtained from charts. Determinants of SES derived from the indi- vidual patient chart were classified as individual markers The aim of the study was to determine whether lower socioeconomic status (SES) was associated with larger nodules on initial presentation. We assessed the rela- tionships between determinants of SES and incidental nodule size. We hypothesized that individuals with mark- ers of lower SES were more likely to present with larger, higher risk incidental nodules. NYC Health + Hospitals/ Woodhull, a community hospital within the New York City Health and Hospitals (NYCHHC) network, was the Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 3 of 10 Table 1 Demographics, comorbidities, and nodule characteristics of individuals with incidental nodules of SES. The United States 2020 Census data were used to obtain median income, educational attainment, percent- age of those living below the federal poverty line, place of birth and unemployment rate according to zip code. A public data repository, City-data [15], was used to obtain data on the air quality index, cost of living index, and vio- lent crime index, according to patient zip code. Determi- nants of SES based on zip code, using census data and the data-repository, were classified as geographic markers of SES. of SES. The United States 2020 Census data were used to obtain median income, educational attainment, percent- age of those living below the federal poverty line, place of birth and unemployment rate according to zip code. A public data repository, City-data [15], was used to obtain data on the air quality index, cost of living index, and vio- lent crime index, according to patient zip code. Determi- nants of SES based on zip code, using census data and the data-repository, were classified as geographic markers of SES. Data reporting Th d The study was designed and reported according to the “Strengthening The Reporting of Observational Stud- ies in Epidemiology” (STROBE) guidelines. A checklist indicating STROBE components and page number is provided in the appendix. Demographics and patient characteristics were reported in Table 1. Results from the logistic regression models were reported in two separate Page 4 of 10 Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Fig. 1 Flow chart of the study, †This includes 239 individuals with nodules and 29 individuals with lung masses Fig. 1 Flow chart of the study, †This includes 239 individuals with nodules and 29 individuals with lung masses Table 3 shows associations of nodule size with various factors using logistic regression analysis, with covariates of age, sex, tobacco use and comorbidities. Table 2 highlights geographical and individual socio- economic determinants. Most individuals were either using federal insurance or uninsured (73%). Geographi- cal factors were also measured and analyzed for associa- tions with nodule size. Increased age was associated with nodules > 8 mm (OR 1.024, p 0.042); however, the effect size was negligible. Male individuals were less likely to have nodules < 6 mm (OR 0.56, p 0.056) and more likely to have nodules between 6–8 mm (OR 2.039, p 0.033). Individuals with noncommercial insur- ance were less likely to have nodules less than 6 mm (OR 0.437, p 0.01). Having noncommercial insurance was also associated with nodules > 8 mm (OR 2.181 p 0.016). Table 2 Socioeconomic determinants according to insurance type and geographical area a NYC care is a unique type of federal insurance offered exclusively to undocumented individuals in New York City Socioeconomic Determinants Insurance Type (%) Commercial 72 (26.9%) Medicare 19 (7.1%) Medicaid 135 (50.3%) Medicare/Medicaid 10 (3.7%) NYC Care a 18 (6.7%) Uninsured 14 (5.2%) Geographical Factors (Median) Violent Crime Index 198 Cost of living Index 123 Air Quality Index 38.15 Birthplace outside US (%) 23.9 Educational Attainment Less than High School/ Equivalent (%) 17.3 Living Below Federal Poverty Level (%) 25.7 Unemployment Rate (%) 8.12 Table 2 Socioeconomic determinants according to insurance type and geographical area Table 4 shows the relationships between geographical SES determinants and nodule size using logistic regression adjusted for age, sex (male reference), history of COPD, and tobacco use. Data reporting Th d Individuals from areas with a higher rate of non- US birthplaces were slightly less likely to have nodules < 6 mm (OR 0.896, p 0.015) and more likely to have nodules > 8 mm (OR 1.078, p 0.051). Individuals from areas with lower edu- cational attainment were more likely to have nodules < 6 mm (OR 1.243, p 0.008) and less likely to have nodules between 6–8 mm (OR 0.795, p 0.027). A lower unemployment rate was associated with smaller nodules < 6 mm (OR 0.754, p 0.045). Individuals from areas with lower income were less likely to have nodules 6–8 mm (OR 0.96, p value 0.02). Discussion Odds ratios were adjusted for covariates including age, smoking status, history of COPD and sex (with male as the reference) Nodule < 6 mm Nodule 6–8 mm Nodule > 8 mm Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Birthplace outside US a 0.896 0.632, 0.953 0.015 1.014 0.826, 1.291 0.776 1.078 0.998, 1.413 0.051 Violent Crime Index 1.016 0.966, 1.112 0.314 0.997 0.923, 1.072 0.877 0.992 0.891, 1.084 0.723 Cost of Living Index 0.933 0.724, 1.005 0.056 1.084 1.002, 1.445 0.048 0.993 0.849, 1.14 0.823 Air Quality Index 0.466 0.006, 5.105 0.309 1.576 0.085, 95.94 0.56 1.848 0.17, 99.31 0.384 Education b 1.243 1.143, 2.382 0.008 0.795 0.37, 0.942 0.027 0.949 0.638, 1.227 0.467 Poverty Level c 0.973 0.767, 1.151 0.543 0.942 0.7, 1.084 0.213 1.084 0.984, 1.469 0.071 Unemployment Rate 0.754 0.276, 0.986 0.045 1.143 0.746, 2.483 0.314 1.114 0.755, 2.178 0.357 Median Income 1.007 0.951, 1.089 0.633 0.96 0.838, 0.991 0.028 1.018 0.977, 1.112 0.208 Table 3 Impact of individual socioeconomic determinants and comorbidities on incidental lung nodule size Nodule < 6 mm Nodule 6–8 mm Nodule > 8 mm Variable Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Demographics Age 0.985 0.918, 1.014 0.073 0.987 0.92, 1.023 0.278 1.024 1.007, 1.107 0.027 Male 0.569 0.076, 1.099 0.056 1.965 1.089, 20.65 0.038 0.965 0.266, 3.192 0.898 Nonwhite race 0.668 0.062, 2.523 0.327 0.899 0.099, 6.209 0.817 1.627 0.469, 20.05 0.242 SES Determinants Undocumented 1.185 0.843, 20.61 0.771 0.795 0.028, 12.62 0.735 1.026 0.095, 11.89 0.962 Noncommercial Insurance 0.437 0.035, 0.637 0.01 1.03 0.208. Discussion Social determinants of health have a significant impact on diverse health outcomes, including malignancy. There is emerging awareness of socioeconomic status Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 5 of 10 Table 3 Impact of individual socioeconomic determinants and comorbidities on incidental lung nodule size Nodule < 6 mm Nodule 6–8 mm Nodule > 8 mm Variable Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Demographics Age 0.985 0.918, 1.014 0.073 0.987 0.92, 1.023 0.278 1.024 1.007, 1.107 0.027 Male 0.569 0.076, 1.099 0.056 1.965 1.089, 20.65 0.038 0.965 0.266, 3.192 0.898 Nonwhite race 0.668 0.062, 2.523 0.327 0.899 0.099, 6.209 0.817 1.627 0.469, 20.05 0.242 SES Determinants Undocumented 1.185 0.843, 20.61 0.771 0.795 0.028, 12.62 0.735 1.026 0.095, 11.89 0.962 Noncommercial Insurance 0.437 0.035, 0.637 0.01 1.03 0.208. 5.495 0.935 2.181 1.544, 2.818 0.016 Comorbidities Tobacco Use 0.971 0.196, 4.446 0.932 1.129 0.221, 7.907 0.76 0.95 0.204, 3.873 0.875 Diabetes 1.113 0.274, 5.998 0.754 0.962 0.159, 5.248 0.92 0.94 0.21, 3.59 0.845 Hypertension 1.32 0.365, 9.863 0.447 0.667 0.066, 2.344 0.306 1.051 0.244, 5.14 0.884 Asthma 0.952 0.208, 3.828 0.878 1.3 0.372, 9.016 0.457 0.867 0.185, 2.805 0.635 COPD 1.107 0.288, 5.534 0.756 0.902 0.153, 4.055 0.776 0.981 0.246, 3.724 0.95 HIV 1.474 0.179, 33.34 0.503 0.546 0.007, 9.099 0.448 1.004 0.078, 13.12 0.994 Table 4 Impact of socioeconomic determinants at the geographic level on incidental lung nodule size a Birthplace outside the US is defined by the percentage of individuals with a place of birth listed as foreign according to the United States 2020 Census data b Education was defined by the percentage of individuals with less than a high school level or equivalent education c Poverty level was defined by the percentage of individuals living below the federal poverty level. Discussion 5.495 0.935 2.181 1.544, 2.818 0.016 Comorbidities Tobacco Use 0.971 0.196, 4.446 0.932 1.129 0.221, 7.907 0.76 0.95 0.204, 3.873 0.875 Diabetes 1.113 0.274, 5.998 0.754 0.962 0.159, 5.248 0.92 0.94 0.21, 3.59 0.845 Hypertension 1.32 0.365, 9.863 0.447 0.667 0.066, 2.344 0.306 1.051 0.244, 5.14 0.884 Asthma 0.952 0.208, 3.828 0.878 1.3 0.372, 9.016 0.457 0.867 0.185, 2.805 0.635 COPD 1.107 0.288, 5.534 0.756 0.902 0.153, 4.055 0.776 0.981 0.246, 3.724 0.95 HIV 1.474 0.179, 33.34 0.503 0.546 0.007, 9.099 0.448 1.004 0.078, 13.12 0.994 Table 3 Impact of individual socioeconomic determinants and comorbidities on incidental lung nodule size Table 4 Impact of socioeconomic determinants at the geographic level on incidental lung nodule size a Birthplace outside the US is defined by the percentage of individuals with a place of birth listed as foreign according to the United States 2020 Census data b Education was defined by the percentage of individuals with less than a high school level or equivalent education c Poverty level was defined by the percentage of individuals living below the federal poverty level. Odds ratios were adjusted for covariates including age, smoking status, history of COPD and sex (with male as the reference) Nodule < 6 mm Nodule 6–8 mm Nodule > 8 mm Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Odds Ratio 95% Confidence Interval p value Birthplace outside US a 0.896 0.632, 0.953 0.015 1.014 0.826, 1.291 0.776 1.078 0.998, 1.413 0.051 Violent Crime Index 1.016 0.966, 1.112 0.314 0.997 0.923, 1.072 0.877 0.992 0.891, 1.084 0.723 Cost of Living Index 0.933 0.724, 1.005 0.056 1.084 1.002, 1.445 0.048 0.993 0.849, 1.14 0.823 Air Quality Index 0.466 0.006, 5.105 0.309 1.576 0.085, 95.94 0.56 1.848 0.17, 99.31 0.384 Education b 1.243 1.143, 2.382 0.008 0.795 0.37, 0.942 0.027 0.949 0.638, 1.227 0.467 Poverty Level c 0.973 0.767, 1.151 0.543 0.942 0.7, 1.084 0.213 1.084 0.984, 1.469 0.071 Unemployment Rate 0.754 0.276, 0.986 0.045 1.143 0.746, 2.483 0.314 1.114 0.755, 2.178 0.357 Median Income 1.007 0.951, 1.089 0.633 0.96 0.838, 0.991 0.028 1.018 0.977, 1.112 0.208 Education was defined by the percentage of individuals with less than a high school level or equivalent education c Poverty level was defined by the percentage of individuals living below the federal poverty level. Discussion While numerous studies have evaluated socioeconomic disparities among individuals enrolled in or eligible for LCS programs, there is a paucity of literature on the clini- cal impact of SES on incidental lung nodule evaluation and follow up [11]. Lower SES is associated with lower LCS adherence, utilization, and guideline concordant care, yet to date, there is minimal data on the impact of SES on ILNs. In the DELUGE study, which prospectively evaluated both LCS and ILN surveillance, individuals from the lung nodule surveillance program were more likely to be diagnosed with lung cancer [10]. Other stud- ies have highlighted the advantages of combined LCS and ILN programs [9, 10, 12, 22] to capture higher-risk groups, which leads to improved early lung cancer diag- nosis and guideline concordant care. However, SES fac- tors in these studies were not consistently reported, and these studies included mostly individuals of White race, which made it challenging to evaluate the effect of the programs when accounting for SES, race, and ethnic inequalities [23]. In our study, we aimed to elucidate the influence of SES on incidental lung nodule characteris- tics. We performed a retrospective analysis of all indi- viduals ≥ 18 years of age with incidental lung nodules on CT chest who were referred to the Pulmonary clinic at an urban, safety net hospital in an underserved area of Brooklyn, New York over a 3-year period. Such hospitals are an essential safety net serving a socioeconomically diverse population, wherein individuals are typically less likely to be included in incidental lung nodule programs or participate in clinical trials [16]. Specifically, we aimed to evaluate the association between incidental nodule size and SES status using common determinants such as education, insurance type and income, as well as less commonly used indicators such as air quality index, cost of living index and immigration status. As mentioned above, there are limited studies evalu- ating the relationship between SES and ILNs. However, several studies have examined the relationship between poverty and lung cancer outcomes, finding that areas of higher deprivation were associated with higher lung cancer incidence and mortality [18, 24]. These subpar outcomes are likely related to high-risk smoking behav- iors and less access to healthcare compared to those with higher SES [25, 26]. Discussion Odds ratios were adjusted for covariates including age, smoking status, history of COPD and sex (with male as the reference) and socioeconomic deprivation as a primary risk fac- tor for both incidence and adverse outcomes in lung cancer [16–18]. SES is comprised of a broad, hetero- geneous yet crucial set of factors used to measure an individual’s social and economic standing, built on parameters such as income, education level, insurance carrier, and geographic location [19, 20]. While there is a gap in literature on studies evaluating the impact of SES on ILN outcomes, robust evidence suggests that individuals with lower SES present with more advanced-stage lung cancer at diagnosis, attenuated response to chemotherapy and obtain less favorable prognosis after diagnosis [16, 18]. There is also a lack of trials evaluating outcomes of ILN programs. How- ever, large trials that investigated lung cancer screen- ing, e.g., the National Lung Screening Trial (NLST) and the Dutch-Belgian lung cancer screening [Ned- erlands–Leuvens Longkanker Screenings Onderzoek (NELSON)] trial, demonstrated a significant mortal- ity reduction of 20–26% [5–7]. Notably, sociodemo- graphic inequalities were present in these trials, and thus there are limitations in generalizing the results Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 6 of 10 to diverse populations with respect to ethnicity, race and income level. Participants in NSLT were pre- dominantly of white race (91%) and had higher edu- cational status (32% with college degrees) and income compared to the general population matched to age and smoking criteria [21]. Therefore, it is uncertain whether the benefits seen in these trials are applicable to those with lower SES. we found that individuals from areas with lower unem- ployment rates presented with CT scans demonstrating smaller nodules < 6 mm (OR 0.754, p 0.045), while those from areas with higher unemployment rates were pre- sented with larger nodules (OR 1.1, p < 0.357). Patients with noncommercial insurance were more likely to pre- sent with larger nodules > 8 mm (OR 2.181, p 0.016) on first presentation and less likely to have smaller nod- ules < 6 mm (OR 0.437, p 0.01). These findings highlight that even with incidental nodules, markers of poverty are associated with larger, inherently higher-risk nodules at initial presentation. Discussion Additionally, individuals with lower SES may be more likely to be exposed to secondhand smoke and other environmental toxins that increase the risk of lung cancer [16]. There is growing evidence evalu- ating the impact of insurance type and lung cancer out- comes. A recent study evaluating ILN surveillance found that underinsured individuals were more likely to be diagnosed with cancer. Individuals with federal insurance are also less likely to complete screening [17, 27] and may not receive full coverage for LDCT. Importantly, patients presenting with ILNs are less likely to be eligible for LCS. It is not yet known if SES disparities in LCS eligibility result in more patients with lower SES presenting with ILNs, rather than participating in LCS programs. This is a notable consideration, since patients with ILNs appear to be at a higher risk for ulti- mately being diagnosed with lung cancer [14]. Dispari- ties in eligibility for LCS due to race have been described [16, 18], but a significant knowledge gap still exists with respect to this relationship between SES and ILNs. There was a period of 11 months for the Centers for Medicare and Medicaid Services (CMS) to expand LCS eligibility criteria (February 2022) to reflect the USPSTF eligibility update (March 2021) [3, 28]. Consequently, most indi- viduals in our study period would not have benefited from the expanded criteria. CMS limits age to 77 years in determining LCS eligibility [28], although most guidelines recommend continued screening up to 80 years old [5–7]. Age is a well-established risk factor for lung cancer [2]. Our study showed that increased age was weakly associ- ated with larger nodules > 8 mm, although with a small effect size. Taken together, these findings suggest that older individuals with federal insurance may be ineligible Socioeconomic status and nodule size Th f d l The impact of SES on nodule size was assessed with logis- tic regression analysis using individual and geographical determinants. Categorization of SES by geographic area has gained traction in recent years with the development of indices such as the area-based deprivation index (ADI) [20]. Geography-based determinants reflect the commu- nity SES, while individual determinants are specific to the patient. After adjusting for age, sex, and tobacco use, Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 7 of 10 for LCS. These limitations may create additional barriers for LCS in individuals with low SES [16, 17]. for LCS. These limitations may create additional barriers for LCS in individuals with low SES [16, 17]. higher (which compromised a significant percentage of NLST participants) [16]. Our assessment was limited by including only one measure of educational attainment, having no comparison group with those of higher attain- ment, and a small sample size. The association between education and ILNs should be further explored, as indi- viduals with ILNs represent a distinct population from those in LCS programs and are less likely to be eligible for LCS. We also evaluated less commonly studied markers of SES, such as the air quality index and cost of living index (COLI). COLI is a relative marker of living expenses com- pared to United States estimates, with values above 100 conferring higher than average costs of living. COLI has not been extensively studied in relation to ILN outcomes; however, prior studies in individuals with hepatocellu- lar cancer found that those with lower COLI presented with more advanced cancers, while higher COLI was associated with improved survival [29]. We found a weak association between individuals from lower COLI areas and smaller nodules < 6 mm (OR 0.933, p 0.056), while higher COLI areas were weakly associated with nodules between 6–8 mm (OR 1.084, p 0.048), the significance of which is undetermined. Individuals with lower SES may be more likely to reside in lower COLI areas; however, New York City (NYC) on average has the highest COLI in the United States, and all individuals in this study resided in areas with a COLI > 100. Studies suggest that increased living costs may compete with other financial burdens, which may disproportionately affect individuals with lower SES, leading to less guideline concordant care [5]. Race/ethnicity and nodule sizeh The distribution of race and ethnicity in our sample was diverse, with mostly nonwhite Hispanic individuals (56%) followed by African American (26.5%) and White (15.3%) individuals. This significantly differs from the NLST, which comprised 91% White individuals [5, 6]. Our study found that nonwhite individuals were 0.67 times less likely to have smaller nodules < 6 mm and 1.63 times more likely to have nodules > 8 mm, although these rela- tionships were not statistically significant. It is crucial to examine the intersection of socioeconomic status (SES) and racial disparities in lung cancer outcomes, given its strong correlation. Although there is limited sociodemo- graphic data on ILN programs in the current literature to date, studies have shown that individuals belonging to racial and ethnic minorities have the lowest SES, result- ing in lower LCS utilization [16, 34]. However, there is a lack of research on how the combination of low SES and minority racial status impacts ILN surveillance, LCS eli- gibility, utilization, or outcomes. Other studies found that African American participants had a lower screening rate than White participants, and unscreened individuals had a lower annual household income [34]. This suggests that African American individuals with low annual household income may have an even lower screening rate. Addi- tionally, our study showed that when adjusting for race/ ethnicity, SES determinants demonstrated stronger asso- ciations with ILN size. Other studies have shown that lower SES and ethnic-minority groups have significantly lower overall lung cancer patient survival rates [2, 17]. These findings suggest that SES represents an important driver of ILN size and ultimately lung cancer risk. The air quality index is a measure of air pollution, standardized based on the Clean Air Act [30]. Metropoli- tan areas such as NYC are required to report air quality daily. In our study, there were no associations with nod- ule size and air quality index, and all individuals resided in areas with satisfactory air quality. The association of the air quality index and ILN size has not been explored, but previous studies have demonstrated significant asso- ciations with a higher air quality index and increased lung cancer incidence [31]. While no relationship was present in our study, it will be interesting to assess how SES and air quality impact ILN size in areas with more pollution. Education level has been assessed as a risk factor for worse lung cancer outcomes. Socioeconomic status and nodule size Th f d l Larger studies utilizing the cost of living index may help further define these relationships in individuals with lung nodules. Race/ethnicity and nodule sizeh Health literacy may be lower in individuals with lower educational attainment [32], and these individuals may face more barriers to screening despite being at higher risk [33]. Our study showed that individuals from areas with educational attainment less than high school were more likely to have smaller nodules (OR 1.243, p < 0.008). There was no asso- ciation between education and nodules > 8 mm. There are limited studies evaluating the relationship between educational status and ILNs, however prior population studies have found that educational attainment lower than high school level was associated with decreased LCS eligibility, in contrast to those with college education or Limitations Our study has several limitations. As with all retrospec- tive studies, our data reveal associations but does not provide evidence for causation. Several SES determi- nants, such as income and poverty level, did not have significant relationships with lung nodule size. This may be due to the overall high rate of deprivation in the popu- lation served by the safety-net hospital, making it more difficult to find significant relationships, since most areas may be similarly deprived. Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Page 8 of 10 Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 As a community hospital located in an area facing a shortage of healthcare professionals during the COVID- 19 pandemic, our center did not have an established for- mal surveillance program for LCS or ILNs during the study period. We included individuals with ILNs who were referred to the pulmonary service for any indica- tion. However, primary care providers in the community may manage ILNs without referring to the pulmonary service. Since our study only included patients referred to the pulmonary services, patients with ILNs managed by PCPs were excluded, which reduced the power of the study. It is conceivable that variability in the medical and socioeconomic history of these excluded patients, com- pared to those referred to the pulmonary service, could have influenced the findings. were more likely to have smaller nodules at presentation. While the association of SES with LCS has been heavily explored, these data suggest that SES also impacts evalu- ation of incidental lung nodules, filling a key knowledge gap in the present literature. The cost of living index and air quality were also investigated as less commonly used determinants of SES. Further studies with these indices may help delineate their relationships with ILN size. Opeyemi Aroyewun p y y Pulmonary and Critical Care Medicine, University of Texas at Austin, Austin, TX, USA Pulmonary and Critical Care Medicine, University of Texas at Austin, Austin, TX, USA Alberto Martinez Alberto Martinez Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY, 11206, USA Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY, 11206, USA Haris Asif Haris Asif Department of Medicine, West Virginia University, Morgantown, WV, USA Prama Rashmi Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY, 11206, USA Acknowledgements The authors would like to thank the Department of Medicine at NYC Health + Hospitals/Woodhull for support and encouragement for publication of this research. Additionally, geographical markers of socioeconomic status were derived from census data according to zip code. These markers do not reflect the individual socio- economic status of patients but rather reflect the depriva- tion faced by their neighborhoods. The individual patient may have markers of higher or lower SES compared to their neighborhood. Ifediba Nwachukwu Ifediba Nwachukwu Pulmonary and Critical Care Medicine, University of Oklahoma, Oklahoma City, USA Ifediba Nwachukwu Pulmonary and Critical Care Medicine, University of Oklahoma, Oklahoma City, USA In conclusion, high-risk individuals presenting with ILNs represent a distinct but complementary at-risk population for lung cancer to those being screened, and combined nodule surveillance and LCS programs lead to improved guideline concordant care. In our unique cohort of patients in a socioeconomically deprived area without a formal LCS program, we found that individu- als with lower SES defined by geographical and individual determinants present with larger incidental nodules. This finding confers higher risk to such patients undergo- ing lung cancer evaluation and underscores the inequity that exists for patients with socioeconomic disadvan- tage. Individuals with markers of higher SES, in contrast, Limitations Abbreviations ADI Area Deprivation Index COPD Chronic obstructive pulmonary disease HIV Human immunodeficiency virus COVID-19 Coronavirus disease 2019 SES Socioeconomic status IRB Institutional review board OR Odds ratio NLST National lung cancer screening trial NELSON Nederlands–Leuvens Longkanker Screenings Onderzoek CT Computed Tomography LDCT Low-dose computed tomography ADI Area Deprivation Index US United States USPSTF United States Preventive Services Task Force ILN Incidental lung nodule LCS Lung cancer screening CMS Centers for Medicare and Medicaid Services NYC New York City NYCHHC New York City Health and Hospitals STROBE Strengthening the reporting of observational studies in epidemiology li Conversely, our study included many patients with small, lower risk ILNs. This distinguishes our study from others that include patients from formal LCS or ILN pro- grams, which generally include nodules at least 6 mm in size. Nonetheless, our sample represents a socially diverse at-risk group that is typically excluded from research studies. A complete case analysis approach was utilized, which resulted in a significant number of patients with nodules being excluded due to missing nodule size or incomplete description. It is unlikely this nodule data were missing at random. It is possible that radiologists were less likely to comment on size for small, less clinically significant nod- ules. Since nodule size was an outcome of interest, and the nodule data were unlikely to be missing at random, the complete case analysis is biased. Olva Bess Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY, 11206, USA Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY, 11206, USA Haris Asif Department of Medicine, West Virginia University, Morgantown, WV, USA Authors’ contributions Conceptualization: SK, MF, CM, BA. Data curation: MF, CM, BA, AG, SB, WRRT. Formal analysis: BA, AD, MF. Funding acquisition: Methodology: SK, MF, BA, AD. Project administration: SK, CM, MF. Supervision: SK. Writing of main manu- script- original draft: MF, SK, CM. Writing, reviewing, and editing manuscripts: MF, SK, CM. All authors read and approved the final manuscript. Page 9 of 10 Fernandes et al. BMC Pulmonary Medicine (2023) 23:469 Availability of data and materials fi Statement. Am J Respir Crit Care Med. 2023;207(6):e31–46. 12. LeMense GP, Waller EA, Campbell C, Bowen T. Development and out- comes of a comprehensive multidisciplinary incidental lung nodule and lung cancer screening program. BMC Pulm Med. 2020;20(1):115. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 13. Brown, D. Metropolitan Anchor Hospital (MAH) Case Study. American Hospital Association. June 2022. Accessed 05/24/2023. https://www.aha. org/case-studies/2022-11-22-nyc-health-hospitals-new-york Funding None. 11. Steiling K, Kathuria H, Echieh CP, Ost DE, Rivera MP, Begnaud A, et al. Research Priorities for Interventions to Address Health Disparities in Lung Nodule Management: An Official American Thoracic Society Research Statement. Am J Respir Crit Care Med. 2023;207(6):e31–46. Author details 1 1 Pulmonary and Critical Care Medicine, Lenox Hill Hospital, Northwell Health, New York, USA. 2 Department of Medicine, NYC Health + Hospitals/Woodhull, New York City Health and Hospitals, 760 Broadway, Brooklyn, NY 11206, USA. 3 Division of Nephrology, SUNY Downstate/Health Sciences Center at Brook- lyn, NY, Brooklyn, USA. 4 Research and Administration, New York City Health and Hospitals, NY, New York, USA. 5 Pulmonary and Critical Care Medicine, New York University Grossman School of Medicine, NY, New York, USA. 19. Kaplan GA. Social Determinants of Health, 2nd Edition. M Marmot and R Wilkinson (eds). Oxford: Oxford University Press, 2006, pp. 376, $57.50. ISBN: 9780198565895. International Journal of Epidemiology. 2006;35(4):1111–2. 20. Sanderson M, Aldrich MC, Levine RS, Kilbourne B, Cai Q, Blot WJ. Neigh- bourhood deprivation and lung cancer risk: a nested case-control study in the USA. BMJ Open. 2018;8(9):e021059. Received: 10 August 2023 Accepted: 20 October 2023 Received: 10 August 2023 Accepted: 20 October 2023 21. National Lung Screening Trial Research T, Aberle DR, Adams AM, Berg CD, Clapp JD, Clingan KL, et al. Baseline characteristics of participants in the randomized national lung screening trial. J Natl Cancer Inst. 2010;102(23):1771–9. Consent for publication 17. Rivera MP, Katki HA, Tanner NT, Triplette M, Sakoda LC, Wiener RS, et al. Addressing Disparities in Lung Cancer Screening Eligibility and Health- care Access. An Official American Thoracic Society Statement. Am J Respir Crit Care Med. 2020;202(7):e95–112. The authors declare no competing interests. 18. Redondo-Sánchez D, Petrova D, Rodríguez-Barranco M, Fernández- Navarro P, Jiménez-Moleón JJ, Sánchez M-J. Socio-Economic Inequalities in Lung Cancer Outcomes: An Overview of Systematic Reviews. Cancers. 2022;14(2):398. Ethics approval and consent to participate 14. 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https://openalex.org/W4378468735	https://www.mdpi.com/1424-8220/23/11/5071/pdf?version=1685009586	English	null	Enhanced Readout from Spatial Interference Fringes in a Point-Source Cold Atom Inertial Sensor	Sensors	2,023	cc-by	9,615	Citation: Wang, J.; Tong, J.; Xie, W.; Wang, Z.; Feng, Y.; Wang, X. Enhanced Readout from Spatial Interference Fringes in a Point-Source Cold Atom Inertial Sensor. Sensors 2023, 23, 5071. https://doi.org/ 10.3390/s23115071 Communication Enhanced Readout from Spatial Interference Fringes in a Point-Source Cold Atom Inertial Sensor Jing Wang, Junze Tong, Wenbin Xie, Ziqian Wang, Yafei Feng and Xiaolong Wang * Key Laboratory of Quantum Precision Measurement of Zhejiang Province, College of Science, Zhejiang University of Technology, Hangzhou 310023, China; 1112002006@zjut.edu.cn (J.W.); 2112009035@zjut.edu.cn (J.T.); 2112109113@zjut.edu.cn (W.X.); 221122090101@zjut.edu.cn (Z.W.); 2112109050@zjut.edu.cn (Y.F.) * Correspondence: xlwang@zjut.edu.cn Abstract: When the initial size of an atom cloud in a cold atom interferometer is negligible compared to its size after free expansion, the interferometer is approximated to a point-source interferometer and is sensitive to rotational movements by introducing an additional phase shear in the interference sequence. This sensitivity on rotation enables a vertical atom-fountain interferometer to measure angular velocity in addition to gravitational acceleration, which it is conventionally used to measure. The accuracy and precision of the angular velocity measurement depends on proper extraction of frequency and phase from spatial interference patterns detected via the imaging of the atom cloud, which is usually affected by various systematic biases and noise. To improve the measurement, a pre-ﬁtting process based on principal component analysis is applied to the recorded raw images. The contrast of interference patterns are enhanced by 7–12 dB when the processing is present, which leads to an enhancement in the precision of angular velocity measurements from 6.3 µrad/s to 3.3 µrad/s. This technique is applicable in various instruments that involve precise extraction of frequency and phase from a spatial interference pattern. Keywords: atom interferometry; principal component analysis; cold atom inertial sensor sensors sensors sensors 1. Introduction Analogous to optical interference, wave-particle duality allows for interference of microscopic particles. Unlike neutrons and electrons, atoms possess distinctive internal energy-level structures and properties such as discrete mass, magnetic moments, and po- larizations, making atomic interference capable of revealing information on physical ﬁelds in detail. Atomic interferometry holds signiﬁcant importance in the domain of precision measurements. Additionally, atom interferometers have been successfully utilized for measuring gravitational acceleration [1,2], ﬁne structure constant [3], angular velocity [4,5], etc., and can be used to verify the equivalence principle and general relativity [6]. In recent years, they have been proposed for applications in gravitational wave detection for better precision than long-path optical interferometers [7]. Academic Editors: Yutaka Shikano and Masazumi Fujiwara Academic Editors: Yutaka Shikano and Masazumi Fujiwara Received: 17 April 2023 Revised: 18 May 2023 Accepted: 24 May 2023 Published: 25 May 2023 Received: 17 April 2023 Revised: 18 May 2023 Accepted: 24 May 2023 Published: 25 May 2023 An atom point-source interferometer (APSI) is a new type of atomic inertial sensor capable of measuring both gravitational acceleration and angular velocity [8]. These instru- ments read the interference phase and contrast in a single measurement cycle, avoiding the need for compensating for velocity-dependent phase shifts. This feature helps to improve the sampling rate and signal-to-noise ratio. For the past decade, APSIs have been applied in atomic gyroscopes [9], gravimeters [10], and gravity gradiometers [11]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Following the point-source model [12], the spatial position and velocity of an atom in a cloud of ultra-cold atoms are coupled after the cloud is tightly focused. Therefore, the velocity-dependent phase is directly reﬂected in the spatial distribution of an atom cloud after free expansion in vacuum, and phase information of APSIs can be obtained by detecting the phase of spatial interference fringes. https://www.mdpi.com/journal/sensors Sensors 2023, 23, 5071. https://doi.org/10.3390/s23115071 2 of 10 Sensors 2023, 23, 5071 To accurately extract the spatial frequency of the interference patterns of an atom cloud in such an interferometer, we have applied speciﬁc methods based on principal component analysis (PCA) [13] in processing raw atomic ﬂuorescence images. This technique effectively ﬁlters out inﬂuences of atomic envelops, stray background light, and other detection noises. The signal-to-noise ratio of the spatial fringes are improved by 5–12 dB, and the fringe contrast is also signiﬁcantly improved. Compared with direct ﬁtting of the fringes from a raw image, the measurement precision of spatial periods is improved by one order of magnitude, leading to about twice-higher precision of angular velocity measurements out of the same atom interferometer. This technique can be combined with a variety of techniques to obtain an increase in measurement performance, for example with LMT for higher sensitivity [14]. 2. Theoretical Methods 2.1. Phase Shear of Atom Point Source Interferometry 2.1. Phase Shear of Atom Point Source Interferometry In an atom interferometer, the interference phase shift rising from the gravitational acceleration g and angular velocity of Earth rotation Ωare expressed as: ∆Φg = −ke f f ·gT2, (1) ∆ΦΩ= 2ke f f ·(Ω× v)T2, (2) ∆Φg = −ke f f ·gT2, (1) (1) ∆ΦΩ= 2ke f f ·(Ω× v)T2, (2) (2) where ke f f is the effective wave-vector of stimulated Raman transition, T is the time interval between Raman pulses, and v is initial velocity of the atom under investigation. In point-source approximation, the atom cloud in the interferometer is focused to a size much smaller compared with its ﬁnal size after free expansion. When the initial cloud before expansion is regarded as a volume-less point with a certain velocity distribution, the inﬂuence of an atom’s initial position on its ﬁnal displacement is neglected and its displacement r = vt is proportional to its initial velocity v during the evolution time t of free expansion. p The displacement–velocity coupling in the point-source model makes the APSI sensi- tive to rotations, even in a single axis interferometer that is conventionally used in grav- itational acceleration measurements. Here is demonstrated the measurement of angular velocity Ωx along the horizontal x-axis as an example. The velocity of the atom cloud along the y-axis is denoted vy, so its displacement at moment t along the y-axis can be expressed as y = vytd according to the point-source model. The spatial fringe frequency caused by mirror rotation can be described by phase gradient κx = ∂∆ΦΩ/∂y = 2ke f f ΩxT2/td, where td is the free expansion time of the atom cloud. The angular velocity is thus obtained by measuring the spatial fringe frequency, which makes the principle of the APSI gyroscope. The APSI works in a way similar to its optical counterpart of a shear interferometer. It is the phase shear added to the interferometer that reveals rich details in the total phase shift in an APSI. As is shown in Figure 1, two types of phase shear can be added in an atom interferometer with a π/2 −π −π/2 Raman pulse conﬁguration, namely the horizontal beam-tilt phase shear ΦH and the vertical timing-asymmetry phase shear ΦV. The horizontal phase shear at a speciﬁc displacement y3, mathematically expressed as ΦH = ke f f δθxy3, is introduced by applying a tilt δθx to the ﬁnal π/2 pulse. When point-source approxi 2.2. Principal Component Analysis rivatives of horizontal and vertical phases can be calculated, and corresponding spatial frequencies of interference fringes are calculated to be: 𝜅𝐻= 𝜕Φ𝐻 𝜕𝑦= 𝑘𝑒𝑓𝑓𝛿𝜃𝑡3 𝑡𝑑 ⁄ , (3) 𝜅𝑉= 𝜕Φ𝑉 𝜕𝑧= 𝑘𝑒𝑓𝑓𝛿𝑇𝑡𝑑 ⁄ , (4) h d h i i h h 3 d R l i li d i f h In actual measurements with an ASPI-type inertial sensor, extraction of interference patterns from a recorded image is itself interfered with by various biases and noises. These extra factors include the envelope the atoms form after free expansion, uneven spatial distribution of detection beams, stray background light, thermal noises in the image sensor, etc. To accurately derive the interference fringes from such an image, an image processing technique based on principal component analysis (PCA) was applied prior to fringe extraction. where 𝑡3 denotes the timing when the 3rd Raman pulse is applied, counting from the fountain launch. 2.2. Principal Component Analysis In actual measurements with an ASPI-type inertial sensor, extraction of interference patterns from a recorded image is itself interfered with by various biases and noises. These extra factors include the envelope the atoms form after free expansion, uneven spatial dis- tribution of detection beams, stray background light, thermal noises in the image sensor, etc. To accurately derive the interference fringes from such an image, an image processing technique based on principal component analysis (PCA) was applied prior to fringe ex- traction. The PCA algorithm is a classic data processing method that is commonly used in g The PCA algorithm is a classic data processing method that is commonly used in dimensionality reduction and feature extraction of multidimensional data sets [15]. With this technique, the recorded raw image containing both the interference patterns and disturbances is considered as the superposition of a low rank and a sparse sub-set [16]. The main task of PCA is to derive a new set of variables of the reduced variable dimensions from the original image. In this way, a series of basis vectors which are known as principal components (PC) are obtained. The PCs are orthogonal to each other and span the original set of multi-pixel images. All recorded images can then be broken down to a sum of projections of the original image onto each PC [17]. These PC basis vectors are ranked in descending order according to the relevance between themselves and the original image. 2.1. Phase Shear of Atom Point Source Interferometry The two separate branches induce horizontal beam-tilt phase shear ΦH and vertical timing-asymmetry phase shear ΦV. Red and blue spheres represent the atom cloud in a ground state \| g⟩and an excited state \| e⟩, respectively. 2.1. Phase Shear of Atom Point Source Interferometry The vertical timing-asymmetry phase shear along the vertical (z) direction ΦV = ke f f vzδT is added by manipulating the asymmetrical timing interval of interferometer pulses, where vz is the velocity of the atom along the z-axis and δT is the difference of the 2 intervals in between 3 Raman pulses. The displacement–velocity coupling in the point-source model makes the APSI sensi- tive to rotations, even in a single axis interferometer that is conventionally used in grav- itational acceleration measurements. Here is demonstrated the measurement of angular velocity Ωx along the horizontal x-axis as an example. The velocity of the atom cloud along the y-axis is denoted vy, so its displacement at moment t along the y-axis can be expressed as y = vytd according to the point-source model. The spatial fringe frequency caused by mirror rotation can be described by phase gradient κx = ∂∆ΦΩ/∂y = 2ke f f ΩxT2/td, where td is the free expansion time of the atom cloud. The angular velocity is thus obtained by measuring the spatial fringe frequency, which makes the principle of the APSI gyroscope. The APSI works in a way similar to its optical counterpart of a shear interferometer. It is the phase shear added to the interferometer that reveals rich details in the total phase shift in an APSI. As is shown in Figure 1, two types of phase shear can be added in an atom interferometer with a π/2 −π −π/2 Raman pulse conﬁguration, namely the horizontal beam-tilt phase shear ΦH and the vertical timing-asymmetry phase shear ΦV. The horizontal phase shear at a speciﬁc displacement y3, mathematically expressed as ΦH = ke f f δθxy3, is introduced by applying a tilt δθx to the ﬁnal π/2 pulse. The vertical timing-asymmetry phase shear along the vertical (z) direction ΦV = ke f f vzδT is added by manipulating the asymmetrical timing interval of interferometer pulses, where vz is the velocity of the atom along the z-axis and δT is the difference of the 2 intervals in between 3 Raman pulses. 2.1. Phase Shear of Atom Point Source Interferometry When point-source approximation (y ≈vytd ≈y3td/t3, z = vztd) is applied, the derivatives of horizontal and vertical phases can be calculated, and corresponding spatial frequencies of interference fringes are calculated to be: κH = ∂ΦH ∂y = ke f f δθ t3/td, (3) κH = ∂ΦH ∂y = ke f f δθ t3/td, (3) Sensors 2023, 23, 5071 3 of 10 κV = ∂ΦV ∂z = ke f f δT/td, (4) (4) where t3 denotes the timing when the 3rd Raman pulse is applied, counting from the fountain launch. IEW 3 of 10 where t3 denotes the timing when the 3rd Raman pulse is applied, counting from the fountain launch. IEW 3 of 10 Figure 1. Schematic diagram of phase shears based on interference sequence of 𝜋/2 −𝜋−𝜋/2 Ra- man pulses. The two separate branches induce horizontal beam-tilt phase shear Φ𝐻 and vertical timing-asymmetry phase shear Φ𝑉. Red and blue spheres represent the atom cloud in a ground state \|𝑔⟩ and an excited state \|𝑒⟩, respectively. Figure 1. Schematic diagram of phase shears based on interference sequence of π/2 −π −π/2 Raman pulses. The two separate branches induce horizontal beam-tilt phase shear ΦH and vertical timing-asymmetry phase shear ΦV. Red and blue spheres represent the atom cloud in a ground state \| g⟩and an excited state \| e⟩, respectively. Figure 1. Schematic diagram of phase shears based on interference sequence of 𝜋/2 −𝜋−𝜋/2 Ra- man pulses. The two separate branches induce horizontal beam-tilt phase shear Φ𝐻 and vertical timing-asymmetry phase shear Φ𝑉. Red and blue spheres represent the atom cloud in a ground state \|𝑔⟩ and an excited state \|𝑒⟩, respectively. Figure 1. Schematic diagram of phase shears based on interference sequence of π/2 −π −π/2 Raman pulses. The two separate branches induce horizontal beam-tilt phase shear ΦH and vertical timing-asymmetry phase shear ΦV. Red and blue spheres represent the atom cloud in a ground state \| g⟩and an excited state \| e⟩, respectively. Figure 1. Schematic diagram of phase shears based on interference sequence of 𝜋/2 −𝜋−𝜋/2 Ra- man pulses. The two separate branches induce horizontal beam-tilt phase shear Φ𝐻 and vertical timing-asymmetry phase shear Φ𝑉. Red and blue spheres represent the atom cloud in a ground state \|𝑔⟩ and an excited state \|𝑒⟩, respectively. Figure 1. Schematic diagram of phase shears based on interference sequence of π/2 −π −π/2 Raman pulses. 3. Experiment and Methods 3. Experiment and Methods When the loading process is finished, the magnetic fields of both 2-D and 3-D MOTs are switched off and atoms are then launched into the interferometer chamber vertically using the moving molasses technique. The 2 pairs of upward and downward cooling laser beams are differentially detuned by ∆𝜈= 3.3 MHz, so that atoms in the MOT region are subject to an upward force along the vertical axis. The atoms are accelerated to 3.63 m/s in about 1.3 ms, enabling them to reach a height of 0.67 m in the fountain. When the loading process is ﬁnished, the magnetic ﬁelds of both 2-D and 3-D MOTs are switched off and atoms are then launched into the interferometer chamber vertically using the moving molasses technique. The 2 pairs of upward and downward cooling laser beams are differentially detuned by ∆ν = 3.3 MHz, so that atoms in the MOT region are subject to an upward force along the vertical axis. The atoms are accelerated to 3.63 m/s in about 1.3 ms, enabling them to reach a height of 0.67 m in the fountain. The atoms are further cooled down when they are in ballistic launch using the polar- ization gradient cooling technique, where cooling laser beams are detuned far, to Δν = −112 MHz gradually, and the intensity is ramped down to 40% in 2.7 ms. After the cool- ing process, the atoms reach a temperature of 5 μK and the size of the cloud is about 1 mm. A total of 36 cm above the MOT, a series of microwave pulses and resonant laser pulses pump and purify atoms into a magnetically insensitive Zeeman sub-state of \|52𝑆1 2 ⁄ 𝐹= 1, 𝑚𝐹= 0⟩ to avoid influences from external magnetic fields on the atoms. The atoms are further cooled down when they are in ballistic launch using the po- larization gradient cooling technique, where cooling laser beams are detuned far, to ∆ν = −112 MHz gradually, and the intensity is ramped down to 40% in 2.7 ms. Af- ter the cooling process, the atoms reach a temperature of 5 µK and the size of the cloud is about 1 mm. 3. Experiment and Methods 3. Experiment and Methods The ASPI-type inertia sensor was implemented in an atomic fountain interferom- eter, as is shown schematically in Figure 2. A combination of a 2-dimensional and a 3-dimensional magneto-optical trap (MOT) captures and cools down 87Rb atoms for the fountain. Cooling laser beams are then red-detuned by 12 MHz from the D-line resonance of 52S1/2F = 2 → 52P3/2F′ = 3 cooling transition. The loading of cold atoms takes 1 s, and an ensemble of 1.6 × 108 87Rb atoms are trapped and cooled for the following fountain process. The ASPI-type inertia sensor was implemented in an atomic fountain interferometer, as is shown schematically in Figure 2. A combination of a 2-dimensional and a 3-dimen- sional magneto-optical trap (MOT) captures and cools down 87Rb atoms for the fountain. Cooling laser beams are then red-detuned by 12 MHz from the D-line resonance of \|52𝑆1 2 ⁄ 𝐹= 2⟩→\|52𝑃3 2 ⁄ 𝐹′ = 3⟩ cooling transition. The loading of cold atoms takes 1 s, and an ensemble of 1.6 × 108 87Rb atoms are trapped and cooled for the following foun- tain process. Figure 2. Schematic diagram of the apparatus. (a) The schematic diagram of phase shear in the atomic interferometer; (b) top view of the Earth rotation compensation stage. (c) The sequence of realizing phase shear in the atomic interferometer. The launching of atoms is marked as 0 ms. Figure 2. Schematic diagram of the apparatus. (a) The schematic diagram of phase shear in the atomic interferometer; (b) top view of the Earth rotation compensation stage. (c) The sequence of realizing phase shear in the atomic interferometer. The launching of atoms is marked as 0 ms. Figure 2. Schematic diagram of the apparatus. (a) The schematic diagram of phase shear in the atomic interferometer; (b) top view of the Earth rotation compensation stage. (c) The sequence of realizing phase shear in the atomic interferometer. The launching of atoms is marked as 0 ms. Figure 2. Schematic diagram of the apparatus. (a) The schematic diagram of phase shear in the atomic interferometer; (b) top view of the Earth rotation compensation stage. (c) The sequence of realizing phase shear in the atomic interferometer. The launching of atoms is marked as 0 ms. When point-source approxi 2.2. Principal Component Analysis The higher-ranked PCs contain more signiﬁcant features of the input image, and the image has larger projections on these high-ranked PCs. g p g y dimensionality reduction and feature extraction of multidimensional data sets [15]. With this technique, the recorded raw image containing both the interference patterns and dis- turbances is considered as the superposition of a low rank and a sparse sub-set [16]. The main task of PCA is to derive a new set of variables of the reduced variable dimensions from the original image. In this way, a series of basis vectors which are known as principal components (PC) are obtained. The PCs are orthogonal to each other and span the original set of multi-pixel images. All recorded images can then be broken down to a sum of pro- jections of the original image onto each PC [17]. These PC basis vectors are ranked in de- scending order according to the relevance between themselves and the original image. The higher-ranked PCs contain more significant features of the input image, and the im- g p j g The process of carrying out PCA is essentially a process of dimensionality reduction and noise reduction. Firstly, the covariance matrix is optimized to obtain its eigenvectors and eigenvalues, which, respectively, represent the PCs and their corresponding variances. Then, different components of the original data set can be obtained using basis transforma- tion. The optimization of the covariance matrix follows two principles. The off-diagonal elements of the covariance matrix are supposed to be as small as possible, because they indicate the correlation between different dimensions. On the contrary, the diagonal ele- ments are supposed to be as large as possible, because they indicate the variance of the corresponding basis vector. Larger variances on most signiﬁcant PCs would lead to lower residue noise, interfering with the detection. Sensors 2023, 23, 5071 4 of 10 lead to 4 of 10 lead to 3. Experiment and Methods 3. Experiment and Methods A total of 36 cm above the MOT, a series of microwave pulses and resonant laser pulses pump and purify atoms into a magnetically insensitive Zeeman sub-state of 52S1/2F = 1, mF = 0 to avoid inﬂuences from external magnetic ﬁelds on the atoms. ⁄ l gi Figure 3 shows the actual apparatus of the atom interferometer. The interferometer operates in a Mach-Zehnder setup that uses 3 velocity-sensitive laser pulses to induce stimulated Raman transitions to split, redirect, and recombine the ultra-cold atoms. Time / Figure 3 shows the actual apparatus of the atom interferometer. The interferometer operates in a Mach-Zehnder setup that uses 3 velocity-sensitive laser pulses to induce stimulated Raman transitions to split, redirect, and recombine the ultra-cold atoms. Time interval between the π/2 −π −π/2 Raman pulses is set at T = 140 ms. To reduce the inﬂuences of non-uniformity of the Raman laser, the light out of a single mode ﬁber is expanded to diameter of 60 mm, collimated, and then truncated with a 25 mm round diaphragm to match the 25 mm diameter of the vacuum fountain tube. The collimator and diaphragm are mounted on top of the whole instrument and shoot the Raman pulse downward to the bottom, where a mirror is mounted on an electrically controlled tilt stage to reﬂect it back to top. The phase shear in the APSI setup can then be applied by Sensors 2023, 23, 5071 5 of 10 n pulse lt stage 5 of 10 n pulse lt stage manipulating the rotation angle or the asymmetrical timing of Raman pulses to induce horizontal or vertical interference fringes. ulating the rotation angle or the asymmetrical timing of Raman pulses to induce horizon- tal or vertical interference fringes. manipulating the rotation angle or the asymmetrical timing of Raman pulses to induce horizontal or vertical interference fringes. ulating the rotation angle or the asymmetrical timing of Raman pulses to induce horizon- tal or vertical interference fringes. Figure 3. Apparatus of the fountain-type inertia sensor in APSI setup. Figure 3. Apparatus of the fountain-type inertia sensor in APSI setup. Figure 3. Apparatus of the fountain-type inertia sensor in APSI setup. Figure 3. Apparatus of the fountain-type inertia sensor in APSI setup. 3. Experiment and Methods 3. Experiment and Methods An additional rotation of equal magnitude but opposite direction, −Ω𝐶, is applied onto the Raman tilting stage. Rough calculation based on the latitude of the lab location gives the direction of the local true north 𝜙𝐸 with respect to the horizon axis x. In the measure- ment, 3 separate tilting steps are applied to the reflecting mirror to achieve the compensa- i f E h’ i Th il i l Ω 𝜙 d Ω 𝑖𝜙 l g g It is necessary to compensate for the rotation of the Earth ΩC to achieve interference patterns sensitive only to the phase shear of the APSI in the laboratory coordinates. An additional rotation of equal magnitude but opposite direction, −ΩC, is applied onto the Raman tilting stage. Rough calculation based on the latitude of the lab location gives the direction of the local true north φE with respect to the horizon axis x. In the measurement, 3 separate tilting steps are applied to the reﬂecting mirror to achieve the compensation of Earth’s rotation. The tilting angles were set at −tΩCcosφE and −tΩCsinφE along the x and y axes, respectively, where t is set to 0 at the ﬁrst Raman pulse for calculation convenience. tion of Earth’s rotation. The tilting angles were set at −𝑡Ω𝐶𝑐𝑜𝑠𝜙𝐸 and −𝑡Ω𝐶𝑠𝑖𝑛𝜙𝐸 along the x and y axes, respectively, where t is set to 0 at the first Raman pulse for calculation convenience. After rough compensation, an extra tilt 𝛿𝜃𝑥 is applied to the last 𝜋/2 pulse to sim- ulate a small angular velocity Ω = 2 𝛿𝜃𝑥𝑡3 ⁄ to be measured, which is proportional to the After rough compensation, an extra tilt δθx is applied to the last π/2 pulse to simulate a small angular velocity Ω= 2δθx/t3 to be measured, which is proportional to the spatial frequency of the horizontal fringes κH, as is shown in Equation (3). Figure 4a demonstrates a series of raw captured images with various rotation angles, with expansion time set to be td = 0.577 s, and the last π/2 pulse at t3 = 0.521 s. spatial frequency of the horizontal fringes 𝜅𝐻 , as is shown in Equation (3). 3. Experiment and Methods 3. Experiment and Methods When the 3-pulse interferometer sequence is finished 0.6 s after fountain launching, a short detection pulse resonant with \|52𝑆1 2 ⁄ 𝐹= 2 →\|52𝑆3 2 ⁄ 𝐹= 3⟩ transition is sent from the top of the sensor to the detection region. At the time of detection, the 5 μK atoms have expanded in overall size and occupy a large area in the vacuum chamber. The colli- mator-diaphragm output port previously used on the Raman laser pulses clamps the pulsed detection beam, leaving only the central part to increase the uniformity of detec- tion. To capture all fringes of interference, a 25 × 36 mm region at the center of the de- tection chamber has to be imaged. To fulfil this, an infrared-enhanced lens of 35 mm focal length couples the detection region onto a global-shutter imaging sensor, 20 cm away from center of the chamber and set along the axis of the rotation to be measured. Exposure time of the camera sensor is set at 2 ms to make distortions due to movement of atoms negligi When the 3-pulse interferometer sequence is ﬁnished 0.6 s after fountain launching, a short detection pulse resonant with 52S1/2F = 2 → 52S3/2 F = 3⟩transition is sent from the top of the sensor to the detection region. At the time of detection, the 5 µK atoms have expanded in overall size and occupy a large area in the vacuum chamber. The collimator-diaphragm output port previously used on the Raman laser pulses clamps the pulsed detection beam, leaving only the central part to increase the uniformity of detection. To capture all fringes of interference, a 25 × 36 mm region at the center of the detection chamber has to be imaged. To fulﬁl this, an infrared-enhanced lens of 35 mm focal length couples the detection region onto a global-shutter imaging sensor, 20 cm away from center of the chamber and set along the axis of the rotation to be measured. Exposure time of the camera sensor is set at 2 ms to make distortions due to movement of atoms negligible. of the camera sensor is set at 2 ms to make distortions due to movement of atoms negligi- ble. It is necessary to compensate for the rotation of the Earth Ω𝐶 to achieve interference patterns sensitive only to the phase shear of the APSI in the laboratory coordinates. 3. Experiment and Methods 3. Experiment and Methods Figure 4a When the Earth’s rotation has been properly compensated, it is also possible to obtain vertical interference fringes by introducing a small asymmetrical timing difference δT into the intervals in between the 3 Raman pulses, namely T1 = T −δT/2 and T2 = T + δT/2. In this case, the fringes are visible on the camera from any horizontal direction. The corresponding images are also shown in Figure 4b with different settings of δT. When phase shear is introduced, readout of the interferometer phase and evaluation of its contrast is feasible within one single measurement cycle. Sensors 2023, 23, 5071 6 of 10 a sion time set to be 𝑡𝑑= 0.577 s, and the last 𝜋/2 pulse at 𝑡3 = 0.521 s. (a) −120 −80 −40 0 40 80 120 Rotation Angle 𝜹𝜽𝒙 ( 𝛍𝐫𝐚𝐝 ) (b) −50 −30 −10 0 10 30 50 Asymmetry Time 𝜹𝑻 ( 𝛍𝐬 ) Figure 4. Spatial interference fringes of the atom cloud captured on x-axis camera. (a) Horizontal spatial fringes caused by beam-tilt phase shear; (b) vertical spatial fringes caused by asymmetrical Raman pulse intervals. Figure 4. Spatial interference fringes of the atom cloud captured on x-axis camera. (a) Horizontal spatial fringes caused by beam-tilt phase shear; (b) vertical spatial fringes caused by asymmetrical Raman pulse intervals. 𝑑 , / p 3 (a) −120 −80 −40 0 40 80 120 Rotation Angle 𝜹𝜽𝒙 ( 𝛍𝐫𝐚𝐝 ) 40 Rotation Angle 𝜹𝜽𝒙 ( 𝛍𝐫𝐚𝐝 ) g −10 0 10 30 50 Asymmetry Time 𝜹𝑻 ( 𝛍𝐬 ) (b) 50 30 Figure 4. Spatial interference fringes of the atom cloud captured on x-axis camera. (a) Horizontal spatial fringes caused by beam-tilt phase shear; (b) vertical spatial fringes caused by asymmetrical Raman pulse intervals. Figure 4. Spatial interference fringes of the atom cloud captured on x-axis camera. (a) Horizontal spatial fringes caused by beam-tilt phase shear; (b) vertical spatial fringes caused by asymmetrical Raman pulse intervals. When the Earth’s rotation has been prop 4. Improvement of the Readout of APSIs When the Earth s rotation has been properly compensated, it is also possible to obtain vertical interference fringes by introducing a small asymmetrical timing difference 𝛿𝑇 into the intervals in between the 3 Raman pulses, namely 𝑇1 = 𝑇−𝛿𝑇2 ⁄ and 𝑇2 = 𝑇+ 𝛿𝑇2 ⁄ . In this case, the fringes are visible on the camera from any horizontal direction. The corresponding images are also shown in Figure 4b with different settings of 𝛿𝑇. When phase shear is introduced, readout of the interferometer phase and evaluation of its con- trast is feasible within one single measurement cycle. 4. Improvement of the Readout of APSIs The measurement of angular velocity on an APSI relies on the accurate derivation of spatial frequency of the interference fringes captured by the imaging sensor However The measurement of angular velocity on an APSI relies on the accurate derivation of spatial frequency of the interference fringes captured by the imaging sensor. However, ﬁtting the raw images directly to the theoretical model is far from accurate because of multiple imperfections of the instrument. For example, uneven distribution of atoms’ velocity and, consequently, their displacement from the center of the cloud is always superposed on the spatial fringes supposed to be measured. Thermal noise of the imaging sensor, residue stray light from detection laser pulses, and reﬂections from the inside of the vacuum chamber also disturb the detection in a way that is difﬁcult to model and predict. To fulﬁll the measurement, a PCA-based pre-ﬁtting procedure was applied to the detected raw image signals. p q y fitting the raw images directly to th l i l i f i f h i 4.1. Pre-Fitting Preparation of Images multiple imperfections of the instrument. For example, uneven distribution of atoms’ ve- locity and, consequently, their displacement from the center of the cloud is always super- posed on the spatial fringes supposed to be measured. Thermal noise of the imaging sen- sor, residue stray light from detection laser pulses, and reflections from the inside of the vacuum chamber also disturb the detection in a way that is difficult to model and predict. To fulfill the measurement, a PCA-based pre-fitting procedure was applied to the detected raw image signals. The PCA-based procedure acts on the detected raw images much like an image ﬁlter separating the desired interference patterns from unwanted background bias and noise. As an unsupervised machine learning algorithm, PCA requires multiple raw images as a training set to derive the eigenvectors in subspace. In the experiment, a training set com- prises 100 images from consecutive detection with identical phase shear settings. Figure 5 shows a series of principal components obtained from an image set of a beam-tilt phase shear experiment, ranked in descending order according to their respective eigenvalues. W 7 of 10 4.1. Pre-Fitting Preparation of Images The PCA-based procedure acts on the detected raw images much like an image filter separating the desired interference patterns from unwanted background bias and noise. As an unsupervised machine learning algorithm, PCA requires multiple raw images as a training set to derive the eigenvectors in subspace. In the experiment, a training set com- prises 100 images from consecutive detection with identical phase shear settings. Figure 5 shows a series of principal components obtained from an image set of a beam-tilt phase shear experiment, ranked in descending order according to their respective eigenvalues. PC 1 PC 2 PC 3 PC 4 PC 5 PC 6 Figure 5. Normalized images of the first 6 principal components obtained from beam-tilt phase shear of 𝛿𝜃𝑥= 60 μrad. Figure 5. Normalized images of the ﬁrst 6 principal components obtained from beam-tilt phase shear of δθx = 60 µrad. ng Preparation of Images A-based procedure acts on the detected he desired interference patterns from u ervised machine learning algorithm, P o derive the eigenvectors in subspace. mages from consecutive detection with i es of principal components obtained f k d i d di d PC 3 PC 4 i like an image filter und bias and noise. 4.2. Extraction of the Spatial Frequency of Fringes 4.2. Extraction of the Spatial Frequency of Fringes When the intensity variance of the detection beam is not taken into account, the ideal spatial distribution of atoms after beam-tilt phase shear should be a superposition of horizontal fringes and a Gaussian number density distribution. The position-dependent probability of ﬁnding an atom on \| F = 1⟩or \| F = 2⟩states is given by P1(x) = A1e −(x−b)2 σ2x [1 2 + c 2sin(kxx + φ)], (5) P2(x) = A2e −(x−b)2 σ2x [1 2 −c 2sin(kxx + φ)], (6) P1(x) = A1e −(x−b)2 σ2x [1 2 + c 2sin(kxx + φ)], (5) (5) P1(x) = A1e σx [2 + 2sin(kxx + φ)], (5) P2(x) = A2e −(x−b)2 σ2x [1 2 −c 2sin(kxx + φ)], (6) P2(x) = A2e −(x−b)2 σ2x [1 2 −c 2sin(kxx + φ)], (6) (6) where σx is the standard deviation of the Gaussian distribution and φ and kx are the phase and frequency of the interference fringes. The probabilities P1 and P2 are orthogonal and differ by half a period. With PCA ﬁltering out inﬂuences caused by the overall distribution of the detection beam, the fringes formed by atoms on the \| F = 1⟩state are rewritten as PFringe(x) = Ale −(x−b)2 σ2x sin(kxx + φ). (7) (7) To ﬁnd the spatial frequency kx, the highest-ranked principal components that contain information of interference patterns are ﬁtted with this function. To ﬁnd the spatial frequency kx, the highest-ranked principal components that contain information of interference patterns are ﬁtted with this function. For a comparison of the accuracy and reliability of raw ﬁtting and PCA, both the averaged raw image and the second principal component derived from PCA were inte- grated along the direction perpendicular to the fringes and subject to parameter ﬁtting according to Formula (7). Figure 6 presents such a comparison for vertical and horizontal interference fringes. As the PCA-based pre-ﬁtting processing signiﬁcantly screened out inﬂuences via unwanted systematic bias and noises, the characteristics of the interference pattern were signiﬁcantly enhanced, and the contrast of the fringe as improved by 7–12 dB, resulting in a more reliable recovery of the spatial frequency and phase from atomic interference fringes. The enhancement is effective in both the vertical and horizontal fringe detection schemes. p q y fitting the raw images directly to th l i l i f i f h i 4.1. Pre-Fitting Preparation of Images ple raw images as a a training set com- ar settings. Figure 5 f a beam-tilt phase PC 6 raw images much unwanted backgrou CA requires multip In the experiment, dentical phase shea rom an image set o di t th i PC 5 shear experiment, ranked in descending order according to their respective eigenvalues. Figure 5. Normalized images of the first 6 principal components obtained from beam-tilt phase shear of 𝛿𝜃𝑥= 60 μrad. Figure 5. Normalized images of the ﬁrst 6 principal components obtained from beam-tilt phase shear of δθx = 60 µrad. In PCA, the corresponding eigenvalue of a specific principal component reflects the proportion of information it contains in the original signal, and the component with a larger eigenvalue is more related to lower-frequency changes in the image [18]. Among the derived principal components shown in Figure 5, the first two principal components contain most of the information of concern on the spatial interference fringes in the image In PCA, the corresponding eigenvalue of a speciﬁc principal component reﬂects the proportion of information it contains in the original signal, and the component with a larger eigenvalue is more related to lower-frequency changes in the image [18]. Among the derived principal components shown in Figure 5, the ﬁrst two principal components Sensors 2023, 23, 5071 7 of 10 7 of 10 contain most of the information of concern on the spatial interference fringes in the image of atom cloud. The third component is mostly related to thermal noise and background bias when illumination on the sensor is weak, and the fourth is related to the overall density distribution of the atom cloud. The lower ranked ﬁfth and sixth components are composed of high-frequency ﬂuctuations caused by the sensor and shutter, and are irrelevant in the extraction of spatial frequencies. 4.3. Enhanced Readout of APSIs (a) and (b) are results f l l it t ith t d ith PCA b d ﬁtti i ( ) d (f) (a) (b) (e) (a) (f) (b) (e) (e) (g) (c) (h) (d) (c) (d) (h) (g) Figure 6. Direct and PCA fitting of spatial fringes. (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c, d, g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the fitted curve from each data set. Figure 6. Direct and PCA ﬁtting of spatial fringes. (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c,d,g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the ﬁtted curve from each data set. W 9 of 10 w n m f s 4 t i i a r (a) As the PCA-based pre-fitting processing significantly screened out influences via un- wanted systematic bias and noises, the characteristics of the interference pattern were sig- nificantly enhanced, and the contrast of the fringe as improved by 7–12 dB, resulting in a more reliable recovery of the spatial frequency and phase from atomic interference fringes. The enhancement is effective in both the vertical and horizontal fringe detection schemes. 4.3. Enhanced Readout of APSIs When the rotation angle in the beam-tilt phase shear scheme or asymmetry time in timing-asymmetry scheme is scanned, spatial frequency extraction with PCA from any image set proves more reliable than that from simple stacking and averaging the images in the set. The comparison is shown in Figure 7. Apart from measurements of very small angles or 𝛿𝑡, where the induced interference frequency is so small that less than one pe- riod can be detected in the imaging region, the signal-to-noise ratio of spatial fringes after PCA is typically improved by 5–10 dB for beam-tilt phase shear and 9–12 dB for asym- metrical timing phase shear. The enhancement is more significant when the frequency of the induced interference fringes is higher in either setup. (a) (b) (c) (d) Figure 7. Derived spatial frequency from phase shears. 4.3. Enhanced Readout of APSIs Red solid lines represent theoretical predic- tions. Red dashed lines represent linear fitting of measured spatial frequencies. (a) and (b) are results from angular velocity measurements without and with PCA-based pre-fitting processing; (c) and (d) are results from asymmetrical timing phase shears. Top chart shows the residuals calculated by subtracting the predicted linear values from the measured data. Figure 7. Derived spatial frequency from phase shears. Red solid lines represent theoretical predic- tions. Red dashed lines represent linear ﬁtting of measured spatial frequencies. (a) and (b) are results from angular velocity measurements without and with PCA-based pre-ﬁtting processing; (c) and (d) are results from asymmetrical timing phase shears. Top chart shows the residuals calculated by subtracting the predicted linear values from the measured data. As the PCA-based pre-fitti wanted systematic bias and no nificantly enhanced, and the co more reliable recovery of the fringes. The enhancement is ef schemes. 4.3. Enhanced Readout of APSIs When the rotation angle i timing-asymmetry scheme is s image set proves more reliable in the set. The comparison is sh angles or 𝛿𝑡, where the induce riod can be detected in the ima (b) reened out influences via un- interference pattern were sig- ved by 7–12 dB, resulting in a se from atomic interference d horizontal fringe detection cheme or asymmetry time in traction with PCA from any ng and averaging the images measurements of very small o small that less than one pe- se ratio of spatial fringes after (d) ng processing significantly sc ses, the characteristics of the ntrast of the fringe as improv spatial frequency and pha ective in both the vertical an n the beam-tilt phase shear s canned, spatial frequency ex than that from simple stacki own in Figure 7. Apart from d interference frequency is s ging region the signal to noi (c) (a) 𝑡, wh d t t (b) less i l (d) nce fr th (c) g g g g p g PCA is typically improved by 5–10 dB for beam-tilt phase shear and 9–12 dB for asym- metrical timing phase shear. The enhancement is more significant when the frequency of the induced interference fringes is higher in either setup. Figure 7. Derived spatial frequency from phase shears. Red solid lines represent theoretical predic- tions. Red dashed lines represent linear fitting of measured spatial frequencies. 4.3. Enhanced Readout of APSIs When the rotation angle in the beam-tilt phase shear scheme or asymmetry time in timing-asymmetry scheme is scanned, spatial frequency extraction with PCA from any image set proves more reliable than that from simple stacking and averaging the images in the set. The comparison is shown in Figure 7. Apart from measurements of very small angles or δt, where the induced interference frequency is so small that less than one period can be detected in the imaging region, the signal-to-noise ratio of spatial fringes after PCA is typically improved by 5–10 dB for beam-tilt phase shear and 9–12 dB for asymmetrical timing phase shear. The enhancement is more signiﬁcant when the frequency of the induced interference fringes is higher in either setup. When compared with the theoretical predictions, the improvement by PCA-based fringe enhancement is obvious. In Figure 7, the solid lines in each bottom chart represent the expected linear relationship following Equations (3) and (4), and the difference between measured and expected values is shown in the top charts. When PCA processing is applied prior to frequency acquisitions, the variation of measured spatial frequency is smaller and in better accordance with the predicted linearity. Sensors 2023, 23, 5071 Sensors 2023, 23, x FOR 8 of 10 8 of 10 (a) (b) (e) (f) (c) (d) (g) (h) Figure 6. Direct and PCA fitting of spatial fringes. (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c, d, g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the fitted curve from each data set. Figure 6. Direct and PCA ﬁtting of spatial fringes. (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c,d,g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the ﬁtted curve from each data set. Sensors 2023, 23, x FOR PEER REVIEW 9 of 10 (a) (b) (e) (f) (c) (d) (g) (h) Figure 6. Direct and PCA fitting of spatial fringes. 4.3. Enhanced Readout of APSIs (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c, d, g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the fitted curve from each data set. As the PCA-based pre-fitting processing significantly screened out influences via un- wanted systematic bias and noises, the characteristics of the interference pattern were sig- nificantly enhanced, and the contrast of the fringe as improved by 7–12 dB, resulting in a more reliable recovery of the spatial frequency and phase from atomic interference fringes. The enhancement is effective in both the vertical and horizontal fringe detection schemes. 4.3. Enhanced Readout of APSIs When the rotation angle in the beam-tilt phase shear scheme or asymmetry time in timing-asymmetry scheme is scanned, spatial frequency extraction with PCA from any image set proves more reliable than that from simple stacking and averaging the images in the set. The comparison is shown in Figure 7. Apart from measurements of very small angles or 𝛿𝑡, where the induced interference frequency is so small that less than one pe- riod can be detected in the imaging region, the signal-to-noise ratio of spatial fringes after PCA is typically improved by 5–10 dB for beam-tilt phase shear and 9–12 dB for asym- metrical timing phase shear. The enhancement is more significant when the frequency of Figure 6. Direct and PCA ﬁtting of spatial fringes. (a) and (e) are averaged raw images of horizontal and vertical fringe detections; (b) and (f) are 2nd highest ranked PC of (a) and (e); lower row of graphs(c,d,g) and (h) are integrations of the corresponding image in the upper row, along the direction parallel to fringes. Red lines depict the ﬁtted curve from each data set. Sensors 2023, 23, x FOR PEER REVIEW 9 of 10 (a) (b) (c) (d) Figure 7. Derived spatial frequency from phase shears. Red solid lines represent theoretical predic- tions. Red dashed lines represent linear fitting of measured spatial frequencies. (a) and (b) are results from angular velocity measurements without and with PCA-based pre-fitting processing; (c) and Figure 7. Derived spatial frequency from phase shears. Red solid lines represent theoretical predic- tions. Red dashed lines represent linear ﬁtting of measured spatial frequencies. 4.3. Enhanced Readout of APSIs (a) and (b) are results from angular velocity measurements without and with PCA-based pre-fitting processing; (c) and (d) are results from asymmetrical timing phase shears. Top chart shows the residuals calculated by subtracting the predicted linear values from the measured data. Figure 7. Derived spatial frequency from phase shears. Red solid lines represent theoretical predic- tions. Red dashed lines represent linear ﬁtting of measured spatial frequencies. (a) and (b) are results from angular velocity measurements without and with PCA-based pre-ﬁtting processing; (c) and (d) are results from asymmetrical timing phase shears. Top chart shows the residuals calculated by subtracting the predicted linear values from the measured data. When compared with the theoretical predictions, the improvement by PCA-based fringe enhancement is obvious. In Figure 7, the solid lines in each bottom chart represent the expected linear relationship following Equations (3) and (4), and the difference be- tween measured and expected values is shown in the top charts. When PCA processing is applied prior to frequency acquisitions, the variation of measured spatial frequency is smaller and in better accordance with the predicted linearity In angular velocity measurements with the fountain-type APSI inertia sensor, the precision of the measurement is evaluated by calculating the variation of measured spatial frequency and converting back into rotation velocity following Equation (3). The evaluation gives a precision of 6.3 µrad/s without PCA-based enhancement and 3.3 µrad/s with enhancement from results of the same measurement. smaller and in better accordance with the predicted linearity. In angular velocity measurements with the fountain-type APSI inertia sensor, the precision of the measurement is evaluated by calculating the variation of measured spatial frequency and converting back into rotation velocity following Equation (3). The evalua- tion gives a precision of 6.3 μrad/s without PCA-based enhancement and 3.3 μrad/s with enhancement from results of the same measurement As for the measurement of very small angular velocity or timing asymmetry where the spatial frequency is too low to be precisely recovered from an image in the limited space within the detection chamber, differential measurement by adding an extra rotation angle or timing difference in opposite directions solves the problem, because the small frequency to be measured is transferred to larger frequencies while maintaining the mean Sensors 2023, 23, 5071 9 of 10 value. The PCA-based processing technique is still appliable and works equally well in such differential measurements. 4.3. Enhanced Readout of APSIs However, because these measurements are not as reliable as those of relatively high frequencies in the same measurement scheme, these inaccurate results are ruled out in the evaluation of the enhancement. 5. Conclusions By implementing PCA-based image processing techniques, measurement of angular velocity on an APSI-type inertial sensor is enhanced. A comparison of measurement results with and without the PCA procedure, and the theoretical predictions according to the dependency of the spatial frequency of interference fringes on angular velocity proves the PCA-based technique capable of improving both the accuracy and precision of measurements. The technique is versatile in various applications of atomic interferometers where the detection is carried out via the imaging of ﬂuorescence from stimulated atoms. Author Contributions: Conceptualization, X.W.; methodology, J.W. and J.T.; software, J.T.; vali- dation, J.W., J.T., W.X. and Y.F.; investigation, J.W. and Z.W.; writing—original draft preparation, J.W.; writing—review and editing, X.W.; visualization, J.W. and Z.W.; supervision, X.W.; project administration, X.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China, grant numbers 61875175 and 61727821. Data Availability Statement: Data underlying the results presented in this paper are not publicly available at this time, but may be obtained from the authors upon request. Data Availability Statement: Data underlying the results presented in this paper are not publicly available at this time, but may be obtained from the authors upon request. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. 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MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
W4283205772.txt	https://journals.wlb-stuttgart.de/ojs/index.php/sh/article/download/3076/3108	de		Erwin Dold, der letzte Kommandant im KZ Dautmergen	Schwäbische Heimat	2,022	cc-by	6,279	Heimat, einmal anders gesehen Immo Opfermann Erwin Dold, der letzte Kommandant im KZ Dautmergen Zu Erwin Dold, dem letzten «Kommandanten» des KZ Dautmergen, schreibt Thomas Seiterich-Kreuzkamp ins Gästebuch der ersten «Wüste»-Ausstellung in Balingen am 12. November 1994: Natürlich decken sich beide Versionen nicht zu hundert Prozent. Dold, auf der Täterseite, Lampin und Colin auf der Opferseite. (...) Gleichwohl bleibt die Geschichte des KZ-Kommandanten von Dautmergen bedenkens- und erinnernswert. Dold beweist, dass die Ausrede «wir konnten nix machen» hohl ist; denn Dold, der sein Leben riskierte und als junger Erwachsener an die Grenzen des ihm Möglichen gegangen ist («aus Anständigkeit», so würde er wohl sagen) ist ein lebendiges Gegenbeispiel zum Mitfunktionieren und zum Kleinmut.1 Andreas Möller nennt im «Schwarzwälder Boten» vom 15./16. Dezember 1973 seinen Beitrag Ich wollte, dass sie leben im Untertitel Bericht über einen Helden 2, und noch Wolfram Wette widmet in dem Sammelband Stille Helden Dold ein eigenes Kapitel 3. Wie wandelte sich ein gläubiger Nationalsozialist 4 zu einem Mann, der als einziger im Rastatter Prozess 1947 wegen erwiesener Unschuld freigesprochen wurde? Was hat diesen Gesinnungswandel bewirkt? Seine Biografie weist Lücken oder Leerstellen auf. Die Bruchstücke, die belegt sind, fußen auf Dokumenten verschiedener Prozesse, in denen Dold als Angeklagter oder Zeuge zu seiner Vita ausgesagt hat. Hitlerjugend und Feldwebel der Luftwaffe – Herbst 1943 über der Krim abgeschossen Erwin Dold, geboren am 16. November 1919, wuchs in einer katholischen Familie mit vielen Kindern in Wagensteig, Gemeinde Buchenbach im Südschwarzwald, auf. Die Eltern betrieben einen Gasthof, eine Landwirtschaft, das Sägewerk, das bis heute mit der Familie Dold in Beziehung steht. Als die Nationalsozialisten an die Macht kamen, war Erwin Dold mit etwas mehr als dreizehn Jahren alt genug für das «Jungvolk», trat, als es möglich war, mit vierzehn Jahren der «Hitler-Jugend» bei, meldete sich, kaum achtzehn Jahre alt geworden und sicherlich beeinflusst von HJ-Schulungen, im Angeklagte im Rastatter Kriegsverbrecher-Prozess 1946/47: Erwin Dold (letzte Reihe, vierter von rechts) hat die Nummer 40. Er wurde als einziger wegen erwiesener Unschuld freigesprochen. Schwäbische Heimat 2010/4 395 Dezember 1937 zur neugegründeten Luftwaffe, deren Selbstbewusstsein Hermann Göring verkörperte. Auf dem Flugplatz Kaufbeuren kam Dold in die Ausbildung als Luft-Aufklärer. Ob er als solcher jedoch bereits nach einem Vierteljahr «den Einmarsch in Österreich» im März 1938 mitmachte, ist fraglich, denn er selbst sagte zu seiner Laufbahn im Rastatter Prozess: Ich wurde im Jahr 1939 eingezogen. Ich habe den Frankreichfeldzug mitgemacht und dann bin ich nach Österreich, Rumänien und Russland gekommen 5. Folgte Dold den Zielen des «Generalobersten Göring», der Deutschland und vor allem die Luftwaffe kriegsfähig machen wollte? Als nach dem Überfall auf Polen 1939 und auf Frankreich 1940 die an den «Blitzkriegen» beteiligte Luftwaffe mit ihrem Chef in eine unkritisch-euphorische Siegesstimmung kam, übertrug sich dies sicher auch auf den Feldwebel Erwin Dold. Im Gegensatz dazu kann vermutet werden, dass nach der desaströs verlaufenen «Luftschlacht um England» auch der Gemütszustand des Fliegers Erwin Dold nicht mehr euphorisch war, denn der privilegierten Luftwaffe wurde Versagen vorgeworfen. Dold war beim Überfall auf die Sowjetunion im Juni 1941 dabei: Machte er sich Gedanken über die Expansionspolitik, der er diente, über Hitlers Raubkrieg, empfand er es als schmählich, dass die Luftwaffe zum «Hilfsinstrument des Heeres degradiert» war, nachdem Hitler den Oberbefehl über das Heer am 19. Dezember 1941 übernommen hatte? Nach der Peripetie des Krieges spätestens in Stalingrad war Erwin Dold in der Uniform der Deutschen Luftwaffe, ca. 1942. 396 die Sowjetunion gegen den Aggressor im Vormarsch: Dold wurde als Jagdflieger im Herbst 1943 über der Krim abgeschossen, obwohl im Juli desselben Jahres die deutsche Luftwaffe noch sehr erfolgreich war. Er überlebte schwer verletzt, war zur Rekonvaleszenz in verschiedenen Lazaretten: Ich war verwundet in Russland und kam dann nach Frankfurt/Oder während der Genesungsabteilung und kam dann nach Freiburg auf den Flugplatz, so die Aussage des Zeugen Dold 1965 zur eigenen Vita, allerdings über zwanzig Jahre nach den Ereignissen. Ich wollte entlassen werden, wir hatten ein kleines Sägewerk zu Hause und wir waren vier Brüder im Krieg und mein Vater hat ein Gesuch eingereicht, daß wenigstens noch einer zu Hause bleibt. (…) Es ist mir aber dann nicht geglückt, ich war wohl d.u. (= dienstunfähig) geschrieben eine Zeitlang. (…) und dann ist es d.v. Heimat (=dienstverwendungsfähig) geworden und dann wurde ich abkommandiert von Freiburg. (…) zu einem Wachkommando nach (…) Haslach im Kinzigtal. Ende 1944 Lagerleiter im KZ Haslach – Sklavenarbeit im Stollen «Vulkan» – Beginn des «Mythos Dold» Nachdem er auf dem Flugplatzkommando Freiburg bereits im Kinzigtal als LKW-Fahrer nach der Genesung durchaus noch «kriegsverwendungsfähig» war, meldete sich Erwin Dold freiwillig nach Haslach als Wachmann eines «Industriewachkommandos», für das auf dem Flugplatz Freiburg geworben worden war. Der Mangel an arbeitsfähigen Männern war so groß, dass auch Mitglieder der Luftwaffe als Wachleute in Frage kamen. Möglicherweise war für Dold das Abgeschossenwerden im Flugzeug und die langwierige Rekonvaleszenz auch ein Lösungsprozess vom Luftwaffen-Mythos. In der Befragung des Landespolizeipostens Kirchzarten vom 30. Juli 1959 sagte Dold zu seiner Ankunft in Haslach im Oktober 1944: Ich unterstand (als Wachmann) einem Oberfeldwebel der Luftwaffe, der das Wachkommando führte. Dieser Oberfeldwebel erkrankte gegen Jahresende 1944 und bat mich, für ihn das Wachkommando zu übernehmen. Dold wurde also Lagerleiter in Haslach, dem befehlsmäßig das ganze Lager unterstand. Haslach war ein KZ, in dem er physisch und psychisch gänzlich heruntergekommene, erbarmungswürdige Häftlinge vorfand. Als Angeklagter im Rastatter Prozess sagte Dold aus: Das, was ich nicht über die Lager wusste, habe ich gesehen und gelernt. Diese Aussage legt nahe, dass Dold der zynisch und euphemistisch benutzte Begriff «Konzertlager» 6 bekannt war, der suggerierte, es handle sich um befristete Vorbeugehaft von Kriminellen. Schwäbische Heimat 2010/4 Reveillon am Neujahrstag des Jahres 1945. Zur Feier des Tages gibt es ein Neujahrsmenü. Gemalt von dem Häftling Ludovic de la Chapelle. Das KZ Haslach, ein Außenkommando von Natzweiler-Struthof 7, wurde am 16. September 1944 eingerichtet, nachdem das Stammlager im Elsass wegen der alliierten Kriegserfolge – am 25. August 1944 wird Paris befreit – Anfang September aufgelöst und die Kommandantur ins badische Guttenbach verlegt worden war. Das KZ-Kommando «Natz Barbe» mit der Bezeichnung «Sportplatz» weist auf den Ort, wo in einer großen Baracke, einem Schuppen der Wehrmacht, bis Februar 1945 ungefähr 250 Gefangene 8 eingesperrt waren. Der Plan des Lagers, gezeichnet von dem ehemaligen Haslach-Häftling Ludovic de la Chapelle, zeigt den improvisierten Charakter des KZ, das in aller Eile seinem Zweck zugeführt werden musste. In der Baracke waren zwei große Schlafsäle, Küche, Waschraum und Krankenstation untergebracht, auffällig ist die vergleichsweise riesige Latrine am Rand des Lagers. Der Appellplatz inmitten des von Stacheldraht umzäunten Rechtecks weist auf die typischen Zählappelle der SS auch hier hin. Der Wachturm zur Kontrolle von oben fehlte ebensowenig. Für den «Lagerältesten» stand laut Plan eine eigene Schlafstätte zur Verfügung. Die SS selbst hatte eine Villa, das Clubhaus, in der Nähe zur Verfügung. Andere Häftlinge, ungefähr 800, mussten im unterirdischen Tunnel «Vulkan» unter schlimmsten Bedingungen schuften, ca. 5 km von Haslach entfernt: die Produktion von Daimler-Benz-Flugzeugmotoren war wegen der alliierten Luftangriffe unter die Erde verlegt worden 9. Dafür gab es ein eigenes Lager «Vulkan», später noch ein weiteres «KinzigSchwäbische Heimat 2010/4 damm»: Insgesamt waren in den drei Haslacher Lagern mindestens 1700 Häftlinge inhaftiert 10. Die Zahl der Häftlinge des Lagers «Sportplatz» differiert zwischen 400 und 600, in jedem Fall war der Wehrmachtsschuppen zu klein für die vielen Gefangenen: Das kam auf mich zu wie eine Lawine. Nicht zwei oder drei Menschen lebten hier in größter Not, sondern Tausende, und täglich sind neue dazugekommen 11. Es herrschten fürchterliche hygienische und medizinische Zustände, weil die Versorgung und Ernährung der KZ-Gefangenen völlig unzureichend war. Die ersten 399 Gefangenen, vor allem «Nachtund-Nebel»-Häftlinge aus Dachau und DachauAllach, waren in der Hauptsache französische Widerstandskämpfer, die vorher aus dem Stammlager Natzweiler abtransportiert worden waren 12. Erwin Dold kam im November 1944, nun Angehöriger der 9. Kompanie des I. Wachsturmbanns des KZ Natzweiler, also Mitglied der Waffen-SS, nach Haslach. Sein direkter Vorgesetzter war bis Dezember 1944 der SS-Oberscharführer Robert Hochhaus, nach dessen Weggang die Leitung von Dold übernommen wurde. Dieser soll den Gefangenen nach der Befehlsübergabe sinngemäß gesagt haben: Ich hoffe, dass ihr diszipliniert und gehorsam seid; in diesem Fall werden wir gut miteinander auskommen und ihr werdet keinen Grund haben, euch zu beklagen 13. In Haslach wurde Dolds Begrüßungssatz positiv aufgenommen, allerdings ex eventu interpretiert. Vielleicht nimmt hier sogar der «Mythos Dold» seinen Anfang, verstärkt dadurch, dass Ludovic de la Chapelle sowohl eine Weihnachts- als auch Neujahrsansprache 44/45 tra397 Häftlinge und Lagerleiter Dold ins KZ Dautmergen – Unmenschliche Zustände für mehr als 3.000 Gefangene – Schlamm, Zelte und schwierige Wasserversorgung diert, in der Erwin Dold davon gesprochen habe, er hoffe, dass das nächste kommende Weihnachtsfest für uns alle in unseren Familien gefeiert werden könne, ebenso, dass das Neue Jahr, das beginnt, das Ende des Krieges und unsere Rückkehr zu unseren Familien 14 bringe. Tatsächlich überliefert der gerettete Häftling sogar das Weihnachts- und Neujahrsmenu und ein Bild von der «Reveillon» vom 1. Januar 45. Der Mut zu diesem gefährlichen Inhalt der Rede ist möglicherweise angedichtet, «defätistisch» ohnehin, jedoch nur durch de la Chapelle bezeugt. Dold hat wohl über das Lager «Barbe» Quarantäne verhängt, auch die Arbeit im Stollen «Vulkan» zeitweise beenden lassen, so dass weniger Tote zu beklagen waren. Der positive, fast schwärmerische Eindruck vom schönen jungen Mann, dem die Lager-SS gehorchte, bleibt. Gustav Steinger, 192. Zeuge im Prozess von Rastatt, sagte für Dold aus: Ich habe ihn oft in meiner Werkstatt getroffen. Man hat ihn mir als Antifaschisten beschrieben. (...) Ich habe ihn studiert und beobachtet, und wir haben uns verstanden. (...) Ich habe ihm Ratschläge gegeben. (...) Er hat die Verpflegung, die Kleidung und das Leben im Lager verbessert. (...) Vier Tage nach der Befreiung sind die Häftlinge zu mir gekommen und haben mir gesagt, daß sie ohne Dold ihr Vaterland nicht wiedergesehen hätten15. Dieser scheint das Prinzip nur Gesunde können arbeiten 16 bereits bei seinem ersten KZ-Einsatz erkannt und verwirklicht zu haben. Nach fünf Monaten wurde das «Lager Sportplatz» am 15. Februar 1945 aufgelöst. Die Gefangenen, völlig geschwächt und typhuskrank, wurden verlegt: einerseits in das «Kranken- und Sterbelager» Vaihingen/Enz, andererseits in das KZ Dautmergen auf Schömberger Gemarkung am Fuß der Schwäbischen Alb, unweit von Balingen. Hier wurde Erwin Dold wie in Haslach letzter Kommandant. Nach seinen eigenen Aussagen hatte er zuvor schon einmal Ende des Jahres 1944 den Lagern in Schömberg einen kurzen Besuch abgestattet, sowohl dem «Bahnhof-KZ» als auch dem KZ Dautmergen. Im Rückblick aus der Distanz von Jahrzehnten jedoch hat es den Anschein, als habe er die Orte, den Zeitpunkt und die Umstände dieser Besuche nicht mehr genau einordnen können. Es gibt mehrere Ungereimtheiten. So konnte Serge Lampin den Kommandanten nicht gekannt haben, wenn dieser erst Ende 1944 in Dautmergen war, vielmehr hat Lampin die Erzählungen seines Freundes René Colin übernommen, der dem «Leichenkommando» in Dautmergen angehörte. Laut Dold selbst war das im Dezember 1944. – Der Lagerführer war nicht da, und ich habe diese Inspektion geführt und das Lagerleben Am 25. Dezember 1944 hat ein britischer Luftaufklärer dieses Luftbild des KZ Dautmergen aufgenommen. Plan des KZ Dautmergen, von Hans Jürgen Schnurr gezeichnet. 398 Schwäbische Heimat 2010/4 beschrieben 17. Die Datierung ist insgesamt schwierig, weil Dold möglicherweise im Herbst 1944 bereits ins Lager Dautmergen abkommandiert worden war, wo auf seine Veranlassung eine Inspektionskommssion zur Überprüfung der hygienischen und medizinischen Situation das Lager besichtigt habe. Erst danach, nämlich zu Weihnachten 1944, müsste Dold dann nach Haslach versetzt worden sein. Genausowenig war er sich sicher, ob er Ende Februar oder Anfang März 1945 das Lager Dautmergen übernommen hatte. Auch wollte er als letzter Lagerkommandant nicht Mitglied der SS gewesen sein, obwohl er im Prozess gegen einen seiner Vorgänger, den SSHauptscharführer Lisken, als SS-Unterscharführer bezeichnet wurde 18. Das KZ Dautmergen, noch auf Schömberger Gemarkung zwischen den Orten Dautmergen und Schömberg, war eine NS-Bezeichnung zur Unterscheidung vom Bahnhofs-KZ in Schömberg, das als erstes KZ an der Wellendinger Straße in Schömberg errichtet worden war. Dautmergen war das größte KZ, in einer Flursenke auf freiem Feld zunächst als Zeltstadt aufgebaut, das vom 23. August 1944 an nach und nach bis ungefähr 3.000 Gefangene aufnehmen musste. Der Höchststand betrug am 1. Februar 1945 3.181 Häftlinge 19. Für Dold, von Natzweiler aus als Lagerleiter von Dautmergen bestimmt 20, begann seine sechs- bis siebenwöchige Zeit als Kommandant. Die Zustände im KZ Dautmergen waren noch unmenschlicher als in Haslach, von verschiedenen Häftlingen in ihren Erinnerungen als «Hölle» wahrgenommen und nachträglich überliefert schlimmer als Auschwitz 21. Das Lager war etwa fünfhundert mal fünfhundert Meter groß, mit einer einfachen Umzäunung, Wachtürmen und Scheinwerfern. (...) Einige Wohnbaracken waren gerade im Bau und konnten noch nicht bezogen werden, ebenso das Krankenrevier und der Schonungsblock. Man blieb in den Zelten. Aber ein paar Tage später wurden wir zu den Baracken befohlen; nach und nach wurden die Zelte abgebaut. Das Revierzelt blieb allerdings noch bis November stehen. Dautmergen war ein Ort von unvorstellbar großem Schrecken. (...) Alles beherrschend war der Schlamm 22. Diese Zustände vor Dolds Ankunft verbesserten sich bis März 1945 nicht wesentlich, ein Tankwagen brachte Wasser oder dieses wurde aus der Schlichem vom Dorf Dautmergen in Kübeln nach oben getragen. Nach dem Aufbau aller Baracken war die Wasserversorgung für Dautmergen und Schömberg gesichert, wie der Kommandoführer für das Bauvorhaben Wüste, ein SS-Untersturmführer, am 17. März 1945 an den Bürgermeister der Stadt Schömberg schrieb, nämlich, dass sowohl das KL Schömberg wie auch das KL Schwäbische Heimat 2010/4 Dautmergen an der Wasserleitung von Schömberg angeschlossen sind und dass zumindest für Entlausungsund Waschzwecke Industriewasser herangeschafft wird, weshalb Wasserversorgungszeiten für beide Lager mitgeteilt wurden 23. Vierzehn Tage vorher hatte die DÖLF (= Deutsche Ölschiefer Forschungsgesellschaft) den Kommandoführer in Kenntnis gesetzt, dass das Wasser der Schlichemtalsperre, die gerade fertig geworden war, durchaus zu Trink- und Kochzwecken verwendet, und zwar abgekocht für die K Lager in Wasserklemmzeiten ohne Gefahr benützt werden (könne). Dagegen kann die Verwendung in einer Gemeindewasserversorgung nicht in Frage kommen, solange nicht eine besondere Filtrier- und ChlorierAnlage eingerichtet wird 24. Der Titel SS-Untersturmführer und das Datum weisen möglicherweise auf Erwin Dold, dem es wichtig war, überhaupt Wasser zu bekommen, obwohl Schlichemwasser der zivilen Bevölkerung von Schömberg nicht zuzumuten war, jedoch durchaus den KZ-Häftlingen. Dass die Entlausungs- und Waschsituation sich verbessert habe, darf bezweifelt werden. Geblieben bis in Dolds Zeit ist der Schlamm des Appellplatzes, der zum Sonntagsvergnügen der SS von Gefangenen umgegraben werden musste, damit weiter Matsch bestehe, in den Häftlinge hineingetrieben wurden: Menschenverachtung einiger Verantwortlicher, die für sich selbst Laufstege hatten bauen lassen, damit ihre Stiefel nicht schmutzig würden 25. Dautmergen größtes KZ-Lager für «Unternehmen Wüste», um aus Ölschiefer Treibstoff zu gewinnen Das KZ Dautmergen wurde als größtes Lager errichtet für das «Unternehmen Wüste», dem spektakulären, wenn auch sinnlosen Versuch, aus schwäbischem Schiefer epsilon in industriellem Maßstab Öl-Treibstoff herzustellen. Schömberg wurde das technische Zentrum der DÖLF, deren Versuchsanlage die Verschwelung im «Meilerverfahren» erprobte. Denn die Tatsachen, dass a) genügend Schiefer, allerdings nur der des wenig ergiebigen Schwarzen Jura, b) eine angeblich günstige Verschwelungsmethode und c) das technische Personal aus Estland vorhanden waren, schienen das Unternehmen «Wüste» zu begünstigen. An der Bahnlinie Tübingen-Rottweil entlang sollten zehn Ölschieferfabriken entstehen. Allerdings waren Ende März 1945, also zur Zeit, in der Dold Kommandant in Dautmergen war, von den 10 im Juli 1944 in Angriff genommenen Ölschiefer399 Dold habe «das Leben im Lager stark verbessert» – Weniger Arbeit, Nahrung und Schuhe besorgt Reine Schikane: Die KZ-Häftlinge müssen in Dautmergen den Appellplatz umgraben. Schwelwerken im Raum Württemberg 5 im Dezember 44 in ihrer Dringlichkeit zurückgestellt und am 2. März 1945 stillgelegt (worden). Dafür wurden die übrigen fünf Werke bevorzugt durchgezogen. Es sind inzwischen angefahren. (...) Werk 2,4, 8 und 9 26. Die Werke 6, 7, 8 (Dormettingen) und 9 (Schömberg, Heimental) konnten durch Fußmärsche der Häftlinge erreicht werden: dafür wurde das KZ Dautmergen an dieser Stelle gebaut. Dass Wüste 9 betriebsfertig war, musste Dold gemerkt haben, denn neue Arbeitskräfte wurden in der Nähe seiner Wohnung in der Dautmerger Straße untergebracht. Die DÖLF «Betrieb 9» beantragte vom 5. bis 18. März 1945 in der «73. Zuteilungsperiode» – die Kriegsbewirtschaftung dauerte an – beim Ernährungsamt für ca. 250 ausländische Zivilarbeiter Raucherkarten; die Russen waren in einer Baracke, möglicherweise in der Nähe des Werkes an der Zimmerner Straße, Italiener, Franzosen, Holländer im «Traube-Saal» in doppelstöckigen Betten 27 zusammengesperrt. Diese Arbeiter kamen aus Stuttgart von Daimler-Benz 28, müssten Dold aufgefallen sein, denn er wohnte in Sichtweite der «Traube». 400 Als KZ-Kommandant hatte sich Dold jedoch mit anderen Dingen zu beschäftigen. Er habe, sagt er im Rückblick, einen Bericht an die Kommandantur in Natzweiler über die Zustände im KZ Dautmergen geschickt; gleicher Bericht ging auch der OT (Organisation Todt) zu 29. Dold scheint demnach geglaubt zu haben, noch etwas durch Beschwerde verändern zu können. Ich konnte es dann durchsetzen, dass die Häftlinge etwa vier Wochen lang nicht zur Arbeit mussten. Die Erinnerungen an drei Wochen Quarantäne wegen einer von ihm zusammengereimten angeblichen Epidemie, deretwegen 1.700 Männer nicht zur Arbeit geschickt werden konnten, beherrscht die Radio- und TV-Features. Ebenso der Wunsch, die Krankenquote so niedrig wie möglich zu halten: die abenteuerähnlichen Geschichten über seine wahnsinnige Kolonne, die aus ausgesuchten Häftlingen verschiedener Nationen bestanden, unter Jagdbomber-Beschuss in Lebensgefahr Nahrung, Decken, Schuhe, Socken, Unterhosen etc. besorgt und dafür z. B. die Wachen im «OT-Lager bei Balingen» mit Schnaps betrunken gemacht habe, scheinen den «Mythos Dold» zu bestätigen. Wahrscheinlich war im NS-Endstadium das Chaos so groß, dass die von ihm festgestellte absolute Desorganisation der KZ-Realität entsprach, sodass er sich selbst Marschbefehle samt Natzweiler-Stempel habe ausstellen und sich nur mit Lüge (habe) durchsetzen können 30. Sein Organisationstalent war auch hier wie in Haslach gefordert. Spielten Erinnerungen an frühere eigene Mentalität während seiner Zeit als Jagdbomberpilot eine Rolle, wenn der Beschuss durch «Jabos» immer zur Standarderzählung gehört? 31 Wie groß war die Empathie mit den Gefangenen? Er habe verhindern wollen, dass jeden Tag 60 in die Grube gekommen seien. Offensichtlich sprach Dold mit den Ärzten Dr. Rohde oder Dr. Steinicke über die Anzahl der Toten, denn diese mussten in der Arzt-Baracke zwischen dem Lager Dautmergen und Schömberg nahe dem Ortseingang gemeldet werden. Die Ärzte schrieben manchmal die für Gefangene typischen Todesursachen auf die Meldescheine, nämlich «Lungentuberkulose», «Darmkatarrh», «Herzmuskelschwäche», «Sepsis», «Phlegmone». Und Dold, der jedes Mal die Arztbaracke auf dem Weg zu seiner Wohnung passierte, muss die Zahlen verglichen haben, denn in seiner Zeit gab es nicht mehr so viele Tote wie in den Monaten November/Dezember 1944 und Januar 1945. Von den 1775 im Schömberger Rathaus beurkundeten Todesfällen sind für 1944 836, für 1945 insgesamt 939 eingetragen 32. Schwäbische Heimat 2010/4 Ein Vergleich der Totenzahlen im Januar mit denen des Februar, März und April 1945 zeigt einen kontinuierlichen Rückgang, jedoch ist ein kausaler Zusammenhang mit der Anwesenheit Dolds und seiner Sorge für die Häftlinge nicht nachzuweisen 33. Dennoch wurde sein Kommen von diesen als Zäsur und Wende zum Positiveren empfunden. Viele ehemalige Dautmerger Häftlinge sagten in verschiedenen Variationen aus: Der Lagerchef Dold hat seit seiner Ankunft das Leben im Lager stark verbessert. – Er hat mir das Leben gerettet. Er hat bei den Zivilisten Essen requiriert und er hat die Appelle abgeschafft. – Obwohl er SSAngehöriger war, war er sehr gut. – Dold war ein guter Mensch 34. Die Aussagen der Zeugen erwecken den Eindruck, als habe er vor allem den Franzosen, die seine Sympathie hatten, das Leben erleichtern wollen 35, oder dadurch, dass er seine Alt-Haslacher glücklicherweise zusammen in der Baracke 2 unterbringen ließ. War Dold also der von Franzosen sogenannte «Weiße Rabe», der sich von anderen fundamental unterschied, weil er Glück hatte und vieles glücklich verändern konnte, sodass ihm zu wünschen ist, dass er lange lebt 36? Die ehemaligen Häftlinge Serge Lampin Baracke «Schonung»: Kranke und entkräftete Häftlinge konnten zeitweise von der Schwerstarbeit befreit werden, allerdings waren sie nicht vor der Willkür der Wachen geschützt. Somit steht «Schonung» auch für den sicheren Tod. Schwäbische Heimat 2010/4 und René Colin, beide Zeugen in Rastatt, stellten in einem Gespräch mit dem Verfasser dar, dass Dolds gutes Leumundszeugnis im Prozess auch von uns evoziert, den Häftlingen also zu verdanken sei, denn er sei kein Peiniger gewesen. Sie, die Zeugen, wollten jedoch nicht Märchen von und über Dold hören, er habe Schläge nicht verhindert, obwohl er der «Chef» gewesen sei, denn er habe keine Autorität gehabt. Ein Pilatus sei Dold nicht 37. Dolds vergeblicher Versuch, einer Exekution zu entgehen – Am 7. April 1945 werden 22 Russen erschossen Zur Beurteilung, ob Dold ein antifaschistischer «Held» war, der Widerstand geleistet habe, lässt sich die «Erschießung im Scheinwerferlicht», die in der Woche nach Ostern 1945 im KZ Dautmergen stattfand, anführen. Das Datum der Exekution ist nicht ganz geklärt, denn sie fand entweder am Donnerstag, 5. April, oder Samstag, 7. April statt 38. Dolds Aussagen zufolge wurden am 30. März diese 22 Gefangenen (...) in einem vergitterten Polizeiauto in das Lager Dautmergen überstellt zur Exekution, die sind verurteilt worden, also ich weiß nicht mehr, als Spione oder als Saboteure. Häftlinge in Zivil auf einem LKW des Sicherheitsdienstes von der SD-Dienststelle im Murgtal. – Man wollte mir 22 Häftlinge übergeben. Ich habe mich geweigert, denn es waren Zivilisten. Es habe eine heftige Debatte mit dem SD-Offizier gegeben, der Dold wegen Befehlsverweigerung (habe) zur Meldung bringen wollen. Dadurch habe es eine Verzögerung von ein paar Tagen gegeben. Weil Dold zunächst den Auftrag erhielt, für den Abend ein Exekutionspeleton zusammenzustellen, weigerte er sich, versuchte die Hinrichtung in andere Hände zu legen und fuhr nachmittags nach Balingen, um der Exekution aus dem Wege zu gehen. Das gelang ihm jedoch nicht, denn bei seiner Rückkehr um 20 Uhr war die Erschießung noch nicht vollzogen, weil vorher noch eine Erhängung stattfinden musste, bei der der Strick riss. Der Unglückliche wurde zum Revier gebracht, wo ihm später, wie Dold sich ausdrückt, der Garaus gemacht wurde. Die verräterische Sprache zeigt den offensichtlichen Widerspruch zur später immer wieder geltend gemachten «Gewissensfrage» Dolds, der sich geweigert habe, Häftlinge als Volksschädlinge zu betrachten. Widersprüchlich ist ebenso Dolds Bericht über seine Flucht, seine Weigerung, selbst Verantwortung zu übernehmen: Er scheint sich insofern durchgesetzt zu haben, dass dann ein Exekutionskommando (...), nur SS-Angehörige, (...) für mich fremde Gesichter zum Lagertor herein(gekommen sei). Die von außerhalb des Lagers Dautmergen zur Exekution 401 Ludovic de la Chapelle hat das Erschießen im Scheinwerferlicht (oberes Bild) und das Erhängen fast fotografisch genau mit malerischen Mitteln festgehalten. bestimmten 22 Russen wurden in zwei Elfer-Gruppen aus dem Waschhaus, wo man sie von Karfreitag an separiert untergebracht hatte, zur Exekutionsstätte zwischen Waschbaracke und einem der Blocks herangeführt und aneinandergefesselt. Der Erschießung im Scheinwerferlicht eines PKWs mussten alle Häftlinge zusehen; die Delinquenten wurden mit Dumdum-Geschossen getötet. De la Chapelle verarbeitete das Geschehen in seinem Bild wie eine Thea402 terinszenierung auf dem Appellplatz, der polnische Dichter Tadeusz Borowski in seiner Erzählung «Das Abendbrot», deren fürchterlich-makabre Pointe das Essen von Gehirnresten durch Mitgefangene beschreibt 39. Vom Lagerbüro am Eingang des KZ, aus 40–60 Metern Entfernung, beobachtete Dold das Geschehen, ging jedoch wieder zu seiner Unterkunft gegenüber dem Eingang, weil es eben eine Sache (war), die einem (den Magen?) umgedreht hat. Wo die Leichen der getöteten Russen auf wessen Anweisung verscharrt wurden, ist nicht genau bekannt, vermutlich in einer der Gruben im «Schönhager Loch» wie alle anderen: vom 7. April 1945 an, dem mutmaßlichen Tag der Erschießung, wurden in den Schömberger Rathauslisten keine KZ-Toten mehr registriert. Der Begriff «Exekution» weist auf ein Gerichtsurteil, nach dem eine «Hinrichtung» vollstreckt werden sollte. Darum ging es, als Erwin Dold 1965 Zeuge im Prozess gegen Hofmann 40 war und gefragt wurde, warum er die Erschießung abgelehnt, ob er sich Gedanken gemacht habe, dass es rechtens sei, was den Menschen geschehe. Er antwortete, dass er nicht beurteilen könne, ob die Russen zurecht verurteilt worden seien. Es sei ein Urteil da gewesen, das ihm in einem Umschlag mit Unterlagen und Akten bei der Überstellung gezeigt worden sei, Genaueres könne er nicht sagen. Schon im Rastatter Prozess von 1946/47 hatte Dold ausgesagt, er könne nicht glauben, dass es etwas anderes war als ein Urteilsspruch, gelesen hat er das Urteil jedoch nicht, sondern es reichte ihm, den Briefkopf zur Kenntnis zu nehmen. Richtigkeit oder Legalität des Urteils hinterfragte er nicht, es hätte ihm auch nichts genützt. Erwin Dold wollte verständlicherweise nur selbst überleben im Chaos der letzten Tage des Regimes, dessen Ende offensichtlich nahte. Wenn die Gefangenen schon fast eine Woche vorher ins KZ Dautmergen zur Erschießung überstellt worden waren, hatte Dold über Ostern 1945 Zeit zu überlegen, wie er selbst unbeschadet aus dem Befehlswirrwarr herauskommen konnte; deshalb die Flucht nach Balingen und das Bemühen, nicht anwesend zu sein. Im Abstand von 20 Jahren zu den Geschehnissen bestätigt Dold 1965 die Meinung des Richters: An erster Stelle stand, dass hier Menschen erschossen werden sollten, und das wollten Sie nicht tun. Eine Exkulpierung ex eventu? Dold entschied sich aus Selbsterhaltungstrieb dafür, ein fürchterliches Urteil nicht zu prüfen, nicht Verantwortung zu übernehmen für etwas, das auch ein KZ-Kommandant nicht mehr hätte verhindern können. Schwäbische Heimat 2010/4 Räumungsbefehl – Dold in französischer Gefangenschaft – Der «Kriegsverbrecher» exhumiert in Schömberg Leichen Zehn Tage nach der Erschießung wurde das Lager Dautmergen am 16. April evakuiert, nachdem bereits vorher Transporte per Bahn nach DachauAllach oder in ein Krankenlager befohlen worden waren. In offenen Waggons sollte dies geschehen, verantwortungslos, weil es regnete und entsprechend der Jahreszeit auch kalt war, bemerkte Dold dazu, verhinderte die Transporte nicht, sondern ließ die Wagen wetterfester machen. Der Räumungsbefehl für das KZ kam per Telefon oder Kurier, nämlich die Häftlinge in Richtung Ravensburg zu Fuß in Marsch zu setzen. Bei diesem Todesmarsch, über den authentische Erinnerungsberichte von Häftlingen vorliegen 41, war Dold nach eigenem Zeugnis mal vorne und (...) einmal hinten mit seinem Motorrad. Er berichtete: Und vor allen Dingen ging es mir darum, dass auch auf dem Marsch, wir wussten ja nicht, was kommt und wohin es gehen soll, dass da wieder für Nahrung gesorgt war für die Leute. Die Erzählung und Erklärung dienten wohl der Vertiefung der Selbsteinschätzung, denn Dold wunderte sich darüber, dass ihm, einem Unteroffizier der Luftwaffe, ein ranghöherer SS-Oberscharführer unterstellt war, dass man die Leute zu einer solchen großen verantwortungsvollen Aufgabe einteilen kann, ohne dass sie die Qualifikation bewiesen haben. Im Subtext meinte Dold natürlich sich, und es hat den Anschein, als ob er habe beweisen wollen, dass er sich trotz seiner Jugend, trotz der eingestandenen «seelischen Probleme» für die Herausforderung, ein KZ-Kommandant zu sein und den Todesmarsch durchzuführen, qualifiziert habe, dass er nicht überfordert gewesen sei. Nach den Zeugnissen von Häftlingen dauerte das Morden der nicht marschfähigen Häftlinge (...) den ganzen Weg nach Garmisch-Partenkirchen 42. Noch im Oberland muss Dold zwei Tage nach der Befreiung durch die Franzosen gefangen genommen, jedoch auf Einspruch von ehemaligen Häftlingen wieder freigelassen worden sein. Er konnte nach Hause gehen, wo er sich bis zu seiner erneuten Verhaftung im Juli 1946 familiären Dingen widmen konnte. Diesmal wurde er in das Internierungslager für «Kriegsverbrecher» nach Reutlingen überstellt. Mittlerweile sollten in Schömberg nach einjährigem Warten 43 die Toten der KZ Dautmergen und Schömberg aus dem «Schönhager Loch» umgebettet und in einer würdigen Grabstätte auf dem «Ehrenfriedhof» erneut begraben werden. Dazu wurden in Schömberg Vorbereitungen getroffen: Eine abgeschlagene Baracke vom Werk 9 musste auf den neuen Friedhof geführt, zwei Notaborte im Auftrag von Capitaine Rabaste angelegt werden, damit die 80 Mann aus dem Kriegsverbrecherlager Reutlingen, die die Umbettungen vornehmen sollten, untergebracht werden konnten, unter ihnen auch Erwin Dold. Dieser war somit zum dritten Mal in Schömberg, erlebte das, Massengrab im «Schönhager Loch» mit 53 Toten, geöffnet am 21. August 1946. Schwäbische Heimat 2010/4 403 was er hatte verhindern wollen, am eigenen Leib, nur graduell anders: Man könnte es als Ironie des Schicksals bezeichnen, dass der letzte Kommandant die KZ-Toten der Zeit vor ihm ausgraben musste, deren Zahl er während seiner kurzen LagerführerZeit zu verringern sich angestrengt hatte. In einem Grab waren einmal 26, die habe ich selber alle freigelegt, und zwar in Sichtweite zum ehemaligen KZ Dautmergen, das 1946 als Lager für deutsche Kriegsgefangene benutzt wurde, für die es unmittelbar nach dem Krieg von damaligen Jugendlichen gesäubert worden war 44. Militärgericht Rastatt: «Freispruch für Nr. 40» – Erwin Dold ein «unschuldiger» KZ-Kommandant? Im November 1946 begann für Dold der Prozess in Rastatt, der mit dem Freispruch für Nr. 40 endet. Dabei wurde deutlich, dass nur die Einmütigkeit der gehörten Zeugen dem Angeklagten Dold zu diesem nichtschuldig verholfen hat, immer im Vergleich zu seinen Vorgängern. Mit dem Richterspruch des Militärgerichts der Besatzungsmacht im Rücken konnte Dold gestärkt in spätere Vernehmungen gehen, bei denen er jedoch vermied, von seiner SS-Mitgliedschaft und Tätigkeit in Lagern des Ostens nach seiner Genesung zu sprechen, denn den guten Ausgang des Rastatter Prozesses wollte er nicht in Frage stellen. Er pochte vielmehr auf seine Einmaligkeit als freigesprochener KZ-Kommandant 45. Primo Levi schreibt in seinem Buch «Die Untergegangenen und die Geretteten» über seine KZErfahrungen in Auschwitz und das Erinnern: Aber es ist ebenso wahr, dass eine Erinnerung, die allzu oft heraufbeschworen und in Form einer Erzählung dargeboten wird, dahin tendiert, zu einem Stereotyp, das heißt zu einer durch die Erfahrung getesteten Form zu erstarren, abgelagert, perfektioniert und ausgeschmückt, die sich an die Stelle der ursprünglichen Erinnerung setzt und auf ihre Kosten gedeiht 46. Dies dürfte auch für die Geschichte des Erwin Dold gelten. Er lebt heute im 91. Lebensjahr in seinem Geburtsort. Der Wunsch einiger Zeugen nach einem langen Leben für ihn ging also in Erfüllung. ANMERKUNGEN 1 Originalzitat im Gästebuch der Ausstellung «Das Unternehmen Wüste», Zehntscheuer Balingen 13. September – 17. November 1994. Im Besitz des Verfassers. – Seiterich-Kreuzkamp hatte im «Publik Forum» 1989 über Dold geschrieben und 1990 mit Serge Lampin und René Colin über diesen in Paris gesprochen. Vgl. Publikforum Nr. 7 vom 13. April 1990, S. 34/35 «Ein Kommandant den der Himmel schickte» und Publikforum Nr. 21 vom 18. Oktober 1991 «Ohne unsere Freundschaft hätten wir nicht überlebt» 404 2 Andreas Möller: «Ich wollte, dass sie leben». Bericht über einen Helden. In: «Der Schwarzwälder am Wochenende» 15./16. Dezember 1973, Spalte zwei oben. Thomas Seiterich übernimmt große Teile dieses Artikels für seinen Beitrag im Publikforum 1990. 3 Wolfram Wette (Hg): Stille Helden. Judenretter im Dreiländereck während des Zweiten Weltkrieges. Freiburg 2005. S. 215 ff. 4 So die Formulierung bei Andreas Möller, vgl. Anm. 2. 5 Alle folgenden, nicht gekennzeichneten Zitate sind Aussagen von Dold aus dem Prozess-Akten, in denen Dold zur eigenen Biografie als Angeklagter oder als Zeuge aussagte. Unautorisierte Übersetzung des Proces Verbal ab 9. Dezember 1946. 419 AR- Z 33 /61, S. 276. Vernehmung Erwin Dold am 7. 9. 1965. Sammlung Unternehmen Wüste Nr. 59, S. 49, des Archivs Zollernalbkreis. Vernehmung Dold Landespolizeiposten Kirchzarten 30. 7. 1959; 218 Sa UW 2 Kreisarchiv Zollernalbkreis. Provenienz Staatsarchiv Ludwigsburg EL III, Bü 1240 (Bd. 4, Bl. 630 – 642) 6 Vgl. Victor Klemperer: «LTI». Die Unbewältigte Sprache. München 1969 (dtv). S. 188 u.ö. 7 Martin Weinmann: Das nationalsozialistische Lagersystem. Frankfurt am Main 2001, S. 175: «CCkdo. Of Natzweiler, located at the «Sportplatz», established Sept.44, in Existence until Jan 45.» 8 Ebenda: «about 250 pris. (original No.book Natzweiler)» 9 Ebenda: «Branch camp of Sicherungslager Schirmeck established in Nov. 44 till April 45 and was disbanded shortly before the arrival of the allied troops. About 800 pris. were employed in the underground galleries of the «Vulcan» quarry about 5 km SE of Haslach installing machines from DaimlerBenz aeroplane motors factory in tunnels». – Ebenda S. 542 wird zu Haslach die Zahl 756 genannt, allerdings als CWC (= Civilian workers camp) mit der Provenienz FNTB (=French National Tracing Bureau). 10 Uwe Schellinger: Haslach im Kinzigtal («Barbe»). In: Der Ort des Terrors. Geschichte der nationalsozialistischen Konzentrationslager. Band 6 Natzweiler, Groß-Rosen, Stutthof. München 2007. S. 106 11 Zeitungsbericht von 1973 (wie Anm.2) zitiert Dold selbst: Die sprachliche Hyperbel nach 40 Jahren entspricht dem damaligen Erschrecken, bezieht sich jedoch vielleicht auf Dautmergen. 12 Uwe Schellinger: Haslach im Kinzigtal («Barbe»). A.a.O. S. 103 ff. Zu dem Begriff «Nacht und Nebel-Häftlinge» schreibt Trygue Wyller selbst NN-Häftling im KZ Erzingen: Denn das Schicksal des N.N.-Gefangenen war es eben, durch eine absolute Nacht und einen undurchdringlichen Nebel in Vergessenheit zu sinken und seine Identität zu verlieren. Niemand sollte wissen, wo sie sich aufhielten. (…) Selbst ihr Tod sollte geheimgehalten werden. Und die Behandlung, der sie ausgesetzt wurden, bedeutete maskiertes Todesurteil. In: Egil A.Wyller: Gestern und Morgen Heute. Henologische Essays zur Europäischen Geistesgeschichte. Würzburg 2005. 13 Ludovic de la Chapelle: Souvenirs d’Allemagne. Hgg. von Marjolaine de la Chapelle, seiner Tochter, die seine Notizen vor seinem Tod mit ihm besprochen hat. Erschienen im Verlag Diotime editions ohne Jahr. – Ludovic de la Chapelle hat die Natzweiler-Nummer 39875: er trifft Dold in Dautmergen wieder. 14 de la Chapelle a.a.O. S. 56/7 15 Gustav Steinger, 192. Zeuge im Prozess von Rastatt. a.a.O. Proces Verbal S. 226 16 SWF-Sendung «Hierzuland» Januar 1990 von Wolfgang Scherer: Freispruch für Nummer 40, ein Radio-Feature zu Erwin Dold. 17 Vgl. Anm. 1. Serge Lampin war vom 21. September bis 21. November 1944 im KZ Dautmergen mit der Natzweiler-Nummer 36008 eingesperrt. Laut Dold selbst war das im Dezember 1944. Der Lagerführer war nicht da, und ich habe diese Inspektion geführt und das Lagerleben beschrieben Proces Verbal S. 276 18 Christine Glauning: «Dautmergen» In: Der Ort des Terrors. Band 6. S. 71 ff. Schwäbische Heimat 2010/4 19 Ebenda 20 Befragung Dolds Kirchzarten 1959 wie Anm. 11. Bei seinem Prozess in Rastatt hatte er gesagt, er sei freiwillig nach Dautmergen gekommen. 21 Immo Opfermann: Das Unternehmen Wüste. Ölschieferwerke und Konzentrationslager entlang der Bahnlinie TübingenRottweil 1944/45. Leitfaden zur Ausstellung. Balingen 1997, S. 63. 22 Floris B. Bakels: Nacht und Nebel. Der Bericht eines holländischen Christen aus deutschen Gefängnissen und Konzentrationslagern. München 1982 (Fischer Taschenbuch) S. 265 ff. «Außenkommando Dautmergen 21. September – 20. November 1944.» 23 StA Schömberg 2 Nr. A 612. Briefkopf des Briefes vom 17. März 1945: Waffen-SS. Der Kommandoführer für das Bauvorhaben «Wüste», Adressat Herr «Giener» zur Kenntnisnahme. Vgl. Immo Opfermann: Schömberg 1918–1946. In: Geschichte der Stadt Schömberg, hgg. von Casimir Bumiller. Schömberg 2005. S. 227f. 24 StA Schömberg 2 Nr. A 612 Schreiben vom 2. 3. 1945 des Wasserwirtschaftsamtes Rottweil, gez. Oberregierungsbaurat Seybold: Ich bitte (...) Ihr K Lager zu verständigen. 25 de la Chapelles Bild «Umgraben des Appellplatzes» ist durch einen persönlichen Brief an den Verfasser verifiziert. 26 Brief Pohls, des SS-Obergruppenführers und Generals der Waffen-SS, an den Reichsführer SS Himmler vom 29. März 1945. 27 Mündlich von Elfriede Zirkel, geb. Koch, der Tochter des Traubenwirtes, Zeugin in Rastatt. 28 Brief vom 5. 4. 1945. Deutsche Ölschiefer-Forschungsgesellschaft m.b.H. Betrieb 9 an das Wirtschaftsamt über Ernährungsamt, Abt. B in Schömberg. StA Schömberg 3, A 1387 Die toten Häftlinge des KZ Dautmergen werden exhumiert. Einer der Grabenden könnte Erwin Dold sein. Schwäbische Heimat 2010/4 29 Befragung Dold Kirchzarten 1959 wie Anm. 13; OT= Organisation Todt als für den Aufbau und die Verpflegung in den KZs verantwortliche Stelle. 1990 sagt Dold, er habe mit Natzweiler telefoniert. 30 Scherers SWF-Feature 1990 und Manfred Bannenbergs NDRTV-Film «Der KZ-Kommandant» 1991. 31 Noch 1991, als Manfred Bannenberg am KZ-Friedhof Schömberg mit Dold zusammentrifft, zeigt er auf das Zementwerk Dotternhausen, dessen Schornstein Angriffsziel für Jabos gewesen sei; tatsächlich wurde im März 1945 der Fabrikschornstein getroffen, für viele Häftlinge ein Symbol des nahen Endes der Quälerei in den Ölschiefersteinbrüchen. – Allerdings bedurfte es einer Erinnerungskorrektur durch den Verfasser, der bei den lokalen Dreharbeiten anwesend war, weil Dold den Ort des KZs Dautmergen nicht mehr lokalisieren konnte, sondern den KZ-Friedhof für das KZ hielt. 32 StA Schömberg Nr. A 1385 33 Chronologischer Etat des décès d’étrangers depuis 2 septembre 1939 établi le 22 septembre 1945, dem Verzeichnis der Todesfälle von Ausländern seit 2. September 1939, aufgestellt am 22. September 1945, unterzeichnet vom Standesbeamten Söll. 34 Proces Verbal, Unautorisierte Fassung: 67. Zeuge Ari Gordon (S. 128), 65. Zeuge Gilbert Ecuyer (S. 124), 66. Zeuge David Billender (S. 126), 70. Zeuge Zelik Gurwiez (S. 130). 35 De la Chapelle, Souvenirs (...) S. 60 …est administré par Dold heureusement qui rassenmblé tous ses «anciens» de Haslach dans la baraque n° 2 36 Der 84. Zeuge Charles Muard sagt dies (S. 147). SWR-Sendung «Hierzuland» 1990. – Der 81. Zeuge Moses Lewin wünscht Dold ein langes Leben, Proces Verbal (S. 142) 37 Video-Film eines Interviews mit Serge Lampin und René Colin, gedreht von Mitgliedern der Geschichts-AG des Verfassers in Rottweil, Parkhotel, Oktober 1992 im Zusammenhang mit den Vorbereitungen zur ersten «Wüste»-Ausstellung in Balingen 1994, in der das Video dem Film «Der KZ-Kommandant» gegenübergestellt wurde. Ausgangspunkt der Fragen an die Zeugen waren die Artikel im Publikforum (vgl. Anm. 1 und 2), von denen sich die Befragten distanzieren wollten. Seiterich-Kreuzkamp scheint entsprechend seinem Eintrag im Gästebuch beide Filme gesehen zu haben. (vgl. Anm. 1 und 2) 38 Andreas Zekorn: Ausbeutung und Tod. Das Schicksal von KZHäftlingen am Beispiel der Lager des Unternehmens «Wüste». In: Opfer des Unrechts. Stigmatisierung, Verfolgung und Vernichtung von Gegnern durch die NS-Gewaltherrschaft an Fallbeispielen aus Oberschwaben. Hgg. von Edwin Ernst Weber im Auftrag des Landkreises Sigmaringen und der Gesellschaft Oberschwaben für Geschichte und Kultur. Stuttgart 2009. S. 193ff. 39 Tadeusz Borowski, Das Abendbrot. In: Borowski: Bei uns in Auschwitz. Frankfurt 2006. S. 337 40 Franz Johann Hofmann, SS-Hauptsturmführer Zivilkommandant von Bissingen. 41 Beispielsweise Lèon Donven, Nr. 2218 in Schörzingen, oder Jerzy Sztanka. In: Wüste 10, Gedenkpfad Eckerwald. Das Südwürttembergische Schieferölprojekt und seine Konzentrationslager. Das Lager Schörzingen und sein Außenkommando Zepfenhan. Hgg. von der Initiative Eckerwald e.V. DeißlingenLauffen. 3. Auflage 2001. S. 63 42 Stanka a.a.O. S. 52 43 Zunächst hatte man den Plan, im «Schönhager Loch» über dem Verscharrungsort der Toten eine «Gedenkstätte» zu errichten, ohne Exhumierung. Dieser Plan wurde im August 1945 verworfen. Ein Jahr später erst begannen die Umbettungen. Der KZ-Friedhof Schömberg wurde am 23. Oktober 1946 seiner Bestimmung übergeben. 44 Verschiedene Dokumente des Stadtarchivs Schömberg vom 10.–27. August 1946 zeigen die Verpflichtungen von Schömbergern durch die Militärregierung. St A Sch. Nr. 1585 45 Proces Verbal Rastatt , S. 354; vgl. Opfermann, Wüste-Broschüre 1997, S. 111 und Kreisarchiv Zollernalbkreis, Sa UW 29 Blatt 1900 als Zeuge im Prozess gegen Hofmann. 46 Primo Levi: Die Untergegangenen und die Geretteten. München 1993, S. 20. 405
https://openalex.org/W2323800198	https://europepmc.org/articles/pmc4607303?pdf=render	English	null	Relation between Water Balance and Climatic Variables Associated with the Geographical Distribution of Anurans	PloS one	2,015	cc-by	11,551	RESEARCH ARTICLE Relation between Water Balance and Climatic Variables Associated with the Geographical Distribution of Anurans Braz Titon, Junior, Fernando Ribeiro Gomes Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, São Paulo, Brazil Braz Titon, Junior, Fernando Ribeiro Gomes Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, São Paulo, Brazil * titonjr.b@hotmail.com * titonjr.b@hotmail.com * titonjr.b@hotmail.com OPEN ACCESS OPEN ACCESS Citation: Titon B, Junior, Gomes FR (2015) Relation between Water Balance and Climatic Variables Associated with the Geographical Distribution of Anurans. PLoS ONE 10(10): e0140761. doi:10.1371/ journal.pone.0140761 Editor: Stefan Lötters, Trier University, GERMANY Received: April 2, 2015 Accepted: September 30, 2015 Published: October 15, 2015 Copyright: © 2015 Titon, Gomes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Titon B, Junior, Gomes FR (2015) Relation between Water Balance and Climatic Variables Associated with the Geographical Distribution of Anurans. PLoS ONE 10(10): e0140761. doi:10.1371/ journal.pone.0140761 j p Editor: Stefan Lötters, Trier University, GERMANY Received: April 2, 2015 Accepted: September 30, 2015 Published: October 15, 2015 Copyright: © 2015 Titon, Gomes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Stefan Lötters, Trier University, GERMANY Received: April 2, 2015 Accepted: September 30, 2015 Published: October 15, 2015 Copyright: © 2015 Titon, Gomes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Titon, Gomes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. such as the Atlantic Forest. These results support the hypothesis that the interspecific varia- tion of physiological traits shows an adaptation pattern to abiotic environmental traits. Data Availability Statement: All data underlying the findings are freely available in the manuscript and supplemental files. Funding: This research was supported by: Fundação de Amparo a Pesquisa do Estado de São Paulo, grant 2006/54699-1, led by FRG, and grants 2008/01917-7 and 2010/16804-3, led by BTJ; Fundação de Amparo a Pesquisa do Estado de São Paulo (website: http://www.fapesp.br/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Amphibian species richness increases toward the equator, particularly in humid tropical for- ests. This relation between amphibian species richness and environmental water availabil- ity has been proposed to be a consequence of their high rates of evaporative water loss. In this way, traits that estimate water balance are expected to covary with climate and con- strain a species’ geographic distribution. Furthermore, we predicted that coexisting species of anurans would have traits that are adapted to local hydric conditions. We compared the traits that describe water balance in 17 species of anurans that occur in the mesic Atlantic Forest and xeric Cerrado (savannah) habitats of Brazil. We predicted that species found in the warmer and dryer areas would show a lower sensitivity of locomotor performance to dehydration (SLPD), increased resistance to evaporative water loss (REWL) and higher rates of water uptake (RWU) than species restricted to the more mesic areas. We estimated the allometric relations between the hydric traits and body mass using phylogenetic gener- alized least squares. These regressions showed that REWL scaled negatively with body mass, whereas RWU scaled positively with body mass. Additionally, species inhabiting areas characterized by higher and more seasonally uniform temperatures, and lower and more seasonally concentrated precipitation, such as the Cerrado, had higher RWU and SLPD than species with geographical distributions more restricted to mesic environments, such as the Atlantic Forest. These results support the hypothesis that the interspecific varia- tion of physiological traits shows an adaptation pattern to abiotic environmental traits. Water Balance, Climate and the Geographical Distribution of Anurans patterns of amphibian species richness with water availability is high permeability of the skin, which is also an important organ of respiratory gas exchange [2,3]. Competing Interests: The authors have declared that no competing interests exist. In amphibians, dehydration affects both sprint and endurance performances, potentially reducing the ability to perform ecologically important behaviors, such as prey capture, escape from predators and search for mates [4]. Additionally, dehydration and body temperature have synergistic effects on locomotor performance [5]. Locomotor performance of both dehy- drated and fully hydrated toads (Anaxyrus americanus) increases proportionally with the rise of body temperature, but maximum performance shifts toward lower temperatures in dehy- drated individuals [5]. Additionally, some studies comparing a few species have pointed to a pattern of interspecific variation of this synergistic effect, with species found in open, hot and dry environments showing lower sensitivity of locomotor performance to dehydration at higher temperatures when compared with species from forested and wet environments [6–8]. Few comparative studies on amphibian water balance are based on a large number of spe- cies, and such studies have associated these physiological parameters with interspecific varia- tion in habit or microhabitat use (arboreal, terrestrial, amphibious, and so forth) [9–12]. Regarding differences in microhabitat use, these studies have shown that arboreal frogs have high resistance to evaporative water loss and, consequently, lower rates of water loss than non- arboreal frogs [9,10]. Moreover, field studies have revealed that microhabitat use and season interact to determine interspecific variation in the hydric state of anurans. This pattern sug- gests that voluntary tolerance to dehydration varies with microhabitat use [11]. When active during the wet season, terrestrial species typically show a lower hydration state than arboreal and amphibious species, whereas amphibious species show a lower hydration state in natural refuges selected during the dry season when compared to terrestrial or arboreal species [11]. Additionally, dehydrated anurans uptake water through the pelvic patch, a specialized region of ventral skin characterized by high permeability and rich vascularization [13]. Rates of rehydration are reported to be higher for terrestrial species than aquatic ones and for species from arid environments when compared to species from mesic environments [14–17]. Other studies have also shown that, when compared to species from mesic environments, species that inhabit arid areas have higher levels of vascularization in the pelvic patch and a greater blood flow in this region when in contact with water after dehydration [18–22]. However, the limited number of species that have data on hydric balance precludes the ability to partition the roles of ecology versus phylogeny in shaping the evolution of rehydration rates. The objective of this study was to investigate the evolution of water balance traits in anurans and the association of these traits with the climatic conditions that characterize the habitats found in their geographic distribution. We compared key traits associated with hydration state in a sample of anuran species that inhabit the Brazilian Atlantic Forest and the savannah area called the Cerrado. We predicted that species living in warmer and dryer areas would show a lower sensitivity of locomotor performance to dehydration, increased resistance to evaporative water loss and higher rates of water uptake than species from more mesic areas. Introduction Amphibian species richness increases toward the equator, and higher diversity occurs in wet tropical forests, such as the Amazon Basin and the Atlantic Forest. This pattern in the distribu- tion of species richness is attributed to two major abiotic factors, water availability and temper- ature [1]. In the literature, a commonly suggested functional cause for this association between 1 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Materials and Methods Collection Localities and Animal Maintenance Collection Localities and Animal Maintenance The animals were collected under license for capture and transport from the “Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renová- veis” (IBAMA, process numbers 17377–1 and 29896–1), and procedures for the collection and use of biological material were performed with the approval of the “Comissão de Ética na Experimentação Animal”, process number 62/08-CEEA, UNESP—Univ Estadual Paulista, Botucatu, Biosciences Institute and “Comissão de Ética no Uso de Animais”, process number 120/2010-CEUA, Biosciences Institute, University of São Paulo. Field work at Estação Ecoló- gica de Assis and Parque Estadual Intervales were conducted under authorization of the “Coor- denadoria de informações técnicas, documentação e pesquisa ambiental” (COTEC, process number 260108–000.000.002.011/0 2008), Instituto Florestal, Secretaria do Meio Ambiente. For the other localities, no specific authorization was required. Collection Localities and Animal Maintenance Males from 17 species of anurans were collected in several localities from Brazil (S1–S17 Figs). Dendropsophus microps (N = 7), Scinax hayii (N = 10), S. crospedospillus (N = 2), Hypsiboas polytaenius (N = 9), H. faber (N = 10), and H. bischoffi (N = 8) were collected at the Estação Biológica de Boracéia, Salesópolis, SP (23°39'13.99"S; 45°53'22.41"W) between 20 and 23 Janu- ary 2009. Leptodactylus notoaktites (N = 3), Physalaemus olfersii (N = 7), P. spiniger (N = 8), Proceratophrys boiei (N = 10), and S. rizibilis (N = 10) were collected at the Parque Estadual 2 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Water Balance, Climate and the Geographical Distribution of Anurans Intervales, Ribeirão Grande, SP (24°16'24.55"S; 48°25'2.27"W) between 14 and 17 November 2009. Rhinella ornata (N = 9) were collected from an artificial pond at the University of São Paulo, São Paulo, SP (23°33'51.6"S; 46°43'48.1"W) between 23 and 24 October 2012. Rhinella icterica (N = 10) were collected at the countryside in São Luiz do Paraitinga, SP (23°10'06"S; 45°17'07"W) between 5 and 10 November 2013. Those collection sites belong to the Atlantic Forest Area [23]. Hypsiboas albopunctatus (N = 9), D. minutus (N = 10) and L. podicipinus (N = 7) were collected at the Estação Ecológica de Assis, Assis, SP (22°34'19.88"S; 50° 24'32.82"W) between 5 and 8 March 2009. Rhinella schneideri (N = 9) were collected at the countryside in Luiz Antônio, SP (21°33'05"S; 47°39'16"W) between 25 and 27 February 2013. Those last two sites belong to the Cerrado area [23]. After collection, the animals were brought to the laboratory in the University of São Paulo and kept in individual plastic containers, where they were exposed to the natural light/dark cycles and temperature and provided with freely available water and some type of shelter. They were fed cockroaches once per week. The mea- surements were performed in sequence (resistance to evaporative water loss, sensitivity of loco- motor performance to dehydration and rates of water uptake) for up to 21 days after the animals arrived at the laboratory. When individuals did not appear visually healthy, the next measurements were cancelled. Consequently, the number of species for which we have data dif- fer for the physiological variables. Collection Localities and Animal Maintenance The animals were collected under license for capture and transport from the “Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renová- veis” (IBAMA, process numbers 17377–1 and 29896–1), and procedures for the collection and use of biological material were performed with the approval of the “Comissão de Ética na Experimentação Animal”, process number 62/08-CEEA, UNESP—Univ Estadual Paulista, Botucatu, Biosciences Institute and “Comissão de Ética no Uso de Animais”, process number 120/2010-CEUA, Biosciences Institute, University of São Paulo. Field work at Estação Ecoló- gica de Assis and Parque Estadual Intervales were conducted under authorization of the “Coor- denadoria de informações técnicas, documentação e pesquisa ambiental” (COTEC, process number 260108–000.000.002.011/0 2008), Instituto Florestal, Secretaria do Meio Ambiente. For the other localities, no specific authorization was required. Intervales, Ribeirão Grande, SP (24°16'24.55"S; 48°25'2.27"W) between 14 and 17 November 2009. Rhinella ornata (N = 9) were collected from an artificial pond at the University of São Paulo, São Paulo, SP (23°33'51.6"S; 46°43'48.1"W) between 23 and 24 October 2012. Rhinella icterica (N = 10) were collected at the countryside in São Luiz do Paraitinga, SP (23°10'06"S; 45°17'07"W) between 5 and 10 November 2013. Those collection sites belong to the Atlantic Forest Area [23]. Hypsiboas albopunctatus (N = 9), D. minutus (N = 10) and L. podicipinus (N = 7) were collected at the Estação Ecológica de Assis, Assis, SP (22°34'19.88"S; 50° 24'32.82"W) between 5 and 8 March 2009. Rhinella schneideri (N = 9) were collected at the countryside in Luiz Antônio, SP (21°33'05"S; 47°39'16"W) between 25 and 27 February 2013. Those last two sites belong to the Cerrado area [23]. After collection, the animals were brought to the laboratory in the University of São Paulo and kept in individual plastic containers, where they were exposed to the natural light/dark cycles and temperature and provided with freely available water and some type of shelter. They were fed cockroaches once per week. The mea- surements were performed in sequence (resistance to evaporative water loss, sensitivity of loco- motor performance to dehydration and rates of water uptake) for up to 21 days after the animals arrived at the laboratory. When individuals did not appear visually healthy, the next measurements were cancelled. Consequently, the number of species for which we have data dif- fer for the physiological variables. PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Sensitivity of locomotor performance to dehydration Locomotor tests were performed at 40% RH, 25°C and five consecutive levels of hydration (100, 90, 80, 75 and 70% of the standard body mass), and the animals were maintained inside an environmental chamber with temperature and humidity controls (FITOTRON 011 –Eletro- lab, São Paulo, São Paulo, Brazil). Prior to testing, animals were maintained in plastic contain- ers (0.13 m×0.13 m×0.11 m) filled with tap water for 1 h at the test temperature. They were then carefully blotted with paper tissue, their bladders were emptied by gently pressing their abdomens, and their body masses were recorded (±0.0001 g). This body mass was considered as the standard mass (hydration level of 100%). The animals were stimulated to jump on the floor of the Fitotron by tapping it gently for six consecutive times. Starting and landing points were marked on the floor, and later, the distance between marks was measured to the nearest 1 cm. The longest jump of the series was used as the best estimate of maximum jumping perfor- mance. Anurans were subsequently dehydrated and locomotor performance tests were per- formed each time the individuals reached one of the intended hydration levels. The evaporative water loss was controlled in order to maintain intervals of 60 min between the locomotor tests. Electric fans were used to accelerate the rates of evaporative water loss when necessary. Conse- cutive tests were performed until anurans reached 75% or 70% of the standard body mass, depending on their general condition and responsiveness to stimuli. Given that toads are char- acterized by high aerobic locomotor capacity [8,24,25], locomotor performance for Rhinella PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 3 / 19 Water Balance, Climate and the Geographical Distribution of Anurans was measured as the distance moved in a circular track (1.5 m diameter) during 10 minutes at each hydration condition. For these animals, consecutive tests of performance at different hydration levels were performed on different days, resulting in a dehydration rate of 10% a day until 80%, and then 5% a day until individuals reached 75% or 70% of the standard body mass. For each individual, graphs were built with the hydration level on the x-axis and the locomotor performance (corrected by the snout-vent length) on the y-axis (S18–S30 Figs). Resistance to evaporative water loss Prior to measuring evaporative water loss, we maintained the anurans in plastic containers (0.13 m×0.13 m×0.11 m) filled with tap water during 1 h at the test temperature. They were then carefully blotted in paper tissue, their bladders were emptied by gently pressing their abdomen and their body masses were recorded (±0.01 g). Animals were then individually put into clear acrylic chambers based on the size of the species (140 mm diameter × 110 mm high for larger animals and 60 mm diameter X 55 mm high for smaller ones) at 25°C. An open flow system was used to measure the rates of evaporative water loss and the resistance to evaporative water loss. Flow rates of 23 cm3 s-1 for the larger chambers and 5 cm3 s-1 for the smaller ones were generated by a set of air pumps connected to a mass flowmeter (SS-3 Subsampler—Sable Systems, Las Vegas, Nevada, USA) that allowed the same flux for each chamber to be sent indi- vidually. Air pumped to the chambers was maintained at a relative humidity of 20% by using a humidity controller (RH/Dewpoint Controller—Sable Systems, Las Vegas, Nevada, USA). At each measurement, airflow passed through three chambers: one empty chamber, one chamber containing a 3% agar model with size and shape approximated to the size and shape of each species, and one chamber containing the animal. The air leaving each chamber was sent to an 8-channel multiplexer (RM8-Intelligent Multiplexer—Sable Systems, Las Vegas, Nevada, USA) and then to a vapor density analyzer (RH-300 RH/Dewpoint Analyzer—Sable Systems, Las Vegas, Nevada, USA). An interface (UI-2 Data Acquisition Interface—Sable Systems, Las Vegas, Nevada, USA) connected to a computer allowed continuous data recording. Only rec- ords corresponding to periods when the animals stayed in a water conservation posture, identi- fied by visual monitoring and constancy of the water vapor density values, were considered for calculations. After maintaining a stable vapor density value for at least 20 minutes, the cham- bers were opened and the surface temperature of both the animal and the agar model was mea- sured by an infrared thermometer (TR-300 –Equitherm, São Paulo, São Paulo, Brazil). Sensitivity of locomotor performance to dehydration Sensitivity of locomotor performance to dehydration (SLPD) was visually interpolated from these graphs and considered as the hydration state that results in 70% of maximum performance [6,8]. where SA means surface area (cm2) and M means body mass (g). Finally, total resistance to Rates of water uptake Dehydrated anurans (approximately 70% of the standard body mass) were maintained in indi- vidual containers filled with water at a depth sufficient to cover only the ventral region of the animal. The animals were taken, carefully blotted with paper tissue and weighed (± 0.0001 g) every 2 minutes for 6 consecutive times. The rates of water uptake were calculated from the regression of the body mass gain against time and expressed as (μg s-1). Rates of water uptake by area (RWU) were calculated by dividing the absolute rates by 1/3 of the total surface area, corresponding to the ventral surface area in contact with water during rehydration [26]. Although the animals were weighed every 2 minutes to obtain the estimates of RWU, they remained motionless during the tests, and the curves of body mass gain by time presented R2 values higher than 0.9. Species occurrence and climatic data Geographical coordinates of occurrence for each species were compiled from the speciesLink Project (S1–S17 Figs) [29]. For each coordinate, mean data from 1950 to 2000 on eight climatic variables (annual mean temperature, maximum temperature of the warmest month, minimum temperature of the coldest month, temperature seasonality, annual precipitation, precipitation of the wettest month, precipitation of the driest month and precipitation seasonality) were extracted from Worldclim with 30 arc-seconds resolution [30,31] using DIVA-GIS [32] version 7.5.0.0. evaporative water loss was calculated from the following formula: r ¼ VDD=CWL r ¼ VDD=CWL where r means the resistance to evaporative water loss (s cm-1), VDD means the difference between the saturated water vapor density (μg cm-3) at the surface of the animal or the agar model, and the air leaving the chamber containing the animal or the agar model and CWL means cutaneous rates of evaporative water loss by area (μg s-1 cm-2) for the animal or the agar model. Saturated water vapor density at the surface of the animal or the agar model was calcu- lated from the ideal gas law using the surface temperature. The resistance to evaporative water loss calculated for the animal and the agar model correspond, respectively, to the total resis- tance to evaporative water loss and the resistance to evaporative water loss of the boundary layer. By subtracting the boundary layer resistance from the total resistance to evaporative water loss, we obtained the skin resistance to evaporative water loss (REWL) [28]. Resistance to evaporative water loss y q The rates of evaporative water loss were calculated from the formula: EWL ¼ FaVDa FeVDe where EWL means absolute rates of evaporative water loss (μg s-1), Fa means airﬂow (cm3 s-1) through the chamber with the animal or the agar model, Fe means air ﬂow (cm3 s-1) through the empty chamber, VDa means water vapor density (μg cm-3) from the chamber with the ani- mal or the agar model, and VDe means water vapor density (μg cm-3) from the empty chamber. Cutaneous rates of water loss by area (CWL) were calculated by dividing the absolute rates by 2/3 of the total surface area, which correspond to the area exposed to air when anurans keep the water conservation posture [26]. Surface area was estimated using the following formula [27]: SA ¼ 9:9M0:56 where SA means surface area (cm2) and M means body mass (g). Finally, total resistance to here SA means surface area (cm2) and M means body mass (g). Finally, total resistance to PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 4 / 19 Water Balance, Climate and the Geographical Distribution of Anurans evaporative water loss was calculated from the following formula: r ¼ VDD=CWL evaporative water loss was calculated from the following formula: r ¼ VDD=CWL PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Phylogenetic relationships A composite tree with divergence time estimates for the 17 species of anurans was compiled (Fig 1), mainly based on [33], which is the most comprehensible current phylogenetic hypothe- sis for the anurans. Phylogenetic information that was not available in [33] was gathered from [34,35], as follows: to include D. microps, a species from the D. parveceps group of species [35], the topological position and divergence time for D. parveceps in [33] was assumed; to include S. hayii, a species that is within a polytomy that includes S. fuscovarius in [34], the topological position and divergence time for S. fuscovarius in [33] was assumed; and to include S. rizibilis, a species from the clade that includes S. berthae in [34], the topological position and divergence time of S. berthae in [33] was assumed. Finally, P. olfersii and P. spiniger were inserted in the phylogeny considering the maximum time of divergence from this genus in [33]. The phyloge- netic information used to build this composite tree was primarily gathered from molecular studies. For the few instances in which morphological data were employed [34], these were 5 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Water Balance, Climate and the Geographical Distribution of Anurans Fig 1. Phylogenetic tree. Composite phylogenetic tree for the 17 anuran species included in the present study, with topology and divergence times based on the literature [33–35]. Fig 1. Phylogenetic tree. Composite phylogenetic tree for the 17 anuran species included in the present study, with topology and divergence times based on the literature [33–35]. doi:10.1371/journal.pone.0140761.g001 probably not related to the physiological variables investigated in the present study. Conse- quently, the information used to build this composite tree and the data analyzed in the present study were confidently independent. There were no topological divergences between the phylo- genetic proposals used to build our composite tree. The number of species included in the study differed for some physiological measurements. Consequently, the trees used to analyze the relations between climatic variables and physiological variables had 16 species for REWL and 13 species for RWU and SLPD. Results Descriptive statistics for phenotypic variables (body mass, REWL, RWU and SLPD) from each species are presented in Table 1 as the mean ± standard deviation. Descriptive statistics for cli- matic variables from the localities of species occurrence related to temperature (annual mean temperature, maximum temperature of the warmest month, minimum temperature of the coldest month and temperature seasonality) and precipitation (annual precipitation, precipita- tion of the wettest month, precipitation of the driest month and precipitation seasonality) are presented, respectively, in Tables 2 and 3 as the mean ± standard deviation. Statistical analyses Descriptive statistics were performed for all physiological and climatic data per species, and data were posteriorly transformed to Log10 for subsequent analyses. For each species, means of the eight climatic variables extracted from each locality were implemented in principal compo- nent analyses (PCA), and the scores from the components with eigenvalues greater than 1.0 were saved for a posteriori analyses. We considered any absolute values higher than 0.65 as a high load. Again, given that the number of species included in the study differed for some physiological measurements, two PCAs were conducted: one containing climatic data for the 16 species from which there were data on REWL, and another containing climatic data for the 13 species from which data on RWU and SLPD were available. 6 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Water Balance, Climate and the Geographical Distribution of Anurans Phylogenetic regressions were used to investigate the relationship between the physiological variables and body mass [36], and the residuals of data phylogenetic corrected by size were saved to be implemented in a posteriori analyses. Phylogenetic regressions [37] were employed to investigate the relationships between physiological variables and climatic data. Physiological variables corrected by size (SLPD, REWL and RWU) were entered into the regression models as dependent variables, and two components from the PCA of climatic variables with eigenval- ues higher than 1.0 were entered as predictors. Additionally, a phylogenetic ANOVA was implemented using the biome where individuals from the different species were collected for physiological measurements (Atlantic Forest and the Cerrado) as a categorical factor. Descriptive statistics and principal component analyses of the climatic variables were per- formed using the software SPSS for Windows version 13.0. Phylogenetic trees were built using Mesquite version 2.75 (build 564). Procedures for phylogenetic size-correction, phylogenetic regressions and phylogenetic ANOVA were conducted with the software R version 3.0.2 (2013-09-25). The phylogenetic regressions were performed using the function gls, from the package nlme to fit a linear model using generalized least squares. The function corPagel from the package ape, was used to determine the structure of Pagel's “lambda” correlation. The com- parison between sites of collection in the Atlantic Forest and the Cerrado was performed using the function phylANOVA, from the package phytools, with 1000 simulations and Bonferroni correction. Relations between climatic variables The principal component analyses performed on climatic variables retained two components, which were the same when using a data set of 16 or 13 species (Table 4). Component 1 explained 66.36% and 75.68% of the total variance observed for the sets of 16 and 13 species, respectively, and represents a direct association between the annual mean temperature, maxi- mum temperature of the warmest month, minimum temperature of the coldest month and precipitation seasonality, which are inversely associated with the temperature seasonality, annual precipitation and precipitation of driest month. Component 2 explained 19.87% and 18.99% of the total variance observed for the sets of 16 and 13 species, respectively, and is mainly associated with precipitation of the wettest month. Water Balance, Climate and the Geographical Distribution of Anurans Table 1. Mean ± standard deviation of the phenotypic variables collected for 17 species of anurans. Species N Body Mass REWL RWU SLPD (g) (s cm-1) (μg cm-2 s-1) (%) Dendropsophus microps 7 0.55 ± 0.03 9.23 ± 0.93 27.80 ± 4.38 76.5 ± 4.3 Dendropsophus minutus 10 0.50 ± 0.03 - 56.59 ± 19.82 83.5 ± 4.0 Scinax rizibilis 10 0.72 ± 0.07 3.76 ± 1.44 - - Scinax crospedospilus 2 1.25 ± 0.01 3.13 ± 0.01 - - Scinax hayii 10 3.51 ± 0.41 4.42 ± 0.74 57.45 ± 12.84 83.8 ± 3.6 Hypsiboas albopuctatus 9 5.86 ± 0.91 2.54 ± 0.72 65.62 ± 20.04 83.7 ± 5.8 Hypsiboas faber 10 38.25 ± 5.09 3.34 ± 0.63 95.42 ± 22.10 76.9 ± 2.1 Hypsiboas bischofﬁ 8 3.50 ± 0.49 5.60 ± 1.57 91.61 ± 22.10 74.0 ± 1.6 Hypsiboas polytaenius 10 1.12 ± 0.11 5.46 ± 1.86 55.23 ± 20.50 78.5 ± 2.1 Proceratophrys boiei 10 11.96 ± 1.87 2.35 ± 0.39 128.36 ± 25.17 77.1 ± 1.6 Leptodactylus notoaktites 3 12.09 ± 1.13 2.53 ± 0.60 - - Leptodactylus podicipinus 7 4.46 ± 1.57 1.70 ± 0.35 33.26 ± 6.74 84.1 ± 3.5 Physalaemus olfersii 7 2.96 ± 0.65 2.85 ± 0.46 20.41 ± 4.70 81.2 ± 4.6 Physalaemus spiniger 8 0.50 ± 0.08 2.63 ± 0.62 - - Rhinella ornata 9 15.20 ± 3.00 4.43 ± 0.51 76.18 ± 23.80 81.7 ± 2.1 Rhinella icterica 10 94.85 ± 21.20 1.16 ± 0.28 182.80 ± 56.09 81.3 ± 4.0 Rhinella schneideri 9 78.48 ± 21.35 1.96 ± 0.71 223.55 ± 56.77 79.2 ± 2.4 N: number of individuals used for data collection; REWL: resistance to evaporative water loss; RWU: rates of water uptake; SLPD: sensitivity of locomotor performance to dehydration, considered as the hydration state that results in 70% of maximum performance. d i 10 1371/j l 0140761 t001 Table 1. Mean ± standard deviation of the phenotypic variables collected for 17 species of anurans. Table 2. Mean ± standard deviation of the climatic variables related to temperature extracted from occurrence data for 17 species of anurans. Allometry and relations between climate and physiological variables The REWL declined as the body mass increased (REWL = 0.608BM-0.185, p = 0.021). In con- trast, RWU increased with body mass (RWU = 1.538BM0.327, p = 0.004). Thus, large species showed a lower REWL but had a higher RWU (Fig 2). A relation between body mass and SLPD was not observed (SLPD = 1.909BM-0.005, p = 0.584). SLPD and RWU were directly affected by component 1 of the climatic PCA (Table 5). Inter- specific variance in anuran REWL showed no association with the climatic components (Table 5). RWU showed a higher phylogenetic signal when compared to REWL and SLPD 7 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 N: number of individuals used for data collection; REWL: resistance to evaporative water loss; RWU: rates of water uptake; SLPD: sensitivity of locomotor performance to dehydration, considered as the hydration state that results in 70% of maximum performance. N: number of occurrence points obtained for each species; AMT: annual mean temperature; MTWM: maximum temperature of warmest month; MTCM: minimum temperature of coldest month; TS: temperature seasonality (considered as the standard deviation of monthly mean temperature) for each species; AMT: annual mean temperature; MTWM: maximum temperature of warmest month; MTCM: th; TS: temperature seasonality (considered as the standard-deviation of monthly mean temperature). Species N AMT MTWM MTCM TS (°C) (°C) (°C) (°C) Dendropsophus microps 55 18.1 ± 2.2 26.5 ± 2.2 8.6 ± 2.7 2.7 ± 0.4 Dendropsophus minutus 579 21.9 ± 2.9 30.2 ± 2.7 12.2 ± 3.4 1.9 ± 0.9 Scinax rizibilis 39 18.8 ± 2.0 27.6 ± 2.0 9.1 ± 2.1 2.9 ± 0.3 Scinax crospedospilus 31 18.8 ± 2.1 26.8 ± 2.2 9.2 ± 2.4 2.4 ± 0.3 Scinax hayii 76 19.1 ± 2.3 26.9 ± 2.4 9.7 ± 2.9 2.4 ± 0.3 Hypsiboas albopuctatus 540 21.7 ± 2.3 29.8 ± 2.3 11.5 ± 2.5 2.0 ± 0.6 Hypsiboas faber 169 20.1 ± 2.3 28.5 ± 2.0 10.8 ± 3.2 2.5 ± 0.5 Hypsiboas bischofﬁ 59 17.9 ± 1.9 26.6 ± 1.8 8.3 ± 2.1 2.9 ± 0.3 Hypsiboas polytaenius 33 19.0 ± 2.2 26.9 ± 2.2 9.2 ± 2.9 2.2 ± 0.2 Proceratophrys boiei 121 18.6 ± 2.0 26.7 ± 2.1 9.2 ± 2.6 2.5 ± 0.4 Leptodactylus notoaktites 29 19.2 ± 2.1 28.0 ± 2.1 9.4 ± 2.2 3.0 ± 0.3 Leptodactylus podicipinus 283 23.4 ± 1.6 31.3 ± 1.6 13.0 ± 2.6 1.9 ± 0.6 Physalaemus olfersii 77 18.1 ± 1.9 26.3 ± 2.2 8.6 ± 2.0 2.6 ± 0.3 Physalaemus spiniger 16 21.8 ± 0.9 30.3 ± 0.8 12.6 ± 1.4 3.0 ± 0.2 Rhinella ornata 113 20.2 ± 1.9 28.1 ± 2.0 10.7 ± 2.4 2.5 ± 0.3 Rhinella icterica 163 18.6 ± 2.2 27.0 ± 2.3 9.1 ± 2.4 2.7 ± 0.4 Rhinella schneideri 143 22.8 ± 1.9 30.7 ± 1.9 12.4 ± 2.3 2.0 ± 0.5 N: number of occurrence points obtained for each species; AMT: annual mean temperature; MTWM: maximum temperature of warmest month; MTCM: minimum temperature of coldest month; TS: temperature seasonality (considered as the standard-deviation of monthly mean temperature). doi:10 1371/journal pone 0140761 t002 es related to temperature extracted from occurrence data for 17 species of anurans. of the climatic variables related to temperature extracted from occurrence data for 17 species of anurans. ble 2. Mean ± standard deviation of the climatic variables related to temperature extracted from occurrence d PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 8 / 19 Water Balance, Climate and the Geographical Distribution of Anurans Table 3. Mean ± standard deviation of the climatic variables related to precipitation extracted from occurrence data for 17 species of anurans. Species N AP PWM PDM PS (mm) (mm) (mm) (mm) Dendropsophus microps 55 1586 ± 372 231 ± 52 58 ± 25 47 ± 15 Dendropsophus minutus 579 1489 ± 295 245 ± 54 32 ± 35 64 ± 24 Scinax rizibilis 39 1514 ± 330 213 ± 43 61 ± 19 42 ± 12 Scinax crospedospilus 31 1624 ± 420 255 ± 46 44 ± 25 59 ± 12 Scinax hayii 76 1765 ± 413 271 ± 44 51 ± 26 57 ± 13 Hypsiboas albopuctatus 540 1411 ± 243 250 ± 47 22 ± 22 72 ± 17 Hypsiboas faber 169 1516 ± 353 222 ± 49 55 ± 33 49 ± 21 Hypsiboas bischofﬁ 59 1612 ± 358 217 ± 42 72 ± 32 39 ± 17 Hypsiboas polytaenius 33 1593 ± 362 273 ± 47 34 ± 27 68 ± 17 Proceratophrys boiei 121 1547 ± 272 245 ± 47 47 ± 20 56 ± 15 Leptodactylus notoaktites 29 1462 ± 202 212 ± 34 56 ± 17 45 ± 13 Leptodactylus podicipinus 283 1345 ± 216 236 ± 35 18 ± 10 72 ± 11 Physalaemus olfersii 77 1614 ± 308 246 ± 47 53 ± 18 52 ± 11 Physalaemus spiniger 16 1856 ± 391 269 ± 66 69 ± 15 45 ± 5 Rhinella ornata 113 1640 ± 430 249 ± 45 50 ± 24 56 ± 11 Rhinella icterica 163 1653 ± 326 234 ± 50 65 ± 32 45 ± 19 Rhinella schneideri 143 1368 ± 225 237 ± 40 21 ± 14 69 ± 12 N: number of occurrence points obtained for each species; AP: annual precipitation; PWM: precipitation of wettest month; PDM: precipitation of driest month; PS: precipitation seasonality (considered as the coefﬁcient of variation of monthly mean precipitation). of the climatic variables related to precipitation extracted from occurrence data for 17 species of anurans. Table 3. Mean ± standard deviation of the climatic variables related to precipitation extracted from occurren N: number of occurrence points obtained for each species; AP: annual precipitation; PWM: precipitation of wettest month; PDM: precipitation of driest month; PS: precipitation seasonality (considered as the coefﬁcient of variation of monthly mean precipitation). (Table 5). : two principal component analyses performed for sets of 16 species and 13 species, according to physiological data available for them. Values with higher loadings (0.65) in each component are highlighted in boldface. The regression line equations stated in the figure represent the conventional linear regression. The equations from phylogenetic regressions are REWL = -0.185BM + 0.608 and RWU = 0.327BM + 1.538. Fulfilled circles represent the mean resistance to evaporative water loss for each species and unfilled circles represent the mean rate of water uptake for each species. Fig 2. Allometric relations between physiological variables and body mass. Regressions of resistance to evaporative water loss (REWL) and rates of water uptake (RWU) as functions of body mass (BM). The regression line equations stated in the figure represent the conventional linear regression. The equations from phylogenetic regressions are REWL = -0.185BM + 0.608 and RWU = 0.327BM + 1.538. Fulfilled circles represent the mean resistance to evaporative water loss for each species and unfilled circles represent the mean rate of water uptake for each species. doi:10.1371/journal.pone.0140761.g002 and Cerrado did not differ in REWL (F = 1.092, p = 0.24) and RWU (F = 0.156, p = 0.65). Oth- erwise, species collected at the Cerrado sites showed significantly higher SLPD than species col- lected at the Atlantic Forest sites (F = 3.636, p = 0.05). and Cerrado did not differ in REWL (F = 1.092, p = 0.24) and RWU (F = 0.156, p = 0.65). Oth- erwise, species collected at the Cerrado sites showed significantly higher SLPD than species col- lected at the Atlantic Forest sites (F = 3.636, p = 0.05). It is also possible to observe two distinct clusters of points in the phylogenetic regres- sion analyses between the scores of the first climatic component derived from the geographical points of species occurrence and the fitted values of both SLPD and RWU (Fig 3). These two clouds correspond to species from which individuals were collected in localities from the Atlantic Forest and the Cerrado, respectively. Species collected at sites in the Atlantic Forest Table 4. Results from two principal component analyses performed on climatic variables extracted from points of occurrence of Brazilian anuran species. Climatic variables 16 species 13 species* C1 C2 C1 C2 Annual Mean Temperature 0.883 -0.062 0.976 -0.126 Max Temperature of Warmest Month 0.812 -0.212 0.954 -0.256 Min Temperature of Coldest Month 0.938 -0.192 0.948 -0.145 Temperature Seasonality -0.921 -0.135 -0.961 -0.112 Annual Precipitation -0.677 0.593 -0.856 0.383 Precipitation of Wettest Month 0.239 0.966 0.169 0.982 Precipitation of Driest Month -0.96 -0.089 -0.969 -0.132 Precipitation Seasonality 0.839 0.44 0.818 0.525 Eigenvalues 5.309 1.59 6.054 1.519 % of Variance Explained 66.36 19.87 75.68 18.99 C1: component 1; C2: component 2; *: two principal component analyses performed for sets of 16 species and 13 species, according to physiological data available for them. Values with higher loadings (0.65) in each component are highlighted in boldface. mponent analyses performed on climatic variables extracted from points of occurrence of Brazilian anuran esults from two principal component analyses performed on climatic variables extracted from points of occ alyses performed on climatic variables extracted from points of occurrence of Brazilian anuran PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 9 / 19 Water Balance, Climate and the Geographical Distribution of Anurans Fig 2. Allometric relations between physiological variables and body mass. Regressions of resistance to evaporative water loss (REWL) and rates of water uptake (RWU) as functions of body mass (BM). The regression line equations stated in the figure represent the conventional linear regression. The equations from phylogenetic regressions are REWL = -0.185BM + 0.608 and RWU = 0.327BM + 1.538. Fulfilled circles represent the mean resistance to evaporative water loss for each species and unfilled circles represent the mean rate of water uptake for each species. Fig 2. Allometric relations between physiological variables and body mass. Regressions of resistance to evaporative water loss (REWL) and rates of water uptake (RWU) as functions of body mass (BM). ates of water uptake; C1: component 1; C2: component 2. Signiﬁcant probabilities (P 0.05) are highlighted Discussion Our interspecific comparative analysis showed that some physiological traits of water balance in anurans, particularly REWL and RWU, show pervasive allometric relations with body mass. Table 5. Results of phylogenetic regression analyses testing the effects of climatic components on anuran physiological variables corrected by size. Variable Phylogenetic signal Factor Slope P Intercept -0.002 0.684 SLPD λ = 0.124 C1 0.01 0.043 C2 0.006 0.193 Intercept 0.01 0.848 REWL λ = 0.175 C1 -0.045 0.361 C2 -0.018 0.717 Intercept -0.002 0.984 RWU λ = 1.119 C1 0.054 0.001 C2 -0.035 0.308 SLPD: sensitivity of locomotor performance to dehydration, considered as the hydration state that results in 70% of maximum performance; REWL: resistance to evaporative water loss; RWU: rates of water uptake; C1: component 1; C2: component 2. Signiﬁcant probabilities (P 0.05) are highlighted in boldface. Table 5. Results of phylogenetic regression analyses testing the effects of climatic components on anuran physiological variables corrected by size. Variable Phylogenetic signal Factor Slope P Intercept -0.002 0.684 SLPD λ = 0.124 C1 0.01 0.043 C2 0.006 0.193 Intercept 0.01 0.848 REWL λ = 0.175 C1 -0.045 0.361 C2 -0.018 0.717 Intercept -0.002 0.984 RWU λ = 1.119 C1 0.054 0.001 C2 -0.035 0.308 SLPD: sensitivity of locomotor performance to dehydration, considered as the hydration state that results in 70% of maximum performance; REWL: resistance to evaporative water loss; RWU: rates of water uptake; C1: component 1; C2: component 2. Signiﬁcant probabilities (P 0.05) are highlighted in boldface. Table 5. Results of phylogenetic regression analyses testing the effects of climatic components on anuran physiological variables corrected by size n analyses testing the effects of climatic components on anuran physiological variables corrected by Table 5. Results of phylogenetic regression analyses testing the effects of climatic components on anuran physiological variables corrected by si e Table 5. Results of phylogenetic regression analyses testing the effects of climatic components on anuran i SLPD: sensitivity of locomotor performance to dehydration, considered as the hydration state that results in 70% of maximum performance; REWL: resistance to evaporative water loss; RWU: rates of water uptake; C1: component 1; C2: component 2. Signiﬁcant probabilities (P 0.05) are highlighted in boldface. PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 10 / 19 Water Balance, Climate and the Geographical Distribution of Anurans Fig 3. Phylogenetic relations between physiological and climatic variables. Discussion Relations between size adjusted sensitivity of locomotor performance to dehydration (A) and rates of water uptake (B) with the PC scores from axis 1. The phylogenetically corrected values for the physiological traits show a positive correlation with the PCA component 1 scores that correspond to a direct association between annual mean temperature, maximum temperature of warmest month, minimum temperature of coldest month and precipitation seasonality, which are inversely associated with temperature seasonality, annual precipitation and precipitation of driest month. Fulfill circles represent species collected at the Cerrado sites and “X” represent species collected at Atlantic Forest sites. Fig 3. Phylogenetic relations between physiological and climatic variables. Relations between size adjusted sensitivity of locomotor performance to dehydration (A) and rates of water uptake (B) with the PC scores from axis 1. The phylogenetically corrected values for the physiological traits show a positive correlation with the PCA component 1 scores that correspond to a direct association between annual mean temperature, maximum temperature of warmest month, minimum temperature of coldest month and precipitation seasonality, which are inversely associated with temperature seasonality, annual precipitation and precipitation of driest month. Fulfill circles represent species collected at the Cerrado sites and “X” represent species collected at Atlantic Forest sites. doi:10.1371/journal.pone.0140761.g003 doi:10.1371/journal.pone.0140761.g003 Additionally, the analyses showed an association of interspecific variation in RWU and SLPD with climatic characteristics associated with geographical distribution. These results, based on phylogenetically informed analyses, suggest a pattern of adaptation of anuran water balance to abiotic conditions. Additionally, the analyses showed an association of interspecific variation in RWU and SLPD with climatic characteristics associated with geographical distribution. These results, based on phylogenetically informed analyses, suggest a pattern of adaptation of anuran water balance to abiotic conditions. REWL declined with an increase in body mass whereas RWU increased with body mass. The relationship between REWL and body mass is likely a consequence of surface area/volume ratio [9,26,38], providing smaller anurans a higher water loss surface. Indeed, the higher toler- ance to evaporative water loss of smaller animals has been interpreted as an adaptation associ- ated with this surface area/volume ratio disadvantage [39]. Our results suggest that a higher REWL might at least partially compensate for the increased rates of water loss associated with the evolution of smaller body masses in anurans. PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Discussion These results differ from those of previous studies that did not find a relation between interspecific variation in anuran REWL and body mass [10,40]. However, these previous studies included species characterized by very high REWL and relatively low body mass, and these species might, at least in part, mask the allome- tric function of REWL. A positive allometric association of RWU with body mass has also been PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 11 / 19 Water Balance, Climate and the Geographical Distribution of Anurans previously suggested to exist to some extent in anurans [19–21]. According to these authors, the required time to reach maximum blood cell flux through the pelvic patch increases with body mass. Otherwise, the magnitude of this flux seems to be related to the environment, given that species from xeric environments show higher flux than species from mesic environments, despite the differences in body mass [19–21]. Our results corroborate the previous positive cor- relation between RWU and body mass and are based on a phylogenetically controlled analysis that includes a larger number of species. This association of RWU with body mass might be functionally related to the fact that anurans characterized by higher body masses show lower surface area/volume ratio, and it might be associated with a lower pelvic patch area. However, large anurans still need absolutely more water to rehydrate. In this way, our results suggest that a higher hydration rate might be selected to compensate for increased body size. The underly- ing mechanisms of the body size-related differences in RWU still remain to be investigated, but they might be associated with differences in vascularization [18] and permeability of the pelvic patch due to the density of aquaporins [41–43]. We also found a clear pattern of association between interspecific variation in RWU and SLPD with climatic variables extracted from the geographical points of occurrence for the dif- ferent species included in this analysis. In particular, the species with geographical occurrence encompassing areas characterized by higher and more seasonally uniform temperatures, and lower and more seasonally concentrated precipitation, had higher RWU and SLPD. Moreover, species collected at sites within the domains of the Atlantic Forest and the Cerrado form two dissociated clusters of data distribution in the phylogenetic regression analyses between these physiological variables and the first climatic component derived from the geographical points of occurrence, reinforcing the pattern that emerged from the continuous covariation. PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Discussion Our present analysis showed the opposite pattern: species with geographical distribution more tightly asso- ciated with mesic environments show lower SLPD than species with geographical distribution encompassing more water-restricted environments. Although our results contradict previous studies, we believe that this opposite pattern to those described for previous studies might be associated with underlying methodological differences. The present study was conducted with a much higher number of species, split into several phylogenetic lineages, and incorporated phylogenetically corrected analyses. Furthermore, our study analyzed the association between the physiological traits and climatic data instead of collecting data exclusively on physiology and associating it to namely characterized environments. In this way, we believe that our results are robust and that this pattern might be associated with selection on temporal patterns of activity in these different environments. In environments characterized by lower and more seasonally concentrated precipitation, such as the Cerrado, individuals might be selected to concentrate reproduction and other general activities, such as foraging, to restricted periods with a higher probability of precipitation. This concentrated pattern of activity might prevent selection from acting on the sensitivity of locomotion to dehydration in the Cerrado. Otherwise, species from environments characterized by higher and seasonally distributed pre- cipitation might maintain continuous activity, with several species showing year-round repro- duction or at least foraging activity [45–49]. This sustained activity at periods of lower relative humidity might allow directional selection on SLPD in the Atlantic Forest. This reasoning remains largely speculative, and comparative studies including the duration of reproductive season on different localities are necessary to test hypotheses along these lines. Previous studies [6–8] also may not be comparable to the present one because they tested SLPD at different tem- peratures and showed that patterns of adaptation and/or acclimatization can shift the curves, resulting in a reduction in the SLPD at temperatures closer to the temperatures of activity for different species in the field. In the present study, we performed tests of SLPD at a single tem- perature. In this way, we need to consider that species from the Atlantic Forest and Cerrado might show different optimum temperatures where the effects of dehydration are reduced, and the comparative analysis of SLPD at these specific temperatures might change the patterns described here. Discussion Although these data clearly show the association between the phenotypic variables and the climate of the occurrence points, these data do not allow verification of the contribution of different processes underlying this phenotypic variation (genetic adaptation or acclimatization). Phenotypic plas- ticity might even play a significant role determining the interspecific physiological variation associated with seasonal acclimatization [10,11], given that individuals from different species were collected and measured at different seasons. The analysis of populations from the same species collected in both biomes, as well as the investigation of the acclimation capacity of these physiological variables, might shed light on these topics. The inclusion of species inhabiting environments characterized by more drastic water restriction, such as arid and semi-arid locali- ties, might also expand the knowledge of patterns of water balance adaptations in anurans. Several authors have previously reported that terrestrial or arid-dwelling species of anurans show higher hydration rates when compared to semi-aquatic species or to those occurring in mesic environments [17,20,21]. These results are consistent with morphological observations showing that species from xeric environments have more vascularized ventral skin [18]. These joint results suggest a pattern of directional selection of individuals able to hydrate more effi- ciently and at faster rates once they find water sources in environments where water represents a scarce resource. Again, our results support these previous results and interpretations through a comparative and phylogenetically informed approach. It is important to highlight that the interspecific variation in RWU reported here was based on measurements from a free water surface, a situation that might not be ecologically relevant for many species. In the field, many species might actually rehydrate more frequently from humid soil, mainly outside of the breed- ing season [44]. In this way, RWU from a free water surface might not be directly selected in nature, but it might be functionally associated with differences in efficiency of rehydration from humid substrates. This hypothesis remains to be tested. Previous comparative studies have found a pattern of negative association between SLPD and the occupation of open and more water-restricted environments [6–8]. These previous PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 12 / 19 Water Balance, Climate and the Geographical Distribution of Anurans results suggest that individuals who are able to maintain behavioral performance at lower states of hydration are under directional selection in water-restricted environments. PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Discussion Our analysis did not recover an interspecific covariation between REWL and the climatic variables associated with geographical distribution, corroborating results from previous studies that attempted to associate variation in REWL with differences in habitat [38,50–54]. These joint results do not corroborate the long-lasting corollary reasoning that high skin permeability would be a direct limitation for the occupation of more water-restricted environments by amphibians and that individuals displaying high REWL and inhabiting these environments would be strongly selected. It is possible that behavioral adjustments, such as on patterns of time of reproduction and microenvironmental selection during activity might also prevent directional selection on REWL. Furthermore, several studies have emphasized a consistent pat- tern of anuran interspecific association between REWL and the habit, with arboreal species dis- playing higher values than terrestrial and semi-aquatic ones [9,10,55]. Although we sampled a relatively high number of species for the present investigation, the interspecific variation in habits are highly skewed through the phylogeny, preventing the analysis of the relationship between REWL and habits in this study. In summary, this comparative analysis of anuran water balance showed that body mass coevolved with REWL and RWU in opposite allometric directions. In this way, species with higher body mass show lower REWL and higher RWU than species with lower body mass. Additionally, species inhabiting areas characterized by higher and more seasonally uniform PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 13 / 19 Water Balance, Climate and the Geographical Distribution of Anurans temperatures, and lower and more seasonally concentrated precipitation, had higher RWU and SLPD than species with geographical distributions that were more restricted to mesic environ- ments. These results suggest that the ability to hydrate faster from a free water surface might indicate an adaptation of anurans to environments characterized by a higher seasonal restric- tion on water availability. These differences in RWU from a free water surface might be, alter- natively, associated with efficiency of water uptake from humid substrates. Otherwise, the higher SLPD displayed by anurans inhabiting the Cerrado might be related to a more intense restriction of activity at the peak of the rainy season, precluding the action of selection on this variable. Supporting Information S1 Fig. Geographical distribution of Dendropsophus microps. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic For- rest and Cerrado domains [23]. (TIF) S2 Fig. Geographical distribution of Dendropsophus minutus. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic For- rest and Cerrado domains [23]. (TIF) S2 Fig. Geographical distribution of Dendropsophus minutus. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic For- rest and Cerrado domains [23]. (TIF) S3 Fig. Geographical distribution of Scinax rizibilis. Collection site of individuals for physio- logical measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S4 Fig. Geographical distribution of Scinax crospedospilus. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S5 Fig. Geographical distribution of Scinax hayii. Collection site of individuals for physiolog- ical measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cer- rado domains [23]. (TIF) S6 Fig. Geographical distribution of Hypsiboas albopuctatus. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic For- rest and Cerrado domains [23]. (TIF) S7 Fig. Geographical distribution of Hypsiboas faber. Collection site of individuals for physi- ological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S8 Fig. Geographical distribution of Hypsiboas bischoffi. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S9 Fig. Geographical distribution of Hypsiboas polytaenius. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 14 / 19 Water Balance, Climate and the Geographical Distribution of Anurans PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 Acknowledgments We would like to thank PhD Paula Hanna Valdujo and PhD Renata Brandt for statistical and methodological discussions. S30 Fig. Locomotor performance of Rhinella schneideri. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S30 Fig. Locomotor performance of Rhinella schneideri. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S1 Text. ARRIVE. Animal Research: Reporting In Vivo Experiments Guidelines Checklist. (DOCX) and Cerrado domains [23]. (TIF) and Cerrado domains [23]. (TIF) S10 Fig. Geographical distribution of Proceratophrys boiei. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S11 Fig. Geographical distribution of Leptodactylus notoaktites. Collection site of individu- als for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S12 Fig. Geographical distribution of Leptodactylus podicipinus. Collection site of individu- als for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S13 Fig. Geographical distribution of Physalaemus olfersii. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S14 Fig. Geographical distribution of Physalaemus spiniger. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S15 Fig. Geographical distribution of Rhinella ornata. Collection site of individuals for phys- iological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S16 Fig. Geographical distribution of Rhinella icterica. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S17 Fig. Geographical distribution of Rhinella schneideri. Collection site of individuals for physiological measures, points of occurrence for the species [29] and areas of Atlantic Forrest and Cerrado domains [23]. (TIF) S18 Fig. Locomotor performance of Dendropsophus microps. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S19 Fig. Locomotor performance of Dendropsophus minutus. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S20 Fig. Locomotor performance of Scinax hayii. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) PLOS ONE \| DOI:10.1371/journal.pone.0140761 October 15, 2015 15 / 19 Water Balance, Climate and the Geographical Distribution of Anurans S21 Fig. Locomotor performance of Hypsiboas albopuctatus. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S22 Fig. Locomotor performance of Hypsiboas faber. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) S23 Fig. Locomotor performance of Hypsiboas bischoffi. and Cerrado domains [23]. (TIF) Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S24 Fig. Locomotor performance of Hypsiboas polytaenius. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S25 Fig. Locomotor performance of Proceratophrys boiei. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S26 Fig. Locomotor performance of Leptodactylus podicipinus. Mean locomotor perfor- mance transformed as a percentage of maximum performance in different hydration levels. (TIF) S27 Fig. Locomotor performance of Physalaemus olfersii. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S28 Fig. Locomotor performance of Rhinella ornata. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) S29 Fig. Locomotor performance of Rhinella icterica. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) S30 Fig. Locomotor performance of Rhinella schneideri. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S1 Text. ARRIVE. Animal Research: Reporting In Vivo Experiments Guidelines Checklist. (DOCX) Acknowledgments We would like to thank PhD Paula Hanna Valdujo and PhD Renata Brandt for statistical and h d l l d S22 Fig. Locomotor performance of Hypsiboas faber. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) S23 Fig. Locomotor performance of Hypsiboas bischoffi. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S24 Fig. Locomotor performance of Hypsiboas polytaenius. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S25 Fig. Locomotor performance of Proceratophrys boiei. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S26 Fig. Locomotor performance of Leptodactylus podicipinus. Mean locomotor perfor- mance transformed as a percentage of maximum performance in different hydration levels. (TIF) S27 Fig. Locomotor performance of Physalaemus olfersii. Mean locomotor performance transformed as a percentage of maximum performance in different hydration levels. (TIF) S28 Fig. Locomotor performance of Rhinella ornata. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) S29 Fig. Locomotor performance of Rhinella icterica. Mean locomotor performance trans- formed as a percentage of maximum performance in different hydration levels. (TIF) References 1. 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https://openalex.org/W2991926155	https://cyberleninka.ru/article/n/the-effect-of-service-quality-on-satisfaction-and-its-impact-on-loyalty-of-cooperative-members/pdf	English	null	THE EFFECT OF SERVICE QUALITY ON SATISFACTION AND ITS IMPACT ON LOYALTY OF COOPERATIVE MEMBERS	Russian journal of agricultural and socio-economic sciences	2,019	cc-by	4,434	THE EFFECT OF SERVICE QUALITY ON SATISFACTION AND ITS IMPACT ON LOYALTY OF COOPERATIVE MEMBERS Arizal N., Associate Professor Seswandi Agus, Lecturer Faculty of Economics, University of Lancang Kuning, Indonesia *E-mail: arizal@unilak.ac.id ABSTRACT The objective of this study is to know empirically the effect of service quality on satisfaction and its impact on the loyalty of the members of savings and credit cooperative. In this study, the research method uses a descriptive method (Survey) with the quantitative research design, data that users are primer and secondary sources of data. A research population includes all members of savings and credit cooperative in Kasikan Kampar regency. In this study, the sample size consists of 95 respondents, which sample size determination based on Slovin's formula with the 10% margin of error. The sampling technique uses non-random sampling, particularly the method of accidental sampling with the help of SPSS assistance. The results of the research, these three dimensions have a significant influence on satisfaction. there is a positive relationship with the satisfaction and the results of research for empathy dimension indicate that there is a positive relationship with a satisfaction, but both dimensions do not have a significant effect on satisfaction, there is a positive relationship to loyalty and has a significant influence. RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 DOI 10.18551/rjoas.2019-11.32 LITERATURE REVIEW The good quality of service provided by companies to customers or members of cooperatives can bring satisfaction. Satisfaction felt by customers or members of the cooperative will be able to bring loyalty from customers, which mean that the level of loyalty is determined by the level of satisfaction and the level of satisfaction obtained by customers, one of which is determined by the quality of service. Tjiptono, et al (2008: 70) service quality reflects the comparison between the level of service provided by the company compared to customer expectations. This means that customer expectations should be the same as the services provided by the company. p p y p y The study of SERVQUAL by parasuraman (1988) in Lupiyoadi; 2006; 182 concluded that there are five dimensions of service quality called SERVQUAL. Those are: p p y p y The study of SERVQUAL by parasuraman (1988) in Lupiyoadi; 2006; 182 concluded that there are five dimensions of service quality called SERVQUAL. Those are: 1. Tangibles (Tangibles), is the ability of a company to provide physical evidence to customers, both in terms of humans, the shape of buildings and equipment used in the service process; p . Reliability (Reliability) is the ability of the company to provide services in accordanc with what was promised accurately and reliably; 3. Responsiveness (Responsiveness), which is the ability of a company to help and provide fast and appropriate services to customers and clear information; 4. Assurance (Assurance) (i.e. in the form of knowledge, politeness, and the ability of the company employees to foster the trust of customers to the company. Its components include communication (communication), credibility (credibility), security (security), competence (competence) and courtesy (courtesy); 5. Empathy (Empathy). Give sincere, individual or personal attention given to customers by trying to understand consumer desires. According to Kotler, satisfaction is the level of one's feelings after comparing the performance or results he feels compared to his expectations (Kotler et al, 2000: 52). Whereas Tse and Wilton (1988) in Lupiyoadi (2004: 349) customer satisfaction or dissatisfaction is the customer's response to the evaluation of the disconfirmation between the previous expectations and the actual performance of the product felt after its use. Wilkie (1990) defines satisfaction as an emotional response to an evaluation of the consumption experience of a product or service. RJOAS, 11(95), November 2019 Good service quality is the expectation of the customer, in this case, is a member of the cooperative, because this will be able to bring satisfaction. Customer satisfaction has become a central concept in business and management discourse (Tjiptono and Chandra, 2005: 192). Customers generally expect the product in the form of goods or services consumed can be accepted and enjoyed with good or satisfying service (Assauri, 2003: 8). Satisfying service can bring loyalty, and this is the hope of all companies both engaged in goods and services. The importance of loyal customers according to Griffin (2003; 223) include: Reducing marketing costs (because low costs can attract new customers); Reducing transaction costs (such as contract negotiation fees, order processing); Reducing customer turnover costs (due to fewer customer changes); Increase cross-sales which will enlarge the company's market share; A more positive of word of mouth assuming that loyal customers also means those who are satisfied. Foster (2008; 175) proposed several indicators of loyalty, namely: doing regular purchases; Buy between lines of products and services; Recommend products to other people (refers other); Demonstrate competitiveness with other competitors. KEY WORDS Service quality, satisfaction, loyalty, public service. The cooperative is a business entity consisting of the person or cooperative legal entity that lies its activity based on the principle of cooperation and also as a people's economic movement based on the principle of kinship (based on the provision of Law No. 25 of 1992 concerning Cooperatives). Furthermore, the Cooperative aims to advance the welfare of members in particular and the community in general and participate in building the national economic order in order to create an advanced, justice and prosperous society based on Pancasila and the 1945 Constitution. The form of business entity should have a large role in the development and welfare of the Indonesian people. p p p In Indonesia, the role of cooperatives in contributing to national income is not yet high; this can be seen from the contribution of presentations from 2014 to 2017. Which is 2017 the contribution of cooperatives in Indonesia was 1,715, in 2015 it developed to 4.41%. In 2016, the role of Indonesian Cooperatives on national income decreased to 3.4%, while in 2017 it increased again by around 4.6%. Based on these data, the role of cooperatives in Indonesia to national income fluctuated from 2014 to 2017 and from these figures, it can be seen that their role is quite small. One of the goals of establishing a cooperative is to be able to improve the welfare of its members. The purpose of the cooperative in its activities can choose various fields, namely the production sector, the field of service provision, the field of goods supply and lending to its members. In running its business, cooperatives can appoint a manager who has the task to carry out cooperative activities and try to achieve cooperative goals and can make cooperatives that are developed and large-scale. Therefore, cooperative products must be able to provide a good level of service quality, which has 5 dimensions according to Kotler in Alma (2005: 284-285) and also Davis in Tjiptono (2016) and according to Parasuraman in Sopiah (2013) namely; Tangible (tangible); Reliability (reliability), Responsiveness (fast response); Assurance (certainty) and Empathy (empathy). 228 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 Customer satisfaction has become a central concept in business and management discourse (Tjiptono and Chandra, 2005: 192). Customers generally expect products in the form of goods or services consumed that can be received and enjoyed with good or satisfying service (Assauri, 2003: 8). Customer satisfaction can shape perceptions and then can position the company's products based on customers. If performance is below expectations, the customer is not satisfied, if performance meets satisfying customer expectations. If performance exceeds expectations, the customer is very satisfied or happy. p p p Loyal service will bring benefits, Griffin (2003; 223), namely;  Marketing costs will be reduced (because the cost to attract new customers is more expensive);  Will reduce transaction costs (such as contract negotiation fees, order processing, etc.);  Customer turnover costs will be reduced (due to fewer customer changes); ( g  Increase cross-sales which will enlarge the company's market share; g p y Word of mouth (a promise from mouth to mouth) is more positive assuming that loy customers also mean those who are satisfied.  Word of mouth (a promise from mouth to mouth) is more positive assuming that loyal customers also mean those who are satisfied. Furthermore, Griffin stated the characteristics of loyal customers, namely; Furthermore, Griffin stated the characteristics of loyal customers, namely;  Routinely use certain types of services from a company;  Use other services from the company;  Willingness to recommend company services to other parties/people; e to reject the company's competitors' offers From the research results of Tuti Hastuti et al 2014, with the title Service Quality, Customer Satisfaction, and Customer Loyalty: SERVQUAL Model Application in Malang Islamic Microfinance Institutions. In this study, the sample used was 112 respondents as customers of Sharia Microfinance Institutions (LKMS) Malang. Research results: Service quality has a significant effect on customer satisfaction, while tangibles and empathy dimensions have no effect on satisfaction. In addition, another result is that service quality has a significant effect on customer loyalty through customer satisfaction. Furthermore, the study entitled The Effect of Service Quality on Customer Satisfaction and Loyalty of PT BPR HOKI in Tabanan Regency by Dewi et al. (2014: 257). The results of statistical analysis show that service quality, customer satisfaction, and loyalty have a positive and significant relationship Research by Dewi et al. RJOAS, 11(95), November 2019 (2014: 257), states that service quality, customer satisfaction, and loyalty have a positive and significant relationship. Likewise, research by (Rizan, 2011) with the title Effect of Product Quality and Service Quality on Customer Satisfaction states product quality and service quality have a significant effect on Suzuki customers. Furthermore, research from (Rachmawati & Azis, 2017) states that consumer satisfaction with the quality of hotel services. Service quality also has a significant effect based on research conducted by (Ersi & Semuel, 2014), (Trisno Musanto, 2004), (Wahyudi, 2015). LITERATURE REVIEW Engel, et al (1990) states that customer satisfaction is an evaluation after the transaction process where the alternative is chosen is at least the same or exceeds customer expectations, while dissatisfaction arises if the results (outcomes) do not meet expectations (Tjiptono, 2004: 349). Customer satisfaction is the response of the customer to the discrepancy between the level of interest before and the actual performance felt after use (Rangkuti, 2002: 30). Customer satisfaction is influenced by perceptions of service quality, product quality, prices and factors that are personal as well as those of a momentary situation (Kotler, 2000: 41). 229 RJOAS, 11(95), November 2019 In this research, the data analysis technique used is the questionnaire analysis method. Each questionnaire was taken from each research indicator and each answer was scored with very good criteria (5), to very poor (1). Analysis of the influence of the variables in this study using analysis tools with SPSS. METHODS OF RESEARCH This type of research is a descriptive method (survey) with a qualitative research design, which is to determine the implementation, the quality of service and satisfaction and loyalty. In addition, quantitative design, namely research not only provides a description of the phenomenon but also explains, tests the hypothesis and gets the meaning of the facts. Types and sources of research data in the form of primary data and secondary data. The population in this study were all members of Maju Bersama savings and credit cooperatives in Kasikan Kampar regency in 2017, totaling 1,492 people. Because of the large population, then in this study took a sample of 5 respondents using Slovin theory with an error rate of 10%. Types of Sampling Techniques. The method used in this research is non-random sampling technique by accidental sampling method where each population found is used as a sample. 230 RESULTS OF STUDY The characteristics used in this study consisted of; gender, age, education, occupation and duration of membership of the cooperative. The following are the results of respondents' characteristic research on Maju Bersama savings and credit cooperatives in the Kasikan Regency. Table 1 – Characteristics of Respondents by Gender Gender Frequency Percentage (Respondents) (%) Male 36 37,90 Female 59 61,10 Total 95 100 Source: Data Processed. Source: Data Processed. Based on the table above, it can be seen that the majority of respondents are women, namely 59 people or 61.10%, while men are 36 or 37.90%. This means that the members of the cooperative are dominated by women. The following are the characteristics of respondents based on their level of education can be seen in the table below. Table 2 – Characteristics of Respondents by Education Level Education Frequency Percentage (Respondents) (%) Junior High School 26 27,40 Senior High School 44 46,30 Associate Degree 12 12,60 Bachelor's degree 13 13,70 Amount 95 100,0 Source: Data Processed. Table 2 – Characteristics of Respondents by Education Level Source: Data Processed. Source: Data Processed. Based on the above table, it can be seen that the majority of respondents are respondents who are educated at the Senior High School level as many as 44 people or 27.40% while those with Junior High School are 26 people or 27.40%, who have a diploma education of 12 people or 12.60 % and those with an undergraduate education of 13 people or 13.70%. Table 3 – Characteristics of Respondents Based on Employment Employment Frequency Percentage (orang) (%) Students 7 7,40 Civil Servants 12 12,60 Entrepreneur 32 33,70 Others 44 46,30 Total 95 100,0 Source: Data Processed. Table 3 – Characteristics of Respondents Based on Employment Source: Data Processed. From the above table, it can be seen that there are 7 people or 7.40% of cooperative members who have worked as students or students, there are 12 people as civil servants or 12.60% and there are 32 people as entrepreneurs. While the remaining 44 people or 46.30% 231 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 are having other jobs. To find out whether each question item is valid, a validity test is carried out where the results can be seen in the following table: Table 4 – Uji Validity Source: Data Processed. Source: Data Processed. From the table above, it can be viewed that all question items from each research variable are valid. Meanwhile, to find out whether a reliable instrument to be used as a data collection tool is used the reliability test. The reliability coefficient is good if> 0.60. y y g From the table above, it can be seen that the results of reliability testing indicate that the reliability coefficient value is greater than the stipulation of 0.60, then the test results indicate that all instruments are reliable. Table 5 – Reliability Test Results Variabel Cronbach’s Alpha Value Status Tangible 0,634513 0,60 Reliable Reliability 0,807193 0,60 Reliable Responsiviness 0,81168 0,60 Reliable Assurance 0,841495 0,60 Reliable Empaty 0,730523 0,60 Reliable Satisfaction 0,716264 0,60 Reliable Loyalty 0,726291 0,60 Reliable 232 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 Table 6 – One-Sample Kolmogorov-Smirnov Test Unstandardized Residual N 49 Normal Parameters a Mean .0000000 Std. Deviation 2.86559103 Most Extreme Differences Absolute .089 Positive .089 Negative -.080 Kolmogorov-Smirnov Z .622 Asymp. Sig. (2-tailed) .834 a. Test distribution is Normal. Table 6 – One-Sample Kolmogorov-Smirnov Test To test the normality of tangible variables, reliability, responsiveness, insurance and empathy for satisfaction. this can be seen from the table above. Wherefrom the Kolmogorov- Smirnov test one-sample table it is known that the significance value is 0.834 and greater than 0.05. Then it can be concluded that the data tested is a normal distribution. Table 7 – One-Sample Kolmogorov-Smirnov Test Standardized Residual N 49 Normal Parameters a Mean .0000000 Std. Deviation .94648472 Most Extreme Differences Absolute .089 Positive .089 Negative -.080 Kolmogorov-Smirnov Z .622 Asymp. Sig. (2-tailed) .834 a. Test distribution is Normal. Table 7 – One-Sample Kolmogorov-Smirnov Test Table 7 – One-Sample Kolmogorov-Smirnov Test a. Test distribution is Normal. a. Test distribution is Normal. From the above table, it can be seen that from the table of the Kolmogorov-Smirnov one-sample test it is known that the significance value is 0.834 and greater than 0.05. Then it can be concluded that the data tested is a normal distribution. RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 The results of the research on the reliability dimension show that there is a negative relationship between reliability and satisfaction level of -0.008 which means that if the provision of physical evidence is increased, the level of negative satisfaction is -0.008. The test results for the significance level is 0.283 greater than 0.05 which means there is no significant effect. The results of the research for the responsiveness dimension show that there is a positive relationship with the satisfaction level of 0.451, which means that if the responsiveness of employees is increased, then the level of satisfaction will positively increase by 0.451. From the test results for the significance level is 0,000 less than 0.05 which means there is a significant influence. g The results of the research for the assurance dimension show that there is a positive relationship with satisfaction of 0.252, which means that if the assurance from the cooperative is improved, then the level of satisfaction will positively increase by 0.252. From the test results for the significance level is 0.05, which means there is a significant influence. The results of research for the empathy dimension show that there is a positive relationship with a satisfaction level of 0.220, which means that if empathy of employees is increased, then the level of satisfaction will positively increase by 0.220. From the test results for the significance level is 0.004 smaller than 0.05, which means there is a significant influence. Based on the results of the study, it can be seen that from the five dimensions studied, three dimensions, namely responsiveness, assurance, and empathy have a partial effect on satisfaction. While the other two dimensions, namely tangible and reliability do not have a significant effect on satisfaction. But simultaneously that dimension of service quality has a significant effect on the satisfaction that is equal to 0.63, which means that an improvement in the services provided by the company will increase consumer satisfaction by 0.63. The results of the test of significance obtained a probability level of 0,000 and smaller than 0.05. On the satisfaction variable, the results of the study of the effect of satisfaction on loyalty from shows that there is a positive relationship of 0.661, which means that a change in satisfaction provided by the company will increase customer loyalty by 0.661. RESULTS AND DISCUSSION The results of research for service quality variables of the five dimensions studied, then there are three dimensions, namely the dimensions of responsiveness, assurance, and empathy partially have a significant effect on satisfaction. While the other two dimensions, namely tangible and reliability do not have a significant effect on satisfaction. However, the dimensions of service quality have a significant simultaneous effect on the satisfaction that is equal to 0.63. This is in line with research Hastuti et al (2014), which states that the variable service quality has a significant effect on customer satisfaction, research Dewi et al (2014) shows that the results of statistical analysis show that service quality, customer satisfaction, and loyalty have a positive and significant relationship. Misbach, et al. (2013;), the results of the study stated that the service quality of Islamic banks has a significant effect on customer satisfaction and on trust. The results of this study indicate that the dimension of satisfaction has a positive and significant effect on the loyalty of 0.661. This is in line with the research of Mohsan et al. (2011), showing that customer satisfaction is correlated or has a positive relationship with loyalty. Khan and Fasih (2014), the results of research that service quality, customer satisfaction, and loyalty have a positive and significant relationship. RJOAS, 11(95), November 2019 From the results of the test of significance obtained the probability level of 0.000 and less than 0.05, which means there is a significant influence. RJOAS, 11(95), November 2019 From the above table, it can be seen that from the table of the Kolmogorov-Smirnov one-sample test it is known that the significance value is 0.834 and greater than 0.05. Then it can be concluded that the data tested is a normal distribution. Figure 1 – Structural Research Model The results of data processing using SPSS, the results obtained for the dimensions of physical evidence, that there is a positive relationship between physical evidence with a satisfaction level of 0.78 which means that if the provision of physical evidence is increased, then the level of satisfaction will positively increase as big as 0.78. The test results for the significance level is 0.283 greater than 0.05 which means there is no significant effect. Tangible Reliability Responsiviness Satisfaction Asurance Empaty Loyalty ,078 -,008 ,451 ,252 ,220 ,661 Figure 1 – Structural Research Model Tangible Reliability Responsiviness Satisfaction Asurance Empaty Loyalty ,078 -,008 ,451 ,252 ,220 ,661 Loyalty Empaty Figure 1 – Structural Research Model The results of data processing using SPSS, the results obtained for the dimensions of physical evidence, that there is a positive relationship between physical evidence with a satisfaction level of 0.78 which means that if the provision of physical evidence is increased, then the level of satisfaction will positively increase as big as 0.78. The test results for the significance level is 0.283 greater than 0.05 which means there is no significant effect. 233 233 RECOMMENDATIONS For the dimensions of responsiveness, assurance, and empathy, the hotel should improve the quality of services provided so as to increase the level of customer satisfaction. Besides that, with increased satisfaction from hotel customers will be able to increase customer loyalty. CONCLUSION The results of the research for the responsiveness dimension show that there is a positive relationship with the level of satisfaction with the assurance dimension. The results 234 RJOAS, 11(95), November 2019 RJOAS, 11(95), November 2019 showed that there was a positive relationship with the satisfaction and the results of the study for the empathy dimension showed that there was a positive relationship with the satisfaction. These three dimensions have a significant influence on satisfaction. Whereas for physical evidence, there is a positive relationship with the satisfaction and the results of research for empathy dimension indicate that there is a positive relationship with a satisfaction, but both dimensions do not have a significant effect on satisfaction. For the satisfaction variable, there is a positive relationship to loyalty and has a significant influence, which means that a change in satisfaction provided by the company will increase customer loyalty by 0.00 and less than 0.05, which means there is a significant effect. REFERENCES 1. Alma, Bukhari., 2005, Manajemen Pemasaran and Jasa, CV Alfa beta, Bandung. 2. Dewi dkk, 2014, Pengaruh Kualitas Pelayanan Terhadap Kepuasan and Loyalitas Nasabah PT BPR HOKI di Kabupaten Tabanan. 2. Dewi dkk, 2014, Pengaruh Kualitas Pelayanan Terhadap Kepuasan and Loyalitas Nasabah PT BPR HOKI di Kabupaten Tabanan. p 3. Ayu, G., Ratih, P., Dewi, K., Nyoman, N., Yasa, K., & Sukaatmadja, P. G. (2014). 4. Pengaruh kualitas pelayanan terhadap kepuasan and loyalitas nasabah pt bpr hoki di kabupaten tabanan, 5, 257–275. 4. Pengaruh kualitas pelayanan terhadap kepuasan and loyalitas nasabah pt bpr hoki di kabupaten tabanan, 5, 257–275. p , , 5. Ersi, D. Y., & Semuel, H. (2014). 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https://openalex.org/W3008842851	https://europepmc.org/articles/pmc7079636?pdf=render	English	null	Novel Smart Textiles	Materials	2,020	cc-by	2,757	Received: 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 Received: 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 Abstract: The sensing/adapting/responding, multifunctionality, low energy, small size and weight, ease of forming, and low-cost attributes of SMART textiles and their multidisciplinary scope offer numerous end uses in medical, sports and fitness, military, fashion, automotive, aerospace, built environment, and energy industries. The research and development for these new and high-value materials crosses scientific boundaries, redefines material science design and engineering, and enhances quality of life and our environment. “Novel SMART Textiles” is a focused special issue that reports the latest research of this field and facilitates dissemination, networking, discussion, and debate. Keywords: smart textiles; textile sensors; e-textiles; visual brain; thermal textile pixels; stretchable electronics; conductive textiles; wearables; stitch-based sensors; biofunctional textiles; ECG; hybrid electrodes; motion tracking; carbon nanotextiles; composites; EMS textiles; electrospun solar cells; embroidered e-textiles; targeted delivery; psychotextiles; energy harvesting; multifunctional A measure of the importance of smart textiles can be realized by its market size which will exceed USD 5.55 billion by 2025, with the healthcare and well-being sectors being a signiﬁcant driving force. The garment sensor-based telemedicine part is expected to exceed 50% CAGR in the next ﬁve years. Research for highly speciﬁc applications is increasing in exploring the opportunities oﬀered A measure of the importance of smart textiles can be realized by its market size which will exceed USD 5.55 billion by 2025, with the healthcare and well-being sectors being a signiﬁcant driving force. Th t b d t l di i t i t d t d 50% CAGR i th t ﬁ Research for highly speciﬁc applications is increasing in exploring the opportunities oﬀered by manipulating textile materials down to the nanoscale for creating new “smart” adaptive/active functionality, and by the development of "E-textiles” oﬀering intelligent ﬂexible integrated systems capable of sensing, actuation and wirelessly communicating in the form of intelligent high-tech fabrics and wearable garments. The development of these systems presents a complex set of interdisciplinary challenges in material design, hierarchical integration, control strategies, and manufacturing. This focused journal collection of highly original papers is underpinning these issues by reporting the latest research progress. The ﬁrst paper by George K. Stylios and Meixuan Chen proposes a new type of SMART fabrics called Psychotextiles [1]. www.mdpi.com/journal/materials Materials 2020, 13, 950; doi:10.3390/ma13040950 George K. Stylios George K. Stylios Research Institute for Flexible Materials, School of Textiles & Design, Heriot-Watt University, Scottish Borders Campus, Galashiels TD1 3HF, UK; G.Stylios@hw.ac.uk materials l Smart Textiles Stylios nstitute for Flexible Materials, School of Textiles & Design, Heriot-Watt University, Scottish Borders Galashiels TD1 3HF, UK; G.Stylios@hw.ac.uk 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 The sensing/adapting/responding, multifunctionality, low energy, small size and weight, ease g, and low-cost attributes of SMART textiles and their multidisciplinary scope offer numerous in medical, sports and fitness, military, fashion, automotive, aerospace, built environment, gy industries. The research and development for these new and high-value materials crosses boundaries, redefines material science design and engineering, and enhances quality of life nvironment. “Novel SMART Textiles” is a focused special issue that reports the latest research ld and facilitates dissemination, networking, discussion, and debate. ds: smart textiles; textile sensors; e-textiles; visual brain; thermal textile pixels; stretchable electronics; ve textiles; wearables; stitch-based sensors; biofunctional textiles; ECG; hybrid electrodes; racking; carbon nanotextiles; composites; EMS textiles; electrospun solar cells; embroidered ; targeted delivery; psychotextiles; energy harvesting; multifunctional asure of the importance of smart textiles can be realized by its market size which will exceed billion by 2025, with the healthcare and well-being sectors being a signiﬁcant driving force. ent sensor-based telemedicine part is expected to exceed 50% CAGR in the next ﬁve years. arch for highly speciﬁc applications is increasing in exploring the opportunities oﬀered ulating textile materials down to the nanoscale for creating new “smart” adaptive/active ity, and by the development of "E-textiles” oﬀering intelligent ﬂexible integrated systems sensing, actuation and wirelessly communicating in the form of intelligent high-tech fabrics ble garments. The development of these systems presents a complex set of interdisciplinary in material design, hierarchical integration, control strategies, and manufacturing. ocused journal collection of highly original papers is underpinning these issues by reporting materials l Smart Textiles Stylios nstitute for Flexible Materials, School of Textiles & Design, Heriot-Watt University, Scottish Borders Galashiels TD1 3HF, UK; G.Stylios@hw.ac.uk 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 The sensing/adapting/responding, multifunctionality, low energy, small size and weight, ease g, and low-cost attributes of SMART textiles and their multidisciplinary scope offer numerous in medical, sports and fitness, military, fashion, automotive, aerospace, built environment, gy industries. The research and development for these new and high-value materials crosses boundaries, redefines material science design and engineering, and enhances quality of life nvironment. “Novel SMART Textiles” is a focused special issue that reports the latest research ld and facilitates dissemination, networking, discussion, and debate. ds: smart textiles; textile sensors; e-textiles; visual brain; thermal textile pixels; stretchable electronics; ve textiles; wearables; stitch-based sensors; biofunctional textiles; ECG; hybrid electrodes; racking; carbon nanotextiles; composites; EMS textiles; electrospun solar cells; embroidered ; targeted delivery; psychotextiles; energy harvesting; multifunctional asure of the importance of smart textiles can be realized by its market size which will exceed billion by 2025, with the healthcare and well-being sectors being a signiﬁcant driving force. ent sensor-based telemedicine part is expected to exceed 50% CAGR in the next ﬁve years. arch for highly speciﬁc applications is increasing in exploring the opportunities oﬀered ulating textile materials down to the nanoscale for creating new “smart” adaptive/active ity, and by the development of "E-textiles” oﬀering intelligent ﬂexible integrated systems sensing, actuation and wirelessly communicating in the form of intelligent high-tech fabrics ble garments. The development of these systems presents a complex set of interdisciplinary in material design, hierarchical integration, control strategies, and manufacturing. ocused journal collection of highly original papers is underpinning these issues by reporting Received: 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 The issues of uncontrolled active molecules in spraying of fabric substrates is addressed in the next manuscript in which deﬁned polymers protect active components enabling controlled drug delivery and regulation of dosage, with promising results for home use and in clothes, and hence creating SMART biofunctional textiles [6]. The problem of surface area that enables better performance of electrically conductive polymer-based textiles has been addressed in the next paper by Lukas Vojtech et al. [7]. They used an electrochemical method to measure the resistance between two electrodes for comparing fabric surface areas. The combination of ECG measurement and motion tracking is achieved by developing a new hybrid soft textile electrode. Systematic measurements have shown that this hybrid textile electrode is capable of recording ECG and motion signals synchronously, which may prove a life changing approach to continuous health monitoring, as reported by Xiang An and George K Stylios [8]. The failure of the performance of composites can have catastrophic consequences and composite manufacturing is compensating for not precise detection by overengineering which is costly. This has been addressed in the paper by G Wang et al. [9], who proposed a carbon nanomaterial SMART ﬁbre sensor capable of in-line monitoring in the manufacture of high-performance composites. With the rapid expansion of the Internet of Things (IoTs) already aﬀecting our work, our homes, and our communications with others, Electromagnetic Shielding (EMS) is important to guard against emissions of Electromagnetic Frequencies (EMF). Beyond electrical reliability, the protection of health and prevention of hacking are high in this agenda and what better for combating this problem than textile fabrics with their high ﬂexibility and formability, as reported in the paper by Mark Neruda and Lukas Vojtech [10]. In the same area of interest, (IoTs), harvesting of energy for all those devices has been a quest that will continue for years to come and the two papers by L Juhasz and I.J Junger [11] and by J Junger et al. [12] shed some new light into textile-based dye-sensitized solar cells. The ﬁrst paper deals with the understanding of the physical processes in the cell and its optimization, and the second paper provides electrospun polyacrylonitrile (PAN) nanoﬁbre mats coated by a conductive polymer as collar cells on their own right. Finally, the paper by B Moradi et al. Received: 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 After studying the direct relationship between design and brain waves, using EEG, the characteristics and attributes of patterns that inﬂuence speciﬁc brain emotions are established, which are in turn designed into four pairs of smart pattern-changing fabrics for investigation. A novel thermochromic process was devised to enable the development of novel yarns which when knitted into jacquard patterned fabrics they can switch from one pattern into another. This process was fundamental in realizing these new types of smart textiles named Psychotextiles. This paper shows for the ﬁrst time how to design speciﬁc patterns for aﬀecting speciﬁc human emotions and discusses how this research can be extended towards colour and touch. The concept of a thermal textile pixel is addressed in the next paper, which is based on a textile structure that shows spatial and temporal thermal contrast and can be used in the context of thermal communication [2]. Textiles are ﬂexible and easy to form around three-dimensional surfaces such as our bodies. Novel electrically conductive textiles for stretchable electronic systems that can be bent or shaped around complex curvatures are being developed and the optimization and properties of these structures is reported in the paper by Christian Dils et al. [3]. E-sensors is the topic of the next two papers. Conductive formulations containing micron-size metal ﬂakes of silver-coated copper Materials 2020, 13, 950; doi:10.3390/ma13040950 2 of 3 Materials 2020, 13, 950 (Cu) and pure silver (Ag) were used as conductors on woven fabrics, and fabric ﬂexural stiﬀness and sheet resistance (Rsh), were investigated for durability, performance and reliability, as reported by Veronica Malm et al. [4]. In the next paper by Orathai Tangsirinaruenart and George K Stylios [5], novel textile-based strain sensors have been developed and their performance was evaluated. These sensors are likely to change the way we measure stresses and strains by using entirely the textile itself, and hence ﬁnding end uses in garments as wearables for physiological wellbeing monitoring such as body movement, heart monitoring, respiration, and limb articulation measurement. The authors show how electrical resistance and mechanical properties of seven diﬀerent textile sensors were optimized and measured, and report on their composition. References 1. Stylios, G.K.; Chen, M. The Concept of Psychotextiles; Interactions between Changing Patterns and the Human Visual Brain, by a Novel Composite SMART Fabric. Materials 2020, 13, 725. [CrossRef] [PubMed] 1. Stylios, G.K.; Chen, M. The Concept of Psychotextiles; Interactions between Changing Patterns and the Human Visual Brain, by a Novel Composite SMART Fabric. Materials 2020, 13, 725. [CrossRef] [PubMed] 1. Stylios, G.K.; Chen, M. The Concept of Psychotextiles; Interactions between Changing Patterns and the Human Visual Brain, by a Novel Composite SMART Fabric. Materials 2020, 13, 725. [CrossRef] [PubMed] 2. Stöhr, A.; Lindell, E.; Guo, L.; Persson, N.-K. Thermal Textile Pixels: The Characterisation of Temporal and 1. Stylios, G.K.; Chen, M. The Concept of Psychotextiles; Interactions between Changing Patterns and the Human Visual Brain, by a Novel Composite SMART Fabric. Materials 2020, 13, 725. [CrossRef] [PubMed] 2. Stöhr, A.; Lindell, E.; Guo, L.; Persson, N.-K. Thermal Textile Pixels: The Characterisation of Temporal and Spatial Thermal Development. Materials 2019, 12, 3747. [CrossRef] [PubMed] Human Visual Brain, by a Novel Composite SMART Fabric. Materials 2020, 13, 725. [CrossRef] [PubMed] 2. Stöhr, A.; Lindell, E.; Guo, L.; Persson, N.-K. Thermal Textile Pixels: The Characterisation of Temporal and Spatial Thermal Development. Materials 2019, 12, 3747. [CrossRef] [PubMed] 2. Stöhr, A.; Lindell, E.; Guo, L.; Persson, N.-K. Thermal Textile Pixels: The Characterisation of Temporal and Spatial Thermal Development. Materials 2019, 12, 3747. [CrossRef] [PubMed] 3. Dils, C.; Werft, L.; Walter, H.; Zwanzig, M.; Krshiwoblozki, M.; Schneider-Ramelow, M. Investigation of the Mechanical and Electrical Properties of Elastic Textile/Polymer Composites for Stretchable Electronics at Quasi-Static or Cyclic Mechanical Loads. Materials 2019, 12, 3599. [CrossRef] [PubMed] 3. Dils, C.; Werft, L.; Walter, H.; Zwanzig, M.; Krshiwoblozki, M.; Schneider-Ramelow, M. Investigation of the Mechanical and Electrical Properties of Elastic Textile/Polymer Composites for Stretchable Electronics at Quasi-Static or Cyclic Mechanical Loads. Materials 2019, 12, 3599. [CrossRef] [PubMed] . Malm, V.; Seoane, F.; Nierstrasz, V. Characterisation of Electrical and Stiﬀness Properties of Conduc Textile Coatings with Metal Flake-Shaped Fillers. Materials 2019, 12, 3537. [CrossRef] [PubMed] g p , , [ ] [ ] 5. Tangsirinaruenart, O.; Stylios, G. A Novel Textile Stitch-Based Strain Sensor for Wearable End Users. Materials 2019, 12, 1469. [CrossRef] [PubMed] 5. Tangsirinaruenart, O.; Stylios, G. A Novel Textile Stitch-Based Strain Sensor for Wearable End Users. Materials 2019, 12, 1469. [CrossRef] [PubMed] 6. Received: 14 February 2020; Accepted: 19 February 2020; Published: 20 February 2020 [13] proposes a more practical solution of connecting e-textiles using the embroidery process and they report how signal propagation control maybe achieved, enabling customized electromagnetic properties such as ﬁltering for wearable electronics. Studying of these research papers, increases our knowledge, enables us to see our own work in context, it empowers us to improve our understanding, it increases the rigour of our research, and encourages us to work collectively. I hope that to some extent the publication of the concentrated eﬀort of these researches in this special issue, can aid us towards a clearer roadmap for our further research advancement. Adding my own observations about the challenges that smart textiles must overcome; I can say that washability along with user safety and reliability are three important factors which need addressing in our research. Traditional textiles working with electrical components need a change of culture, which is time consuming and diﬃcult. The supply chain is not yet ready to embrace fast changes that are much needed, and designers and engineers must learn to work together. On the other hand, we have a tired textile industry that is producing consumer products at pence per minute blamed for damaging the environment. I believe that there are untapped opportunities for this industry and 3 of 3 Materials 2020, 13, 950 that smart textiles must converse with traditional textiles. My own hope for the future of this ﬁeld is not to poise for incremental changes but to force its way for a transformational change. that smart textiles must converse with traditional textiles. My own hope for the future of this ﬁeld is not to poise for incremental changes but to force its way for a transformational change. Acknowledgments: I cannot end this editorial by not thanking all authors for their hard work, discipline and rigour, and to encourage them to continue pushing the boundaries of this fascinating ﬁeld. The exposure and promotion of our work in this special issue has been facilitated by the editorial and production oﬃce of MPDI. Conﬂicts of Interest: The author declares no conﬂict of interest. Conﬂicts of Interest: The author declares no conﬂict of interest. References Lis Arias, M.J.; Coderch, L.; Martí, M.; Alonso, C.; García Carmona, O.; García Carmona, C.; Maesta, F. Vehiculation of Active Principles as a Way to Create Smart and Biofunctional Textiles. Materials 2018, 11, 2152. [CrossRef] [PubMed] 7. Vojtech, L.; Neruda, M.; Reichl, T.; Dusek, K.; De la Torre Megías, C. Surface Area Evaluation of Electrically Conductive Polymer-Based Textiles. Materials 2018, 11, 1931. [CrossRef] [PubMed] 8. An, X.; Stylios, G.K. A Hybrid Textile Electrode for Electrocardiogram (ECG) Measurement and Motion Tracking. Materials 2018, 11, 1887. [CrossRef] [PubMed] 9. Wang, G.; Wang, Y.; Luo, Y.; Luo, S. Carbon Nanomaterials Based Smart Fabrics with Selectable Characteristics for In-Line Monitoring of High-Performance Composites. Materials 2018, 11, 1677. [CrossRef] [PubMed] 10. Neruda, M.; Vojtech, L. Electromagnetic Shielding Eﬀectiveness of Woven Fabrics with High Electrical Conductivity: Complete Derivation and Veriﬁcation of Analytical Model. Materials 2018, 11, 1657. [CrossRef] [PubMed] 11. Juhász, L.; Juhász Junger, I. Spectral Analysis and Parameter Identiﬁcation of Textile-Based Dye-Sensitized Solar Cells. Materials 2018, 11, 1623. [CrossRef] [PubMed] 12. Juhász Junger, I.; Wehlage, D.; Böttjer, R.; Grothe, T.; Juhász, L.; Grassmann, C.; Blachowicz, T.; Ehrmann, A. Dye-Sensitized Solar Cells with Electrospun Nanoﬁber Mat-Based Counter Electrodes. Materials 2018, 11, 1604. [CrossRef] [PubMed] 13. Moradi, B.; Fernández-García, R.; Gil, I. E-Textile Embroidered Metamaterial Transmission Line for Signal Propagation Control. Materials 2018, 11, 955. [CrossRef] [PubMed] © 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4224526108	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/A8251264BA9288143F95BB84723D06AB/S1866980822000084a.pdf/div-class-title-comprehension-of-different-types-of-novel-metaphors-in-monolinguals-and-multilinguals-div.pdf	English	null	Comprehension of different types of novel metaphors in monolinguals and multilinguals	Language and cognition	2,022	cc-by	19,724	†The title has been changed since publication. Abstract It has been suggested that multilingualism can lead to increased cognitive flexibility and creativity. No studies to date, however, have investigated whether this advantage leads to a greater propensity to find meaning in different kinds of novel metaphors. This article reports a self-paced reading study that focuses on whether such an increased propensity is displayed by multilingual English speakers, as opposed to monolingual English speakers. The article explores the difference between two broad types of novelty in metaphorical expressions, which are distinguished by how readily they conform to existing metaphorical schemata. The results indicate that both monolinguals and multilinguals find novel metaphors that conform readily to an existing schema easier to comprehend those that do not. They also take longer to seek meaning in metaphors that conform readily to an existing schema. Multi- linguals are more likely than monolinguals to find meaning in both types of novel metaphor. The theoretical distinction drawn between metaphors that conform readily to an existing schema and those that do not highlights the variability of meaning in novel metaphors. It also focuses attention on the different extents to which hearers seek rich meanings as opposed to less rich but more easily derived ones. Keywords: novel metaphor; multilingualism; cognitive flexibility; metaphor comprehension; high-conforming novel metaphor; low-conforming novel metaphor Language and Cognition (2022), 14: 3, 401–436 doi:10.1017/langcog.2022.8 Language and Cognition (2022), 14: 3, 401–436 doi:10.1017/langcog.2022.8 Comprehension of different types of novel metaphors in monolinguals and multilinguals† 1University of Osijek; 2University of Bologna; 3University of Birmingham Corresponding author. Email: m.bolognesi@unibo.it 1University of Osijek; 2University of Bologna; 3University of Birmingham Corresponding author. Email: m.bolognesi@unibo.it (Received 07 September 2021; Revised 25 March 2022; Accepted 28 March 2022) (Received 07 September 2021; Revised 25 March 2022; Accepted 28 March 2022) © The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 1. Introduction This work originated in two different but interacting interests: (1) the ways in which being multilingual rather than monolingual affects one’s comprehension of novel metaphor; and (2) the diverse ways in which metaphorical utterances can be novel, and how this diversity affects comprehension of the utterances. There is evidence to suggest that being multilingual leads to increased cognitive flexibility (Bialystok, 2011), although, as we will see below, this idea has been © The Author(s), 2022. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al 402 challenged. Prompted by this, we ask whether such cognitive flexibility effects arise in novel metaphor comprehension. Specifically, we investigate whether multilinguals more readily comprehend novel metaphorical utterances than monolinguals do. That is, we explore whether multilinguals are more inclined to see meaning in novel metaphorical utterances than monolinguals are. At the same time, we are interested in whether any such effect differs between two different types of metaphor novelty that we introduce, based partly on work by Barnden (2015), and that we call here ‘high-conforming novelty’ and ‘low-conforming novelty’. These types of novelty reflect differing extents to which novel metaphors that conform to an existing schema. The article proceeds as follows. The next section provides theoretical and experi- mental background, and motivates the particular hypotheses for experimental inves- tigation in our study. Section 3 presents the detailed nature of the experiment. Sections 4 and 5 report and discuss the results, showing how they broadly support our hypotheses concerning the impact of metaphor novelty type on comprehension difficulty and the tendency for multilingualism to enhance comprehension for both novelty types. Finally, the Supplementary Document1 explains why, in the experi- ment, we classified particular metaphorical examples in the way that we did. 1Accessible at https://osf.io/ek4q8/?view_only=faa82d8334fd478cb8f99fd15f107597. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2. Background, approach and hypotheses 2.1. Multilinguals, monolinguals and metaphor One might hypothesise that multilingual individuals are more flexible than mono- linguals when seeking to comprehend novel metaphor. There is a substantial body of literature indicating that multilinguals are, generally speaking, more cognitively flexible than monolinguals. Classic empirical studies show that multilinguals appear to outperform monolinguals on cognitive control tasks (Bialystok, 2001a, 2001b; Bialystok et al., 2014). Cognitive control is the ability to deal with potentially conflicting sources of information, to ignore irrelevant sources and to deliberately switch between sources. Multilinguals’ superior performance suggests that learning another languagerendersourcategorisation systems moreflexible (Bialystok & Martin, 2004; Jacques & Zelazo, 2001; Martin-Rhee & Bialystok, 2008). In addition, multi- linguals appear to develop stronger metalinguistic skills than monolinguals (Bialystok, 2001a; Bialystok et al., 2014), and this has a washback effect on their first language (L1) ability (Jarvis, 2003; Murphy & Pine, 2003; Yelland, Pollard, & Mercuri, 1993). y p y The issue of multilinguals’ higher cognitive flexibility, compared to monolinguals, is not free from controversy and has, in recent years, been at the centre of a replication crisis. Recent meta-analyses of studies that claim to have found cognitive advantages for multilinguals conclude that some differences in executive control tasks may have been overstated (Lehtonen et al., 2018; Paap & Greenberg, 2013; Papageorgiou et al., 2019). Other meta-analyses suggest that such differences may be linked to the publication bias phenomenon, in which empirical studies that observe a significant difference are more likely to be published than studies that report no statistical effect (de Bruin, Bak, & Della Sala, 2015; de Bruin, Dick, & Carreiras, 2021; de Bruin, Treccani, & Della Sala, 2015a, 2015b). It therefore remains unclear whether, to what extent, and in what ways mono- linguals and multilinguals differ in their repertoire of cognitive and linguistic https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 403 Language and Cognition abilities. As a step towards removing some of this lack of clarity in the field, we focus specifically on metaphor, and test the hypothesis that multilingual speakers display greater flexibility in dealing with novel metaphor, compared to monolinguals. This general hypothesis is supported by empirical literature in metaphor studies, show- ing, for instance, that on acquiring a second language (L2), people more frequently produce new metaphors in their L1 (Kecskés & Papp, 2000). 2. Background, approach and hypotheses 2.1. Multilinguals, monolinguals and metaphor These authors suggest that L2 acquisition engenders greater ability to construe events in different ways: it leads to a common underlying conceptual base for the two language channels, which constantly interact. Presumably, therefore, multilinguals can incorporate metaphorical associations from both languages, access them in either language and switch between them easily. Other studies have suggested strong links between divergent thinking (namely, the tendency to make fast associative connections between distant ideas aimed at exploring potential solutions; see Runco & Acar, 2012), fast automatic holistic processing and creative metaphor production and comprehension, particularly in L2s (Birdsell, 2018a, 2018b; Littlemore, 2001, 2010; Littlemore & Low, 2006a, 2006b). In addition, people are more likely to find meaning in novel metaphor when operating in their L2 than when operating in their L1 (Littlemore, 2010). These studies suggest that multilinguals will be more flexible in comprehending novel metaphor, in the sense of being more likely to find meaning in it. g It is less clear how multilingualism might affect the speed with which novel metaphorical expressions are processed. Fast metaphor meaning identification cor- relates with holistic processing (Littlemore, 2001), suggesting that it relies largely on loose associative networks and coarse semantic processing (Beeman, 1998). The fact that multilinguals have more elaborate associative networks than monolinguals may mean that it takes them longer to search these networks for meaning. This tentative hypothesis is indirectly supported by robust findings in lexical retrieval tasks where multilinguals tend to perform more slowly than monolinguals, and exhibit more effortful language processing (Bialystok et al., 2009). https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.2.1. Conventionality Usually, a metaphor is considered to be conventional if it has an established meaning that is widely shared by L1 speakers of the language.2 The matter can differ between different theorists, notably because of different views of what counts as metaphorical. Another complication is that the degree of familiarity a language user might have with the conventional term and its meaning(s) can vary between users and between expressions. Nevertheless, there are many expressions, such as ‘at the back of one’s mind’, which have an established meaning that most theorists would regard as metaphorical and that would be easily discerned by L1 speakers. Following Müller (2009), we take conventional metaphor to come in two broad subtypes, ‘transparent’ and ‘opaque’, with the latter subtype equated by Müller with the type that should truly be said to be ‘dead’ or ‘historical’ (e.g., ‘broken heart’). The transparent metaphors, which are the only ones of explicit interest in our study, are those where L1 speakers are equipped to see how the metaphorical meaning fits with extant literal meanings of the words in the metaphor. In Section 3.1.1, we comment further on our particular criteria for selecting conventional metaphorical terms for our particular study. p y In line with Müller’s (2009) claims that conventional metaphorical meaning, if sufficiently common and standard, is easily retrievable by hearers, and in line with evidence about the speed of comprehending conventional metaphor, even when transparent and out of context (see, e.g., Holyoak & Stamenković, 2018), we assume that conventional metaphorical meanings are usually simply retrieved, in much the same way as literal meanings are generally assumed to be. However, Müller (2009) mentions various types of special circumstance where something more than straight- forward retrieval can happen, including circumstances where the term in question is accompanied by pictures, hand gestures or other linguistic metaphorical terms using the same source subject matter.3 However, even without a special circumstance, we should 2The notion of conventionality is rarely as precisely defined in the metaphor research literature as one might wish, although it is necessarily operationalised in particular ways in the many psycholinguistic studies that feature conventional metaphor. Our description of it incorporates the portrayal of conventional metaphor in Müller, 2009 (see especially p. 181 and elsewhere in Ch. 2The notion of conventionality is rarely as precisely defined in the metaphor research literature as one might wish, although it is necessarily operationalised in particular ways in the many psycholinguistic studies that feature conventional metaphor. Our description of it incorporates the portrayal of conventional metaphor in Müller, 2009 (see especially p. 181 and elsewhere in Ch. 6), and accords with the notion of conventionality as familiarity (i.e., being repeatedly experienced) in Holyoak & Stamenković’s (2018) survey of major empirically investigated theories of metaphor. 2.2. Conventionality and two types of novelty Numerous studies have investigated differences between novel and conventional metaphors (see below for further information on how we define these terms), comparing, for instance, the speed with which they are comprehended. The broad conclusion from such studies appears to be that novel metaphors take longer to process (Cacciari et al., 2011; Cardillo et al., 2012). Blasko & Connine (1993) and Blasko & Briihl (1997) demonstrated a clear effect of familiarity on how metaphors are comprehended (see also Bambini et al., 2019; Columbus et al., 2015; Mashal & Faust, 2009). Similarly, Bowdle & Gentner (2005) presented their career of metaphor hypothesis, whereby novel metaphors are comprehended through intricate analogy building, but conventional metaphors (at least of the A-is-B type) are comprehended through a less effortful process of categorisation. g p g In such studies, there are some gradations of novelty and conventionality, and these qualities are generally regarded as occupying different regions of a single, simple scale. There have, however, been few attempts to explore whether qualitatively different ways of being novel have a bearing on comprehension difficulty, and if so, whether this relates to mono/multilingualism. We mention here two studies that doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al 404 have investigated phenomena that are related to these issues. First, Littlemore et al. (2018) studied the role that ‘optimal innovation’ plays (Giora et al., 2004) in metaphor comprehension, and found that ‘optimally innovative’ metaphors were more likely to be deemed to be of a higher quality than metaphors that were ‘too’ creative and therefore not optimally innovative. Second, Werkmann Horvat, Bolognesi, & Kohl (2021)) explored the ways in which easy-to-interpret and difficult-to-interpret meta- phors were received by people with and without multilingual experience. They found that people with multilingual experience were more likely to say that the metaphor makes sense than those without multilingual experience. However, neither of these studies discussed the differences between different types of novelty in any depth. Before further addressing novelty, we must address conventionality. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.2.2. Novelty in general 2.2.2. Novelty in general We take the common approach of defining novelty in metaphor as a lack of conventionality – the fewer widely shared meanings that a metaphorical expression has, the more novel it is. The more novel it is, the less likely it is that a hearer can just retrieve a familiar, relevant metaphorical meaning, and so the more likely it is that he/she must work out a metaphorical meaning in some other way, a ‘beyond retrieval’ way as we will say. To the extent that the comprehension of novel metaphor involves a beyond-retrieval process, we are faced with a range of different processes that have been proposed by different metaphor theories. We are also faced with the possibility that different types of processing, whether drawn from the same theory or from different theories, are useful in different types of metaphorical phrases, different contexts and so forth. In defining different varieties of meta- phorical novelty, we do not wish to be bound by the limits imposed by particular theories of metaphor. We therefore abstract two general forms of beyond-retrieval processing that are discernible in different specific forms in different theories, and that can therefore plausibly be assumed to be used by hearers in some way, irrespective of what one’s specific theory of metaphor comprehension is. These general forms are: (1) the use of bridges between source and target subject matters; and (2) the following of within-source connections. These are discussed separately in the following two subsections. 2.2.3. Metaphoric bridges: mappings, superordinate categories etc. p g pp g p g The comprehension of metaphor must involve some way in which the hearer can take aspects of the source subject matter to relate suitably to aspects of the target subject matter. Different metaphor theories have different versions of this, but we generalise by using the term ‘[metaphoric] bridge’ for whatever device is being used to relate source to target aspects. Some prominent theories are in some way reliant on bridges in the form of ‘mappings’. Consider first a metaphor theory that proposes that the hearer finds an analogy entirely from scratch between the source and target subject matters as the central part of comprehension. A salient example here is the structure-mapping theory (Gentner, 1988; Gentner & Wolff, 1997; Wolff & Gentner, 2011); also incorporated as one part of the career of metaphor theory proposed by Bowdle & Gentner (2005). matter has clear boundaries.) We also say the subject matters are on the source side and target side, respectively. 4See also Casasanto & Gijssels (2015) and Hampe (2017) for commentary on some of the evidence amassed by various authors about the source subject matter of metaphors being activated, and Barnden (2020) for further commentary and a discussion of what such activation means for metaphor theory in general. 2.2.1. Conventionality 6), and accords with the notion of conventionality as familiarity (i.e., being repeatedly experienced) in Holyoak & Stamenković’s (2018) survey of major empirically investigated theories of metaphor. j p y g p 3The target subject matter is the one that is being addressed, and the source subject matter is the one being used to address it. For instance, for ‘firm belief’, the source subject matter can be taken to be that of physical objects and their properties, and the target subject matter can be taken to be mental states. (Neither subject https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 405 Language and Cognition be open to the possibility that something more than simple retrieval happens. Holyoak & Stamenković (2018) postulate that some conventional metaphors may be compre- hended by a constrained, non-cognitively weighty analogy mechanism.4 matter has clear boundaries.) We also say the subject matters are on the source side and target side, respectively. 4See also Casasanto & Gijssels (2015) and Hampe (2017) for commentary on some of the evidence amassed by various authors about the source subject matter of metaphors being activated, and Barnden matter has clear boundaries.) We also say the subject matters are on the source side and target side, respectively. 4 2.2.2. Novelty in general The analogy consists of postulated map- pings between aspects of the source and aspects of the target. For instance, if an academic department is talked of metaphorically as a solar system, the hearer might be theorised to create a mapping between the most prominent researcher and the star of the solar system, mappings between other academic staff members and https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2022.8 Published online by Cambridge University Press Werkmann Horvat et al 406 planets, a mapping between the relationship of working under someone and gravitational attraction to a heavier object, a mapping between academic prowess and physical size of solar-system objects and so forth. In such a theory, the ‘bridges’ are the mappings created. pp g Another possibility is that the hearer already knows of an analogy, and extends it for the purposes of comprehending a specific utterance. For instance, we can regard a conceptual metaphor (Lakoff & Johnson, 1980, 1999), such as LOVE RELATIONSHIP AS A JOURNEY as a body of already-known analogy (Holyoak & Stamenković, 2018). That is, it is an already-known set of mappings between love items and journey items (e.g., between the lovers and the journey companions, and between the progress of the relationship and the progress of the journey). However, the comprehension of a particular sentence that relies on such a conceptual metaphor, for example, ‘John and Mary were on a lazy tropical cruise together’ when this is taken to be metaphorically about John and Mary’s love relationship rather than literally about a cruise, might involve the creation of new mappings. An example would be a mapping that puts physical effort (a lack of which is suggested by ‘lazy’) in correspondence with emotional and mental effort aimed at sustaining the relationship. Similar points apply, though with great differences of specific detail, if the already known mappings are of much more generic sorts such as in the primary metaphors of Grady (1997) (e.g., ORGANISATION IS PHYSICAL STRUCTURE) or in the correspondences proposed by the ATT-Meta theory (Barnden, 2015, 2016; Barnden & Lee, 2001). In the theories of the sort discussed in this paragraph, we take both the already-known mappings and the newly constructed ones as bridges. Other theories propose other ways whereby hearers relate source aspects to target aspects. Most saliently, consider categorisation-based theories. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.2.2. Novelty in general We include here not just Glucksberg’s (2001) theory, but also proposals from Relevance Theory, such as in Carston & Wearing (2011) and Sperber & Wilson (2008). Such theories propose the finding and/or construction of categories (often called ‘super- ordinate’ categories) that include a mentioned source category and also the target item. Take, for instance, the widely discussed (Glucksberg, 1998) example ‘My job is a jail’. Here a category jail* of situations that are behaviour-limiting and unpleasant may be retrieved or invented. This category contains the (real-)jail category as a subcategory, but is also broad enough to include at least some jobs or types of jobs. We count this superordinate category as the bridge used between source and target. As we have already indicated, such a superordinate category may be either already known or freshly invented. An intermediate possibility is that the hearer knows of a superordinate category jail* that includes real jails, but finds it to be too narrow or too broad to fit the specific job with optimal aptness, and so constructs a new category jail** that is broader or narrower, as needed. This makes the point that metaphor comprehension may involve the refinement of an existing bridge as opposed to the construction of an entirely new one. Alternatively, instead of theorising in terms of superordinate categories, one can do so in terms of shared properties. In the job/jail example, the shared properties would be behaviour limitation and unpleasantness. These properties would be aspects of both target and source, and would serve as a type of bridge. yp g The types of bridges so far mentioned are drawn from the main types of theories surveyed by Holyoak & Stamenković (2018), namely detailed fresh-analogy-finding accounts, conceptual metaphor theories (here considered as a type of known-ana- logy-cum-fresh-analogy-finding theory) and categorisation theories. Their survey 407 Language and Cognition covers those theories that are about adult metaphorical comprehension and that have been subjected to major psychological investigation aimed at support or rebuttal. Their survey also mentions some further possibilities, for example, a weaker, simpler form of analogising. Nevertheless, the sense in which source and target aspects are related is similar to what was already described above. 2.2.2. Novelty in general It should be noted that their survey also omits some major theories, notably blending theory (or conceptual integration theory; Fauconnier & Turner, 1998, 2008), presumably on the grounds that it has mainly been subject to linguistic theorising rather than psychological experimentation. The melding of different conceptual items within blend spaces can be seen as a type of bridging that is different from the types above, but in fact such melding is tied to the existence of (already known or newly stipulated) mappings between subject matters as well. An aspect of our relative neutrality with respect to different theories is the way we hypothesise the existence of particular familiar bridges. Although we have been influenced by specific bridges that existing theories have postulated as familiar, we do not rely only on these postulations. Instead, as we will see below, in our study, we use conventional expressions (including those in our materials as specified in Section 3, but also others) directly as evidence for the existence of bridges. For instance, we used the conventional metaphoricity of ‘firm belief’ to evidence a familiar bridge between beliefs and physical objects (the Supplementary Document contains more examples of this). p In summary, bridges are the constructs proposed by theories as constituting the relationships that the hearer sees between source and target subject matters and that help to comprehend the metaphorical utterance at hand. The notion of bridge is not intended as a new idea, but just as a convenient way of abstracting what is common from well-known proposals by previous researchers. 2.2.4. Within-source inferencing and other connection-following Beyond-retrieval metaphor comprehension can also benefit from something orthogonal to the use of bridges, namely the following of inferential or other connections within the source subject matter, whereas bridges are between the target subject matter and the source subject matter. As an example, let us assume that, when something is being metaphorically viewed as a source of physical light, there is a familiar bridge between (on the source side) the physical brightness of the item and (on the target side) the item’s usefulness and strikingness. Thus, one way to comprehend ‘high-wattage idea’ is to assume that the idea is being viewed as an illumination source and then to infer from its high wattage that it is especially bright. This especially strong brightness then plausibly suggests, via the bridge, that the idea is especially useful and striking. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.2.2. Novelty in general However, the inference to the especially strong brightness from the high wattage is entirely within the source subject matter. Connections followed within the source subject matter do not need to be a matter of inference. For instance, they might be a matter of negation, opposition or lack. Suppose an idea that has so far been considered to be ‘bright’ is said to be ‘extin- guished’. This might be comprehended by taking the idea to be an initially bright light source as above that at some point has its shining stopped. The resulting non-shining could be interpreted as non-usefulness, using the same bridge as above. There is certainly an inference here from extinguishing to non-shining, but to use this to Werkmann Horvat et al 408 exploit the bridge between shining and usefulness, there needs to be a step taken from non-shining to shining. The use of inferencing or other connection-following within the source subject matter, or ‘on the source side’ as we will also say, is discernible in many theories of metaphor, but is especially notable in relevance theory proposals (Carston & Wear- ing, 2011), ATT-Meta (cited above) and the approach taken by Ruiz de Mendoza and colleagues (Ruiz de Mendoza & Galera, 2014, especially pp. 108ff). Within-source connection-following (inferencing etc.) is also involved in the use of so-called ‘entailments’ of conceptual metaphors (Lakoff & Johnson, 1980, 1999; Lakoff & Turner, 2009). It therefore spans a number of different theories of metaphor comprehension. 2.2.5. Two types of metaphorical novelty: high-conforming and low-conforming The particular constellations of beyond-retrieval processing types (based on existing bridges, refinement of existing bridges, construction of new bridges and within- source connections) that are proposed by different theories of metaphor compre- hension could have different effects on speed of processing and on the results of meaning construction. Furthermore, for a given expression, markedly different processing mixes might be used in different contexts or by different hearers. In essence, there is an extensive, intricate landscape of different beyond-retrieval processing scenarios that could be proposed to account for the novelty of metaphor- ical expressions and the ways in which they are processed, and derivatively we can talk of there being different types of novelty on the basis of such scenarios.5 The points discussed here are somewhat expanded upon in the Supplementary Discussion. 5A slight complication in our discussion is that beyond-retrieval processing could in principle be used for transparent conventional metaphor as well as for novel metaphor. Empirical results such as those sum- marised in Holyoak & Stamenković (2018) suggest that this does not generally happen, and our main interest in the working-out is in application to novel metaphor, but as a matter of principle and of thoroughness, we should not restrict the possibility of beyond-retrieval processing solely to novel metaphor. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 6Henceforth, for brevity, we will count such refinement as a type of construction of new bridges. 7The Supplementary Materials contain more examples of how we decided whether a novel pair was high- conforming or low-conforming. 2.2.2. Novelty in general p y By contrast, a novel metaphorical expression is low-conforming if, when the expression is presented out of context, the following holds: • in order to derive a distinctive metaphorical meaning for the expression tha p g p • exploits distinctive source-side concepts raised by the expression, p p y p • the hearer needs to: creatively entertain special contexts, AND/OR y p • construct new bridges (or refine old ones6), AND/OR g ( ) • follow difficult or not generally applicable within-source connections. We place ‘curved hope’ in this category, because of the probable need to entertain a special context, and because of the relative difficulty of making useful within-source inferences from curvedness. Now, a hearer might infer from the curvedness that the hope is probably being viewed as a solid physical object, and then infer that it has at least a normal level of robustness. However, this meaning, relying only on familiar bridges and easy within-source inferencing, does not exploit anything very distinctive about being curved as opposed to being straight. One possible meaning would be to take being curved as opposed to straight as implying greater visual appeal – especially if, let us imagine, the attractiveness of hopes is under discussion in a rather unusual context – and to use some new or old bridge between visual appeal and more abstract attractiveness.7 Note that the low-conforming type of novel metaphor is not simply the negation of the high-conforming type – it involves a fairly strong departure from high conformity. Novel metaphors can conform to any degree, and can lie between the two types. These types are intended to lie relatively near either end of the scale of conformity to what is familiar, generic and straightforward. High-conforming novel metaphor covers at least some of what is commonly called extended metaphor, although the more difficult cases of extended metaphor would be low-conforming. g p g In our characterisations of high conformity and low conformity, we have sought to build in matters other than the question of whether new bridges are needed, in order to respect the point that hearers of metaphorical expressions may derive meanings of different richness depending on how strongly they exploit distinctive source-side features, what special contexts they entertain and what relatively difficult or special within-source inferencing or other connection-following they do. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.2.2. Novelty in general They require much more extensive development than is possible in this paper, but as they stand, they suggest that we can usefully pick out a relatively easily comprehended, high-conforming type of novel metaphor. We deem a nonconven- tional metaphorical expression to be high-conforming if, when the expression is presented out of context, the following holds: • major distinctive aspects of source-side concepts raised by the expression can be used, i h i l i i i l • major distinctive aspects of source-side concepts raised by the expression can be used, • without creatively entertaining any special context, y g y p • to provide distinctive aspects of a metaphorical meaning for the expression, • by means only of bridges that can plausibly be suggested to be familiar to hearers together with • easy, generally applicable within-source connections. In dubbing an expression as high-conforming, we assume that different metaphor theories would agree that the expression can be comprehended in the way indicated, even if the particular bridges and within-course connections deployed might be importantly different, and even if the distribution of effort between the use of bridges 5A slight complication in our discussion is that beyond-retrieval processing could in principle be used for transparent conventional metaphor as well as for novel metaphor. Empirical results such as those sum- marised in Holyoak & Stamenković (2018) suggest that this does not generally happen, and our main interest in the working-out is in application to novel metaphor, but as a matter of principle and of thoroughness, we should not restrict the possibility of beyond-retrieval processing solely to novel metaphor. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 409 Language and Cognition and the use of within-source connections is different. Given the comments above, the expression ‘high-wattage idea’ is arguably high-conforming as a novel metaphorical expression. In the process imagined above, all that was needed was a simple, generally applicable within-source inference from the distinctive source-side feature of high wattage to especially high brightness, together with the use of a familiar bridge between degree of brightness and degree of strikingness and usefulness; and no special context needed to be creatively entertained. 2.2.2. Novelty in general g g y We should point out that what counts as high conforming or low conforming depends on what counts as a bridge and what counts as within-source inferencing or other connection-following. A metaphor theory that proposes different types of bridges of connection might identify different boundaries between high-conforming https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al 410 and low-conforming novel metaphors. Additional types of existing knowledge, beyond knowledge of possible contexts, bridges and within-source connection- following possibilities would also play a role in making this distinction. The study below is confined to our current notions of high conforming and low conforming. However, given that it takes into account the main empirical approaches, as surveyed in Holyoak & Stamenković (2018), it should be reasonably robust in the context of current developments in metaphor theory. and low-conforming novel metaphors. Additional types of existing knowledge, beyond knowledge of possible contexts, bridges and within-source connection- following possibilities would also play a role in making this distinction. The study below is confined to our current notions of high conforming and low conforming. However, given that it takes into account the main empirical approaches, as surveyed in Holyoak & Stamenković (2018), it should be reasonably robust in the context of current developments in metaphor theory. We also need to stress that the high-conforming/low-conforming distinction may well be language-specific in that speakers who know different languages may to some extent deploy different bridges and know different within-source connec- tions. The classifications of expressions in our study are from the point of view of English. Finally, we do not assume that the process of constructing or using bridges, following inferential or other connections on the source side, or considering possible contexts, needs to be conscious. Nor do we assume that a meaning that is derived for the expression is clearly apparent in consciousness. These points greatly affected our methodology below. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2.3. Hypotheses We divide the hypotheses for our experiment into two sets. The first set is centred on the differences between monolinguals and multilinguals in making sense of novel metaphor, irrespective of novelty type (high conforming versus low conforming), whereas the second set is centred on the way in which those two different types of novelty affect readers’ attempts to make sense of an expression, irrespective of the readers’ mono/multilingualism. We had no specific expectation as to whether there would be an interaction whereby multilinguals and monolinguals act more differ- ently from each other on one type of novelty than on the other. y yp y Our main hypotheses concern the likelihood of someone taking a novel meta- phorical expression to make sense (be meaningful), as one aspect of the person’s degree of cognitive flexibility. As a subsidiary matter, we also include hypotheses about the time taken to make those meaningfulness judgements, as a way of illuminating the amount of effort involved, potentially giving more detailed insight into cognitive flexibility. g y We include both literal and conventional metaphorical expressions in our study as something to contrast novel metaphorical expressions to, but the study does not aim to explore differential effects from being literal as opposed to conventional meta- phorical. Different theories may differ on whether an expression is literal or con- ventional metaphorical, and as indicated above, it is reasonable to claim that conventional metaphorical meanings are typically found by simple retrieval from memory, on a par with literal meanings of words. We thus assume that people make sense of conventional metaphorical expressions about as easily as literal expressions, in line with findings from previous research (Werkmann Horvat, Bolognesi, & Lahiri, 2021) However, there is no guarantee that a meaning that could reasonably be claimed to be conventional metaphorical is obtained simply by memory retrieval (see Section 2.2.1), so we still mention literal and conventional metaphorical phrases separately in our hypotheses. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 411 Language and Cognition 3.1. Hypotheses concerning monolinguals vs. multilinguals Hypothesis A. Multilinguals are more likely to take novel metaphorical expressions to make sense than monolinguals are, regardless of whether the expressions are high- conforming or low-conforming. g g Hypothesis B. Multilinguals take longer than monolinguals to assess whether novel metaphorical expressions make sense, whether they are high-conforming or low-conforming. These hypotheses follow from the discussion in Section 2.1. 2.3. Hypotheses Since the multilinguals as well as the monolinguals in our experiment are L1 English speakers, the differences could be subtle. In particular, as regards Hypothesis B, the factors affecting timing might be too mixed in their individual effects to show a generalisable overall effect. The extra information potentially possessed by multilinguals could tend to slow them down, while possibly speeding them up in cases where it allows considerably easier comprehension of an expression. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 3. Methods The experiment, in which sentences containing literal and metaphorical expressions were presented to participants, was a self-paced reading study combined with a YES/NO meaningfulness judgement. We recorded answers to the meaningfulness judgement, the time it took to make the judgement and the time needed to read each region of the sentence. g This type of task was chosen because we were interested in seeing possible comprehension difficulties upon encountering a certain phrase, which this task allowed partly through measuring the reading time in certain sentence regions, as well as observing the polarity of the answer to the meaningfulness question and measuring the time taken to make the answer. We assume that reading time here acts as a proxy for processing difficulty, and therefore we assume that larger reading times can be interpreted as reflecting difficulty in finding meaning, since other effects that might cause processing difficulties are controlled for. Thus, we con- sidered the notion of ‘making sense’ of an expression to involve not just the making of the meaningfulness judgement, but also processes undertaken while reading the sentence. Notably, we did not ask participants to report meanings that they discerned for the sentences. As discussed further in Section 5.3, the main reason for this was that we did not wish to assume that participants were necessarily conscious of the meanings, or at any rate to have a clear and full enough conscious awareness of them to allow useful reporting of them under the pressure of an experiment (even though we imposed no time limit on answering the meaningfulness question). This generates some problems in interpreting results (see Section 5.3), but, on the other hand, it properly allows for the fact that some of our novel examples, especially but not exclusively some low- conforming ones, are difficult to assign a specific meaning to; and even when one does have a specific meaning consciously in mind, it can be difficult to express. Further- more, given the relatively high indeterminacy of meaning of novel metaphor, a participant may detect a range of different meanings, and therefore face a difficult task if required to choose between them or to summarise them in the service of reporting a meaning. 2.3.2. Hypotheses concerning effect of novel metaphoricity Hypothesis C. Monolinguals and multilinguals are (a) more likely to take expressions to make sense when they are literal or conventional metaphorical than when they are novel metaphorical (whether in a high-conforming or low-conforming way); and (b) more likely to take novel metaphorical expressions to make sense when they are high-conforming than when they are low-conforming. Hypothesis D. Monolinguals and multilinguals (a) take less time in assessing whether literal or conventional metaphorical expressions make sense than whether novel metaphorical ones (of either type) do so; and (b) take different amounts of time in assessing whether low-conforming as opposed to high-conforming novel expres- sions make sense. Hypotheses C(a) and D(a) are based on the suspicion that conventional meta- phorical expressions are typically comprehended by simple retrieval of metaphorical meanings, and on findings from experiments suggesting that comprehension of novel metaphors is relatively taxing (Gentner & Wolff, 1997; Lai & Curran, 2013; Lai, Curran, & Menn, 2009; Rutter et al., 2012; Werkmann Horvat, Bolognesi, & Kohl, 2021). Hypothesis C(b) is motivated by the special work needed for rich comprehension in low-conforming cases. Nevertheless, the hypothesis may be invalidated if enough participants settle for less-rich comprehension of some low-conforming expressions, or enough participants fail to comprehend some high-conforming expressions. As regards hypothesis D(b), there are considerations that pull in different direc- tions as regards processing speed. Low-conforming expressions are more likely than high-conforming ones to need, for rich comprehension, the creative entertainment of special contexts, the construction of new metaphoric bridges (e.g., new/refined mappings or superordinate categories) or the finding of unusual within-source inferential or other connections. Therefore, attempts at rich comprehension of a low-conforming expression, exploiting distinctive aspects of the source-side concepts raised, may often be slower than for a high-conforming one (recall our discussion of ‘curved hope’ in Section 2.2.5). However, precisely because of the relative difficulty of rich comprehension in low-conforming cases, participants may, more frequently than in high-conforming cases, give up trying to comprehend at all or settle for less- rich, quick-to-derive comprehension that ignores distinctive aspects of source-side Werkmann Horvat et al 412 concepts and eschews special contexts or new metaphoric bridges. A faster response than in high-conforming cases could even arise. Nevertheless, due to the assumed major differences in comprehension, we predict that there will be an overall speed difference. 2.3.2. Hypotheses concerning effect of novel metaphoricity It was our aim to explore the direction of this difference in this study, as a basis for further studies. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 3. Methods These points also mean that a meaningfulness ‘judgment’ by a participant might not be anything like a considered judgment (result of deliberation) but might simply be based on a feeling of comprehending or failing to comprehend. g y g g g All data, stimuli, analyses and supplementary materials are stored in an online repository on the Open Science Framework (OSF): https://osf.io/ek4q8/?view_only= faa82d8334fd478cb8f99fd15f107597. The research was reviewed by, and received ethics approval through, the Univer- sity of Oxford Central University Research Ethics Committee (Ethics Approval Reference: R56945/RE001). https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 2022.8 Published online by Cambridge University Press 413 Language and Cognition Language and Cognition 3.1.1. General characteristics The metaphorical expressions were English adjective–noun pairs (A–N pairs; henceforth, referred to simply as pairs), as in ‘firm belief’ and ‘hazy hope’. We assumed that the most likely metaphorical meanings would arise from the noun being taken literally, with only the combination with the adjective making the expression metaphorical. For instance, we assumed a ‘shaky price’ would be taken as meaning a financial price that is metaphorically shaky, not as a metaphorical price (e.g., diminution of health) that is (metaphorically) shaky. Hence, in the application of the notions of high conforming and low conforming to the novel pairs, the noun and adjective were assumed to indicate the target and source subject matters, respectively. p y The critical stimuli consisted of 96 pairs (see Table 1), based on 24 different nouns (four for each of six target subject matters: mind, time, economics, weather, geog- raphy and food-and-drink). For each noun, there were four different pairs, for four different metaphoricity conditions: a literal pair and three metaphorical pairs – a conventional one (in an operationalised sense explained below), a high-conforming novel one and a low-conforming novel one. No adjective was used with more than one noun. For instance, the four pairs using idea were ‘simple idea’ (literal), ‘bright idea’ (conventional metaphorical), ‘grey idea’ (high-conforming novel metaphorical) and ‘damp idea’ (low-conforming novel metaphorical), and the four adjectives here appeared with no other noun. Table 1. Critical nouns and adjectives Adjectives Nouns Literal Conventional High-conf Low-conf Idea Simple Bright Grey Damp Pride Ethnic Foolish Wise Edible Hope False Faint Hazy Curved Belief Sincere Firm Thick Fitted Year Rainy Golden Brass Locked Night Chilly Lengthy Fat Pointed Week Current Quiet Muted Liquid Hour Crucial Peak Bottom Milky Loan Useful Flexible Stiff Purple Fee Rental Fixed Loose Pale Tax Local Heavy Slim Wet Price Fair Stable Shaky Melted Storm Wintry Raging Kicking Blond Cloud Ugly Angry Furious Loyal Rain Freezing Gentle Excited Witty Wind Warm Fierce Rude Married Creek Tiny Roaring Howling Drunk Hill Steep Lonely Troubled Tied River Shallow Lazy Tired Dizzy Lake Salty Calm Eager Ripe Beer Fizzy Strong Tough Folded Tea Fragrant Weak Fragile Keen Wine Tasty Bold Humble Silent Soup Instant Hearty Shy Shouting Table 1. Critical nouns and adjectives https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 414 Werkmann Horvat et al Each of our 96 pairs was embedded in a short sentence. 3.1.1. General characteristics These critical sentences were read by participants in the self-paced reading task. The sentences were partitioned into four nonoverlapping lists, each of which contained 24 of the 96 critical sentences, plus 72 filler sentences containing only nouns and adjectives not used in the critical sentences. Each list contained all 24 critical nouns, hence each noun only once. A given noun appeared in different metaphoricity conditions in different lists. A given participant saw just one of the four lists. The metaphoricity conditions were evenly spread across each list (six critical sentences for each of the four conditions), and the 48 participants were evenly spread across the lists. y For each of the four pairs involving a given noun, the surrounding wording in the sentences containing the pairs was the same. See the examples in Table 2. g p p The reason for embedding the pairs in sentences was to provide reasonably natural syntactic contexts and to avoid possible special processing effects at the start and end of reading a word sequence. We acknowledge that including the adverb at the end of the sentence might affect participants’ judgements about the A/N pair. However, the adverbs were always the same across the four conditions for any given noun, which somewhat controls for this effect. The final adverb was added to track possible spillover effects from reading the noun. Note that the definitions of high-conforming and low-conforming novelty in Section 2.2.5 are predicated on the expression being presented out of context. The presentation of our pairs therefore departs slightly from such pure presentation. However, we endeavoured to keep the wording outside the pairs in the sentences semantically generic so that it would give participants relatively little help towards finding particular meanings for the pairs, under the above assumption that the participants would take the nouns literally. For instance, in Table 2, the ‘idea’ is ‘suggested quickly’ and the ‘loan’ is ‘got easily’: whatever the nature of the idea, it might be suggested quickly under suitable circumstances, and whatever the nature of the loan, it might be got easily under suitable circumstances. The wording may direct participants towards certain interpretations, but we felt the sentences were a suitable compromise between the ideal of reasonably natural syntactic contexts and the ideal of null semantic contexts. As shown in Table 2, each sentence was divided into six regions. 3.1.1. General characteristics The presentation of each sentence was followed by the meaningfulness question, ‘Does this sentence make sense?’ with a forced choice between YES and NO. The adjectives were balanced for frequency and length across the four conditions. A single-factor analysis of variance showed that there were no significant differences for frequency and length. See Table 3 for details. Table 2. Examples of sentences Condition Region 1 Region 2 Region 3 Region 4 (Adj) Region 5 (Noun) Region 6 (Adv) Literal John suggested a simple idea quickly Conventional John suggested a bright idea quickly High-conf John suggested a grey idea quickly Low-conf John suggested a damp idea quickly Literal Cath got a useful loan easily Conventional Cath got a flexible loan easily High-conf Cath got a stiff loan easily Low-conf Cath got a purple loan easily Table 2. Examples of sentences https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Language and Cognition 415 Table 3. Lexical measures for adjectives Table 3. Lexical measures for adjectives CELEX freq Length Mean lit 44.69 (SD = 59.19) 5.75 (SD = 1.22) Mean conv 43.01 (SD = 40.66) 5.54 (SD = 1.18) Mean high-conf 29.78 (SD = 25.76) 5.29 (SD = 1.36) Mean low-conf 31.02 (SD = 28.25) 5.29 (SD = 1.20) F (3, 92) = 0.88 (3, 92) = 0.76 p 0.452 0.519 3.1.2. Literal and conventional metaphorical pairs 3.1.2. Literal and conventional metaphorical pairs We checked the following when judging a pair to be literal: that our chosen dictionaries,8 taken collectively, gave a sense for the adjective and a sense for the noun such that: these senses were current, and not dependent on specialised know- ledge; neither sense was listed as metaphorical, figurative, dialect, slang and so forth; the adjective’s sense applied directly and easily to the noun’s sense and the resulting composed meaning for the pair was directly usable in the sentence the pair was embedded in. Our conventional pairs were not required to be conventional metaphorical terms in themselves, although some were. Rather, we dubbed a pair as a conventional metaphorical one for the purposes of our study when the adjective by itself had a current, nonspecialised conventional metaphorical sense that applied directly and easily to a current, nonspecialised literal sense for the noun, and delivered a com- posed meaning for the pair that was directly usable in the embedding sentence. We also required a minimum corpus frequency of 10 for operational reasons. 8The Chambers Dictionary, 2003, the Oxford English Dictionary Online, full version, accessed in 2020 and 2021, and Webster’s Third New International Dictionary, unabridged, 1961. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 3.1.1. General characteristics We judged an adjective to have a particular conventional metaphorical meaning when that meaning was listed in one or more of our dictionaries and we judged it to be metaphorical. In a few cases, our judgment was aided by a sense being marked by the dictionary as metaphorical or figurative. In either case, we required the meaning to be transparent (in our judgment) in the sense of Section 2.2.1. p ( j g ) We included conventional pairs in our study because we were interested in seeing whether at least some conventional pairs, even though transparent, were easier and quicker to comprehend by our participants than the high-conforming novel pairs. If so, we would have evidence that the conventional pairs were being comprehended through simpler processing so that novel metaphoricity was actu- ally having an effect. It was partly to maximise the possibility of obtaining this evidence that we also included literal pairs and ensured that our conventional pairs were, overall, roughly as familiar as our literal pairs. We therefore counted corpus occurrences of the pairs in the 45-billion-token English Web Corpus (enTenTen) using SketchEngine. We found that our literal and conventional pairs occurred in similar numbers overall. A paired t-test showed that the difference between the literal and conventional conditions was nonsignificant (MLit = 2,844.5, SD = 4,290.45; MConv = 1,898.08, SD = 2,778.01; t = 0.94; p = 0.355). Note that a literal or conventional pair in our study could be lexically novel in the sense that it did not occurr frequently. In particular, for a literal pair to be quickly and easily https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al 416 comprehended by a hearer it was not necessary for it to ever have been encountered before by that hearer; a similar latitude also needed to apply to the conventional pairs.9 Therefore, the contrast in metaphorical novelty between (1) the literal and conventional pairs, on the one hand, and (2) the novel pairs, on the other hand, was not on the basis of lexical novelty, but on the basis that the pairs in did not contain metaphorical novelty: the literal pairs were intended to be nonmetaphorical, and the metaphoricity of a conventional pair was intended to be nonnovel metaphori- city arising from its adjective. 9Of course, normally, the adjective will frequently have been used with its conventional metaphorical meaning in other phrases before. Recall also that we required at least 10 corpus occurrences, although this did not enforce a marked difference to the literal phrases as the minimum frequency of these turned out to be 6. 3.1.3. Novel metaphorical pairs and occurrence frequencies b f h l d A basic requirement for the novel pairs, as used in our sentences, was that they were not classed as literal or conventional metaphorical as above. We made a judgment about whether a novel pair (that was metaphorical in our judgment) was low- conforming or high-conforming on the basis of: (1) evidence afforded by conven- tional metaphorical expressions about the existence of familiar bridges (see Section 2.2.3); and (2) our judgments about what special contexts, if any, needed to be considered and what within-source inferencing or other connection tracing was needed. Some detailed examples of our decision making are given in the Supple- mentary Document. y As regards corpus frequency, we considered that metaphorical novelty would be most likely to be present if the pair was lexically rare. We therefore severely limited the number of allowed occurrences in the English Web Corpus (as determined by SketchEngine). In the low-conforming case, we did not allow there to be any occurrences in the corpus at all. In the high-conforming case, we considered that it was acceptable for there to be a few occurrences, because of the familiarity of bridges (e.g., mappings) used, the straightforwardness of within-source inferencing or other connection-following, and there being no need for considering unusual contexts. However, for safety, we still required occurrences to number below 10. Overall, the mean level of occurrence was M = 1.58 (SD = 1.98). Note, however, that low- conforming and high-conforming novel pairs do not need to have these degrees of rarity in general. y g Although our nonnovel pairs were not required to be frequently occurring, their frequencies in the English Web Corpus (accessed via SketchEngine) were in fact much higher overall than those of the novel pairs. The mean frequencies as above were: 2,844.5 for literal, 1,898.08 for conventional, 1.58 for high-conforming novel as noted above and of course zero for low-conforming novel. Since the standard deviations in the literal and conventional cases were also large, the ranges of frequency need to be considered. The important comparison here is between the highest frequencies for high-conforming pairs and the lowest frequen- cies for literal and conventional ones. The high-conforming pairs had a maximum frequency of only eight occurrences, reached by one pair (‘rude wind’), with all the remaining pairs at four occurrences or below. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 3.1.1. General characteristics Having said this, the literal and conventional pairs were in fact, overall, much more frequent in the corpus than the novel pairs, as will be explained in Section 3.1.3. 10We assumed that for ordinary English speakers, the word ‘understand’ would be more accessible and natural than the word ‘comprehend’ used in the text of the present paper. We take these words meanings to be closely similar. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 3.1.3. Novel metaphorical pairs and occurrence frequencies b f h l d In fact, 10 of the 24 high-conforming 9Of course, normally, the adjective will frequently have been used with its conventional metaphorical meaning in other phrases before. Recall also that we required at least 10 corpus occurrences, although this did not enforce a marked difference to the literal phrases as the minimum frequency of these turned out to be 6. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 417 Language and Cognition pairs had no occurrences at all. By contrast, although we allowed conventional pairs to have a frequency as low as 10 (this number chosen operationally to ensure a break with high-conforming pairs; see the Supplementary Document for more information on this), the 24 conventional pairs in fact had a minimum frequency of 21 (‘lengthy night’), with only 2 other pairs having 100 occurrences or fewer (‘bold wine’ with 44 and ‘angry cloud’ with 97 occurrences). The 24 literal pairs had a minimum frequency of 6 (‘useful loan’), which is comparable to the maximum of 8 for high- conforming pairs, but only 3 other literal pairs had 100 occurrences or fewer (24, 65 and 88 occurrences). Although it has been fairly common in previous metaphor studies to assess the degree of conventionality or novelty of metaphorical pairs by acquiring ratings from participants (separate from those taking part in the main experiment), in this work, we moved away from this method. The main reason for this is that we could not expect nonexperts to assess whether a metaphorical pair was novel in a high- conforming way or in a low-conforming way. With respect to the distinction between metaphorically novel and nonnovel pairs, we suggest that is more reliable to rely on (non)novelty as revealed by dictionary definitions than on participants’ within- experiment and conscious opinions about the matter. This is because such opinions might be unduly influenced by lexical novelty, whereas our notion of novelty only indirectly involves lexical novelty. 3.1.4. Norming study concerning comprehension difficulty low-conf hi-c = 3.20 (SD = 0.92) lo-c = 1.89 (SD = 0.35) <0.0001 7.34 1.88 Table 4. Results of the norming study (paired T-test) https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 11We acknowledge that the term native speaker is complex and layered (Davies, 2003); nevertheless, we decided that this term sounds most familiar and straightforward for naïve speakers with no linguistic training. Therefore, in the questionnaires, they were asked about their native language(s), rather than their first language. 11We acknowledge that the term native speaker is complex and layered (Davies, 2003); nevertheless, we decided that this term sounds most familiar and straightforward for naïve speakers with no linguistic training. Therefore, in the questionnaires, they were asked about their native language(s), rather than their first language. The term fluency was also used as a term that tends to be meaningful for naïve speakers, meaning the ability to use a given language independently and skilfully in a variety of contexts. This was the definition given to potential participants if they were not sure about what the term encompasses. 3.1.4. Norming study concerning comprehension difficulty 3.1.4. Norming study concerning comprehension difficulty We naturally expected L1 English speakers in general to find low-conforming novel A–N pairs to be more difficult to comprehend than high-conforming ones, and the latter to be more difficult than nonnovel (literal or conventional) ones. Accordingly, as a further, indirect, check on our classification of our metaphorical pairs, we normed the pairs with L1 English speakers using Qualtrics, after all materials had been created and classified. We included literal pairs for completeness. We recruited 130 L1 English speakers (different from any in the main part of the experiment), who each saw all 96 pairs. They were asked to assess the comprehen- sibility of each pair, on a Likert scale. The instructions were: ‘For each phrase mark how easy is it to understand the phrase on a scale from 1 to 7: ‘For each phrase mark how easy is it to understand the phrase on a scale from 1 being the most difficult, 7 being the easiest’.10 As Table 4 shows, there was a highly significant difference in participants’ ratings of literal and conventional versus both high-conforming and low-con- forming novel pairs, and in the ratings of high-conforming versus low-conforming pairs. The difference between literal and conventional was also significant although small (the literal pairs were slightly more comprehensible than the conventional ones). 10We assumed that for ordinary English speakers, the word ‘understand’ would be more accessible and natural than the word ‘comprehend’ used in the text of the present paper. We take these words meanings to be closely similar. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al Werkmann Horvat et al 418 Table 4. Results of the norming study (paired T-test) Conditions Mean P(T < = t) two-tail t-Stat Cohen’s d Lit vs. conv lit = 6.62 (SD = 0.30) conv = 6.32 (SD = 0.54) 0.021 2.48 0.68 Lit vs. high-conf lit = 6.62 (SD = 0.30) hi-c = 3.20 (SD = 0.92) <0.0001 17.23 4.99 Lit vs. low-conf lit = 6.62 (SD = 0.30) lo-c = 1.89 (SD = 0.35) <0.0001 57.68 14.51 Conv vs. high-conf conv = 6.32 (SD = 0.54) hi-c = 3.20 (SD = 0.92) <0.0001 14.29 4.13 Conv vs. low-conf conv = 6.32 (SD = 0.54) lo-c = 1.89 (SD = 0.35) <0.0001 35.79 9.73 High- vs. The term fluency was also used as a term that tends to be meaningful for naïve speakers, meaning the ability to use a given language independently and skilfully in a variety of contexts. This was the definition given to potential participants if they were not sure about what the term encompasses. 3.2.1. Participants https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 419 Language and Cognition Since our multilingual group included speakers of different languages, it was difficult to test the language knowledge of all participants. Although we acknowledge possible issues with self-reporting such as subjectivity and possible problems with comparability (Tomoschuk, Ferreira, & Gollan, 2019), self-report questionnaires tend to be quick and practical, and also have been shown to correlate with more objective measures, for example, cloze task scores (Sabourin, Brien, & Burkholder, 2014; Tomoschuk, Ferreira, & Gollan, 2019). According to the self-reporting, some multilinguals were balanced bilinguals, while some spoke more than one non-English language fluently. Thus, our multi- linguals were very similar to what Butler (2013) calls multilanguage users, who are either bilingual or fluent in more than two languages. This is appropriate to the current study, which is into how knowledge of at least one non-English language might affect comprehension. Language questionnaires. To define our two groups, we administered a language questionnaire before the self-paced reading experiment. It was adapted from one used in the ERPLing Lab at the University of Ottawa (Sabourin et al., 2016).12 In the monolingual group, the average age was 22 (min 18, max 46, SD = 6.86), with 5 males and 19 females. All members identified English as their only native language, and as their parents’ native language. Four identified their strongest L2 to be at an intermediate level, with the rest stating it was at a low or very low level. The average age at which they began learning their strongest L2 was 10.5 years, and the average daily use of it was 0.2 hours. The average number of non-English languages they reported knowing was 1.9. y p g As for the multilinguals, the average age was 28 (min 19, max 52, SD = 9.52) with again 5 males and 19 females. All identified English as one native language, whereas 10 identified a further native language. Fourteen reported having a parent whose native language was not English or who was multilingual. All the multilinguals, except one, identified their strongest L2 (or in some cases their second L1) to be at advanced, near-native or native level. One reported two native languages but marked one as being at an intermediate level. Out of all the multilinguals, two participants reported knowing three languages at an advanced level. 12The questionnaire that was used can be found at: https://osf.io/ek4q8/?view_only= faa82d8334fd478cb8f99fd15f107597. 3.2.1. Participants p General characteristics and group classification. All 48 participants were L1 speakers of English, aged 18–55, recruited in Oxford, UK, right-handed, with normal or corrected-to-normal vision and no known language-related, neurological or hearing disorders. They were divided into two groups: those that spoke another language fluently (n = 24), and those who did not (n = 24). The latter group we call monolingual throughout the paper, whereas the former we call multilingual, which we take to include bilingual. The same group of participants participated in, and was similarly described in, Werkmann Horvat, Bolognesi, & Kohl (2021)). The number of participants was determined based on the number of the experimental stimuli lists and on previous studies where two groups of participants are compared (Chen & Husband, 2018), but also on the feasibility of recruiting participants with the desired language history. Forming such groups of participants is a challenging task. One challenge in Europe is finding a true, minimalist monolingual, someone who has only ever been exposed to one language. Moreover, we recruited mainly Oxford University students, who have usually been exposed to at least one non-English language in their primary education. On the other hand, for multilingualism, we did not wish to take the maximalist line of requiring full, L1-like proficiency in one or more non-English languages. q g p y g g g We formed the groups based on self-report. All participants needed to report as being L1 English speakers. The multilinguals and monolinguals were then those who self-reported as, respectively, speaking at least one further language fluently or not doing so. In the advertisement recruiting the participants, we requested participants whose native language is English and who either spoke another language fluently or did not speak another language fluently.11 11We acknowledge that the term native speaker is complex and layered (Davies, 2003); nevertheless, we decided that this term sounds most familiar and straightforward for naïve speakers with no linguistic training. Therefore, in the questionnaires, they were asked about their native language(s), rather than their first language. g g The term fluency was also used as a term that tends to be meaningful for naïve speakers, meaning the ability to use a given language independently and skilfully in a variety of contexts. This was the definition given to potential participants if they were not sure about what the term encompasses. 3.2.1. Participants The average age of acquisi- tion of strongest L2 was 5.5 years, with average daily use at 2.2 hours. On average, the multilinguals reported knowing 3.1 languages beyond English. Non-English lan- guages that the participants reported knowing to an advanced level included: French (6); German (6); Mandarin (2); Portuguese (2); Spanish (2) and Arabic, Bahasa, Cantonese, Fuzhou, Korean and Russian (1 each). https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al The self-paced reading experiment lasted about 10 minutes, depending on the participants’ speed. The instructions were: The words will appear one by one, as you press the button A. This means that you are controlling the reading pace. After every read word, press A to see the next word. Continue with this until the end of the sentence. After that, a question will appear on the screen: Does this sentence make sense? You will have to press either a YES button or a NO button. 4. Results and statistical analysis 4.1. Analysis methods We analysed the self-paced sentence-reading times, meaningfulness answers and times taken to give the meaningfulness answers of the two participant groups: monolinguals and multilinguals. There were four critical conditions for presented sentences: literal; conventional metaphorical; high-conforming novel metaphorical and low-conforming novel metaphorical. There were six sentence regions (see Table 2) plus the YES/NO answer region. We analysed how long participants took to read the noun region (Region 5) and the adverb region (Region 6). We also analysed the times taken to answer the question Does this sentence make sense? (question/answer region RT, labelled as RTQ below), and the answers themselves (YES/NO). Regions 1–4 were not analysed: Regions 1–3 were the same across conditions (for any given noun), and in Region 4, only the adjective of the adjective–noun pair had appeared, so the pair’s metaphoricity was not clear yet. We excluded responses in the answer region where no answer button was pressed. This resulted in exclusion of 2.6% of data in the answer region. All fillers were excluded from the analysis. The data were analysed using a linear mixed-effects model for RTs and a general linear model with a binomial distribution for mean- ingfulness answers with the lme4 package, version 1.1-26 (Bates et al., 2015) in R, version 4.0.4. (R Development Core Team, 2011). The fixed effects were condition and group with random effects of participant and item. Initially, the models included by-participant varying intercepts and by-participant varying condition slopes, and by-item varying intercepts and by-item varying group slopes (Winter, 2019). For the noun and answer regions, the model failed to converge with random slopes, and therefore, it was simplified. In the adverb region, an error message suggested the model was overfitted, so the random slopes were also simplified. Contrasts between conditions were analysed using the emmeans R package, version 1.5.4 (Lenth, 2021), and F-tests for main effects were analysed using the lmerTest R package, version 3.1-3 (Kunzetsova, Brockhoff, & Christensen, 2017; Lenth, 2021). 3.2.2. Procedure h The experiment was conducted in a soundproof room in the Language and Brain Laboratory at the University of Oxford. It used the Presentation® software, and the sentences were shown on a Dell Latitude 7480 laptop screen. The participants used a Logitech Gamepad F310 joystick to progress from one sentence to the next, and for each sentence pressed buttons to read through the words at their own pace and then answer the YES/NO meaningfulness question. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 420 Werkmann Horvat et al https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 4.2. Results Figs. 1 and 2 display the average reaction times in different regions for the mono- lingual group (Fig. 1) and for the multilingual group (Fig. 2). The two graphs show partially different trends, which suggest slightly different processing patterns for the two groups. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Language and Cognition 421 Fig 1. Average RTs in different regions for the monolingual group (SE error bars). Language and Cognition 42 Fig 1. Average RTs in different regions for the monolingual group (SE error bars). Fig 1. Average RTs in different regions for the monolingual group (SE error bars). Fig 2. Average RTs in different regions for the multilingual group (SE error bars). Fig 2. Average RTs in different regions for the multilingual group (SE error bars). Fig 2. Average RTs in different regions for the multilingual group (SE error bars). https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 422 Werkmann Horvat et al In the noun region (Region 5), there were no significant main effects of condition (F(3, 1,080.9) = 0.50, p = 0.69) or group (F(1, 48) = 0.05, p = 0.83). The interaction was also nonsignificant (F(3, 1,080.9) = 0.65, p = 0.58). In other words, the different stimuli were processed in very similar ways, and the different groups performed in very similar ways. y y In the spillover region, that is, the adverb region (Region 6), there was a significant main effect of condition (F(3, 1,080.8) = 4.71, p = 0.003), but the main effect of group (F(1, 48) = 1.61, p = 0.21) and the interaction (F(3, 1080.8) = 0.51, p = 0.68) were nonsignificant. To identify the specific differences between processing times for the stimuli, we ran a post-hoc analysis for the significant main effect of condition. The Tukey’s honestly significant difference test with the Kenward–Roger degrees for freedom method showed significant contrasts between high-conforming and con- ventional (t(1,087) = 2.58, p = 0.049) and between low-conforming and conven- tional (t(1,087) = 3.47, p = 0.003), with high-conforming and low-conforming being slower in each case.13 In the answer region, there was again a significant main effect of condition (F(3, 1,052.68) = 8.70, p < 0.001), but no significant differences for group (F(1, 48.06) = 1.59, p = 0.21) or interaction (F(3, 1,052.68) = 1.28, p = 0.28), as in the previous case. 13The contrast method for multiple comparisons was pairwise. For factor levels A (conventional), B (high- conforming), C (literal) and D (low-conforming), emmeans pairs() function generates the comparisons A–B, A–C, A–D, B–C, B–D and C–D. 14For main effects, we used R’s anova (glm, test = “Chisq”) function because it is recommended in the literature to use a chi-square instead of the F-distribution for binomial distributions (Baayen, 2008, p. 218). https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 13The contrast method for multiple comparisons was pairwise. For factor levels A (conventional), B (high- conforming), C (literal) and D (low-conforming), emmeans pairs() function generates the comparisons A–B, A–C, A–D, B–C, B–D and C–D. 14For main effects, we used R’s anova (glm, test = “Chisq”) function because it is recommended in the literature to use a chi square instead of the F distribution for binomial distributions (Baayen 2008 p 218) 4.2. Results In a post-hoc analysis for the significant main effect of condition, the Tukey test as above showed significant contrasts between high-conforming and literal (t(1,058) = 3.81, p = 0.0009), between high-conforming and conventional (t(1,058) = 4.83, p < 0.0001) and between high-conforming and low-conforming (t(1,059) = 3.11, p = 0.01). All models’ results are reported in Table 5. p p The percentages of YES and NO meaningfulness responses for groups and conditions are shown in Table 6. We ran a generalised linear model with a binomial distribution (Table 7). The model14 showed a significant main effect of condition (χ2(3, N = 1,118) = 479.38, p < 0.0001) and group (χ2(1, N = 1,117) = 15.86, p < 0.0001), while the overall interaction was nonsignificant (χ2(3, N = 1,114) = 3.06, p = 0.38). p<0.05; p<0.01; *<0.001. p p p Based on these main effects, we ran simple contrast tests with the Tukey method for p-value adjustment. The results are given on the log odds ratio scale. For condition, the only nonsignificant contrast was conventional-vs-literal (z = 1.45, p = 0.47). The other contrasts were highly significant: literal-vs-high-conforming (z = 9.51, p < 0.0001), literal-vs-low-conforming (z = 14.19, p < 0.0001), conven- tional-vs-high-conforming (z = 9.13, p < 0.0001), conventional-vs-low-conforming (z = 14.38, p < 0.0001) and low-conforming-vs-high-conforming (z = 7.68, p < 0.0001), with more YES answers in the high-conforming condition. For group, there was a significant monolingual-vs-multilingual contrast (z = 3.03, p = 0.002), with multilinguals more frequently answering YES. g q y g As noted, the condition-group interaction was nonsignificant, and so one would not normally run a post-hoc test contrasting how the different groups performed on the different conditions. In this case, we feel it is justified to look at these contrasts, not with reference to interaction, but to see for which of the four conditions a difference https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press est Std. teres te 8 0 4.2. Results error df t value Pr(> 28.01 69.35 15.45 <0.001 17.61 1,080.88 1.27 0.21 17.61 1,080.88 0.29 0.77 17.61 1,080.88 0.02 0.98 39.15 66.60 0.23 0.82 24.90 1,080.88 0.61 0.54 24.90 1,080.88 0.03 0.98 24.90 1,080.88 0.78 0.43 74.35 73.99 7.60 <0.001 52.33 1,080.84 1.65 0.10 52.33 1,080.84 0.004 0.99 52.33 1,080.84 2.10 0.04 104.51 72.63 0.80 0.43 74.00 1,080.84 0.26 0.80 74.00 1,080.84 1.18 0.24 74.00 1,080.84 0.503 0.61 82.76 107.26 8.75 <0.001 76.12 1,051.82 3.20 0.001 76.12 1,051.60 0.87 0.39 75.97 1,051.46 0.06 0.95 115.14 103.68 1.44 0.15 107.88 1,052.30 0.33 0.74 107.74 1,051.49 0.21 0.83 108.55 1,053.92 1.58 0.11 Pr( <0.00 0.2 0.77 0.98 0.82 0.54 0.98 0.43 <0.00 0.10 0.99 0.04 0.43 0.80 0.24 0.6 <0.00 0.00 0.39 0.95 0.15 0.74 0.83 424 Werkmann Horvat et al Werkmann Horvat et al Table 6. YES or NO meaningfulness answers in percentages for groups and conditions Monolingual Multilingual YES NO YES NO Literal 92.20 (130) 7.80 (11) 94.29 (132) 5.71 (8) Conventional 88.81 (127) 11.19 (16) 90.91 (130) 9.09 (13) High-conf 43.97 (62) 56.03 (79) 59.71 (83) 40.29 (56) Low-conf 7.80 (11) 92.20 (130) 27.82 (37) 72.18 (96) Table 6. YES or NO meaningfulness answers in percentages for groups and conditions Table 7. Generalised linear model for answers to the meaningfulness question Predictor Estimate Std. error z value Pr(>\|z\|) (Intercept) 2.07 0.27 7.81 <0.001* Conditionhigh-c 2.31 0.32 7.35 <0.001* Conditionliteral 0.40 0.41 0.97 0.33 Conditionlow-c 4.07 0.37 10.98 <0.001*** Groupmultilingual 0.23 0.39 0.59 0.56 Conditionhigh-cGroupmultlingual 0.40 0.46 0.88 0.38 ConditionliteralGroupmultilingual 0.10 0.62 0.16 0.87 Conditionlow-cGroupmultilingual 0.81 0.51 1.59 0.11 p<0.05; p<0.01; *<0.001. Table 7. Generalised linear model for answers to the meaningfulness question between how the monolinguals and multilinguals answers contributed significantly to the main effect of group. The Tukey post-hoc test shows that there are significant monolinguals-vs-multilinguals contrasts in both the high-conforming (z = 2.62, p = 0.009) and low-conforming (z = 3.23, p = 0.001) conditions, with multilinguals more frequently answering YES in both conditions (cf. Table 6). In the literal (z = 0.69, p = 0.49) and conventional (z = 0.59, p = 0.58) conditions, there is no significant contrast between the groups. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.1. Comparison of results to the hypotheses Overall, the results suggest that being multilingual makes an important difference to the comprehension of novel metaphor of both types (high-conforming and low- conforming), and that the distinction between these two types of novel metaphor is empirically important. In stating this, we should re-emphasise the point made in Section 2.2.5 that the classification in our study of metaphorical expressions as novel or conventional, and, when novel, as high-conforming or low-conforming, was influenced by the nature of English, and that the notions of monolingual and multilingual in our study were relative to English. g y g Before we consider our specific hypotheses (Section 2.3), we can see that the YES/NO meaningfulness answer profile across the different conditions was highly consistent with the norming study of perceived comprehension easiness (see Table 4). As per Table 6, for both monolinguals and multilinguals, most answers (90% or so) were YES for literal and conventional pairs, fitting the easiness of around 6.5 out of 7 in the norming. Roughly, half (40% for monolinguals, 57% for multi- linguals) of the answers were YES for high-conforming, fitting with the medium easiness of 3.2/7 in the norming. Relatively, few answers (around 10% for 425 Language and Cognition Language and Cognition monolinguals, 27% for multilinguals) were YES for low-conforming, fitting the low easiness of 1.9/7. 5.1.1. Hypotheses A and B, concerning the effect of multilingualism yp , g g Hypotheses A and B concern the effect of multilingualism as opposed to monolin- gualism on meaningfulness judgements about novel metaphorical expressions (whether high-conforming or low-conforming). Hypotheses A and B concern the effect of multilingualism as opposed to monolin- gualism on meaningfulness judgements about novel metaphorical expressions (whether high-conforming or low-conforming). g g g Hypothesis A was supported. The hypothesis is that multilinguals are more likely than monolinguals to take novel metaphor (of either type) to make sense. The outcome is consistent with previous literature that suggests greater cognitive flexi- bility in multilinguals (see discussion in Section 2.1). Our results show not only that this advantage applies in the particular task of processing novel metaphor, but that it applies both to the processing types used for high-conforming expressions and to the partially different types for low-conforming ones. This therefore suggests that this flexibility in multilinguals is at least present in linguistic tasks, even if our results were taken not to contribute directly to evidence about general cognitive flexibility. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.1. Comparison of results to the hypotheses This supported our expectation that Werkmann Horvat et al 426 high-conforming novel metaphorical expressions would be processed at a different speed not just from the nonnovel conditions (an aspect of part (a)), but also more interestingly from low-conforming novel metaphors (part (b)). It also answers our question (in Section 2.3.2) about whether high-conforming novel metaphors would be processed more quickly or more slowly than low-conforming novel metaphors. However, the answer-region results did not support the aspect of Hypothesis D(a) where we predicted that low-conforming novel metaphors would be processed at a different speed from literal and conventional metaphorical expressions. The results for the adverb region show partially supportive results for D(a), with high-conforming and low-conforming being significantly slower than conventional. However, the literal condition, surprisingly, did not differ significantly from the novel conditions. Hypothesis D(b) as applied to the adverb region was not supported, as there was no significant difference between the two novel conditions. Despite the incompleteness of support for hypothesis D as applied to the adverb and answer regions, separately, the results for these regions taken together make it plausible that high-conforming novel expressions are more effort-involving than low-conforming ones, and that, in line with previous literature (see Section 2.3.1), novel expressions of both our types are more effort-involving than both literal and conventional expressions. This is promising for future research aimed at strength- ening this plausibility, while raising specific issues needing further exploration, such as the speed of assessing the meaningfulness of low-conforming expressions versus literal and conventional ones. As noted, in the answer region, high-conforming novel metaphors were processed significantly more slowly than low-conforming novel metaphors. One might have expected high-conforming novel metaphors to be faster to process, because they should not require new bridges (mappings, superordinate categories etc.), unusual within-source inferencing or other connection-following, or entertainment of special contexts. However, that only suggests a speed advantage over low-conforming expressions when rich comprehension is achieved in low-conforming as well as high-conforming cases. As suggested in the discussion of Hypothesis D in Sec- tion 2.3.2, many participants may have opted for less-rich comprehension in low- conforming cases, reducing or omitting work on new bridges, unusual within-source connection-following or special contexts. Indeed, many participants may have quickly rejected some low-conforming expressions as meaningless, rather than being more attentive and seeking rich meaning. 5.1. Comparison of results to the hypotheses Hypothesis B is that multilinguals take longer than monolinguals to assess whether novel metaphorical expressions make sense, irrespective of whether these expressions are high-conforming or low-conforming. This was not supported. In our results, the time taken to give a YES/NO answer for novel metaphorical expressions did not differ significantly between the two groups. The same lack of significant difference applies to the noun-region or adverb-region reading times. The possible factors that might have played a distinctive role in multilinguals’ reaction times were perhaps not strong enough to make a significant difference because both groups were composed of L1 English speakers, as previously suggested in Section 2.3.1. 5.1.2. Hypotheses C and D, concerning the effect of metaphoricity condition Hypotheses C and D concern the effect of novel metaphoricity on meaningfulness judgements (regardless of whether the judgements are by monolinguals or multi- linguals). 5.1.2. Hypotheses C and D, concerning the effect of metaphoricity condition Hypotheses C and D concern the effect of novel metaphoricity on meaningfulness judgements (regardless of whether the judgements are by monolinguals or multi- linguals). Hypothesis C was supported. Part C(a) of the hypothesis is that novel expressions (of either type) are less likely to be taken to make sense than conventional and literal ones are, and indeed we found them to be significantly less likely to be so. According to Hypothesis C(b), high-conforming novel expressions are more likely to be taken to make sense than low-conforming ones are, and we did find them significantly more likely to be so. The support for Hypothesis C(b) underscores the importance of addressing different types of novelty, and, in particular, provides some support for the distinctive way we have done this. y Hypothesis D(a) is that people take less time to assess whether literal or conven- tional metaphorical expressions make sense than novel metaphorical ones (whether low- or high-conforming). Hypothesis D(b) is that people will take different amounts of time to assess whether low-conforming and high-conforming novel expressions make sense. We found relevant significant timing differences for the adverb region and the answer region, though not in the noun region, as follows. g g g The results for the answer region supported Hypothesis D(b) and partially supported Hypothesis D(a). High-conforming novel metaphorical expressions attracted significantly slower meaningfulness judgements than all other conditions, but there were no other significant differences. 5.1. Comparison of results to the hypotheses The meaningfulness results show that participants took the pairs to made sense less often in the low-conforming cases. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.2. The issue of condition/group interaction Our results show no statistical interaction between metaphoricity condition and membership of the monolingual or multilingual group, whether as regards the polarity of meaningfulness judgements or the time taken to make them or read the sentences, despite the pattern of YES/NO answers in Table 6. Indeed, we had no clear basis on which to hypothesise an interaction. Our main interest as regards differences between multilinguals and monolinguals was to establish that they exhibit both some difference in the low-conforming condition and some difference in the high-con- forming condition, rather than to show that such differences on individual conditions did or did not themselves differ from each other. 427 Language and Cognition Nevertheless, having observed that multilinguals and monolinguals performed very similarly to each other in both the literal and conventional conditions, while performing differently in the novel conditions, it would be interesting to see whether a future, larger study would show a statistically significant condition/group inter- action, at least involving the nonnovel/novel difference, if not the difference between the two novel conditions. In our study, the significant contrasts on novel conditions were presumably not strong enough to drive an interaction effect towards signifi- cance, due to an extremely striking similarity between the literal and conventional conditions. An interaction of mono/multilingualism with the two types of novelty might arise if knowing additional languages, in general, or knowing specific additional languages, has an effect on differences in processing high-conforming versus low-conforming expressions (with high/low conformity here still being from the English viewpoint). For instance, it could make the person familiar with more metaphoric bridges (mappings, superordinate categories etc.). This would probably make more of a difference to low-conforming expressions that could now benefit from these bridges than to high-conforming expressions. It would effectively turn some English-wise low-conforming expressions into high-conforming ones from a certain other lan- guage’s viewpoint. Nevertheless, for a study to reveal this, it would need to be systematic about, for instance, which additional languages multilinguals know, and it would need to include some expressions that engage bridges that are peculiar to the additional languages. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.3. Some limitations of the current study As with all linguistic materials in experiments, but especially when materials are novel and potentially puzzling, there is the complication of possible multiple inter- pretations. It was beyond the scope of the present study to filter out cases where participants constructed nonmetaphorical meanings for the pairs we classified as metaphorical, as we did not ask participants to state meanings that occurred to them. Nor, of course, could we check whether metaphorical meanings that they did discern exploited distinctive features of the adjectives’ meanings, as was required for richest comprehension. As noted in Section 3’s preamble, our main reason for not asking participants to provide meanings for the pairs was that we did not assume that a participant’s judgement that a sentence made sense would necessitate them having a particular meaning that was clear in their conscious mind and that therefore could be usefully reported during the experiment. A meaning could be largely absent from con- sciousness except in very broad strokes, or might be present but difficult to express; or there might be a range of alternative meanings that the participants would be hard pressed to choose from or to summarise. These possibilities are particularly salient for low-conforming expressions. For instance, it is difficult enough for us as metaphor researchers to state what a ‘curved hope’ might be, even given considerable time to think about it, let alone for a non-metaphor researcher to be able to formulate a meaning during an experiment. An additional reason for not asking for meanings was that we wished to avoid the theoretically highly contentious process of judging whether stated meanings were reasonable, metaphorical ones. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 428 Werkmann Horvat et al Furthermore, in the case of our novel materials, it was inappropriate to use the common technique in metaphor studies of asking participants for lists of features or for their choices from lists of features or meanings provided by experimenters. This was because of the subtlety and structural complexity of possible interpretations, especially as different interpretations could have been divided largely by matters of degree. For instance, a ‘curved hope’ (one of our low-conforming novel examples) could be taken to mean a hope that was somewhat more precisely defined and pleasing than a ‘hazy hope’ (the corresponding high-conforming example), rather than differing from a hazy hope in a more black-and-white way. 5.3. Some limitations of the current study g y y A related limitation is that in our interpretation of the results in Section 5.1, we have taken YES answers on meaningfulness to mean the participants did have a genuine feeling of comprehension (whether or not because of consciously discerning clear, specific meanings), rather than feeling that they failed to comprehend but nevertheless wishing to save face by not appearing ignorant. However, note that if such face-saving were frequent enough to substantially invalidate our interpretations, we would have an interesting potential phenomenon of multilinguals engaging in it more than monolinguals, and of high-conforming expressions attracting it more often than low-conforming ones. Such possibilities are interesting targets for future research. Because of the various limitations, and also for reasons of sample size, we were not able to make confident, separate analyses of reaction times for cases when partici- pants said YES and cases where they said NO. It would naturally be beneficial in future work to try to surpass this limitation and the previously noted ones. The findings from our study suggest that high-conforming novel phrases are comprehended more slowly than low-conforming ones. It would be intriguing in future work to filter out, in the low-conforming case, the YES responses that are based on less-rich, quick-to-derive meanings that do not exploit distinctive aspects of the phrases, thereby proceeding as if the expressions were high-conforming. If a speed advantage for low-conforming over high-conforming still remained after such filtering of YES answers, it might indicate that, for instance, the within-source inferencing or other connection-following and the use of familiar metaphoric bridges (mappings, superordinate categories etc.) in low-conforming cases tend to be less careful than in, or to hit obstacles earlier than in, actual high-conforming cases. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.5. Further general discussion: novelty and user relativity There are a number of things to note concerning our approach to metaphor novelty. (We should stress that we continue with this article’s focus on what novelty amounts to from the hearer’s point of view. See Hidalgo-Downing (2020) for an overview of metaphoric creativity and novelty, one that encompasses speakers and hearers, context-sensitivity and multimodality.)15 On the one hand, we have downplayed the role of lexical novelty: we have presumed in our study that an expression is, as a practical matter, more likely to be novel in our sense if it has only appeared rarely in corpora, but lexical novelty is not in fact part of our conception of metaphoric novelty. Moreover, in principle, an expression could have been frequently used with a particular meaning, but this meaning nevertheless may not have become entrenched in the sense of being simply retrievable from memory. Equally, in principle, it could happen that a very infrequently used expression could have a metaphorical meaning that becomes quickly entrenched for some special reason. q y p Furthermore, there is a hearer-relativity issue whereby different L1 hearers of a language may approach a given metaphorical expression in different ways. This is ultimately why we have defined our types of novelty on the basis of how hearers might process the expressions at hand, rather than trying to define them in a more traditional, hearer-neutral way. The characterisations of our types of novelty are not strongly based on any specific theory of metaphor, but only require a very general notion of bridges between subject matters, together with the notion of drawing inferences within the source subject matter or following other sorts of connection within it, and the possibility that a hearer creatively (though perhaps unconsciously) entertains a special context when no helpful context is provided that points to a specific meaning. At the same time, our approach emphasises the point that the particular, detailed way in which an expression is novel as metaphor is ultimately a theory- relative issue. For instance, one theory might argue that a metaphor is novel because it requires new bridge construction, whereas another might say that a metaphor is novel because it requires unusual within-source inferencing, and a yet a third theory might say it requires both. In addition, these matters are, as we have already pointed out, language-relative. 15Note that Hidalgo-Downing there lists ‘novel metaphors’ separately from ‘creative elaborations of conventional metaphors’ and ‘elaborating and expanding source domains’. We take her to be using ‘novel metaphors’ to mean new ways of putting target and source subjects in correspondence – new bridges in our terms. Her other two categories are also types of novelty in our own view. 5.4. Further general discussion: cognitive flexibility and multilinguals Our experimental results indicate that multilinguals tend to accept novel metaphors more than monolinguals do, which is in line with previous research on the ‘cognitive flexibility’ of multilinguals. Although we acknowledge the controversial nature of this claimed flexibility (Lehtonen et al., 2018; Paap & Greenberg, 2013; Papageorgiou et al., 2019), our findings suggest that differences in the way monolinguals and multilinguals process linguistic input exist, and need to be investigated, analysed and interpreted with different methods and tools. Although the vague and generic labels ‘cognitive advantage’ or ‘cognitive flexibil- ity’ may be easily misinterpreted, more fine-grained labels and specific effects of multilingualism on language processing remain an open and fertile field of investi- gation. Our current contribution goes in this direction, suggesting a specific sector in which monolinguals and multilinguals appear to perform in different ways, that is, a 429 Language and Cognition possible linguistic flexibility: namely, in comprehending two different types of novel metaphors. This suggests that, if there is a cognitive advantage to multilingualism, it may relate more closely to divergent thinking (Runco & Acar, 2012) than to other kinds of flexibility that may be less closely related to novel metaphor comprehension. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.5. Further general discussion: novelty and user relativity For example, literal translation of a metaphorical expression from one language to another can change whether it is metaphorically novel or not, or the precise way in which it is novel, partly because different bridges between source and target might be familiar to hearers. However, this issue itself interacts with what metaphor theory is postulated, because, for instance, the less https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press Werkmann Horvat et al 430 socio-culturally specific the types of bridge proposed by a given theory are, the less they might tend to vary between languages. Our approach may shed new light on the different ways in which we might go beyond theories such as the career of metaphor theory as in Bowdle & Gentner (2005). In particular, this theory suggests a relatively straightforward gradation between conventional and novel metaphors, reflecting two different processing strategies: metaphor comprehension by means of construction of cross-domain mappings from scratch between source and target domains for the more novel metaphors, and metaphor comprehension by means essentially of the categorisation approach (if mere retrieval of meaning is not adequate) for the more conventional metaphors. Such a clear-cut distinction between metaphor types and processing strategies may need to be abandoned in favour of a more sophisticated set of distinctions and a rich array of different mixes of types of processing. Our distinction between different types of novelty addresses the fact that different levels of richness can be found for a given metaphorical expression, according in part to different extents to which the hearer exploits or ignores distinctive aspects of source-side concepts raised. Such a tendency to exploit or to ignore these aspects affects the extent to which the hearer on a specific comprehension occasion needs to, is inclined to, or is able to conjure up new mappings or other types of bridge between source and target, perform unusual within-source inferencing or other connection-following or enter- tain special contexts. These factors in turn can interact with each other. We therefore assume that one cannot distinguish types of novelty on any simple ground such as the binary distinction between requiring or not requiring new bridges, or measuring degree of novelty on any simple, unitary basis. Despite these complications, we found that the high/low-conformity distinction did make a significant difference to meaningfulness judgements and the timing of comprehension attempts. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.5. Further general discussion: novelty and user relativity A fertile area for further research would be to try to tease apart the different roles here of familiar-bridge availability, of possibilities for within-source inferencing or other connection-following, and of entertainment of special contexts. Moreover, in reality, there is a spectrum of degrees of conform- ity between what we have called high and low conformity, so it would be beneficial to investigate the dependence of timings and meaningfulness judgments on the whole range of degrees. How to measure the degrees would itself need to be a focus in such work. Although we have sought to be as theory-neutral as possible in our study, our approach does chime especially well with potential metaphor comprehension theories that are eclectic and flexible in that: existing bridges can be used as they are, or refined when necessary; new bridges can be created when necessary; bridges may be of different types (e.g., mappings or superordinate categories) for different purposes (unless it can be shown that just one type works well under all circum- stances); within-source connections of various types can be followed and the mixing and ordering of these various types of processing is flexible, dynamically arising and highly responsive to circumstances such as the context in which the discourse takes place, the linguistic context surrounding the expression currently being comprehended, and the purposes, interests, world/language experience and condition (mood, level of interest, alertness, distractedness, intellectual capacity etc.) of the hearer. From this point of view, the most promising metaphor theories are those that embed the question of metaphor within the question of semantic/ pragmatic processing more broadly, or indeed cognition as a whole, such as 431 Language and Cognition blending theory (Fauconnier & Turner, 1998, 2008), the cognitive-operation approach of Ruiz de Mendoza and colleagues (Ruiz de Mendoza, 2020; Ruiz de Mendoza & Galera, 2014), proposals within Relevance Theory, especially when incorporating inference about the literal, source-side scenario suggested by the expression at hand (Carston & Wearing, 2011) and the view of metaphor as dynamic/ecological performance (Gibbs Jr., 2019). We now more explicitly address hearer relativity. It has long been generally recognised that the more novel a metaphor is, the more open-ended is the range of meanings that the hearer can justifiably and relevantly extract, and that different hearers and different circumstances can favour different meanings. 5.5. Further general discussion: novelty and user relativity In particular, individual differences in cognitive ability can have an effect on metaphor compre- hension – see especially Stamenković, Ichien, & Holyoak (2020), whose study concentrated on literary metaphors, ones thus tending to the novel, such as ‘Nerves after a quarrel are frozen leaves in winter’. These authors note, however, that relatively few studies have explored this matter of individual ability differences. Relatedly, we argue that the hearer relativity of metaphoricity in general, and of conventional and novel metaphoricity in particular, needs to be more explicitly recognised when talking about conventional and novel metaphors from the hearer point of view. (Compare the comment by Hidalgo-Downing, 2020, that a linguistic expression will not be metaphoric or creative per se, but instead this will depend on how and in what context the expression is used.) It is not that, objectively, some expressions are or are not metaphorical, or are or are not conventional as metaphor or are or are not of this or that type of novelty as metaphor. Rather, the central point is that under particular circumstances, hearers process expressions in particular metaphoric ways, and these can involve (to differing extents) lexical retrieval, the creation or activation of old or new bridges, old or new within-source inferencing, and so forth. From this, as a practically useful but nevertheless crude and derivative abstraction, one can say that some expressions are conventional metaphorical ones because they have been frequently used with a particular fixed meaning (or, more probably in fact, with a cluster of highly context-sensitive but closely related meanings). However, such expressions might at any point be treated by a hearer in a way that partially or entirely ignores such meanings, and seeks meaning(s) afresh, although this does not preclude the hearer from landing on a meaning similar to established ones. Going back to the discussion in Section 2.2.1, such potential divergence from established meanings, or doing something more than just retrieving an established meaning, may normally occur due to pressure arising from an unusual linguistic or world context or accompanying multimodal communication, but equally may be a more general phenomenon. Or, it could sometimes be just because of the particular hearer’s current condition or even random whim. https://doi.org/10.1017/langcog.2022.8 Published online by Cambridge University Press 5.5. Further general discussion: novelty and user relativity On the other hand, an expression that has rarely before been used metaphorically and that potentially has rich meaning could be validly treated by a given hearer in a relatively crude way, and distinctive aspects of possible meaning may be ignored. Therefore, categories such as ‘conventional’, ‘novel’ and ‘creative’ metaphors could usefully be renamed as ‘conventionally amenable’, ‘novelty-enabling’ or ‘cre- ativity-inspiring’ or some such, when we are considering the hearer side of commu- nication. ‘High conforming’ and ‘low conforming’ could more accurately be renamed as ‘closely conformable’ and ‘distantly conformable’. It is, in the end, at best a preliminary and approximate venture to conduct experiments exploring hearers’ treatment of novel or conventional expressions as if these categories had specifiable Werkmann Horvat et al 432 nonhearer-relative natures that govern how hearers comprehend them. Instead, the direction of governing is more the opposite. There are different types of metaphoric processing available to hearers in a circumstance-specific way, and the way these are used on a given occasion governs the designation of a particular hearer’s processing as involving metaphoricity or not, novelty or not and a particular type of novelty or not, on that occasion. The patterns of such processing across different hearers and over time govern the assignment of heuristically useful, but over-simplified and derivative, labels, such as conventional, novel etc., to metaphorical expressions themselves. 6. Conclusion This study looked at two distinct, but connected, research areas: the nature of metaphorical novelty, and the ability of multilinguals as contrasted with monolin- guals to comprehend novel metaphorical language. Our experimental results indicate that multilinguals tend to accept novel metaphors more than monolinguals do, which is in line with previous empirical indications of other sorts of ‘cognitive flexibility’ of multilinguals, and thus tending to show that multilinguals do have some such flexibility advantage despite some controversy on this point. We addressed novel metaphor of two types, carved out from the opposing ends of what is in reality a complex spectrum of novelty. One of the types was high- conforming novelty, where distinctive meaning based on distinctive features of the source items can straightforwardly be found without having to invent new mappings or other bridges between source and target subject matters, and without having to entertain special contexts or do unusual inferencing or other following of connec- tions within the source subject matter. The other type was low-conforming novelty, where rich comprehension requires marked use of such types of processing. This particular way of distinguishing types of novelty may be of value to other researchers. Our results also suggest that, although people find high-conforming novel meta- phorical phrases easier to comprehend than low-conforming ones, they spend more time on the former. This may suggest that in the presence of low-conforming novelty, where special processing is needed, people tend to give up quickly. p p g p p g p q y The language-cognition relationship may be influenced by the nature and on-the- fly condition of the individual participants, the historical and geographical context, the existence of familiar source/target bridges (mappings etc.) in the cognitive underpinnings of the other languages that they speak and other contingent factors. Indeed, given the complex and dynamic nature of the relationship between language and cognition, much further research, both theoretical and empirical, is suggested by our study. Acknowledgments. 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https://openalex.org/W4361941179	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_from_Artificial_Intelligence-Assisted_Serial_Analysis_of_Clinical_Cancer_Genomics_Data_Identifies_Changing_Treatment_Recommendations_and_Therapeutic_Targets/22486649/1/files/39938234.pdf	English	null	Supplementary Figure from Artificial Intelligence-Assisted Serial Analysis of Clinical Cancer Genomics Data Identifies Changing Treatment Recommendations and Therapeutic Targets	null	2,023	cc-by	262	0 200 400 600 800 1000 1200 TP53 PCLO APC KRAS KMT2D RELN ARID1A EGFR NOTCH1 PRKDC ATM BRCA2 NCOR2 NF1 SPEN PIK3CA IRS2 SETD2 RECQL4 MSH3 # of cases All others 5th 4th 3rd 2nd 1st Supplemental Figure 8. The most frequently mutated genes and variants across all cases. The total number of cases that harbor the mutated gene (grey) and the five most common variants within the gene (colored) are shown. 0 200 400 600 800 1000 1200 TP53 PCLO APC KRAS KMT2D RELN ARID1A EGFR NOTCH1 PRKDC ATM BRCA2 NCOR2 NF1 SPEN PIK3CA IRS2 SETD2 RECQL4 MSH3 # of cases All others 5th 4th 3rd 2nd 1st Supplemental Figure 8. The most frequently mutated genes and variants across all cases. The total number of cases that harbor the mutated gene (grey) and the five most common variants within the gene (colored) are shown. 0 200 400 600 800 1000 1200 TP53 PCLO APC KRAS KMT2D RELN ARID1A EGFR NOTCH1 PRKDC ATM BRCA2 NCOR2 NF1 SPEN PIK3CA IRS2 SETD2 RECQL4 MSH3 # of cases All others 5th 4th 3rd 2nd 1st Supplemental Figure 8. The most frequently mutated genes and variants across all cases. The total number of cases that harbor the mutated gene (grey) and the five most common variants within the gene (colored) are shown. All others 5th 4th 3rd 2nd 1st Supplemental Figure 8. The most frequently mutated genes and variants across all cases. The total number of cases that harbor the mutated gene (grey) and the five most common variants within the gene (colored) are shown
https://openalex.org/W3013839928	https://digital.csic.es/bitstream/10261/233040/1/Mechanistic_investigation_POWER_2020.pdf	English	null	Mechanistic investigation of silicon-graphite/LiNi0.8Mn0.1Co0.1O2 commercial cells for non-intrusive diagnosis and prognosis	Journal of power sources	2,020	cc-by	9,678	Manuscript Click here to view linked References Manuscript Click here to view linked References Mechanistic investigation of silicon-graphite/LiNi0.8Mn0.1Co0.1O2 commercial cells for non-intrusive diagnosis and prognosis Research paper (Revised Version) (Revised Version) D. Anseán1,, G. Baure2, M. González1, I. Cameán3, A.B. García3, and M. Dubarry2, 1 Department of Electrical Engineering, Polytechnic School of Engineering, University of Oviedo, 33204, Gijón, Asturias, Spain 2 Hawai'i Natural Energy Institute, University of Hawai'i at Mānoa, 1680 East-West Road, POST 109, Honolulu, HI 96822, USA 2 Hawai'i Natural Energy Institute, University of Hawai'i at Mānoa, 1680 East-West Road, POST 109, Honolulu, HI 96822, USA 1 1 Abstract 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Due to their high energy density, lithium-ion batteries with blended silicon-graphite (Si-Gr) anodes and nickel-rich (NMC) cathodes have been regarded as one of the most promising technologies for next-generation consumer electronics and electric vehicles. However, there are still several technical challenges to overcome for successful wide-spread adoption; in particular, deciphering the degradation phenomena remains complex and challenging, as the blended nature of the electrode creates a new paradigm, with the Si/Gr ratio likely changing with aging. Although ex-situ techniques have been used, a set of in-operando tools that enable diagnosis and prognosis on this technology has yet to be developed. Herein, we present a mechanistic investigation that generates a complete degradation mapping coupled with proposed aging features of interest, to attain accurate diagnosis and prognosis. The mechanistic model allows the visualization of analyzing aging modes that displays incubation periods as a potential prelude to thermodynamic plating, and the identification via incremental capacity of unique silicon features that change predictably as it degrades. A comprehensive look-up table summarizing key features is provided to provide support both to scientists and engineers on designing next-generation battery management systems for this technology. Keywords Silicon-graphite; Nickel-rich, NMC 811; Incremental capacity; Mechanistic model simulations; Battery degradation mapping; 2 1. Introduction Lithium-ion batteries (LiB) performance is steadily improving year after year thanks to the advancement of manufacturing processes and to the introduction of superior electrode materials [1–3]. Among the most promising new materials recently introduced are silicon- graphite (Si-Gr) negative electrodes (NE) and nickel-rich nickel manganese cobalt oxide LiNi0.8Mn0.1Co0.1O2 (NMC811) positive electrodes (PE) [4,5]. 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Energy-dense Si-Gr blended NE, are recognized as one of today’s most promising battery technologies capable of meeting the ever-increasing requirements of LiB systems. Although the preliminary research stage of Si-based anodes dates back to the late 1990s [6,7], its market introduction is very recent due to the performance and reliability issues found throughout its development stages [4]. Nowadays, the issues that hampered Si-based anodes cyclability – mainly, large volumetric changes and subsequent solid-electrolyte interphase (SEI) formation – have been partly addressed by using several approaches, [8–12] one of which being the use of blended electrodes containing silicon and graphite [13–15]. To take full advantage of the NE increase of energy, the PE should also exhibit high capacity. This desired capacity can be attained using nickel-rich materials [5,16–18]. In particular, LiNi0.8Mn0.1Co0.1O2 (NMC811) is currently used in last-generation commercial Si–Gr based cells [19,20]. In total, due to these technological advances, today’s commercial 18650 batteries with Si-Gr/NMC811 configuration exhibit capacities as high as 3.5 Ah, with the Si accounting just for 3 to 4% of the electrode weight [19–21]. 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 Recent studies on Si-based anodes have been focused on understanding the complexity of the degradation phenomena from a material science perspective [20,22–24]. As shown, the underpinning degradation of Si-based anodes has been attributed by unstable SEI and severe volumetric changes of over 300% that lead to particle cracking and electrical isolation [15]. These degradation phenomena correspond to the aging modes loss of lithium inventory (LLI) 48 49 50 51 52 53 54 55 56 57 58 59 60 Recent studies on Si-based anodes have been focused on understanding the complexity of the degradation phenomena from a material science perspective [20,22–24]. 1. Introduction As shown, the underpinning degradation of Si-based anodes has been attributed by unstable SEI and severe volumetric changes of over 300% that lead to particle cracking and electrical isolation [15]. These degradation phenomena correspond to the aging modes loss of lithium inventory (LLI) 3 and loss of active material (LAM), respectively [25]. Despite the valuable insights attained in those studies, their methodology (based on ex-situ, surface-science advanced techniques) is not applicable for direct use in battery management systems (BMS), which require non-invasive techniques. This issue is becoming a critical aspect that needs to be addressed, as these new battery technologies are emerging and are expected to be deployed in consumer electronics and next-generation electric vehicles (EVs) [26,27]. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 In this study, we adapted the non-invasive battery diagnosis and prognosis mechanistic framework to Si-Gr/NMC811 batteries. The use of mechanistic models to attain diagnosis and prognosis on battery performance has been demonstrated to be effective for most current technologies [28–31], but is yet to be developed for the Si-Gr/NMC811 configuration. In particular, the use of a blended material (i.e., Si-Gr) on the NE creates a new paradigm, since silicon is likely to age independently and at faster rates than graphite, all within the same electrode [21,23,32]. Hence, aging modes LLI, and LAM on graphite and on silicon, can take place at different rates due to the blended nature of the electrode. This phenomenon adds an additional complexity level to accurate diagnoses that was never investigated before. Although studies on blended PE were undertaken [33–35], herein, we introduce for the first time the emulation of a blended NE to properly address each active material from an individual perspective. We further expand in more detail the model construction, so it can be applied to either PE or NE blended materials. By combining the knowledge of our established mechanistic approaches with the information obtained from stand-alone Si-based experiments, the fractional degradation of each blended material can be estimated. 1. Introduction We also present for the first time the degradation mapping of this cell technology, together with the main features of interest (FOIs) to enable proper sensibility analysis [30,36] and to facilitate future degradation studies on commercial Si-Gr/NMC811 systems. The knowledge gained from this study shall help to further understand degradation of commercial Si–Gr batteries with the use of non-invasive, 4 4 in-situ techniques, which can be applied for the development of accurate, next-generation BMSs. in-situ techniques, which can be applied for the development of accurate, next-generation BMSs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 SEM and EDX were carried out using the Helios Nanolab 660 DualBeam FIB-SEM (FEI, now Thermo Fisher Scientific) at the Advanced Electron Microscopy Center at the University of Hawai‘i at Mānoa. Powder diffraction data was collected at the X-ray Atlas lab at the Hawai‘i Institute of Geophysics and Planetology, using a Bruker D8 Advance high resolution powder diffractometer, equipped with 2kW CuKa X-ray source and LYNXEYE XE detector. The source was configured in the Bragg-Brentano parafocusing geometry. The data was collected using standard sample mount, in a theta-2 theta mode, with spinning the sample around the phi goniometer axis at 15 revolutions per minute. The scanning range was 5 to 85 deg, with a step on 0.02 and 0.5 s exposure per step. The data was analyzed using DIffrac.Eva software. Simulation tests Computer simulations were undertaken using the ‘alawa battery diagnosis and prognosis toolbox, developed at the University of Hawai‘i [43]. The toolbox used the model described in detail in Ref. [25]. 3. Results Electrochemical characterizations To develop an accurate full-cell reconstruction for implementation in the mechanistic model, four electrode datasets are needed. This includes the harvested PE and NE from a Si-based commercial cell, in this case NMC811 vs. Li and Si-Gr vs. Li, together with graphite vs. Li and silicon vs. Li. to reconstruct the blended anode. For this study, 55 INR18650-35E cells from Samsung-SDI were purchased from an online vendor. According to the manufacturer, this cell exhibits a standard discharge capacity ≥ 3,350 mAh, when discharged at C/1 within the voltage limits (4.2 V charge, 2.65 V cut-off). In this work, a representative cell was subjected to a set of standard conditioning tests [28], including tests at C/25. A multichannel, high-precision series Arbin LBT was used for testing. The cell was placed at constant 23 ºC in a Memmert environmental chamber. This representative cell was later disassembled to harvest the PE and NE. Standard operation procedures (SOPs) were followed for cell disassembling and half-cell construction, as discussed in Ref. [37]. These SOPs were developed based on previous works [38,39], our laboratory experience [31,37,40] and recent recommendations on the topic [41,42] to ensure truthful experimental data, a critical factor to attain accuracy in the half-cell reconstruction and the quantification of the degradation effects. The standard Gr vs. Li dataset was taken from the ‘alawa [43] database library of electrodes, whereas the Si vs. Li dataset was taken from Ref. [44]. Non-electrochemical characterizations 5 5 Additional characterization, including scanning electron microscopy (SEM) with energy- dispersive x-ray spectroscopy (EDX) as well as X-ray diffraction (XRD), were carried out to validate electrode composition. 3. Results S1-S2) for the four constructed half-cells and the experimental full cell (Figs. S3). In addition to the electrochemical tests, EDX mapping showed the intermingled nature of the negative electrode with two sets of grains of different morphologies (see Supplementary material Fig. S4). The above results confirmed that the NE is a blended Si-Gr electrode. The graphite grains are in the 20 μm range, whereas the Si grains are in the 5-10 μm range, as shown in Fig. 2c,f. EDX analysis calculated a Si content of ~3-4 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For the harvested NE, the IC curves exhibit six peaks (Fig. 1d,e, black curves). Peaks ❶ to ❺ can be attributed to Gr (cf. Fig. 1d,e, red curves) [45], whereas a distinctive peak ❻ appeared at a higher potential of ~0.45 V. This peak can be attributed to Si (cf. Fig. 1d,e, gold curves for standard micro-porous Si signature). The standard Gr and the harvested NE profiles are proportional within a potential window below ~0.23 V (see Fig. 2d, red and black, respectively). Above that potential, the harvested NE shows noticeable differences, including the appearance of an additional plateau, showcasing the characteristics of Si-Gr blended electrodes, as shown by Yao et al. [22], and confirmed with the electrochemical profile of standard micro-porous Si vs. Li (Fig. 1d,e, gold color). The delithiation process is shown to reveal the characteristic high potential peak ca. 0.45–0.49 V that created peak ❻ in the harvested, Si-Gr electrode (Fig. 1d,e, black curve). During delithiation, Li+ ion extraction occurs first from graphite particles that fully delithiate within the 0.01–0.23 V range, then from Si within the 0.23–1.0 V range [22,23,32], a phenomenon observed in Fig. 1d (black curve). It is well known that the (de-)lithiation of Si electrodes show large potential hysteresis, together with less pronounced plateaus [23,46,47], while standard graphite shows comparatively minor hysteresis and plateau changes. Supplementary material presents representative charge/discharge profile curves (Figs. S1-S2) for the four constructed half-cells and the experimental full cell (Figs. S3). 3. Results 1. Half-cell experimental data 48 49 50 51 52 53 54 55 56 57 Half-cell experiments are essential to build a virtual replicate of the full cell and construct the mechanistic model. Fig. 1 is the half-cell data voltage profiles and the associated incremental capacity (IC) signature for a C/25 charge of the harvested PE (Fig. 1a,b), and NE (Fig. 1d,e). 6 6 The harvested PE IC curve (Fig. 1b) presented four distinctive redox peaks (noted ①-④) located at ~3.6 V, 3.7 V, 4 V and 4.2 V, which corresponds to the unique signature of Ni-rich NMC811 [16]. This material was further analyzed by XRD and EDX, confirming the dominance of nickel (Supplementary material Fig. S5). 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 For the harvested NE, the IC curves exhibit six peaks (Fig. 1d,e, black curves). Peaks ❶ to ❺ can be attributed to Gr (cf. Fig. 1d,e, red curves) [45], whereas a distinctive peak ❻ appeared at a higher potential of ~0.45 V. This peak can be attributed to Si (cf. Fig. 1d,e, gold curves for standard micro-porous Si signature). The standard Gr and the harvested NE profiles are proportional within a potential window below ~0.23 V (see Fig. 2d, red and black, respectively). Above that potential, the harvested NE shows noticeable differences, including the appearance of an additional plateau, showcasing the characteristics of Si-Gr blended electrodes, as shown by Yao et al. [22], and confirmed with the electrochemical profile of standard micro-porous Si vs. Li (Fig. 1d,e, gold color). The delithiation process is shown to reveal the characteristic high potential peak ca. 0.45–0.49 V that created peak ❻ in the harvested, Si-Gr electrode (Fig. 1d,e, black curve). During delithiation, Li+ ion extraction occurs first from graphite particles that fully delithiate within the 0.01–0.23 V range, then from Si within the 0.23–1.0 V range [22,23,32], a phenomenon observed in Fig. 1d (black curve). It is well known that the (de-)lithiation of Si electrodes show large potential hysteresis, together with less pronounced plateaus [23,46,47], while standard graphite shows comparatively minor hysteresis and plateau changes. Supplementary material presents representative charge/discharge profile curves (Figs. wt% on the harvested NE (see Supplementary material Fig. S4), which is in concordance literature on equivalent cell chemistry and cell format commercial Si-Gr batteries [20,21]. wt% on the harvested NE (see Supplementary material Fig. S4), which is in concordance literature on equivalent cell chemistry and cell format commercial Si-Gr batteries [20,21]. 3. Results In addition to the electrochemical tests, EDX mapping showed the intermingled nature of the negative electrode with two sets of grains of different morphologies (see Supplementary material Fig. S4). The above results confirmed that the NE is a blended Si-Gr electrode. The graphite grains are in the 20 μm range, whereas the Si grains are in the 5-10 μm range, as shown in Fig. 2c,f. EDX analysis calculated a Si content of ~3-4 7 wt% on the harvested NE (see Supplementary material Fig. S4), which is in concordance literature on equivalent cell chemistry and cell format commercial Si-Gr batteries [20,21]. wt% on the harvested NE (see Supplementary material Fig. S4), which is in concordance literature on equivalent cell chemistry and cell format commercial Si-Gr batteries [20,21]. 1. (a) Voltage profile of harvested NMC811 vs. Li and (b) corresponding IC curves. (d) and (e) show the voltage and IC signatures for half-cell harvested Si-Gr (black), standard Gr (red) and porous Si (gold). All were cycled vs. Li. (c) and (f) show SEM images at high magnification of the fresh Si-Gr anode, where the bright grain is silicon (f) Fig. 1. (a) Voltage profile of harvested NMC811 vs. Li and (b) corresponding IC curves. (d) and (e) show the voltage and IC signatures for half-cell harvested Si-Gr (black), standard Gr (red) and porous Si (gold). All were cycled vs. Li. (c) and (f) show SEM images at high magnification of the fresh Si-Gr anode, where the bright grain is silicon (f) wt% on the harvested NE (see Supplementary material Fig. S4), which is in concordance literature on equivalent cell chemistry and cell format commercial Si-Gr batteries [20,21]. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Voltage (V) Capacity (Ah) NMC 811 (Harvested) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Standard Si Si-Gr (Harvested) Standard Gr 0.0 0.1 0.2 0.3 3.5 3.7 3.9 4.1 4.3 4.5 IC (mAh/V) Voltage (V) NMC 811 (Harvested) - IC 0 1000 2000 3000 4000 5000 0.0 0.2 0.4 0.6 IC (%Q/V) Voltage (V) Standard Si Si-Gr (Harvested) - IC Standard Gr - IC ① ② ③ ④ ① ② ③ ④ ❻ - ❻ - ❷ - ❺ ❶ ❶ ❷ - ❺ 7 μm a) d) b) c) e) f) 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Voltage (V) Capacity (Ah) NMC 811 (Harvested) 0.0 0.1 0.2 0.3 3.5 3.7 3.9 4.1 4.3 4.5 IC (mAh/V) Voltage (V) NMC 811 (Harvested) - IC ① ② ③ ④ ① ② ③ ④ a) b) 2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Standard Si Si-Gr (Harvested) Standard Gr ❻ - ❷ - ❺ ❶ d) 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Voltage (V) Capacity (Ah) NMC 811 (Harvested) ① ② ③ ④ a) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 0.0 0.1 0.2 0.3 3.5 3.7 3.9 4.1 4.3 4.5 IC (mAh/V) Voltage (V) NMC 811 (Harvested) - IC ① ② ③ ④ b) 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 f) 7 μm c) f) 8 Fig. 3.2. Emulations and full-cell reconstruction 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 For the full-cell emulation process, a virtual mechanistic reconstruction that behaves as close as possible to the commercial cell is required. The full-cell reconstruction is to be accomplished in three steps; (i) reconstruct the harvested blended NE, (ii) reconstruct the full cell, both with the reconstructed NE and the harvested PE, and (iii), fine-tune kinetic adjustments to accurately match the experimental cell kinetics. In step (i), the reconstruction of the blended NE is attained by determining the capacity contributions of the individual standard micro-porous Si and standard Gr electrodes (i.e., %Gr + %Si). The obtained reconstructed NE shall match the signature of the harvested NE. In step (ii), the full cell is constructed from the combination of reconstructed NE and the harvested PE. Here, the loading ratio (LR) and offset (OFS) [25,37] are the parameters that are adjusted to obtain a reconstruction of the experimental full-cell data. To complete the emulation model (step iii), fine-tune kinetic adjustments are carried out. This is required as experimental full-cell differs from the reconstructed mechanistic model due to several kinetic-related phenomena [48]. The mechanistic model is obtained from half-cell electrode testing vs. Li, while the full-cell configuration internally combines its working electrodes, which introduces differences in cell pressure, separator and electrolyte composition [41,42]. Hence, adjustments to the kinetics parameters of the reconstructed model are necessary. All the above steps and parameter modifications can be performed in the ‘alawa toolbox. Fig. 2a-b compares the voltage profiles and IC curves, respectively, of the harvested NE with the ‘alawa emulated blended Si-Gr NEs with the Si contribution varied from 0% to 40% of the 9 total NE capacity. It is observed that increasing the Si capacity ratio in the blended electrode produces significant changes, particularly on its characteristic high voltage peak ❻. Using peak ❻ as the main feature for comparison with the harvested electrode (Fig. 2b, inset figure), an estimation of the capacity contribution of 10% of Si and 90% of Gr was obtained. This estimation corroborates with EDX results since Si provides more capacity per weight than Gr. 3.2. Emulations and full-cell reconstruction These capacity ratios are also in good agreement with those found in the literature on equivalent cell chemistry and cell format [19,21]. The slight shape difference between reconstructed and harvested NE observed on peak ❻ (Fig. 2b, inset) is to be modified on the last full-cell adjustment stage by accounting for the difference in electrode kinetics. total NE capacity. It is observed that increasing the Si capacity ratio in the blended electrode produces significant changes, particularly on its characteristic high voltage peak ❻. Using peak ❻ as the main feature for comparison with the harvested electrode (Fig. 2b, inset figure), an estimation of the capacity contribution of 10% of Si and 90% of Gr was obtained. This estimation corroborates with EDX results since Si provides more capacity per weight than Gr. These capacity ratios are also in good agreement with those found in the literature on equivalent cell chemistry and cell format [19,21]. The slight shape difference between reconstructed and harvested NE observed on peak ❻ (Fig. 2b, inset) is to be modified on the last full-cell adjustment stage by accounting for the difference in electrode kinetics. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Fig. 2c presents the final cell reconstruction, where the IC curves of the experimental full cell (gray) are compared versus the reconstructed full-cell model (green), showing good agreement between the two. Peak numbering corresponds to the convolution of the NE (circled, black) with the PE (circled, white). The cell reconstruction parameters that best match the experimental cell were a LR of 0.9 between the NE and the PE and an OFS of 10%. These cell parameters match with values from literature on cells with equivalent chemistries and formats [19,21]. A kinetic modification of 0.1 on the reconstructed NE was found to be the best value to match with the experimental cell (Fig. 2c, inset figure) [25,48]. 3.2. Emulations and full-cell reconstruction Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results 3.2. Emulations and full-cell reconstruction (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results 0 100 200 300 400 500 3.0 3.2 3.4 3.6 3.8 4.0 4.2 IC (%Q/V) Voltage (V) Full-cell (Experimental) Full-cell reconstruction (Emulated) ❻① ❶④ ❶③ ❷② ❺① c) ❺① ❻① 0 100 200 300 400 500 3.0 3.2 3.4 3.6 3.8 4.0 4.2 IC (%Q/V) Voltage (V) Full-cell (Experimental) Full-cell reconstruction (Emulated) ❻① ❶④ ❶③ ❷② ❺① c) ❺① ❻① 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. 3.2. Emulations and full-cell reconstruction 10 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Si-Gr (40%-60%) Si-Gr (30%-70%) Si-Gr (20%-80%) Si-Gr (10%-90%) Si-Gr (Harvested) Standard Gr 0 1000 2000 3000 4000 5000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 IC (%Q/V) Voltage (V) ❻ - ❷-❺ ❶ ❶ ❷-❺ ❻ - ❻ - a) b) 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Si-Gr (40%-60%) Si-Gr (30%-70%) Si-Gr (20%-80%) Si-Gr (10%-90%) Si-Gr (Harvested) Standard Gr ❻ - ❷-❺ ❶ a) ❶ 0 1000 2000 3000 4000 5000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 IC (%Q/V) Voltage (V) ❶ ❷-❺ ❻ - ❻ - b) ❶ ❻ 11 11 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. 4. Discussion 3 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 With the emulated cell defined in Fig. 2c, it is possible to simulate the degradation map of this Si-Gr/NMC811 cell at certain degradation rates at C/25 for this Si-Gr/NMC811 cell to reach 10% capacity loss, Fig. 3. The IC curves are shown during charge due to better on-board controllability in charger systems, and because the charging process renders each IC peak accurate presence because of more pronounced plateaus [23,46,47]. Low-rate degradation can be described with three degradation modes, the loss of lithium inventory (LLI, Fig. 3a), the loss of active material (LAM) on the delithiated (de) PE (Fig. 3b) and LAM on NE (Fig. 3d,f). In addition, concurrent loss of lithium and LAM will be referred as liPE (Fig. 3c) and liNE (Fig. 3e,g) for the PE and NE, respectively. In this work, since the negative electrode is blended, LAM on Gr (Fig. 3d,e) and on Si (Fig. 3f,g) were distinguished. The approach described here, although specifically focused on Si-Gr blended NE can be generalized to other blended materials. 12 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 In case of degradation from only LLI, Fig. 3a and from left to right, peaks ❻① and ❺① are shifting towards higher potentials, peak ❷② is disappearing, peak ❶③ is shifting towards lower potentials and peak ❶④ intensity is slightly increasing. In case of degradation from LAMdeNMC (Fig. 3b), peaks ❻①, ❺① and ❷② are shifting towards lower potentials while the intensity of peaks ❺① is increasing. The peak intensities of ❶③ and ❶④are decreasing. In the case of LAMdeGr (Fig. 3d), peak ❻① is unaffected, peak ❺① is shrinking towards lower potentials, peak ❷② is shifting towards lower potentials, peak ❶③ intensity is slightly increasing and peak ❶④ intensity is significantly decreasing. For LAMdeSi (Fig. 3f), peak ❻① is shrinking towards higher potentials, peaks ❺①, ❷② and ❶③ are shifting towards lower potentials, and peak ❶④ is shrinking towards higher potentials. The lithiated losses (Fig. 4. Discussion The thick lines are the initial emulated signatures, the thin lines are the emulated signatures at 10% capacity loss, and the dashed lines are the signatures at 2% intervals in between those two cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 F li d w m m L d li L e b a F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Fig. 3. Degradation map for the Si-Gr/NMC811 commercial cell with peak indexation and proposed FOIs. The thick lines are the initial emulated signatures, the thin lines are the emulated signatures at 10% capacity loss, and the dashed lines are the signatures at 2% intervals in between those two cases 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 14 From the degradation map, it is also possible to extract the trend in capacity loss associated with the different degradation modes, Fig. 3h. This figure provides critical information towards making a degradation mechanisms diagnosis. It is particularly effective in revealing which mode is inducing capacity loss. First of all, the capacity loss associated with degradation for LLI, LAMliNMC and LAMliGr are overlapping. This implies that capacity loss from these degradations are not additives and that the capacity loss is solely associated with the loss of lithium either alone (LLI) or within the active materials. 4. Discussion 3c,e, and g) are amalgams of the signatures of LLI and the corresponding non-lithiated loss with some changes cancelling each other. In case of LAMliNMC (Fig. 3c), peak ❻① is unaffected, peaks ❺①, ❷② and ❶③ are shrinking towards higher potentials, and peak ❶④ is shrinking slightly towards lower potentials. For LAMliGr (Fig. 3e), peak ❻① is shrinking towards higher potentials, peaks ❺① and ❷② are shifting towards higher potentials, peak ❶③ is shrinking towards higher potentials, and peak ❶④ is unaffected. Upon LAMliSi (Fig. 3g), peak ❻① is shrinking towards higher potentials, peaks ❺① are ❷② are shifting towards higher potentials, and peaks ❶③ and ❶④ are unaffected. 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 Fig. 3. Degradation map for the Si-Gr/NMC811 commercial cell with peak indexation and proposed FOIs. 4. Discussion Second, LAMdeNMC, LAMdeGr and LAMdeSi all have an incubation period in which the capacity loss is minimal despite some electrode degradation. This is induced by the positive SOC OFS that protects against LAMdeNMC by shifting the NE towards higher potential and by the fact that the LR is above 1 for LAMdeGr and LAMdeSi which provides a reservoir of Li ions that compensates for the LAMNE. Analyzing Fig. 3e and further, for one of the LAMNEs to induce capacity loss, the final voltage must go 14 below 4.2 V. This will lead to lithium plating. As long as peak ❶④ is present, the capacity loss cannot be induced by some LAMNE. In case of capacity loss induced by LAMPE, some capacity is pushed out of the lower potential window, Fig. 3b. Therefore, if peak ❻① is still fully in the potential window, no capacity loss can be associated with LAMPE. If peak ❶④ and peak ❻① are both still fully in the potential window, the capacity loss can then be unambiguously associated with LLI alone. In terms of diagnosis, some important features of interest (FOI) can also be extracted from this degradation map and the analysis of their capacity loss. First, peak ❻①, if fully in the potential window, is only sensitive and proportional to LAMSi and thus can be used to quantify that degradation mode (FOI1 on Fig. 3b). Second, if peak ❶④ is still present, the intensity of the local minimum at its front (in charge) is a direct indicator of LAMdeNMC (FOI2 on Fig. 3b). Lastly, with 3 out of 4 degradation modes quantified (one from capacity loss and the others from FOI1 and 2), the position and intensity of peak ❺①, FOI3, could be used to quantify either LLI or LAMGr by simple fitting from the simulation of the position and intensity of the peak with the three quantified parameters. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Table I is a detailed look-up table for Si-Gr/NMC811 cell technology with a summary of the changes to the main features discussed above. The look-up table provides a simple, yet reliable tool for rapid evaluation of degradation modes, avoiding extensive and complex electrochemical analyses, therefore facilitating degradation identification for BMS integration. 4. Discussion The description of the symbols in the lookup table is as follows: vertical arrows (↓ / ↑) indicates IC peak reduction or peak increase, respectively. The horizontal arrows (→ / ←) indicates IC voltage shifts. Diagonal arrows (↙ / ↗ / ↖ / ↘) indicate a voltage shift accompanied with IC reduction or increase. Double headed arrows (↡ / ↟ / ↞ / ↠) indicate largest IC changes within the aging mode. 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Table I: Look-up table of the main aging modes of the Si-Gr/NMC811 cell during charge Aging modes Incremental Capacity Main Features (peak number) Main features to evaluate for diagnosis and prognosis ❻① ❺① ❷② ❶③ ❶④ Features of Interest (FOI) Incubation period Li- plating trigger Cap. fade rate LR evolution OFS evolution LLI ↠ ↠ ↘ ← ↑ FOI 3 No No Fast Unvaried Large LAMdeNMC ↞ ↖ ← ↓ ↓ FOI 2&3 Yes No Medium Increase Large LAMliNMC = ↘ ↘ ↘ ↙ – No No Fast Increase Unvaried LAMdeGr = ↙ ← ↑ ↡ – Yes Yes Medium Degrease Unvaried LAMliGr ↘ ↠ → ↘ = FOI 3 No No Fast Decrease Large LAMdeSi ↙ ← ← ← ↡ FOI 1 Yes Yes Slow Minor Unvaried LAMliSi ↡ → ↘ = = FOI 1 No No Slow Minor Minor 5. Conclusion With new electrode configurations emerging in next-generation Li-ion batteries, it is important to provide the battery research and industry community with adaptable, in-situ diagnosis and prognosis tools that have been proved successful in previous generation materials. We sequentially presented the framework to construct an accurate mechanistic model, from a commercial representative Si-Gr/NMC811 battery. The constructed model allowed us to emulate via the ‘alawa toolbox the degradation modes, including individual degradation rates of silicon and graphite within the blended negative electrode, that the battery could experience under real-life operating conditions. Both the incremental capacity curves and the full-cell capacity losses versus the degradation modes, which are required to analyze and further decipher the underpinning degradation phenomena, were presented to attain accurate battery diagnosis and prognosis analyses. 4. Discussion The details of the simulations allowed us to postulate that three aging modes (i.e., 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Table I: Look-up table of the main aging modes of the Si-Gr/NMC811 cell during charge Aging modes Incremental Capacity Main Features (peak number) Main features to evaluate for diagnosis and prognosis ❻① ❺① ❷② ❶③ ❶④ Features of Interest (FOI) Incubation period Li- plating trigger Cap. fade rate LR evolution OFS evolution LLI ↠ ↠ ↘ ← ↑ FOI 3 No No Fast Unvaried Large LAMdeNMC ↞ ↖ ← ↓ ↓ FOI 2&3 Yes No Medium Increase Large LAMliNMC = ↘ ↘ ↘ ↙ – No No Fast Increase Unvaried LAMdeGr = ↙ ← ↑ ↡ – Yes Yes Medium Degrease Unvaried LAMliGr ↘ ↠ → ↘ = FOI 3 No No Fast Decrease Large LAMdeSi ↙ ← ← ← ↡ FOI 1 Yes Yes Slow Minor Unvaried LAMliSi ↡ → ↘ = = FOI 1 No No Slow Minor Minor Table I: Look-up table of the main aging modes of the Si-Gr/NMC811 cell during charge 5. Conclusion 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 With new electrode configurations emerging in next-generation Li-ion batteries, it is important to provide the battery research and industry community with adaptable, in-situ diagnosis and prognosis tools that have been proved successful in previous generation materials. We sequentially presented the framework to construct an accurate mechanistic model, from a commercial representative Si-Gr/NMC811 battery. The constructed model allowed us to emulate via the ‘alawa toolbox the degradation modes, including individual degradation rates of silicon and graphite within the blended negative electrode, that the battery could experience under real-life operating conditions. Both the incremental capacity curves and the full-cell capacity losses versus the degradation modes, which are required to analyze and further decipher the underpinning degradation phenomena, were presented to attain accurate battery diagnosis and prognosis analyses. 16 The details of the simulations allowed us to postulate that three aging modes (i.e., LAMdeNMC, LAMdeGr and LAMdeSi) have incubation periods. Full-cell capacity loss due to these aging modes is negligible compared to the LLI during initial cycling, but after a certain point in 16 aging will become considerable. In addition, the results show that both LAMdeGr and LAMdeSi modes may trigger thermodynamic plating in the cell, hence revealing a plausible impact of silicon on degradation in these blended electrodes. On the resulting IC curves, we highlight the importance of analyzing peak ❻① as this feature directly correlates to the influence of silicon. We set up a series of key Features of Interest (FOIs) that are sensitive to degradation and must be analyzed in detail to understand and deconvolute concurrent aging modes. As there exists a total of seven aging modes acting on four IC peaks yielding multiple possibilities of degradation, we created a look-up table that shall ease analysis of standard Si-Gr/NMC811 batteries. Due to the intrinsic nature of look-up tables, the features could be embedded on a microcontroller-based architecture. In a broader perspective, this paper aims to impart the capability and know-how to battery scientists and engineers, to facilitate the integration of degradation diagnosis and prognosis tools for battery management systems (BMS) operating this novel technology. Bernard Lestriez for the Si vs. Li data. References [1] N. Nitta, F. Wu, J.T. Lee, G. Yushin, Li-ion battery materials: present and future, Mater. Today. 18 (2015) 252–264. doi:10.1016/j.mattod.2014.10.040. [2] S. Goriparti, E. Miele, F. 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Power Sources. 440 (2019) 227117. doi:10.1016/j.jpowsour.2019.227117. 20 Table(s) - provided separately Aging modes Incremental Capacity Main Features (peak number) Main features to evaluate for diagnosis and prognosis ❻① ❺① ❷② ❶③ ❶④ Features of Interest (FOI) Incubation period Li- plating trigger Cap. fade rate LR evolution OFS evolution LLI ↠ ↠ ↘ ← ↑ FOI 3 No No Fast Unvaried Large LAMdeNMC ↞ ↖ ← ↓ ↓ FOI 2&3 Yes No Medium Increase Large LAMliNMC = ↘ ↘ ↘ ↙ – No No Fast Increase Unvaried LAMdeGr = ↙ ← ↑ ↡ – Yes Yes Medium Degrease Unvaried LAMliGr ↘ ↠ → ↘ = FOI 3 No No Fast Decrease Large LAMdeSi ↙ ← ← ← ↡ FOI 1 Yes Yes Slow Minor Unvaried LAMliSi ↡ → ↘ = = FOI 1 No No Slow Minor Minor Main features to evaluate for diagnosis and prognosis Figure(s) - provided separately Figure(s) - provided separately g ( ) p p y Click here to download Figure(s) - provided separately: Fig. References 1.docx 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Voltage (V) Capacity (Ah) NMC 811 (Harvested) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Standard Si Si-Gr (Harvested) Standard Gr 0.0 0.1 0.2 0.3 3.5 3.7 3.9 4.1 4.3 4.5 IC (mAh/V) Voltage (V) NMC 811 (Harvested) - IC 0 1000 2000 3000 4000 5000 0.0 0.2 0.4 0.6 IC (%Q/V) Voltage (V) Standard Si Si-Gr (Harvested) - IC Standard Gr - IC ① ② ③ ④ ① ② ③ ④ ❻ - ❻ - ❷ - ❺ ❶ ❶ ❷ - ❺ 7 μm a) d) b) c) e) f) 3.2 3.4 3.6 3.8 4.0 4.2 4.4 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Voltage (V) Capacity (Ah) NMC 811 (Harvested) ① ② ③ ④ a) 2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Standard Si Si-Gr (Harvested) Standard Gr ❻ - ❷ - ❺ ❶ d) ❻ Normalized capacity (%) 0 1000 2000 3000 4000 5000 0.0 0.2 0.4 0.6 IC (%Q/V) Voltage (V) Standard Si Si-Gr (Harvested) - IC Standard Gr - IC ❻ - ❶ ❷ - ❺ e) p y ( ) 0.0 0.1 0.2 0.3 3.5 3.7 3.9 4.1 4.3 4.5 IC (mAh/V) Voltage (V) NMC 811 (Harvested) - IC ① ② ③ ④ b) f) f) 7 μm ) c) Figure(s) - provided separately g ( ) p p y Click here to download Figure(s) - provided separately: Fig. References 2.docx 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Si-Gr (40%-60%) Si-Gr (30%-70%) Si-Gr (20%-80%) Si-Gr (10%-90%) Si-Gr (Harvested) Standard Gr ❻ - ❷-❺ ❶ a) 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 Voltage (V) Normalized capacity (%) Si-Gr (40%-60%) Si-Gr (30%-70%) Si-Gr (20%-80%) Si-Gr (10%-90%) Si-Gr (Harvested) Standard Gr 0 1000 2000 3000 4000 5000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 IC (%Q/V) Voltage (V) 0 100 200 300 400 500 3.0 3.2 3.4 3.6 3.8 4.0 4.2 IC (%Q/V) Voltage (V) Full-cell (Experimental) Full-cell reconstruction (Emulated) ❻ - ❷-❺ ❶ ❶ ❷-❺ ❻ - ❻ - ❻① ❶④ ❶③ ❷② ❺① a) b) c) ❺① ❻① 0 1000 2000 3000 4000 5000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 IC (%Q/V) Voltage (V) ❶ ❷-❺ ❻ - ❻ - b) ❻ 0 100 200 300 400 500 3.0 3.2 3.4 3.6 3.8 4.0 4.2 IC (%Q/V) Voltage (V) Full-cell (Experimental) Full-cell reconstruction (Emulated) ❻① ❶④ ❶③ ❷② ❺① c) ❺① ❻① Figure(s) - provided separately Figure and Table Caption(s) - provided separately Click here to download Figure and Table Caption(s) - provided separately: Figure and Table Captions.docx Figure and Table Caption(s) - provided separately Click here to download Figure and Table Caption(s) - provided separately: Figure and Table Captions.docx Fig. 1. (a) Voltage profile of harvested NMC811 vs. Li and (b) corresponding IC curves. (d) and (e) show the voltage and IC signatures for half-cell harvested Si-Gr (black), standard Gr (red) and porous Si (gold). All were cycled vs. Li. (c) and (f) show at high magnification SEM images of the fresh Si-Gr anode, where the bright grain is silicon (f) Figure and Table Caption(s) - provided separately Click here to download Figure and Table Caption(s) - provided separately: Figure and Table Captions.docx Fig. 1. (a) Voltage profile of harvested NMC811 vs. Li and (b) corresponding IC curves. (d) and (e) show the voltage and IC signatures for half-cell harvested Si-Gr (black), standard Gr (red) and porous Si (gold). All were cycled vs. Li. (c) and (f) show at high magnification SEM images of the fresh Si-Gr anode, where the bright grain is silicon (f) Fig. 1. (a) Voltage profile of harvested NMC811 vs. Li and (b) corresponding IC curves. Supplementary Materials Supplementary Materials Click here to download Supplementary Materials: Supplementary Material - Revised.docx References (d) and (e) show the voltage and IC signatures for half-cell harvested Si-Gr (black), standard Gr (red) and porous Si (gold). All were cycled vs. Li. (c) and (f) show at high magnification SEM images of the fresh Si-Gr anode, where the bright grain is silicon (f) Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results Fig. 2. (a) Voltage profile and (b) its corresponding IC curves, for standard Gr (red), harvested Si-Gr (black), and emulated Si-Gr (gray) with Si capacity contribution varied from 0% to 40%. Inset image in (b) shows the significant differences of the emulated blended (gray) on voltage peak ❻. (c) Compares the IC curves at C/25 for experimental full cell data (black) and for the emulated reconstructed full cell (green, markers), showing on inset figure details of the kinetic adjustments results Fig. 3. Degradation map for the Si-Gr/NMC811 commercial cell with peak indexation and proposed FOIs. The thick lines are the initial emulated signatures, the thin lines are the emulated signatures at 10% capacity loss, and the dashed lines are the signatures at 2% intervals in between those two cases Table I: Look-up table of the main aging modes of the Si-Gr/NMC811 cell during charge
https://openalex.org/W4381951687	https://ojs.wiserpub.com/index.php/JEEE/article/download/2835/1426	English	null	DC and RF Performance of an N-channel Monolayer Black Phosphorus Nanoribbon Transistor	Journal of electronics and electrical engineering	2,023	cc-by	7,104	Received: 13 April 2023; Revised: 14 May 2023; Accepted: 22 May 2023 Received: 13 April 2023; Revised: 14 May 2023; Accepted: 22 May 2023 Abstract: Two-dimensional black phosphorus is a relatively new discovery. There are numerous studies on black phosphorus two-dimensional transistors that focus on analog and RF performance. However, the RF performance of black phosphorus nanoribbon transistors is yet to be explored. We use a four-band tight binding Hamiltonian in conjunction with a non-equilibrium Green’s function quantum transport simulator to investigate both the DC and RF performance of a monolayer black phosphorus nanoribbon transistor. We found that electron intra-band tunneling is responsible for current flow in the off-state, while in the on-state, the electrons flow over the top of the channel barrier potential. With a VDD of 0.4 volt and a gate length of 5 nm, our black phosphorus nanoribbon transistor has DC performance metrics of 510 µA/µm on-state current, 105 on/off current ratio, and 65 mV/dec inverse subthreshold slope. The device’s RF performance characteristics are as follows: cut-off (unity current gain) frequency of 772.84 GHz, maximum oscillation (unity power gain) frequency of 1.15 THz, and open circuit voltage gain of 26.7 dB with transistor operating in the on-state. The RF performance of the device is found to be significantly impacted by the source and drain contact resistances. With source and drain resistances set to zero, the cut-off frequency increases to 995.23 GHz and the unity power gain frequency increases to 4.16 THz. The device shows unconditional stability above 893 GHz and it is conditionally stable below this frequency. eywords: DC performance, RF performance, black phosphorus, nanoribbon transistor, NEGF, tight bindin Journal of Electronics and Electrical Engineering https://ojs.wiserpub.com/index.php/JEEE/ Khairul Alam Khairul Alam Department of Electrical and Electronic Engineering, East West University, Dhaka, Bangladesh E-mail: kalam@ewubd.edu 1. Introduction Due to their unique electrical, mechanical, and thermal properties, two-dimensional (2D) materials have garnered considerable interest in the scientific community [1–4]. Even at the short channel limits, the 2D field- effect transistors (FETs) show great mobility, high on/off current ratio, and minimal leakage [5–9]. Graphene [5,8,9] and transition metal dichalcogenides (TMDs) [7] are two of the most investigated materials on the extensive list of 2D candidates. However, graphene has a zero band gap, and the poor mobility of TMDs restricts their usefulness [10]. Black phosphorus (BP) is a new and promising addition to the list. Its puckered shape and layer-dependent direct band gap, which varies from ~0.35 eV (bulk) to ~1.85 eV (monolayer) [11], make it an interesting material to study. This material also possesses notable in-plane anisotropic properties [12,13]. Because of its high mobility, controlled band gap, and anisotropic band structure, BP is an excellent option for use in electronic as well as optoelectronic applications [12]. On BP 2D FETs, a significant amount of research has been done [14–21]. It has been said that the performance of BP transistors can be higher than that of their MoS2 equivalents [16]. Applications of BP as radio-frequency (RF) transistors have also been investigated alongside nanotransistors [19,22–28]. Theoretically estimated high RF figures of merit have a unity 86 \| Khairul Alam. 86 \| Khairul Alam. Journal of Electronics and Electrical Engineering power gain frequency of fmax = 950 GHz [27]. Experiments have shown that a device with a channel length of 300 nm can achieve a fmax of 20 GHz [22]. Despite substantial research on 2D BP FETs, one-dimensional (1D) black phosphorus nanoribbons (NRs) and nanotubes (NTs) have received less attention [29–34]. Both experiments and theoretical first-principles simulations have been used to investigate the structural [29] and thermal [35,36] stability, band gap fluctuation [37], and characterization [38] of 1D BP NRs. Feng et al. [30] experimentally showed complementary BP NR top gate FETs. According to their findings, the BP NR 1D FETs outperform the BP 2D FETs by an order of magnitude in terms of on-state current, five times in terms of subthreshold slope, and four times in terms of peak transconductance. However, we realize that the RF performance of BP 1D FETs is yet to be explored. 1. Introduction This study uses an in-house developed quantum transport code and a four-band tight binding Hamiltonian to theoretically investigate the DC and RF performance of a monolayer 5 nm gate length BP NR transistor. The transistor exhibits a high on/off current ratio with an inverse subthreshold slope of 65 mV/dec when operated at a DC bias. In the off-state, electron intra-band tunneling regulates the current flow, and in the on-state, thermionic emission over the channel potential barrier does the same. An equivalent circuit is utilized in order to assess the radio-frequency performance. The parameters of the circuit are established by the use of numerical simulation. The RF performance parameters calculated are as follows: a unity current gain frequency of 772.84 GHz, a unity power gain frequency of 1.15 THz, and an open circuit voltage gain of 26.7 dB. Journal of Electronics and Electrical Engineering 2. Device Structure and Simulation Approach With gate length LG = 5 nm, effective oxide thickness EOT = 0.45 nm, and VDD = 0.4 V, which are near the specifications of 2027 low-power logic [39], we simulate a double-gate monolayer BP NR 1D FET. tox = 2.15 nm and ox = 19 represent the physical oxide thickness and dielectric constant, respectively, that we employ in our simulation. Figure 1 depicts the cross-section of the device. The lengths of the source and drain extensions, LS and LD, are both set to 10 nm. Both the source and the drain are doped with a donor concentration of ND = 1.25 × 1013 cm-2, making the channel intrinsic. In order to align the source conduction band with the source Fermi level, we selected this doping density. Figure 1. (a) Monolayer BP nanoribbon. x and y axes represent the armchair and the zigzag directions, respectively. The red rectangle shows the 4 atoms unit cell, and the vertical blue lines show the nanoribbon unit cell used in recursive Green’s function calculation. (b) The two-dimensional cross-section of x −z plane of the BP NR 1D FET used in this study. Figure 1. (a) Monolayer BP nanoribbon. x and y axes represent the armchair and the zigzag directions, respectively. The red rectangle shows the 4 atoms unit cell, and the vertical blue lines show the nanoribbon unit cell used in recursive Green’s function calculation. (b) The two-dimensional cross-section of x −z plane of the BP NR 1D FET used in this study. A four-band tight binding Hamiltonian is used to model BP [11] A four-band tight binding Hamiltonian is used to model BP [11]     ij i j i j H t c c (1)     ij i j i j H t c c (1) (1) me 2 Issue 1\|2023\| 87 Journal of Electronics and Electrical En Volume 2 Issue 1\|2023\| 87 In this equation, i and j refer to the lattice sites,  ijt is the in-layer hopping parameter between sites i and ,j and  ic and jc are the operators for creating and destroying electrons at sites i and ,j respectively. Note that the on-site energies have been set to zero. The model has been validated against the ab initio self-consistent 0 GW results [11]. The red rectangle in Figure 1(a) represents the four atoms that make up the BP monolayer’s unit cell. 2. Device Structure and Simulation Approach Our recursive Green’s function algorithm [34,40,41] uses nanoribbon unit cell depicted by the blue vertical lines in Figure 1(a). The layer (or unit cell) Hamiltonian Lh and the layer-to-layer (or unit cell-to-unit cell) coupling matrices , 1  L L h and , 1  L L h have sizes that are 4 times the number of unit cells along the y (zigzag) direction. Lh and , 1  L L h can be written as [42] † † † † † 0 0 0 0 0 0                                     u uW uWF uW u uW uWF L uWF uW u uW uWF uW u h (2) , 1 0 0 0 0 0 , 0 0 0 0 0 0                                 uL uLF uLB uL uLF L L uLB uL uLF uLB uL h (3) (2) (3) and † , 1 , 1.    L L L L h h The block matrices , u , uW , uWF , uL uLF and uLB are 4 × 4 matrices, and are provided in Ref. [42]’s Eqs. (2–5). There in Table 1, the hopping parameters are also provided. and † , 1 , 1.    L L L L h h The block matrices , u , uW , uWF , uL uLF and uLB are 4 × 4 matrices, and are provided in Ref. [42]’s Eqs. (2–5). There in Table 1, the hopping parameters are also provided. We have the required three matrices, , Lh , 1  L L h and , 1  L L h in hand. We can now apply the recursive Green’s function approach to determine the electron density from the diagonal portions of the source and drain spectral functions [34,40,41], , , 1 ( , , ) ( , , , ) ( , ) ( , , , ) ( , ) . 2. Device Structure and Simulation Approach 2            S D L L L S L L D dE n x y z diag A E x y z f E A E x y z f E (4) (4) (4) In this equation, ,  f and S stand for grid volume, Fermi distribution function, and Fermi energy levels at the source and drain, respectively. The spectral functions are given by † , ,1 1,1 1, ,   S L L L L A G G and   † , , , , ,    D S L L L L L L L L A i G G A where the broadening function is supplied by   † 1,1 1,1 1,1     i and the decimation method [43,44] is used to get the boundary self-energy 1,1 1,0 0,0 0,1.  h g h The required blocks of full Green’s function are determined by sequentially solving the following equations.     1 , , 1 1, 1 1, 1 1,1 1 1 1,1 1,2 2,2 2,1 , , , , 1 1, 1 1, , ,1 , , 1 1,1                         L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L g E h U h g h G E h U h g h G g g h G h g G g h G (5) (5) ,1 , , 1 1,1    L L L L L L G g h G Poisson’s solver is used to calculate the layer’s potential energy, . L U The recursive calculation starts from the right contact with  L N and , , 1 1, 1 1, .       N N N N N N N N h g h Potential profile is obtained by solving a three-dimensional Poisson’s equation in Cartesian coordinates . Journal of Electronics and Electrical Engineering 2. Device Structure and Simulation Approach The intrinsic component of the device potential is updated using the computed current in the following way:   i GS GS D S V V I R and  .    i DS DS D S D V V I R R These newly calculated voltages serve as the boundary condition when Poisson’s equation is solved using the data from the most recent computation of charge density. The loop remains active until convergence is reached. Anderson mixing [48] is used to hasten the convergence. After the loop has reached convergence, the results are saved, and then we move on to calculating the next bias value. The simulation software is developed internally using open-source Julia programming language [49]. Journal of Electronics and Electrical Engineering 2. Device Structure and Simulation Approach                                      V V V x x y y z z (6) (6) ronics and Electrical Engineering 88 \| Khairul Alam. g 88 \| Khairul Alam. 88 \| Khairul Alam. The potential profile and location dependent dielectric constant are represented by ( )  V U qV and , respectively. ( )   D N n is the charge density. To solve the Poisson’s equation, a finite difference discretization with Newton-Rapshon approach is utilized. At the gate metal, potential is maintained constant, while all other boundaries have their normal electric field component set to zero. We use a contact resistance value of 130 m     S D R R [34]. Experimentally, it has been possible to attain a contact resistance value for BP FETs of 135 m   with a Ni contact [45] and 310 m   with a Ti contact [46]. Theoretically, a ceiling of 14 m   has been expected [47]. The contact resistance used in this work is thus not too far from becoming achievable. The first step in the self-consistent loop is to make an educated guess about the potential profile. After that, we compute the charge density, Eq. 4, as well as the current [40,41]    † 1,1 1,1 1,1 1,1 1,1 tr A . 2            D S D q dE I G G f f (7) (7) The intrinsic component of the device potential is updated using the computed current in the following way:   i GS GS D S V V I R and  .    i DS DS D S D V V I R R These newly calculated voltages serve as the boundary condition when Poisson’s equation is solved using the data from the most recent computation of charge density. The loop remains active until convergence is reached. Anderson mixing [48] is used to hasten the convergence. After the loop has reached convergence, the results are saved, and then we move on to calculating the next bias value. The simulation software is developed internally using open-source Julia programming language [49]. Volume 2 Issue 1\|2023\| 89 3. Simulation Results and Discussions Figure 2 depicts the E-k dispersions of monolayer BP nanoribbon with a width of W = 2.3 nm that we used in our simulation. The energy reference is located in the band gap’s midpoint. Seven 4-atom unit cells (Figure 1), or 28 atoms, make up the nanoribbon unit cell. The transport route is in the armchair direction (x in Fig. 1), whereas the quantization direction (nanoribbon width) is along the zigzag direction (y in Figure 1). The nanoribbon has a 1.918 eV direct band gap. The top valence subbands merge as a result of a significantly heavier hole in the quantization (zigzag) direction [18], but the bottom conduction subbands are closely spaced. Figure 2. A few bands of monolayer BP NR. The width of the nanoribbon W = 2.3 nm. kx is along the ribbon armchair direction. Energy is zero at the center of the band gap. Figure 2. A few bands of monolayer BP NR. The width of the nanoribbon W = 2.3 nm. kx is along the ribbon armchair direction. Energy is zero at the center of the band gap. Figure 3 displays the simulated  I V characteristics of an n-channel double gate nanoribbon field-effect transistor. The  D D I V characteristic is similar to that of a standard MOSFET. In order to get the  D G I V characteristics, we began by setting the drain bias at 0.4 volts and then sweep the gate bias across a broad range. The off-state current is then adjusted by shifting the  D G I V characteristics to a value of 3 5 10 A / m     when Volume 2 Issue 1\|2023\| 89 Journal of Electronics and Electrical Engineering the gate bias is at zero. The calculated current is in A.  Therefore, in order to obtain the current measured in A / m   , we divided it by the width of the nanoribbon. The device features a 65 mV / dec subthreshold slope, a 510 A / m   on-state current, and a 1.02×105 on/off current ratio. Experiments have shown that an optimized few-layer BP 2D FET doped with lithium has a high on-state current of 773 A / m   and an on/off current ratio of 7.2 × 105 [17]. Journal of Electronics and Electrical Engineering 3. Simulation Results and Discussions It is important to note that these values were determined by conducting measurements at a low temperature of 200 K for a device that had a long channel and a gate bias of −4 volts. Figure 3. (a) nFET ID - VG characteristics at VD = 0.4 V. VG = 0 represents the off-state, while VG = 0.4 volt represents the on-state. (b) ID - VD characteristics at VG = 0.4 volt. Figure 3. (a) nFET ID - VG characteristics at VD = 0.4 V. VG = 0 represents the off-state, while VG = 0.4 volt represents the on-state. (b) ID - VD characteristics at VG = 0.4 volt. In order to acquire a better understanding of electron transport, we depict the energy profile of current, denoted by JE, overlaid on the conduction band profile in Figure 4(a) at a gate bias of 0.31 volt. The Fermi level at the source is used as the zero energy reference. The dashed line indicates that the top of the conduction band is located at 0.063 eV. This value represents the barrier energy for electrons that are injected from the source contact. Any injected electrons with energies lower than this barrier energy will be able to “tunnel” through it and reach the drain terminal. This tunneling current can be determined by integrating JE from −0.4 to 0.063 eV (barrier top), which accounts for 49.5% of the total current when the gate bias is 0.31 volt. In the remaining 50.5% of the total current, known as the thermal current, electrons pass over the barrier top. The current is broken down into its tunneling component and its thermal component across the entire voltage range in Figure 4(b). Clearly, electron tunneling dominates in low bias, whereas thermionic emission predominates at higher biases. Current in the off-state is 83% tunneling, while current in the on-state is 74% thermionic. 90 \| Khairul Alam. Journal of Electronics and Electrical Engineering Figure 4. Current energy density, labeled by JE, is plotted over the conduction band profile, labeled by EC, at VG = 0.31 volt. The conduction band top located at 0.063 eV is depicted by a dashed horizontal line. Source Fermi level is set to have zero energy. Figure 4. Current energy density, labeled by JE, is plotted over the conduction band profile, labeled by EC, at VG = 0.31 volt. Journal of Electronics and Electrical Engineering 3. Simulation Results and Discussions The conduction band top located at 0.063 eV is depicted by a dashed horizontal line. Source Fermi level is set to have zero energy. After having determined the DC figures of merit (on-state current, inverse subthreshold slope, and on-off current ratio) and having gained an understanding of the mechanism underlying electron transport, we moved on to evaluating the device’s RF performance parameters. In order to accomplish this, we will be looking at the small signal equivalent circuit that is depicted in Figure 5. The following is how we determine the transconductance, denoted by m g , and the output conductance, denoted by 0. g Figure 5. Equivalent small signal circuit of the simulated NR FET for RF performance analysis. Figure 5. Equivalent small signal circuit of the simulated NR FET for RF performance analysis. Figure 5. Equivalent small signal circuit of the simulated NR FET for RF performance analysis. Journal of Electronics and Electrical Engineering Volume 2 Issue 1\|2023\| 91 0     D m DS GS D GS DS I g V V I g V V (8) (8) For m g calculation, we set DS V to 0.4 volt and sweep GS V from 0 to 0.4 volt. Similarly, GS V is set to 0.4 volt and DS V is swept from 0 to 0.4 volt to calculate 0. g The computed conductances are shown in Figure 6. Transconductance m g is a figure of merit that determines the amplification delivered by a transistor. It increases with gate bias, and its value in on-state is 6.7 mS/ m.  In linear region 0 g is large due to uniform channel, and it decreases with drain bias due to channel length modulation. In on-state 0 0.31 mS/ m,   g that is, the output resistance is 0 0 1/ 3.2 / m.     r g k Figure 6. (a) Transconductance gm vs VG at VD = 0.4 volt. (b) Output conductance g0 vs VD at VG = 0.4 volt. Figure 6. (a) Transconductance gm vs VG at VD = 0.4 volt. (b) Output conductance g0 vs VD at VG = 0.4 volt. The capacitances from the gate to the source and from the gate to the drain can be estimated as follows 2 2 2 . Journal of Electronics and Electrical Engineering 92 \| Khairul Alam. 3. Simulation Results and Discussions 2     ox qs gs ox qs ox qd gd ox qd C C C C C C C C C C (9) (9) /   ox ox ox C t represents the oxide capacitance per unit area, and the factor 2 compensates for two identical gates [50]. The quantum capacitances, qs C and , qd C are computed as       ch qs s ch qd s D Q C Q C V (10) (10) onics and Electrical Engineering 92 \| Khairul Alam. 92 \| Khairul Alam. where ch Q refers to the channel charge and s refers to the surface potential. Figure 7 shows the computed capacitances, gs C and . gd C These capacitances play crucial roles for analyzing RF performance, such as the cutoff frequency, Tf and the maximum oscillation frequency, max. f Figure 7. (a) Gate to source capacitance Cgs vs VG at VD = 0.4 volt. (b) Gate to drain capacitance Cgd vs VD at VG = 0.4 volt. Figure 7. (a) Gate to source capacitance Cgs vs VG at VD = 0.4 volt. (b) Gate to drain capacitance Cgd vs VD at VG = 0.4 volt. To determine the RF performance metrics, we first derive the admittance matrix of the intrinsic part of the equivalent circuit, Figure 5, as follows   1 1 1 0 2 2 2 ( ) .                                 gs gd gd m gd gd jw C C jwC i v v Y g jwC g jwC i v v (11) (11) Now the voltage relation between extrinsic part (including , g R s R and d R ) and intrinsic part can be written Now the voltage relation between extrinsic part (including , g R s R and d R ) and intrinsic part can be written s Now the voltage relation between extrinsic part (including , g R s R and d R ) and intrinsic part can be written as as   1 1 1 2 2 2 1 1 1 2 2 . 3. Simulation Results and Discussions                                            s g s s s d s g s s s d R R R v v i R R R v v i R R R i i Y R R R i i (12) (12) That is, the admittance matrix of the full equivalent circuit is That is, the admittance matrix of the full equivalent circuit is is, the admittance matrix of the full equivalent circuit is   1 1                    s g s s s d R R R Y Y R R R (13) (13) From the admittance matrix, we can calculate the short circuit current gain, 21 11 / ,  iA Y Y open circuit voltage gain, 21 22 / ,  vA Y Y and the unilateral power gain [51] as From the admittance matrix, we can calculate the short circuit current gain, 21 11 / ,  iA Y Y open circuit voltage gain, 21 22 / ,  vA Y Y and the unilateral power gain [51] as   2 21 12 11 22 12 21 . 4 Re( )Re( ) Re( )Re( )    Y Y U Y Y Y Y (14) (14) Volume 2 Issue 1\|2023\| 93 The voltage gain at DC can be written as 0 ( 0) /   v m A f g g [52]. After substitution of m g and 0 g in on- state we get (0) 26.7 dB.  vA Figure 8 depicts the on-state short circuit current gain and the on-state unilateral power gain. In the calculations, we use 65 m     s d R R [34] and 4.4 m    g R [51,53]. The plot allows us to determine that the cut-off frequency, also known as the unity current gain frequency (0 dB), is equal to 772.84 GHz.  Tf In a similar vein, the frequency at which the power gain is equal to one, also known as the highest oscillation frequency, is equal to max 1.12 THz. 3. Simulation Results and Discussions  f For the nanoribbon FET, the source and drain contact resistances have significant effects on Tf and max. f If we set the contact resistances, s R and d R , to zero then Tf can be analytically derived as [52]. Figure 8. Plots of unilateral power gain and short circuit current gain against frequency in the on-state. Figure 8. Plots of unilateral power gain and short circuit current gain against frequency in the on-state. 2 , 2 2    m T gs gs gd g f C C C (15) (15) and its value becomes 995.23 GHz.  Tf Under this condition, max f can be analytically approximated as [25,53] max 0 , 2 2   T g T gd g f f g R f C R (16) (16) and its value becomes 4.16 THz. Clearly, the contact resistances have significant effects on Tf and max , f of which, max f is primarily affected. In our NR FET, we see that max .  T f f That means the current is saturated with increasing drain bias and this results in smaller 0. g The opposite relation is confirmed in graphene FET [54], that is, max  T f f due to semimetalic nature of graphene. So, BP NR FET is an attractive candidate for high frequency electronics. and its value becomes 4.16 THz. Clearly, the contact resistances have significant effects on Tf and max , f of which, max f is primarily affected. In our NR FET, we see that max .  T f f That means the current is saturated with increasing drain bias and this results in smaller 0. g The opposite relation is confirmed in graphene FET [54], that is, max  T f f due to semimetalic nature of graphene. So, BP NR FET is an attractive candidate for high frequency electronics. Finally, we look into the stability issue of our nanoribbon FET. For this purpose, we calculate the stability factor K as follows [51] 11 22 12 21 12 21 2Re( )Re( ) Re( ) .   Y Y Y Y K Y Y (17) (17) since the components of the admittance matrix are a function of frequency, so is the stability factor. Figure 9 is a frequency-dependent plot of the stability factor. Journal of Electronics and Electrical Engineering 4. Conclusion To summarize our findings, we used a four-band tight binding Hamiltonian and a non-equilibrium Green’s function quantum transport code to investigate the transport mechanism and DC performance of a monolayer black phosphorus nanoribbon transistor. When in the off-state, electron transport is controlled by the intra-band tunneling, and when in the on-state, it is controlled by thermionic emission over the channel potential barrier. The device exhibits a current density of 510 µA/µm in its on state and a slope of 65 mV/dec in its subthreshold region, with an on/off current ratio of 105. Next, we use the admittance matrix of a two-port network with environmental influences to assess the device’s RF performance. The gate-source terminals serve as the input port, while the drain-source terminals serve as the output port. With a maximum oscillation frequency of 1.15 THz, a cut-off frequency of 772.84 GHz, and an open-circuit voltage gain of 26.7 dB, this device boasts excellent RF performance metrics. The device is completely stable above 893 GHz and partially stable below that frequency. 3. Simulation Results and Discussions Stability ensures that passive loads connected to the input or output ports cannot accidentally induce oscillations in the network. To accomplish this, K must be greater than 1. The network is said to be conditionally stable if the value of K falls within the range −1< K<1, which means that it is stable when the source and load impedances are set up in certain ways. A K value below −1 makes the network unstable. From the results we see that the device is unconditionally stable above 893 GHz. That is, the Journal of Electronics and Electrical Engineering 94 \| Khairul Alam. 94 \| Khairul Alam. system will not show any oscillation above 893 GHz for any passive load at the input or output ports. Below 893 GHz operation, the network shows stable behavior for certain combinations of passive loads. Figure 9. Stability factor K vs. frequency plot in on-state. Figure 9. Stability factor K vs. frequency plot in on-state. Figure 9. Stability factor K vs. frequency plot in on-state. Conflict of Interest There is no conflict of interest for this study. Journal of Electronics and Electrical Engineering References [10] Li, W.; Zhang, G.; Guo, M.; Zhang, Y.-W. Strain-tunable electronic and transport properties of MoS2 nanotubes. Nano Res. 2014, 7, 518–527, https://doi.org/10.1007/s12274-014-0418-y. [11] Rudenko, A.N.; Yuan, S.; Katsnelson, M.I. Toward a realistic description of multilayer black phosphorus: From GW approximation to large-scale tight-binding simulations. Phys. Rev. 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https://openalex.org/W3096646307	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0238731&type=printable	English	null	The interdependency structure in the Mexican stock exchange: A network approach	PloS one	2,020	cc-by	15,172	Introduction In this paper we investigate the interdependency structure of daily returns in the Mexican stock exchange market. To this end, we build a database of free and publicly available time series of main stocks for the period 2000-2019 and conduct our study in stages that are then put together to give a unified treatment to our main topic of interest here which is the interde- pendency structure of daily log-returns in the Mexican stock exchange. Editor: Ning Cai, Beijing University of Posts and Telecommunications, CHINA Editor: Ning Cai, Beijing University of Posts and Telecommunications, CHINA In the first stage we focus on the estimation of partial correlations of log returns of daily prices. The reasons for focusing on partial correlations are the following. Given a collection of Gaussian series A1, . . ., An, a zero partial correlation between A1 and A2 implies that A1 and A2 are conditionally independent, meaning that A1 and A2 could still be (unconditionally) corre- lated but only through a third factor adapted to the other series A3, . . ., An. There are of course different methods of estimating a covariance/correlation/concentration matrix and we have selected a estimation based on a specific class of Markovian Random Fields (MRF) which in the statistical literature is well known under the name Gaussian Graphical models (GGm). The adjective “graphical” emphasizes the fact that attached to the probabilistic model there is a graph in which edges express conditional dependencies, from which a very convenient visual representation is obtained. There are three reasons for working with this model. First of all, the benefit of the already mentioned visual representation provided by the model. The second is that we have decided to study the period 2000-2019 on a yearly basis. There is a trade-off to this treatment. On the one hand, short periods of time reduce problems with heavy tails. On the other, the number of stocks in each year is a significant proportion of the available observa- tions. Hence, a lasso-regularized estimation is useful in this context which is inbuilt in the esti- mation of a GGm. Third, we want an estimation that filters out a “noisy” correlation selecting only clear relationships between two series, again this is provided by the lasso-regularized Copyright: © 2020 Erick Treviño Aguilar. PLOS ONE RESEARCH ARTICLE Erick Treviño AguilarID* Unidad Cuernavaca del Instituto de Matema´ticas, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Mexico * erick.trevino@im.unam.mx OPEN ACCESS Citation: Treviño Aguilar E (2020) The interdependency structure in the Mexican stock exchange: A network approach. PLoS ONE 15(10): e0238731. https://doi.org/10.1371/journal. pone.0238731 Abstract Our goal in this paper is to study and characterize the interdependency structure of the Mex- ican Stock Exchange (mainly stocks from Bolsa Mexicana de Valores) for the period 2000- 2019 which provide a one shot big-picture panorama. To this end, we estimate correlation/ concentration matrices from different models and then compute centralities and modularity from network theory. * erick.trevino@im.unam.mx PLOS ONE PLOS ONE Introduction This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The data underlying the results presented in the study is freely available from Yahoo.Finance (https://finance.yahoo.com/). It can be directly taken from that source. The authors confirm they had no special access privileges to the data others would not have. Funding: The author received no specific funding for this work. Competing interests: No competing interests. Competing interests: No competing interests. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 1 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange estimation. Loosely speaking, we follow a partial correlations selection approach which con- ceptually is comparable to a covariance selection approach [1]. Once partial correlations matri- ces have been estimated we provide a list of stylized facts from them. Then, taking the graphs constructed from the matrix of partial correlations as its adjacency matrix, we compute eigen-, between-, and degree- centralities. In a second stage we compute networks based on matrices of Tail-dependence coefficients ([2]) of every pair of log returns. This coefficient quantifies the relationship of lower tails and captures dependencies in the events of negative returns. In the third stage we estimate correlation matrices of time series (estimated through a Mul- tivariate Dynamic Conditional Correlation GARCH specification). Then, we apply a technique from network-theory based on those correlation matrices known as the maximization of a modularity objective function. This procedure will provide a community structure. As part of our main goal of studying the interdependency structure of the Mexican stock exchange we contribute to the existing literature on financial networks concerning the follow- ing aspects. First of all, many papers focus on financial networks constructed from Pearson correlation matrices but much less papers focus on financial networks constructed from partial correlations and/or Tail-dependence matrices, as we do here. Moreover, from the few papers focusing on partial correlation matrices, we are not aware of any of them applying the Gauss- ian Graphical model we consider in this paper. As a consequence no paper has previously compared network-structures from the three afore-mentioned different matrices (Pearson correlation, partial correlations and Tail-dependence) as we do in the present paper. The premise is that different underlying matrices yield different network-topologies. Introduction Many papers study aggregated financial indices and do not go into the details of analyzing at the level of stocks in the selected market, hence missing the point of analyzing interdepen- dency at the level of individual stocks, where the network perspective could represent an advantage to support financial decisions; see e.g., [3, pp. 8, inmediately before the section “Fac- tor models”]. For example, we find that the main index in the Mexican stock exchange denoted IPC (not to be confused with the Index of Consumer Price level.) is “influential” with respect to degree- and eigen- centrality but the intensity varies with respect to which matrix the net- work is based on. However, it is not influential with respect to betweenness-centrality. Thus, the index does not convey all the information in the market; compare e.g., again with [3, pp. 10, first paragraph]. Few papers focus on the case of Mexico, a representative market in the region which some studies have found to be a connecting node between Latin American and US markets, hence playing a key role; see [4]. For example, [5] is an early paper studying stock market integration between Latin American countries and the US. This includes Mexico, but only as part of the region with no particular focus. Background The classical Markowitz theory of portfolio selection illustrates the relevance of asset correla- tion matrices for financial decisions. However, the nontriviality of correlation estimation from empirical data has been known for a long time, see e.g., [6]. Moreover, in contexts where sparse correlation (specially for partial correlation) matrices are expected, it is desirable to have a systematic method to discard “non-clear correlations” and account for a parsimonious model as motivated by [1]. As we mentioned in the introduction, in this paper we choose to apply a GGm for a parsimonious estimation of concentration/partial-correlation matrices. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 2 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Estimation of Tail-dependence coefficients are based on the non-parametric estimator in [7]. Pearson-correlation matrices are estimated from a multivariate GARCH model. Estimation of Tail-dependence coefficients are based on the non-parametric estimator in [7]. Pearson-correlation matrices are estimated from a multivariate GARCH model. Beyond the estimation problem, it is useful to have tools that, starting from matrices, are able to generate metrics providing snapshots of the market from which quick but trustable diagnosis are available. Situations in which this is desirable include, from the point of view of an investor, the decision to rebalance a portfolio, and from the point of view of a regulator, interventions in the market in order to lessen the contagion of a shock in a specific sector. We find those tools in the theory of random graphs, specially in the form of metrics (in this paper, degree-, eigen- and betweenness- centralities) which classify the interconnectedness of stocks and a global metric (the modularity computed from correlation matrices) to detect com- munities of stocks. The approach described integrates into the outline of [8] and is a very active research area; see e.g., the survey in [9]. However for the Mexican stock exchange there has been little research in this direction. In the next section we present related literature. Note however that we do not pretend to provide an exhaustive revision of this active topic which deserves a survey by its own, but to give a brief panorama of current research in this area. Stock markets from GGm, random graphs, and network-theory approaches Gaussian graphical models, Random graphs, and Network theory approaches in a financial context is an active research area attracting more and more attention with an increasing num- ber of papers; see e.g., the survey in [9]. The following is a non exhaustive list merely describing different approaches and applications. Papers in finance reporting an approach related to a graphical model include [10, 11] and [12]. However, none of these papers focus on asset prices. Theoretical background on graphi- cal models can be found in [13, 14] and [15]. Papers studying financial networks based on partial correlations include [4, 16, 17] and [3]. Papers with applications based on a network approach in a financial context include (a) spill- over effects and shocks contagion, [17–20] and [21] (b) portfolio selection, [22, 23] and [24] (c) detection of stock prices manipulation [25] and (d) portfolio diversification [26]. Papers studying financial networks based on the distributions’ tails of prices/log-returns include [22, 27–30] and [31]. Some studies determine power laws for degree connectedness defined by assets correlation matrices; see [16, 26, 32–34] and [35]. Papers studying community detection in a financial context include [16, 36–38] and [39]. See [40] for a survey on methods for community detection. Minimal Spanning Trees applied to financial market ranking include [4, 35, 41, 42] and [43]. Random matrix theory for correlation matrices has been presented in [17, 36, 44] and [45]. Subprime crisis According to [46] there indeed existed an impact from the 2007-2008 subprime crisis on the Mexican economy. Mainly due to two shocks, first, a decline in Mexico’s exports and second, a constrained access to international financial markets, thus evidencing an integration of the Mexican stock exchange with the US market. A phenomenon documented by some authors; see e.g., [47–49]. Fig 1 illustrates price levels for the main IPC index in the Mexican stock mar- ket for the years 2006, 2007 and 2008. It can be argued the presence of a bullish market on 2006 while on the second semester of 2008 the market turned bearish. Not surprising and reported by some authors [50] and [51]. Later we will go beyond a visual examination and PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 3 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 1. IPC index. Time series for the period 2006-2008. https://doi org/10 1371/journal pone 0238731 g001 confirm by a multivariate GARCH model through a shift from positive to negative intercepts on log returns of each time series of the period. However, quite interesting, we will show that the partial-correlations interdependency structure of the Mexican financial market does not exhibit a drastic change as consequence to that shock, see Fig 3. Fig 1. IPC index. Time series for the period 2006-2008. https://doi.org/10.1371/journal.pone.0238731.g001 Fig 1. IPC index. Time series for the period 2006-2008. https://doi.org/10.1371/journal.pone.0238731.g001 https://doi.org/10.1371/journal.pone.0238731.g001 confirm by a multivariate GARCH model through a shift from positive to negative intercepts on log returns of each time series of the period. However, quite interesting, we will show that the partial-correlations interdependency structure of the Mexican financial market does not exhibit a drastic change as consequence to that shock, see Fig 3. confirm by a multivariate GARCH model through a shift from positive to negative intercepts on log returns of each time series of the period. However, quite interesting, we will show that the partial-correlations interdependency structure of the Mexican financial market does not exhibit a drastic change as consequence to that shock, see Fig 3. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Data We constructed a database of daily closing prices from free and publicly available information at Yahoo.Finance website which we downloaded through the R package quantmod. The com- plete list of analyzed stocks can be found in the S1 Table. The frequency of data is daily in a span of time comprising 01-01-2000 to 31-12-2019. We have considered throughout the paper time series of log returns: RtðiÞ ¼ log Stþ1ðiÞ StðiÞ where St(i) is the price level at time t of stock labeled i. Data is organized in windows of one year (from january to december) and we applied a fil- tering process in two steps. In the first step, for each year, stocks in the market with the most complete information were selected. The criterion was that only stocks with more than 90% of all the available dates were selected. Then, in a second step, stock prices not having a minimum of variance in moving windows spanning 30 dates were discarded. This filtering process already presents the interesting fact of a positive evolution of the Mexican stock exchange in PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 4 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Table 1. Industrial sectors. Sector No. Stocks Description 1 Basic consumming 21 Manufacturers and distributors, food and beverage companies 2 Energy 2 Energy producers, equipment, services and distribution 3 Financial services 25 Includes banks, financial and insurance firms 4 Health 4 Care providers, equipment, supplies and pharmaceuticals 5 Industry 36 Include companies providing equipment and services in the productive chain 6 IPC index 1 Main index in the Mexican stock exchange 7 IT 1 Software and Hardware for information technologies 8 Materials 22 Include companies providing input materials in the productive chain 9 Non basic consumming 19 Includes retailers, consumer service providers and consumer durables 10 Telecomm 9 Includes wireless providers, internet service providers and satellite companies https://doi.org/10.1371/journal.pone.0238731.t001 https://doi.org/10.1371/journal.pone.0238731.t001 the sense of an increase in activity. Indeed, as we go forward through the years, more and more time series of stock prices satisfy the filtering process, evidencing an evolution in terms of more activity in the market with more variability of prices and more quotes. Visual evidence can be found in Fig 3. An important aspect of this work will be to consider how industrial sec- tors are interconnected. Data Here we consider a list of sectors obtained from the Bolsa Mexicana de Valores (BMV) classification. These are listed in Table 1. Fig 2 presents an estimated net- work in which stocks can be identified in its sector. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Gaussian graph model Markovian random fields. In this section we start with the basic definition of a MRF which is the fundamental probabilistic concept from which a GGm is defined. Let us introduce a graph G = (V, E) with a set of nodes V = {1, . . ., n} and edges E. Recall that a complete sub- graph of G is called a clique. We denote by C the class of maximal cliques of the graph G. Let us start with a given a random vector ~X ¼ ðX1; . . . ; XnÞ with multivariate cumulative distribution function p. Then, the vector ~X has a Gibbs distribution compatible with the graph G if its distri- bution has a representation pðx1; . . . ; xnÞ ¼ 1 Z Y C2C cCðxCÞ; where fccgC2C are suitable functions and xC denotes a vector in which only the indexes of C appear. Gibbs distributions are characterized through different Markov properties. To this end, we need a notation. For A V, A ¼ ðAi1; . . . ; AikÞ, the notation ~XA denotes the vector ðXi1; . . . ; XikÞ. The following list provides the Markov properties: 1. ~X is a MRF with respect to G if it has the Markov property: For any pair i, j 2 V with i 6¼ j and non adjacent in the graph G, the random variables Xi and Xj are conditionally indepen- dent on all the other variables. We denote this conditional independency by: 1. ~X is a MRF with respect to G if it has the Markov property: For any pair i, j 2 V with i 6¼ j and non adjacent in the graph G, the random variables Xi and Xj are conditionally indepen- dent on all the other variables. We denote this conditional independency by: Xu ? Xv j ~XV=fu;vg: Xu ? Xv j ~XV=fu;vg: 5 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 2. A network in which stocks can be identified in their sectors. Fig 2. A network in which stocks can be identified in their sectors. https://doi.org/10.1371/journal.pone.0238731.g002 2. ~X is locally a MRF with respect to G if: For each v 2 V, the random variable Xv is condition- ally independent of all other variables which are not neighboors (they are not adjacent). PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Gaussian graph model We denote this by 2. ~X is locally a MRF with respect to G if: For each v 2 V, the random variable Xv is condition- ally independent of all other variables which are not neighboors (they are not adjacent). We denote this by Xv ? ~XV=neighborhoodðvÞ j ~XneighborhoodðvÞ: 3. ~X is globally a MRF if: For two disjoint subsets A, B V, the vectors ~XA, ~XB are condition- ally independent on a separating set S V. We denote this by: 3. ~X is globally a MRF if: For two disjoint subsets A, B V, the vectors ~XA, ~XB are condition- ally independent on a separating set S V. We denote this by: ~XA ? ~XB j ~XS: We continue with a fundamental equivalence result; see [52, Chapter 7]. Theorem 1 (Hammersley-Clifford). Assume that the cumulative distribution p of ~X is defined in a finite state space and is positive valued. Then p is a Gibbs distribution if and only ~X satisfies any of the above listed Markov properties. 6 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange For a list of Gibbs distributions see [13, Section 3]. In this paper we will work with the fol- lowing specific Gibbs distribution (hence, specific MRF and specific GGm) pyðxÞ ¼ exp y x þ 1 2 X n i¼1 X n i¼1 Yi;jxixj AðyÞ ( ) : ð1Þ ð1Þ where n is the dimension of the random vector ~X, and A() is a normalizing constant; see [13, Example 3.3] for more details. The MRF model in (1) also specifies the GGm we will work with. Indeed, (1) does not apriori specify any graph, but from the set of parameters Yi;j 2 R we derive a partial correlation matrix which indeed can be seen as the adjacency matrix of a weighted graph. Covariance selection. Let Σ ¼ ðSi;jÞ be the covariance matrix of a random vector ~R ¼ ðR1; . . . ; RnÞ with multivariate Gaussian distribution. A zero component Si,j = 0 expresses marginal independence between Ri and Rj. The inverse matrix J ≔Σ1 is the so-called concen- tration matrix. It has the property that a zero component Ji,j = 0 expresses conditional indepen- dence; see e.g., [53, Thm. 9.2.1] or for complex distributions [15, Thm 7.1 p. 117]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Gaussian graph model It is possible from further considerations that many components Ji,j are expected to be zero. In this case, it is desirable to have a statistical procedure to estimate the distribution taking into account such information. One such procedure is the so-called covariance selection in [1]. Grounded on maximum likelihood, it provides a framework to test for zero partial correla- tions. Recent research on covariance selection focuses on sparse large dimensions in which the number of variables is large but there are also many variables which are conditionally indepen- dent; see [54]. Thus, for such structures the concentration matrix is sparse and the lasso (least absolute shrinkage and selection operator; also lasso or LASSO) method introduced by [55] is fundamental for statistical estimation and variable selection. Indeed, the Gaussian Graphical model that we are going to use is nodewise estimated through a lasso procedure. For the lasso implementation we use the R package mgm that builds on the package glmnet. The estima- tions of this last package are based on the algorithm of [56]. Then, the collection of nodewise regressions are combined through an AND rule to give a unique estimation of a multivariate vector. This approach is naturally based on the asymptotic consistency results due to [54]. In particular, the estimation yields a concentration matrix J. Systematic presentations for graphi- cal models can be found in [13, 14] and [15]. The GGm. Now we explain the specification of the GGm we are going to estimate. Let S be the covariance matrix of the log returns time series R(1), . . ., R(n). Denote by J the concen- tration matrix, J ≔S−1. The components of the matrix J are given in terms of the coefficients Θi,j in Eq (1). Denote by ρi,j the partial correlation of R(i) and R(j). Consider the linear regres- sions defining partial correlations: RðiÞ mðiÞ ¼ X j6¼i bi;jðRðjÞ mjÞ þ ðiÞ ð2Þ ð2Þ where μi is the unconditional mean of R(i) and (i) is a residual. Then where μi is the unconditional mean of R(i) and (i) is a residual. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE PLOS ONE The interdependency structure in the Mexican stock exchange Table 2. Links in the rank (0.3, 1] for the period 2000-2009. tick1 tick2 weight year tick1 tick2 weight year ELEKTRA ICA 0.98 2000 ICHB SIMECB 0.44 2012 FEMSAUBD MXX 0.4 2000 AMXA MXX 0.83 2013 AZTECACPO SORIANAB 0.31 2001 FEMSAUBD MXX 0.42 2013 AMXA MXX 0.37 2002 GMEXICOB MXX 0.48 2013 AZTECACPO MXX 0.38 2002 ICHB SIMECB 0.34 2013 CEMEXCPO MXX 0.38 2002 MXX WALMEX 0.31 2013 FEMSAUBD MXX 0.45 2002 CEMEXCPO MXX 0.33 2014 MXX SORIANAB 0.32 2002 FEMSAUBD MXX 0.56 2014 AMXA MXX 0.59 2003 ASURB GAPB 0.36 2015 AZTECACPO MXX 0.31 2003 CEMEXCPO MXX 0.36 2015 CEMEXCPO MXX 0.36 2003 FEMSAUBD MXX 0.42 2015 FEMSAUBD MXX 0.35 2003 GFNORTEO MXX 0.37 2015 MXX WALMEX 0.73 2003 GMEXICOB MXX 0.33 2015 AMXA MXX 0.72 2004 ICHB SIMECB 0.36 2015 CEMEXCPO MXX 0.49 2004 CEMEXCPO MXX 0.45 2016 MXX WALMEX 0.43 2004 FEMSAUBD MXX 0.59 2016 CEMEXCPO MXX 0.44 2005 GFNORTEO MXX 0.34 2016 MXX WALMEX 0.4 2005 MXX WALMEX 0.35 2016 CEMEXCPO MXX 0.49 2006 ASURB GAPB 0.33 2017 FEMSAUBD MXX 0.41 2006 CEMEXCPO MXX 0.75 2017 GMEXICOB MXX 0.34 2006 FEMSAUBD MXX 0.42 2017 MXX WALMEX 0.77 2006 GEOB HOMEX 0.36 2017 CEMEXCPO MXX 0.5 2007 GFNORTEO MXX 0.47 2017 GAPB OMAB 0.35 2007 GMEXICOB MXX 0.3 2017 GMEXICOB MXX 0.31 2007 ICHB SIMECB 0.37 2017 MXX WALMEX 0.56 2007 ASURB GAPB 0.32 2018 GAPB OMAB 0.31 2008 CEMEXCPO MXX 0.74 2018 ICHB SIMECB 0.41 2008 FEMSAUBD MXX 0.87 2018 MXX WALMEX 0.37 2008 GFNORTEO MXX 0.65 2018 CEMEXCPO MXX 0.37 2009 GIGANTE LIVEPOL1 0.32 2018 GAPB OMAB 0.34 2009 MXX WALMEX 0.41 2018 ICHB SIMECB 0.34 2009 ASURB GAPB 0.31 2019 MXX WALMEX 0.35 2009 CEMEXCPO MXX 0.39 2019 CEMEXCPO MXX 0.35 2010 FEMSAUBD GFNORTEO -0.36 2019 ICHB SIMECB 0.34 2010 FEMSAUBD MXX 0.89 2019 MXX WALMEX 0.36 2010 GAPB OMAB 0.33 2019 FEMSAUBD MXX 0.35 2011 GFNORTEO MXX 0.81 2019 MXX WALMEX 0.33 2011 GMEXICOB MXX 0.55 2019 HOMEX URBI 0.32 2012 MXX WALMEX 0.56 2019 https://doi.org/10.1371/journal.pone.0238731.t002 Table 2. Links in the rank (0.3, 1] for the period 2000-2009. In Tables 4 and 5 we provide information concerning most persistent links in the observed period. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Gaussian graph model Then bij ¼ ri;j ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi varððiÞÞvarððjÞÞ p : ð3Þ ð3Þ PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 7 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 7 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange It is also true that It is also true that ri;j ¼ Ji;j ffiffiffiffiffiffiffi JiiJjj p : ri;j ¼ Ji;j ffiffiffiffiffiffiffi JiiJjj p : The adjacency matrix P = (Pi,j) is defined by The adjacency matrix P = (Pi,j) is defined by Pi;i ¼ 0 and Pi;j ¼ ri;j: ð4Þ ð4Þ Remark 1 Let us emphasize now that the estimation of the GGm (1) will ultimately result in the matrix P and this matrix is our main input for the network based on the GGm. Results from GGm estimation: Stylized facts. In this section we report the results of esti- mating a GGm for each year in the period 2000-2019 using (1). From this estimation exercise, we get a partial correlation matrix P for each year in the period 2000-2019, hence twenty matrices in total. A graphical representation of partial correlations is displayed in the panel of Fig 3. In Table 2 we display partial correlations in absolute value above the threshold 0.3. Partial correlations in absolute value in the interval (0.2, 0.3] are displayed in Table 3. Fig 3. Graphs associated with partial correlation matrices, by year, in the period 2000-2019. For a given edge the green color (respectively red color) represents a positive (respectively negative) relationship. Edge’s width represents the strength of correlation. Vertexes are grouped according to industrial sector. https://doi.org/10.1371/journal.pone.0238731.g003 Fig 3. Graphs associated with partial correlation matrices, by year, in the period 2000-2019. For a given edge the green color (respectively red color) represents a positive (respectively negative) relationship. Edge’s width represents the strength of correlation. Vertexes are grouped according to industrial sector. https://doi.org/10.1371/journal.pone.0238731.g003 https://doi.org/10.1371/journal.pone.0238731.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 8 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange PLOS ONE Table 3. Links in the rank (0.2, 0.3] for the period 2000-2009. PLOS ONE tick1 tick2 weight year tick1 tick2 weight year CEMEXCPO MXX 0.27 2000 ASURB GAPB 0.24 2010 GFINBURO MXX 0.21 2000 AXTELCPO GFAMSAA 0.21 2010 GFNORTEO MXX 0.23 2000 GCARSOA1 GFINBURO 0.26 2010 MXX SORIANAB 0.26 2000 GFNORTEO MXX 0.21 2010 ARA GFNORTEO 0.26 2001 GMEXICOB MXX 0.29 2010 AZTECACPO ELEKTRA 0.2 2001 KUOA LIVEPOL1 0.22 2010 CEMEXCPO FEMSAUBD 0.3 2001 ALFAA MXX 0.22 2011 ALFAA MXX 0.2 2002 CEMEXCPO ICA 0.28 2011 AZTECACPO ELEKTRA 0.22 2002 CEMEXCPO MXX 0.21 2011 GFINBURO MXX 0.29 2002 ELEKTRA MXX 0.22 2011 GFNORTEO MXX 0.26 2002 GFNORTEO MXX 0.3 2011 ARA MXX 0.22 2003 GMEXICOB MXX 0.29 2011 GFINBURO MXX 0.21 2003 HOMEX URBI 0.28 2011 MXX SORIANAB 0.26 2003 CEMEXCPO MXX 0.27 2012 ALFAA MXX 0.29 2004 FEMSAUBD MXX 0.28 2012 AMXA CEMEXCPO -0.23 2004 GMEXICOB MXX 0.26 2012 AZTECACPO MXX 0.2 2004 MXX WALMEX 0.22 2012 BIMBOA MXX 0.2 2004 ALFAA MXX 0.29 2013 GFINBURO MXX 0.23 2004 AMXA FEMSAUBD -0.22 2013 GMEXICOB MXX 0.26 2004 CEMEXCPO MXX 0.22 2013 MXX SORIANAB 0.24 2004 GFNORTEO MXX 0.26 2013 ALFAA MXX 0.23 2005 GMEXICOB PE&OLES 0.22 2013 AMXA MXX 0.23 2005 HOMEX SAREB 0.22 2013 ARA URBI 0.21 2005 ALFAA MXX 0.24 2014 FEMSAUBD MXX 0.22 2005 ALSEA CULTIBAB 0.24 2014 GMEXICOB MXX 0.25 2005 ASURB GAPB 0.2 2014 KIMBERA MXX 0.21 2005 GFNORTEO MXX 0.26 2014 ALFAA MXX 0.22 2006 GMEXICOB MXX 0.29 2014 AMXA MXX 0.25 2006 MFRISCOA-1 PE&OLES 0.27 2014 GFINBURO MXX 0.28 2006 ALFAA MXX 0.22 2015 GFNORTEO MXX 0.2 2006 GFINBURO MXX 0.22 2015 MXX PINFRA 0.21 2006 AC BIMBOA 0.2 2016 BIMBOA MXX 0.23 2007 ALFAA AZTECACPO 0.27 2016 GFNORTEO MXX 0.23 2007 ALFAA GFINBURO 0.22 2016 HOMEX MXX 0.2 2007 ASURB GAPB 0.21 2016 ICA MXX 0.24 2007 GENTERA PINFRA 0.23 2016 MXX URBI 0.25 2007 GMEXICOB MXX 0.21 2016 ALFAA ARA 0.3 2008 MFRISCOA-1 PE&OLES 0.27 2016 ALFAA AXTELCPO 0.26 2008 MXX ORBIA 0.2 2016 AMXA MXX 0.23 2008 ALFAA ALPEKA 0.24 2017 CEMEXCPO MXX 0.26 2008 CEMEXCPO GFNORTEO -0.24 2017 FEMSAUBD MXX 0.26 2008 GAPB OMAB 0.2 2017 GCARSOA1 MXX 0.21 2008 GFNORTEO GMEXICOB -0.22 2017 GMEXICOB MXX 0.28 2008 HOMEX URBI 0.28 2017 HOMEX MXX 0.2 2008 MXX WALMEX 0.23 2017 MXX PE&OLES 0.25 2008 CEMEXCPO FEMSAUBD -0.27 2018 AMXA MXX 0.21 2009 FEMSAUBD GFNORTEO -0.26 2018 (Continued) Table 3. Links in the rank (0.2, 0.3] for the period 2000-2009. PLOS ONE p We obtain the following stylized facts: We obtain the following stylized facts: • First of all we see in Fig 3 a stable continuous evolution of partial-correlations interdepen- dency structure. At this stage of initial visual inspection, if an impact of global crisis episodes 9 / 31 PLOS ONE Table 3. (Continued) tick1 tick2 weight year tick1 tick2 weight year AMXA RCENTROA 0.21 2009 GAPB OMAB 0.22 2018 BIMBOA MXX 0.22 2009 GMEXICOB MXX 0.26 2018 FEMSAUBD MXX 0.21 2009 ICHB SIMECB 0.22 2018 GCARSOA1 MXX 0.22 2009 GFNORTEO GMEXICOB -0.26 2019 GMEXICOB MXX 0.26 2009 HCITY UNIFINA 0.2 2019 HOMEX MXX 0.25 2009 https://doi.org/10.1371/journal.pone.0238731.t003 indeed existed (e.g., dot.com bubble, the subprime crisis and the European soveregin debt crisis) it doesn’t seem to produce large variations in network interdependency structures. indeed existed (e.g., dot.com bubble, the subprime crisis and the European soveregin debt crisis) it doesn’t seem to produce large variations in network interdependency structures. • There are several edges with a weight (partial-correlation) above the threshold 0.2 which fre- quently include the main index from the Mexican stock Exchange BMV denominated IPC (quoted as MXX in Yahoo.Finance); see the Tables 2 and 3. • As we move forward in time, the market grows (with more nodes of stocks consistently quoted by year). However, it does not seem to be evidence that interconnectedness in the market changes drastically from one year to the next, even for the subprime crisis period. • Connections must be due to exogenous factors to the market, but inherent to each stock, since the graph is based on partial correlations. However, for edges that include the IPC node, the other node may be a stock used in the construction of the index. • A large number of links between stocks in different sectors wich is an empirical fact reported for other markets; see e.g., [16], and to our best knowledge, not previously documented for the Mexican stock market. Nonetheless, intrasectorial partial-correlations are also present. • There are persistent links between pairs of stocks that appear frequently, but not systemati- cally, over the twenty year period; see Tables 4 and 5. • Negative partial-correlations appear only seldomly. • For the year 2000 we see a partial correlation of 0.98 between ICA and ELEKTRA which appears to be an odd finding, but it is actually supported by data; see Fig 4. Table 4. Persistent links in the rank (0.1, 1] for the period 2000-2009 (1/2). PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 10 / 31 The interdependency structure in the Mexican stock exchange https://doi.org/10.1371/journal.pone.0238731.t003 PLOS ONE PLOS ONE The interdependency structure in the Mexican stock exchange Table 5. Persistent links in the rank (0.1, 1] for the period 2010-2019 (2/2). 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 ALFAA-MXX 0.22 0.20 0.29 0.24 0.22 AMXA-MXX 0.12 0.83 CEMEXCPO-MXX 0.35 0.21 0.27 0.22 0.33 0.36 0.45 0.75 0.74 0.39 FEMSAUBD-MXX 0.35 0.28 0.42 0.56 0.42 0.59 0.42 0.87 0.89 GFINBURO-MXX 0.14 0.22 0.13 GFNORTEO-MXX 0.21 0.30 0.19 0.26 0.26 0.37 0.34 0.47 0.65 0.81 BIMBOA-MXX 0.18 0.16 0.18 0.15 0.13 0.18 MXX-WALMEX 0.36 0.33 0.22 0.31 0.20 0.19 0.35 0.23 0.41 0.56 GMEXICOB-MXX 0.29 0.29 0.26 0.48 0.29 0.33 0.21 0.30 0.26 0.55 ASURB-GAPB 0.24 0.17 0.16 0.16 0.20 0.36 0.21 0.33 0.32 0.31 ICHB-SIMECB 0.34 0.44 0.34 0.11 0.36 0.37 0.22 htt //d i /10 1371/j l 0238731 t005 Table 5. Persistent links in the rank (0.1, 1] for the period 2010-2019 (2/2). • The strongest links above 0.3 are those typically having as one of its nodes the IPC. Also for the rank [0.2, 0.3] links with IPC as a node dominate but with a small decline in frequency in contrast with the interval (0.3, 1]. • FEMSA indeed has a persistent relationship with the IPC index with partial correlations above 0.3; see Tables 4 and 5. In this same tables we do not see an important stock such as AMX. This is an interesting confirmation of the strength of the GGm, since it captures real facts (see for example news stories from the mexican magazines expansion and el economista). In Fig 5 we see a panel of barplots for degree-centralities separated into different ranges for all stocks in their respective period. Links with negative values are few in quantity and magni- tude as more precisely illustrated in Fig 5(a). In Fig 5(b) we see a quite homogenous distribu- tion in the range [0.01, 0.1]. An analogous situation is appreciated in Fig 5(c) in the interval Fig 4. In purple the time series for ELEKTRA and in blue the time series for ICA. Prices in logarithmic scale for the year 2000. Source: Yahoo.Finance. https://doi.org/10.1371/journal.pone.0238731.g004 Fig 4. In purple the time series for ELEKTRA and in blue the time series for ICA. Prices in logarithmic scale for the year 2000. Source: Yahoo.Finance. https://doi.org/10.1371/journal.pone.0238731.g004 Fig 4. In purple the time series for ELEKTRA and in blue the time series for ICA. PLOS ONE 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 ALFAA-MXX 0.17 0.20 0.29 0.23 0.22 AMXA-MXX 0.16 0.37 0.59 0.72 0.23 0.25 0.23 0.21 CEMEXCPO-MXX 0.27 0.14 0.38 0.36 0.49 0.44 0.49 0.50 0.26 0.37 FEMSAUBD-MXX 0.40 0.18 0.45 0.35 0.19 0.22 0.41 0.19 0.26 0.21 GFINBURO-MXX 0.21 0.16 0.29 0.21 0.23 0.28 0.17 GFNORTEO-MXX 0.23 0.26 0.20 0.23 0.20 BIMBOA-MXX 0.11 0.17 0.20 0.17 0.23 0.14 0.22 MXX-WALMEX 0.73 0.43 0.40 0.77 0.56 0.37 0.35 GMEXICOB-MXX 0.26 0.25 0.34 0.31 0.28 0.26 ASURB-GAPB 0.17 0.13 ICHB-SIMECB 0.41 0.34 Table 4. Persistent links in the rank (0.1, 1] for the period 2000-2009 (1/2). PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 11 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 11 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 [0.1, 0.5]. Only in the range [0.5, 1] we see in Fig 5(d) a more heterogenous situation with some dominating stocks. [0.1, 0.5]. Only in the range [0.5, 1] we see in Fig 5(d) a more heterogenous situation with some dominating stocks. Before we continue with a discussion of results in this section, we estimate metrics (centrali- ties) from network theory to see a possible effect of global financial crisis. PLOS ONE Prices in logarithmic scale for the year 2000. Source: Yahoo.Finance. https://doi.org/10.1371/journal.pone.0238731.g004 https://doi.org/10.1371/journal.pone.0238731.g004 12 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 5. A comparison of degree centralities by year at different ranges. h //d i /10 1371/j l 0238731 005 Fig 5. A comparison of degree centralities by year at different ranges. Fig 5. A comparison of degree centralities by year at different ranges. Fig 5. A comparison of degree centralities by year at different ranges. Fig 5. A comparison of degree centralities by year at different ranges. Fig 5. A comparison of degree centralities by year at different ranges. Centralities from partial correlations Centrality is a metric designed in such a way that a vertex with high centrality can be consid- ered highly influential. The first concept of centrality we use is the degree-centrality which for a vertex in a weighted network is the sum of weights of the connecting edges. For our graphs of partial correlations, the degree centrality gives information on the pattern of a shock’s trans- mission. The idea is that if an influential (i.e., with high centrality) stock in the financial net- work is having a bad day, it will be accompanied by many other stocks in similar situations. Note that there is no causality claimed here. The second measure of centrality that we estimate is the eigen-centrality. This is a global measure in that scores for each node are assigned via a PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 13 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange comparison of the quality of its links. For example a node with just one link to another influen- tial node could have a highest eigen-centrality than a node with two or more links. The com- putation of eigencentralities transfers to a spectral analysis of the adjacency matrix and in crucial steps is substantiated by Perron-Frobenius theory (see e.g., [57, Chapter 17]). The third concept that we estimate is betweenness-centrality for the absolute values of weights. For each vertex, it gives the proportion of shortest paths passing through it. Shock transmissions. Let us explain in more detail eigencentrality and at the same time also clarify shock transmissions. Let V = {1, . . ., n} index our set of stocks and recall the matrix P defined in (4). The eigencentrality is a function f : V ! R satisfying f ðvÞ ¼ r X w2NðvÞ Pv;wf ðwÞ; v 2 V; ð5Þ ð5Þ where r is a non negative constant and N(v) denotes the neighbors of v. Note that f ðvÞ ¼ r X w2V Pv;wf ðwÞ; f ðvÞ ¼ r X w2V Pv;wf ðwÞ; since by definition w 2 N(v) if and only if Pv,w 6¼ 0. Now this can be written in matricial nota- tion as since by definition w 2 N(v) if and only if Pv,w 6¼ 0. Now this can be written in matricial nota- tion as f ðVÞ ¼ rPf ðVÞ T; where f(V) = (f(1), . . ., f(n)). Centralities from partial correlations Hence, f(V) is an eigenvector of P attached to r as its eigenvalue. To continue we follow the discussion in [17], returning to the coefficients βi,j in Eq (3). The matrix of coefficients B = (βi,j) with βii = 0 is then connected to the adjacency matrix as B ¼ diagðJÞ 1 2PdiagðJÞ 1 2. We can write the linear regression in a compact matricial notation as ~R m ¼ Bð~R mÞ þ ¼ diagðJÞ 1 2PdiagðJÞ 1 2ð~R mÞ þ : ð6Þ Let ~RðiÞ ≔RðiÞ mðiÞ and ~R ¼ ðRð1Þ mð1Þ; . . . ; RðnÞ mðnÞÞ. Then, diagðJÞ 1 2~R ¼ PdiagðJÞ 1 2ð~R mÞ þ diagðJÞ 1 2: ~R m ¼ Bð~R mÞ þ ¼ diagðJÞ 1 2PdiagðJÞ 1 2ð~R mÞ þ : ð6Þ ð6Þ Let ~RðiÞ ≔RðiÞ mðiÞ and ~R ¼ ðRð1Þ mð1Þ; . . . ; RðnÞ mðnÞÞ. Then, diagðJÞ 1 2~R ¼ PdiagðJÞ 1 2ð~R mÞ þ diagðJÞ 1 2: Hence the vector ~X ≔diagðJÞ 1 2~R satisfies Hence the vector ~X ≔diagðJÞ 1 2~R satisfies ~X ¼ P~X þ Z PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 where Z ≔diagðJÞ 1 2. Now assume that between times t0 and t1 there is a shock Δ = (0, . . ., 0, δ, 0, . . ., 0) affecting X(i). Then, ~X at time t1 is given by Pð~X þ DÞ þ Z and the change is then PΔ. Note that PΔ does not need to be a scalar of Δ, meaning that the shock originally affecting X(i) has also an impact on other components, indicating that the shock propagates. The spectral decomposition of P helps in assessing the scale of propagation and rationalizes the definition of eigencentrality. Let W1, . . ., Wn be the set of eigenvectors of P and Λ = {λ1, . . ., λn} the corresponding set of eigenvalues, which we assume is decreasingly ordered with respect to its modulus. Here is a common assumption: there is a unique eigenvalue attaining the spectral radius. This means \|λ1\|>\|λ2\|\|λ2\|. . . \|λn\|. If the matrix P has only non-negative components, then the Perron-Frobenious theory guarantees we are in this situation and other PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 14 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 6. Network centralities from partial correlations. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year, always attained by the IPC index. The red line is the average of degree-centralities, respectively the red dashed line is the average of absolute value of degree-centralities. The gray line represents the maximum betweenness-centrality of absolute values. In the upper panel, time series are shown in their original scale, while in the lower panel time series have been rescaled by its maximum. Fig 6. Network centralities from partial correlations. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year, always attained by the IPC index. The red line is the average of degree-centralities, respectively the red dashed line is the average of absolute value of degree-centralities. The gray line represents the maximum betweenness-centrality of absolute values. In the upper panel, time series are shown in their original scale, while in the lower panel time series have been rescaled by its maximum. https://doi.org/10.1371/journal.pone.0238731.g006 https://doi.org/10.1371/journal.pone.0238731.g006 properties besides; see e.g., [57, Chapter 17]. Represent Δ by Δ = ∑i αi Wi. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 where Z ≔diagðJÞ 1 2. Then, for k 2 N PkD ¼ lk 1 a1W1 þ X n i¼2 li l1 k Wi ( ) : Hence Then, as time passes the leading term indicating the effect of the initial shock Δ takes the form lk 1a1W1. Then, as time passes the leading term indicating the effect of the initial shock Δ takes the form lk 1a1W1. Results of estimation. In Fig 6 we see estimated centralities for our networks. The blue line is the largest modulus per year of eigenvalues. The green line represents the maximum degree-centrality for each year and unsurprising this maximum is always attained by the IPC index. The red (respectively red and dashed) line represents the average of each node’s degree- centrality (respectively the average of each node’s absolute value degree-centrality). The gray PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 15 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 7. Eigenvalue modulus per year. https://doi.org/10.1371/journal.pone.0238731.g007 Fig 7. Eigenvalue modulus per year. https://doi.org/10.1371/journal.pone.0238731.g007 https://doi.org/10.1371/journal.pone.0238731.g007 line represents the maximum of betweenness-centrality which has been computed for absolute values of weights with the R package igraph. line represents the maximum of betweenness-centrality which has been computed for absolute values of weights with the R package igraph. These are the findings we observe from Fig 6: These are the findings we observe from Fig 6: • The spectral radius is approximately bounded by two, which coincides with the range docu- mented for other markets; see e.g., [16]. • The red line and the red dashed lined are almost indistinguishable. This happens as a conse- quence of the fact that almost all partial-correlations are non-negative. We also observe the stability on the metric represented by this line. • The patterns of the green and blue lines are similar. As we mentioned, the green line is attained by the IPC index, so it could be expected that the blue line is also related to this index. Although we do not investigate this claim, assuming it is correct, in order to capture effects beyond the IPC index it might be necessary in this case to complement with the sec- ond eigenvalue together with its eigenvector for centrality and the analysis for a shock conta- gion. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 where Z ≔diagðJÞ 1 2. Indeed, Fig 7 shows that in many cases the dominant eigenvalue has a multiplicity of two or more, and in other cases that the second eigenvalue turns out to be close to the first. 16 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Certainly, the idea of considering beyond the dominant eigenvector for eigencentrality is not new; see [58]. Analysis for the Mexican case will be addressed elsewhere. Certainly, the idea of considering beyond the dominant eigenvector for eigencentrality is not new; see [58]. Analysis for the Mexican case will be addressed elsewhere. Certainly, the idea of considering beyond the dominant eigenvector for eigencentrality is not new; see [58]. Analysis for the Mexican case will be addressed elsewhere. • There is indeed variability for centralities, but changes from one year to the next are indeed relatively small. Thus, changes are subtle. For example, for the subprime crisis period, we see a small upwards jump from 2005 to 2006 of around 0.4 and then from 2007 to 2008 a down- wards jump of around 0.25. Small jumps are also observed for max degree-centrality on the green line. • Continuing with the previous point, we see an abrupt upwards movement of the green line which we assume is associated with the dot.com bubble’s crisis: From the year 2001 to 2002. • Continuing with the previous point, we see an abrupt upwards movement of the green line which we assume is associated with the dot.com bubble’s crisis: From the year 2001 to 2002. Discussion. The financial networks of partial correlations illustrated in Fig 3 present low degree centrality and are sparse. Below we construct financial networks based on Pearson cor- relation matrices and we will also compute their centralities; see Fig 12. A comparison of Figs 6 and 12 yield evidence that partial-correlations generate sparser networks, understood by the fact that comparing year by year, centralities for partial correlations are significantly lower than for Pearson correlations. This was expected and agrees with the findings in [16] and [17] which also compare networks based on Pearson and partial-correlations. Interestingly, estima- tions of matrices are done by different methods and different data, thus, providing evidence that sparsity of partial-correlation based networks are robust with respect to statistical proce- dures and data. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 where Z ≔diagðJÞ 1 2. Another paper that also compares networks based on Pearson and partial cor- relations is [4]. The authors conclude that networks based on Pearson correlations show different structures than partial correlation matrices, and in particular a different clustering structure. However, they construct Minimum Spanning Trees and the comparison of sparsity is unclear. They also compute betweenness-centrality. Although they report differences, these are not as marked as the ones presented here. We will elaborate on betweenness-centrality fur- ther after we also analyze Tail-dependence networks. The IPC index has been found to be a vertex where edges consistently present their highest weight (partial-correlation). Indeed, the maximum of degree- and eigen- centralities are always attained at this vertex. If the analysis of the mean variance portfolio of [23] holds true also for partial correlations, then this would mean that in such a portfolio, the IPC seen as an asset on its own would receive a lower weight. Thus, any Exchange-Traded Fund (ETF) tracking the IPC index does not diversify investments from the point of view of the classical Markowitz portfolio theory and achieve a lower proportion of portfolio value. Whether the negative rela- tionship found in [23] also holds true for partial correlations can be the subject of future research, however we find that the IPC index also has high degree- and eigen- centralities for networks based on Tail-dependence and Pearson correlation matrices. In the period 2000-2019 there are three important financial episodes: The dot.com bubble, the subprime crisis and the European sovereign debt crisis. The time series of centralities in Fig 6 exhibit several local maximum which may connect with those episodes. Now here is a trade-off. Partial correlations and the lasso estimation of the GGm indeed result in a stringent sieve in which only the most significant and “clear” relationships pass through and as a conse- quence the centrality time series are quite stable. However, the aforementioned financial epi- sodes are captured by centralities of partial-correlations and show moderate increases. We will see that Tail-dependence networks exhibit a more sensible topology for those market condi- tions. This is reasonable due to the symmetric nature of distributions in GGm while on Tail- dependence networks the emphasis is on lower tails. 17 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Tail-dependence networks For two random variables X and Y the Tail-dependence coefficient ([2]) is the limit lL ¼ lim q!0 PðX F1 X ðqÞ j Y F1 Y ðqÞÞ where FX represents the cumulative distribution of X, and similarly for FY. It is clear that λL quantifies the relationship of lower tails between X and Y. In this section we focus on net- works based on matrices whose components are the coefficients λL for pairs of stock time series. We estimate the Tail-dependence coefficient through the non-parametric estimator in [7] which is implemented in the R package FRAPO (Financial Risk Modelling and Portfolio Optimisation). In Figs 8 and 9 the Tail-dependence networks for years during the period 2006-2009 are illustrated. In Fig 8 vertex size is a function of betweenness-centrality while on Fig 9 it is a function of eigen-centrality. The illustrated structure repeats for the years over the period 2000-2019. The time series of centralities are illustrated in Fig 10. This is a list of stylized facts: Fig 8. Tail-dependence networks for the years 2006-2009. Vertex size as a function of betweenness-centrality. The pink shaded area shows the highest 95% quantile. The colors of the edges are blue for weights in absolute value in the interval [0.2, 0.3], and red in the interval (.3, 1]. Otherwise they are gray. https://doi.org/10.1371/journal.pone.0238731.g008 Fig 8. Tail-dependence networks for the years 2006-2009. Vertex size as a function of betweenness-centrality. The pink shaded area shows the highest 95% quantile. The colors of the edges are blue for weights in absolute value in the interval [0.2, 0.3], and red in the interval (.3, 1]. Otherwise they are gray. Fig 8. Tail-dependence networks for the years 2006-2009. Vertex size as a function of betweenness-centrality. The pink shaded area shows the highest 95% quantile. The colors of the edges are blue for weights in absolute value in the interval [0.2, 0.3], and red in the interval (.3, 1]. Otherwise they are gray. https://doi.org/10.1371/journal.pone.0238731.g008 https://doi.org/10.1371/journal.pone.0238731.g008 18 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 9. Tail-dependence networks for the years 2006-2009. Vertex size as a function of eigen-centrality. The colors of the edges are blue for weights in absolute value in the interval [0.2, 0.3], and red in the interval (.3, 1]. Otherwise they are gray. Fig 9. Tail-dependence networks for the years 2006-2009. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Tail-dependence networks They find that the banking sector is at the core of systemic risk between 2008 and 2010. In con- trast, the insurance companies are less relevant for systemic risk. Their empirical results exhibit growing interconnectedness during the period of a financial crisis. A financial network based on Tail-distributions is studied by [30]. Data comes from a selection of 51 time series of large European banks and 17 sovereigns bonds during the period from 2006 through 2013. Their empirical results show that network densities increase from the intensity of the (sub- prime) financial crisis. More precisely, network densities increases from 2006 up to its (local) maximum around the peak of the global financial crisis and then decreases. A different approach based on entropy is studied by [28]. The empirical result reports that “node strengths peak in times of crisis”. papers in this regard. A financial network from 100 selected stocks of financial institutions in the US is studied in [29]. Emphasis is on tail events from the point of view of systemic risk. They find that the banking sector is at the core of systemic risk between 2008 and 2010. In con- trast, the insurance companies are less relevant for systemic risk. Their empirical results exhibit growing interconnectedness during the period of a financial crisis. A financial network based on Tail-distributions is studied by [30]. Data comes from a selection of 51 time series of large European banks and 17 sovereigns bonds during the period from 2006 through 2013. Their empirical results show that network densities increase from the intensity of the (sub- prime) financial crisis. More precisely, network densities increases from 2006 up to its (local) maximum around the peak of the global financial crisis and then decreases. A different approach based on entropy is studied by [28]. The empirical result reports that “node strengths peak in times of crisis”. Hence, there exists an empirical fact manifest along a variety of methods applied to different data: estimated network-interconnectedness increases at some point in the development of a crisis, it might indeed affect one sector more than other, yet it is going to be globally observ- able. We evidence this empirical fact for the Mexican stock exchange in Fig 10. This is already interesting, and going deeper into the details, we mention two subtleties about timing and the different centralities. Tail-dependence networks Vertex size as a function of eigen-centrality. The colors of the edges are blue for weights in absolute value in the interval [0.2, 0.3], and red in the interval (.3, 1]. Otherwise they are gray. https://doi.org/10.1371/journal.pone.0238731.g009 https://doi.org/10.1371/journal.pone.0238731.g009 https://doi.org/10.1371/journal.pone.0238731.g009 1. The IPC index is also important with respect to eigen-centrality, just as it was for partial- correlation networks. 2. For betweenness-centrality the 95% quantile is quite dynamic concentrated in a few stocks and consistently does not include the IPC index. 3. The 95% quantile is more distributed for eigen-centrality than for betweenness-centrality. 4. Fig 10 clearly shows that time series of centralities experience an increase in activity associ- ated with the subprime and the European sovereign debt crisis. Discussion. In Fig 10 we see an illustration of financial network’s interdependency-evolu- tion for the Mexican stock exchange. The first fact to note are the peaks in the years 2008 and 2011. It is reasonable to associate such increments in interconnectedness (as measured by degree- and eigen-centrality) to the subprime financial crisis and the European sovereign debt crisis. This is coherent with findings in the literature. It is worth emphasizing that in this litera- ture, other data have been analyzed with different methods. For comparison, let us recall a few PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 19 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 10. Network centralities from Tail-dependence networks. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year. The red line is an average of degree-centrality. The gray line represents the maximum betweenness-centrality of absolute value weights. In the upper panel, time series are shown in their original scales, while in the lower panel time series have been rescaled by their maximums. https://doi.org/10.1371/journal.pone.0238731.g010 Fig 10. Network centralities from Tail-dependence networks. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year. The red line is an average of degree-centrality. The gray line represents the maximum betweenness-centrality of absolute value weights. In the upper panel, time series are shown in their original scales, while in the lower panel time series have been rescaled by their maximums. https://doi.org/10.1371/journal.pone.0238731.g010 https://doi.org/10.1371/journal.pone.0238731.g010 papers in this regard. A financial network from 100 selected stocks of financial institutions in the US is studied in [29]. Emphasis is on tail events from the point of view of systemic risk. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 Tail-dependence networks We have observed in partial-correlation networks that degree- and 20 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange eigen-centrality show the same pattern. This similarity in patterns happens also for the Tail- dependence networks analyzed in this section; compare Figs 6 and 10. It will be observed again for networks based on (filtered) Pearson correlations; see Fig 12 below. However, betweenness-centrality exhibits a different pattern, more so for Tail-dependence networks; see [59] for a similar finding with different data and statistical estimation. A relevant difference worth emphasizing concerns the timing of local extrema. Whether this difference is indeed robust with respect to data and statistical procedures certainly is an interesting question for future research. Differences in patterns, discernible in the time series, is also visible in the networks in Figs 8 and 9. In the former, in which node size is a function of betweenness-centrality, the most influential nodes are linked exclusively through gray edges. These nodes have a lot of links although all of them have small weights. On the latter graph, where node size is a function of eigen-centrality, the most influential nodes are connected together by red links. This indicates weights in the interval (.3, 1]. https://doi.org/10.1371/journal.pone.0238731.t007 Network theory: Community detection DCC multivariate Garch model. Let y ≔fytg N t¼1 denote a one dimensional time series with N observations. A GARCH specification for its volatility usually starts with a flux of infor- mation determined by a filtration fF tg N t¼1 in which F t is a σ-algebra representing information at time t and y follows the dynamic yt ¼ E½yt j F t1 þ tðyÞ: Here θ is a parameter vector whose definition specifies the model, while μ(θ) is the condi- tional mean of the time series at time t, usually modeled through an ARMA time series. For example an ARMA(1,1) (as we will consider here) is specified by ð7Þ mt ¼ m þ εt þ mt1 þ cεt1; ð7Þ where ϕ, ψ are parameters to be estimated and ε is white noise, i.e. an uncorrelated centered time series. The residual (θ) captures the conditional volatility of y: where ϕ, ψ are parameters to be estimated and ε is white noise, i.e. an uncorrelated centered time series. The residual (θ) captures the conditional volatility of y: varðyt j F tÞ ¼ E½ðyt mtðyÞÞ 2 j F t ¼ E½ðtðyÞÞ 2 j F t ¼ varðtðyÞÞ: Its specification is the essence of a GARCH model. We will consider the standard GARCH (1,1) model: t ¼ stzt ð8Þ s2 t ¼ a0 þ a12 t1 þ b1s2 t1; ð9Þ t ¼ stzt ð8Þ ð8Þ s2 t ¼ a0 þ a12 t1 þ b1s2 t1; ð9Þ s2 t ¼ a0 þ a12 t1 þ b1s2 t1; ð9Þ where fztg N t¼1 is white noise. where fztg N t¼1 is white noise. where fztg N t¼1 is white noise. Now consider a set of univariate time series y(1),. . .,y(n). A class of models in the multivari- ate GARCH literature known as Dynamic Conditional Correlation (DCC) was introduced by [60] and [61]. The DCC class builds upon univariate GARCH models and then specifies the dynamic of time varying conditional covariance matrix of the time series y(1),. . .,y(n). It has the general dynamics Ht ¼ DtRtDt: Here Dt is a diagonal matrix of time varying standard deviations from univariate GARCH models and Rt is a time varying correlation matrix. For estimation, the matrix Rt is PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 21 / 31 The interdependency structure in the Mexican stock exchange PLOS ONE PLOS ONE Table 6. The coefficient μ for the year 2006. Stock mu value Stock mu value 1 ALFAA 0.0006 GISSAA 0.0011 2 ALSEA 0.0026 GMD 0.0036 3 AMXA 0.0017 GMEXICOB 0.0021 4 ARA 0.0027 HERDEZ 0.0016 5 AXTELCPO 0.0013 HOMEX 0.0027 6 AZTECACPO 0.0006 ICA 0.0026 7 BACHOCOB 0.0012 ICHB 0.0036 8 BIMBOA 0.0018 KIMBERA 0.0011 9 CEMEXCPO 0.0013 MXX 0.0021 10 CMOCTEZ 0.0016 PAPPEL 0.0018 11 CMRB 0.0013 PASAB -0.0015 12 CYDSASAA 0.0016 PE&OLES 0.0027 13 ELEKTRA 0.0017 PINFRA 0.0065 14 FEMSAUBD 0.0024 RCENTROA 0.0039 15 GCC 0.0022 SORIANAB 0.0021 16 GFINBURO 0.0007 URBI 0.0018 17 GFNORTEO 0.0033 WALMEX 0.0023 https://doi.org/10.1371/journal.pone.0238731.t006 Table 6. The coefficient μ for the year 2006. decomposed as where Q is specified in [62, Equation (2)]. where Q is specified in [62, Equation (2)]. p q Means for the years 2006 and 2008. In Table 6 we report the coefficient μ in the specifica- tion (7) for each stock in the year 2006, and analogously for the Table 7 in the year 2008. The p q Means for the years 2006 and 2008. In Table 6 we report the coefficient μ in the specifica- tion (7) for each stock in the year 2006, and analogously for the Table 7 in the year 2008. The tion (7) for each stock in the year 2006, and analogously for the Table 7 in the year 2008. The Table 7. The coefficient μ for 2008 year. Stock mu value Stock mu value Stock mu value 1 AC -0.0013 ELEKTRA 0.0006 IDEALB-1 -0.0008 2 ALFAA -0.0019 FEMSAUBD 0.0019 KIMBERA 0.0002 3 ALSEA -0.0013 FINDEP -0.0036 LAMOSA -0.0016 4 AMXA -0.0023 FRAGUAB 0.0004 MAXCOMA -0.0028 5 ARA -0.0018 GAPB -0.0017 MEDICAB -0.0001 6 ASURB -0.0014 GCARSOA1 -0.0003 MEGACPO -0.0028 7 AUTLANB 0.0037 GCC -0.0032 MXX -0.0010 8 AXTELCPO -0.0053 GFAMSAA -0.0023 OMAB -0.0024 9 AZTECACPO 0.0000 GFINBURO 0.0008 PAPPEL -0.0047 10 BACHOCOB -0.0020 GFNORTEO -0.0003 PASAB -0.0030 11 BIMBOA 0.0002 GIGANTE -0.0026 PE&OLES -0.0010 12 CABLECPO 0.0000 GISSAA -0.0009 PINFRA -0.0016 13 CEMEXCPO -0.0030 GMD -0.0053 POCHTECB -0.0048 14 CIEB -0.0023 GMEXICOB -0.0032 SAREB -0.0033 15 CMOCTEZ -0.0005 GRUMAB -0.0001 SIMECB 0.0007 16 CMRB -0.0005 HOMEX 0.0007 SORIANAB 0.0010 17 CULTIBAB -0.0001 ICA -0.0006 TMMA -0.0040 18 CYDSASAA -0.0029 ICHB 0.0010 URBI -0.0018 Table 7. The coefficient μ for 2008 year. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 22 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange estimation of these coefficients provides further support to the claim made after the visual evi- dence of Fig 1. Modularity. Assume we are given an undirected and unweighted graph G with vertexes V = {1, . . ., n} and edges E. Community structure in the graph means that there exists a parti- tion of V in groups of vertices in such a way that within groups vertices are highly connected and more edges exist among them, while at the same time, edges between groups are less observed; see [40] for a survey of methods in community detection. The aforementioned description presents a general idea and to make it operative, it is necessary to use a more quan- titative formulation. A popular approach is through the famous concept of modularity as introduced by [63] and further developed in [64]. Following the notation of [64] we introduce the following objects. Let A = (Ai,j) be the adjacency matrix of G and let m ¼ 1 2 P iki, where ki denotes the degree of vertex i, so ki = ∑j Ai,j. Further denote by s 2 {1, . . ., n}n a vector having the same dimension as A, and representing an allocation of vertexes to communities. Thus, si represents the community assigned to vertex i. Now the idea is to compare the graph G with a graph G0 having no community structure. A group Vk = {i 2 V j si = k} possesses an accumu- lated weight of P i;j2VkAi;j. Now for G0, assuming it is a random instance of an Erdős-Re´nyi graph, the set Vk should have an accumulated weight of P i;j2Vk kikj 2m. Hence, the difference P i;j2VkAi;j kikj 2m quantifies how distant is the immersion of community Vk in the graph G from G0. The modularity function is defined as the sum of these differences over all communities: QðsÞ ≔ X k X i;j2Vk Ai;j kikj 2m ¼ X i;j2V Ai;j kikj 2m dðsi; sjÞ; where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. As such, the modularity function Q() is defined for unweighted, undirected graphs. In par- ticular, for graphs obtained from a correlation matrix, which indeed is weighted, the modular- ity function Q() needs to be adjusted. Moreover, the null model (the graph G0) is critical for the well-functioning of modularity; see the discussion in [40]. Hence, to deal with this prob- lem, we choose to work with the formulation of [36] where the correlation matrix is filtered and modularity is adjusted for the right “null model” G0. The analysis is again based on a spectral analysis as we now explain. Let C be a correlation matrix and consider the set of eigenvalues λ1, . . ., λn which we assume are displayed in increasing order. Let v1, . . ., vn be the corresponding eigenvectors. Moreover, let T be the number of observations and λ−, λ+ be the critical values l ≔ 1 ffiffiffiffin T r 2 ; lþ ≔ 1 þ ffiffiffiffin T r 2 : The values λ−, λ+ are parameters for Marcenko-Pastur distribution in random matrix the- ory which is given by rðlÞ ¼ T n ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðlþlÞðllþÞ p 2pl . Let us introduce the matrices Cr ≔ X lilþ livtr i vi ð10Þ ð10Þ Cg ≔ X lþ<li<ln livtr i vi ð11Þ ð11Þ PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 23 / 31 23 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Cm ≔lnvtr n vn: ð12Þ Cm ≔lnvtr n vn: ð12Þ We have a decomposition of the correlation matrix C given by We have a decomposition of the correlation matrix C given by ð13Þ C ¼ Cm þ Cg þ Cr: ð13Þ From the ordering of the eigenvalues, the matrix Cr represents random noise, Cm a global signal which in our financial context is attached to the market as a whole and Cg represents information in a mesoscopic scale between Cr and Cm. Next, we explain how the modularity function Q() is adjusted. where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. Accordingly, focusing on the matrix Cg, and taking into account the decomposition (13), the null model is Cr + Cm and the modularity function takes the form Q3ðsÞ ≔ 1 Cnorm X i;j ½Ci;j Cr i;j Cm i;jdðsi; sjÞ ¼ 1 Cnorm X i;j C g i;jdðsi; sjÞ ð14Þ ð14Þ for Cnorm = ∑i,j Ci,j a normalizing constant. However, the set of eigenvalues λi satisfying λ+ < λi < λn could be empty (as we will find for some years in our sample). In this case the matrix Cg will be undefined and it makes no sense to consider it. For those cases we will consider a decomposition C = Cs + Cr with Cs ≔P lþ<lilivtr i vi and then the modularity is defined by for Cnorm = ∑i,j Ci,j a normalizing constant. However, the set of eigenvalues λi satisfying λ+ < λi < λn could be empty (as we will find for some years in our sample). In this case the matrix Cg will be undefined and it makes no sense to consider it. For those cases we will consider a decomposition C = Cs + Cr with Cs ≔P lþ<lilivtr i vi and then the modularity is defined by Q2ðsÞ ≔ 1 Cnorm X i;j ½Ci;j Cr i;jdðsi; sjÞ ¼ 1 Cnorm X i;j Cs i;jdðsi; sjÞ: ð15Þ ð15Þ Hence, in this section we maximize the modularity functions Q2 and Q3 in order to define communities and report on them. It is known that the maximization of modularity functions is a NP-hard problem; see [65]. Consequently, the optimization is approached through several heuristic algorithms. We implement the popular Louvian algorithm, adjusted as described by [36] according to the modularity functions Q2 and Q3. Modularity function Q2. In Fig 11 we see the resulting communities obtained with the Louvian algorithm applied to the modularity function Q2 defined in (15). During all the years of the period there are two communities. The first community is a “giant component” and the other community consists of a small number of isolated vertices. Hence, at this scale our proce- dure does not detect a complex community structure. This is unsurprising, since Q2 is based on the matrix Cs which includes the “market mode”. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. Note however the stylized fact: • The turmoil at the subprime financial crisis and the European sovereign debt crisis periods are captured by a visually evident increase in interconnectedness. This can also be observed from the time series of centralities in Fig 12. • The turmoil at the subprime financial crisis and the European sovereign debt crisis periods are captured by a visually evident increase in interconnectedness. This can also be observed from the time series of centralities in Fig 12. Modularity function Q3. For the definition of the modularity function Q3 the matrix Cg is necessary and should not be a null matrix. For our data, this is the case for only a few years: 2000, 2010, 2016, 2018 and 2019. For them, a representation of communities can be seen from Fig 13. This is what we observe: 24 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 11. Communities obtained from modularity function Q2. The color of the nodes represent community, which is equivalently represented by the vertex’ shape. For clarity, only edges with weights in absolute value in the interval [.3, 1) are shown. Only weights above 0.5 in absolute value are distinguished in the edge’s width. https://doi.org/10.1371/journal.pone.0238731.g011 Q Th l f th d t it hi h i Fig 11. Communities obtained from modularity function Q2. The color of the nodes represent community, which is equivalently represented by the vertex’ shape. For clarity, only edges with weights in absolute value in the interval [.3, 1) are shown. Only weights above 0.5 in absolute value are distinguished in the edge’s width. https://doi.org/10.1371/journal.pone.0238731.g011 Fig 11. Communities obtained from modularity function Q2. The color of the nodes represent community, which is equivalently represented by the vertex’ shape. For clarity, only edges with weights in absolute value in the interval [.3, 1) are shown. Only weights above 0.5 in absolute value are distinguished in the edge’s width. https://doi.org/10.1371/journal.pone.0238731.g011 • First of all, in each year, there are only two communities as can be seen from the color of the vertexes, or equivalently from their shape. Interestingly, there is no clear larger community. • First of all, in each year, there are only two communities as can be seen from the color of the vertexes, or equivalently from their shape. Interestingly, there is no clear larger community. where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. • First of all, in each year, there are only two communities as can be seen from the color of the vertexes, or equivalently from their shape. Interestingly, there is no clear larger community. • Second, for our data the industrial sector is non determinant for the community assignment. More clearly, each industrial sector has vertexes in each community. This fact should be compared with the finding based on partial correlations where there also existed intersector- ial links. • This is our explanation for the years in which there existed a non-trivial matrix Cg. First of all recall that this matrix represents structure between the scales of individual stocks and the market as a whole, while in crisis periods this last structure is what prevails since stocks tend to be highly correlated at those times. In the year 2000 the peak of the dot.com bubble is located and for the years 2001 and 2002 bearish markets prevailed. What we see from Fig 11 for the network constructed from the matrices Cs, is an increase in interconnectedness, while in Fig 13, we see that in the period 2000:2002, there existed a mesoscopic structure for the year 2000, in which there is a “local minimum” for graph interconnectedness. The same PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 25 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 12. Network centralities from filtered Pearson networks based on the matrix Cs. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year. The red line is an average of degree-centrality. The gray line represents the maximum betweenness-centrality. In the upper panel, time series are shown in their original scale, while in the lower panel time series have been rescaled by its maximum. https://doi.org/10.1371/journal.pone.0238731.g012 Fig 12. Network centralities from filtered Pearson networks based on the matrix Cs. The blue line is the largest eigenvalue. The green line is the maximum degree-centrality by year. The red line is an average of degree-centrality. The gray line represents the maximum betweenness-centrality. In the upper panel, time series are shown in their original scale, while in the lower panel time series have been rescaled by its maximum. https://doi.org/10.1371/journal.pone.0238731.g012 occurs, analogously for the year 2010 in Fig 13, which coincides with a local minimum in Fig 11 for the “extended” subprime crisis period 2007-2010. where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. occurs, analogously for the year 2010 in Fig 13, which coincides with a local minimum in Fig 11 for the “extended” subprime crisis period 2007-2010. • Now we compare the years 2016, 2018 and 2019 in Figs 11 and 13. Those are years in which various global events occurred, some of them: The Brexit (starting from its referendum in june 2016), the US elections for the period 2017-2020, the China-US trade conflict starting from july 2018. However, none of these seems to be comparable to the magnitudes of the dot.com bubble and the subprime crisis. In particular for the Mexican stock market they didn’t have a sufficient impact to hide the effects of a mesoscopic structure, inducing all stocks to move as a result of a common factor. • Now we compare the years 2016, 2018 and 2019 in Figs 11 and 13. Those are years in which various global events occurred, some of them: The Brexit (starting from its referendum in june 2016), the US elections for the period 2017-2020, the China-US trade conflict starting from july 2018. However, none of these seems to be comparable to the magnitudes of the dot.com bubble and the subprime crisis. In particular for the Mexican stock market they didn’t have a sufficient impact to hide the effects of a mesoscopic structure, inducing all stocks to move as a result of a common factor. Discussion. In this section we apply the methodology presented by [36]. The authors of that paper obtain the result that for stocks in the S&P500 index, the maximization of modular- ity without any sort of adjustment results in a unique community. This is uninteresting and also happens in our case with data from the Mexican stock exchange under the modularity function Q2. It is based on the Pearson correlation matrix after noise has been filtered out according to Random Matrix Theory. This is the matrix Cs which includes, as we mentioned before, the “market mode” from which the result of a unique community is unsurprising. In PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 26 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Fig 13. Communities from the modularity function Q3. The color of nodes identifies membership to the same community and equivalently for the vertex’ shape. For clarity, only the edges with weights in absolute value in the intervals [.05, 1) are shown. where δ(si, sj) = 0 unless si = sj in which case δ(si, sj) = 1. Only weights above 0.5 in absolute value are distinguished via the edge’s width. https://doi.org/10.1371/journal.pone.0238731.g013 Fig 13. Communities from the modularity function Q3. The color of nodes identifies membership to the same community and equivalently for the vertex’ shape. For clarity, only the edges with weights in absolute value in the intervals [.05, 1) are shown. Only weights above 0.5 in absolute value are distinguished via the edge’s width. https://doi.org/10.1371/journal.pone.0238731.g013 https://doi.org/10.1371/journal.pone.0238731.g013 the paper [36] communities under Q3 are also determined. They obtain five communities for stocks in the S&P500 index. In our case there are only two communities, but it is also true that year by year we are considering on average less than one-fifth of the number of stocks in the S&P500 index. In this sense, magnitude in the number of communities seems to be coherent. The configuration of community-structure on both cases shares two properties: (a) communi- ties are multisectorial and (b) negative links joining nodes belonging to different communities are mostly negative. This is already interesting for the objective of understanding the interde- pendency structure in a single trustable snapshot. It also provides information for applications. For example, the fact that inter-communities links are negative is a useful taxonomy for the tasks of portfolio-allocation and hedging. Conclusion In global crisis periods, price levels of stocks in the Mexican stock exchange indeed present obvious changes which are visually evident and confirmed by econometric models. We have shown this fact here and it is also documented by other authors. However, the interdepen- dency structure is a more complex phenomenon and much less studied. Our findings show that as long as partial-correlations are concerned, the interdependency structure is quite stable 27 / 31 PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 PLOS ONE The interdependency structure in the Mexican stock exchange and centrality metrics from network theory have the sensibility to quantify small variations. Degree- and eigen-centralities indeed present variations, an upwards jump at the peak of the crisis and then a downwards jump when the shock of the crisis has been absorbed in the mar- ket. Another interesting finding from studying interdependency structure from partial-corre- lations is that only a small number of negative partial correlations which are also in magnitude small are present. We argue this is an indicator of a positive synergy of an integrated market. Reinforcing this claim, we find that industrial sectors are strongly interconnected even at the level of partial correlations. This is a less studied property, in general and in particular for the Mexican case. Estimation of networks based on different matrices successfully captures different aspects of interdependency. Tail-dependence networks and their centralities maxima have been shown to give the most concise timing for the crisis’s heights (for the subprime and European debt crises). Interdependency from the point of view of (“full”) correlations confirms findings from partial correlations. It also provides evidence of an integrated market for the Mexican case. Indeed, this is what we learned from the estimation of modularities which determined com- munity structure without separating industrial sectors. From filtered matrices with noise fil- tered out (the matrices Cs), a single giant component emerged. Moreover, here the effect of global episodes for interdependency structure was clear even by simple visual inspection. This is what we learned in Fig 11 and is perfect as evidence for the modeling strength. Indeed, cor- relations are more sensitive to trading activity than partial-correlations and capture relation- ships among stocks due to such activity which is even more pronounced at crisis periods. We also studied community structure from the matrices Cg, which are the correlation matrices after noise and the global market mode have been filtered out. Supporting information S1 Table. List of analyzed stocks. (CSV) S1 File. (PDF) S1 Table. List of analyzed stocks. (CSV) S1 File. (PDF) Conclusion At this scale it happens that only a few observed years present a mesoscopic structure. For the years 2000 and 2010 in which mesoscopic structure is present, we observe a “local minimum” for interconnectedness in Fig 11. 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Formal analysis: Erick Treviño Aguilar. Investigation: Erick Treviño Aguilar. Methodology: Erick Treviño Aguilar. Software: Erick Treviño Aguilar. Validation: Erick Treviño Aguilar. Data curation: Erick Treviño Aguilar. Formal analysis: Erick Treviño Aguilar. Investigation: Erick Treviño Aguilar. Methodology: Erick Treviño Aguilar. Software: Erick Treviño Aguilar. Validation: Erick Treviño Aguilar. Data curation: Erick Treviño Aguilar. Validation: Erick Treviño Aguilar. PLOS ONE \| https://doi.org/10.1371/journal.pone.0238731 October 29, 2020 28 / 31 PLOS ONE The interdependency structure in the Mexican stock exchange Visualization: Erick Treviño Aguilar. Writing – original draft: Erick Treviño Aguilar. Writing – review & editing: Erick Treviño Aguilar Visualization: Erick Treviño Aguilar. Visualization: Erick Treviño Aguilar. Writing – original draft: Erick Treviño Aguilar. Writing – review & editing: Erick Treviño Aguilar. Writing – original draft: Erick Treviño Aguilar. 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https://openalex.org/W4382583444	https://www.chimia.ch/chimia/article/download/2023_390/6236	English	null	Enantioselective Switch and Potential Applications in Biocatalysis	Chimia	2,023	cc-by	3,752	390 390 Biocatalysis CHIMIA 2023, 77, No. 6 Chimia 77 (2023) 390–394 © L. Robustini, F. Paradisi doi:10.2533/chimia.2023.390 Correspondence: Prof. Dr. F. Paradisi, E-mail: francesca.paradisi@unibe.ch Dept. Chemistry, Biochemistry and Pharmaceutical Sciences. University of Bern, Freiestrasse 3, CH-3012 Bern Lucia Robustini and Francesca Paradisi Abstract: Enantioselectivity has always been a key feature of enzymatic synthesis. In some cases, when enzymes are not strictly enantioselective, it is possible to induce an enantioselective switch by tuning the reaction condi- tions. A transaminase from Halomonas elongata (ω-HeWT), while generally S-selective, could be shifted towards generating the R-enantiomer at higher concentrations of amino acceptor or ionic strength, for example. Other en- zymes are reported to have a similar behavior, and here we discuss some of them and their potential applications. Keywords: Biocatalysis · Enantiopreference · Enzymes · Transaminase Fig. 1. Example of enantiomers with different features. On the left, S-(+)-carvone mentholated, spicy aroma with bready notes and medium strength. On the right, R-(–)-carvone, has herbal, minty and sweetish medium-strength fragrance. Francesca Paradisi is the Chair of Sus- tainable Pharmaceutical Chemistry at the University of Bern since 2019. Biocatalysis as a sustainable approach to the synthesis of valuable products is the focus of her re- search. Her group developed several en- zyme-based processes in continuous flow, with immobilized biocatalysts, reducing the gap between academic discovery and industrial application. Fig. 1. Example of enantiomers with different features. On the left, S-(+)-carvone mentholated, spicy aroma with bready notes and medium strength. On the right, R-(–)-carvone, has herbal, minty and sweetish medium-strength fragrance. Lucia Robustini is a PhD candidate in the group of Prof. Francesca Paradisi at the University of Bern since April 2020. She graduatedin2015asaFoodBiotechnologist from the University of Ferrara (Italy), with a thesis in Biocatalysis at the University of Freiburg (Germany). Tas, PDB 6GWI) are pyridoxal-5'-phosphate (PLP)-dependent en- zymes that catalyze the formal reductive amination of prochiral substrates, such as ketones to the corresponding chiral amines and are widely recognized for their high enantioselectivity.[7,8] Steric discrimination at the active site is accepted as the explanation for the specificity of this enzyme class.[9] Implementing chiral amine synthesis from small prochiral substrates using Halomonas elon- gata transaminase, an enantioselectivity inversion was observed under specific reaction conditions (Scheme 1).[10–12] Introduction Enantioselectivity is a highly regarded advantage of biocata- lysts, often portrayed as an almost unbeatable feature when com- pared with less performing synthetic chiral catalysts.[1] This key characteristic, which is inherent in a vast majority of biological catalysts, often avoids tedious separations and enables easy reac- tion workup under mild conditions. Downstream purification of the desired product from contaminants, including heavy metals, side-products, solvents, and unwanted stereoisomers, is one of the most significant parameters that influence the cost of a process.[2] The presence of the unwanted enantiomer reduces the reac- tion yield making it a critical step in the production of Active Pharmaceutical Ingredients (APIs)[3,4] or in the fragrance industry, where one or the other enantiomer differ for olfactive properties such as character or intensity (Fig. 1).[5,6] Scheme 1. Oxidative transamination of pro-chiral substrate from Halo- monas elongata Transaminase (HeWT). Scheme 1. Oxidative transamination of pro-chiral substrate from Halo- monas elongata Transaminase (HeWT). In nature, other enzymes were reported to have similar enantio- switching behavior: lipases from Candida rugosa (CRL),[13] ethi- onamide monooxygenase (EthA),[14] or phenylacetone monooxy- genase (PAMO)[14] to name some of them. The enantioselectivity can clearly be influenced by several parameters, such as reaction time, ionic strength, temperature, solvent polarity (logP), water activity (aw), pH, substrate concentration,[15] or biocatalyst con- centration.[16] Enzymes, being chiral in nature, are usually highly selective when applied to their natural substrates. This selectivity tends to remain when the same biocatalysts are tested with non-natural substrates. In some cases, however, enzymes can be tested with particularly challenging substrates, which are not so easily recog- nized by the biocatalyst in an optical sense. ω-Transaminases (ω- Enantioselective Switch and Potential Applications in Biocatalysis Lucia Robustini and Francesca Paradisi* Enantioselective Switch Triggered by Co-/Substrate Concentration The enantiopreference switch was first observed during the scale up of the THF-ketone amination reaction. In this case, the ee % switched from 11% (S) to 17% (R) when the concentration of the amino acceptor was increased from 10 mM to 300 mM, with 5 equivalents of amino donor. To determine whether the switch was due to one of the substrates or their combination, further studies were done. The concentration of amino donor (isopropyl amine, IPA) was first kept constant at 50 mM, while the amino acceptor was increased from 10 to 300 mM. The result showed that the (S)-preference of HeWT was enhanced by increasing the concentration of THF-ketone. However, when the amino donor concentration was increased from 50 mM to 1.5 M, the selectivity curiously shifted from (S) to (R) (Table 1). Fig. 2. Conversion and ee (%) of THF-ketone into THF-amine catalysed by different concentrations of HeWT. Samples of the reactions were ta- ken after (A) 3 hours and (B) 24 hours. donor. This bulky diamine cyclizes upon reaction and therefore the issue of requiring a large excess of amino donor in the reac- tion to shift the equilibrium is resolved.[22] Moreover, this low-cost substrate was used in previous study, resulting in an enhanced ee with excellent conversions.[23] Fig. 3 shows how the behavior of the biocatalyst is similar, when increasing concentration of either cadaverine (Fig. 3A) or IPA (Fig. 3B). In both cases, as the ratio of amino donor vs amino acceptor increases leading to a racemic mixture (Fig. 3A), the concentration of the (R)-enantiomer increases, or even is preferred over the (S) (Fig. 3B). This shared trend indicates that the concen- tration of amine in solution strongly affects the enantiopreference of the HeWT, independently of the specific amine donor. Enantioselective Switch Triggered by Biocatalyst Concentration The enantiopreference of HeWT has been previously tested also with cyclic prochiral ketones.[17] The standard reaction condi- tions include 1 mg/mL of purified enzyme, 10 mM tetrahydrofu- CHIMIA 2023, 77, No. 6 391 391 Biocatalysis Fig. 2. Conversion and ee (%) of THF-ketone into THF-amine catalysed by different concentrations of HeWT. Samples of the reactions were ta- ken after (A) 3 hours and (B) 24 hours. A B A ran-1-one (THF-ketone), 10 mM (S)-α-methylbenzylamine (S-MBA) and 0.1 mM PLP (Scheme 2). ran-1-one (THF-ketone), 10 mM (S)-α-methylbenzylamine (S-MBA) and 0.1 mM PLP (Scheme 2). Scheme 2. Transamination of THF-ketone into THF-amine with HeWT. Reaction conditions: 10 mM THF-ketone, 50 mM amine (S-MBA or iso- propyl amine (IPA), 1 mg/mL HeWT, in KPi buffer (50 mM, pH 8.0), and 30 °C. A Scheme 2. Transamination of THF-ketone into THF-amine with HeWT. Reaction conditions: 10 mM THF-ketone, 50 mM amine (S-MBA or iso- propyl amine (IPA), 1 mg/mL HeWT, in KPi buffer (50 mM, pH 8.0), and 30 °C. The ee (%), calculated after three hours, indicated a decrease in enantiopreference from 18 to 6% when the concentration of HeWT was increased from 0.1 to 5 mg/mL. In fact, as reported by Hanefeld et al.[18] and Gröger et al.[19] the thermodynamic prod- uct, the racemate in this case, is predominant in reaction with high biocatalyst concentrations or when the reaction is performed for longer time. The explanation proposed involved the dissipation of the initial driving forces toward one enantiomer rather than the other one, and the equilibrium established between the two enan- tiomers. On the other hand, the kinetic product, (S)-enantiomer in our case, is favored when the reaction occurs in short period of time or at low enzyme concentration, as the enantiopreference, even if not absolute, is predominant (Fig. 2A). Furthermore, after 24 h with 2 mg/mL of catalyst, the product in solution was already converted into a racemic mixture (Fig. 2B). It was postulated that a plausible reason for this behavior is related to a thermodynamic equilibrium, even though the mechanism at molecular level is not yet fully understood. B B Enantioselective Switch Triggered by Alternative Substrates To better understand the mechanism behind the inversion, al- ternative amino donors and amino acceptors were screened.[20] Specifically, 2-butanone and cadaverine were tested as potential alternatives. The rationale behind screening alternative substrates was primarily related to the potential interaction between the sub- strates and the catalytic residues in the pocket.A similar trend was shown in Humicola lauginosa lipase, where the enantioselectivity changed accordingly to the bulkiness of the substrate: specifically, from an (R)-enantiomer, with 2-phenoxypropanoic acid ester, to an (S)-enantiomer analogue ester with longer acyl moieties.[21] In line with this principle, HeWT was tested with 2-butanone as an alternative amino acceptor to THF-ketone. The results showed that the (S)-selectivity of the enzyme was enhanced by 3 to 11-fold compared to THF-ketone (Table 2). Table 1. Enantioselectivity inversion of HeWT at different substrate concentration. Substrate Concentration (M) ee% THF-ketonea 0.01–0.3 5–15 (S) IPAb 0.05–1.5 10 (S)–7 (R) aIPA constant at 0.05 mM; bTHF-ketone constant at 0.1 mM Table 1. Enantioselectivity inversion of HeWT at different substrate concentration. Substrate Concentration (M) ee% THF-ketonea 0.01–0.3 5–15 (S) IPAb 0.05–1.5 10 (S)–7 (R) aIPA constant at 0.05 mM; bTHF-ketone constant at 0.1 mM Table 1. Enantioselectivity inversion of HeWT at different substrate concentration. In addition, the effect of alternative amino donors was also investigated. Along with IPA, cadaverine was screened as amino aIPA constant at 0.05 mM; bTHF-ketone constant at 0.1 mM 392 Biocatalysis CHIMIA 2023, 77, No. 6 Scheme 3. Transesterification catalysed by AoP between N-acetyl-phe- nyl alanine 2-chloroethyl ester and 1-propanol. Table 2. Alternative amino acceptor: at the same concentration of 10, 100 and 300 mM the (S)-selectivity of the enzyme is enhanced when 2-bu- tanone is used as substrate. The amino donor (IPA) and cofactor were fixed at 50 mM and 100 mM respectively, in KPi buffer (50 mM, pH 8). Substrate Concentration (M) Substrate THF- ketone (ee%) Substrate 2-Butanone (ee%) 10 6 (S) 69 (S) 100 9 (S) 58 (S) 300 14 (S) 53 (S) Scheme 3. Transesterification catalysed by AoP between N-acetyl-phe- nyl alanine 2-chloroethyl ester and 1-propanol. N-acetyl-l-or d-phenyl alanine 2-chloroethyl ester and 1-propa- nol in presence of 18 different anhydrous solvents (Scheme 3).[25] Mesiano et al. observed a similar behavior of the protease, interestingly, also in relation to changes in pressure.[26] This en- zyme in fact (together with subtilisin which was also included in the study) becomes more stereoselective as the pressure increased. Other Parameters with a Key Role in the Behavior As mentioned above, the protease from Aspergillus oryzae inverts its enantiopreference in response to changes in pressure. A similar case was reported for benzoylformate decarboxylase and respective mutants, where the hydrostatic pressure pro- moted the production of the (R)-enantiomer.[27] Other enzymes, however, are reported to exhibit a change in their stereoprefer- ence when other, less obvious, reaction parameters are changed. Thermoanaerobiurn brockii sec-ADH[28] is an enzyme that cata- lyzes the reduction of aliphatic acyclic ketones, resulting in the formation of alcohols such as 2-butanol. Interestingly, the enan- tioselectivity of the resulting alcohol depends on the temperature at which the reaction takes place. Above 26 °C, the enzyme pref- erentially produces the (R)-enantiomer, while below this temper- ature, the (S)-enantiomer is favored. Another thermo-depending enantiospecificity was presented in 2022 by Alphand et al. for a type II Baeyer-Villiger monooxygenase (BVMO) that loses enan- tiopreference at low temperatures.[29] It is also worth mentioning another curious case of an iminoreductase from Amycolatopsis orientalis (AoIRED).[30] Its specificity towards the (S)-enantiomer is extremely high, with ees up to >99%, when the enzyme is fresh- ly prepared, but this changes in favor of the (R)-product when the enzyme is 74 hours old. As mentioned above, the protease from Aspergillus oryzae inverts its enantiopreference in response to changes in pressure. A similar case was reported for benzoylformate decarboxylase and respective mutants, where the hydrostatic pressure pro- moted the production of the (R)-enantiomer.[27] Other enzymes, however, are reported to exhibit a change in their stereoprefer- ence when other, less obvious, reaction parameters are changed. Thermoanaerobiurn brockii sec-ADH[28] is an enzyme that cata- lyzes the reduction of aliphatic acyclic ketones, resulting in the formation of alcohols such as 2-butanol. Interestingly, the enan- tioselectivity of the resulting alcohol depends on the temperature at which the reaction takes place. Above 26 °C, the enzyme pref- erentially produces the (R)-enantiomer, while below this temper- ature, the (S)-enantiomer is favored. Another thermo-depending enantiospecificity was presented in 2022 by Alphand et al. for a type II Baeyer-Villiger monooxygenase (BVMO) that loses enan- tiopreference at low temperatures.[29] It is also worth mentioning another curious case of an iminoreductase from Amycolatopsis orientalis (AoIRED).[30] Its specificity towards the (S)-enantiomer is extremely high, with ees up to >99%, when the enzyme is fresh- ly prepared, but this changes in favor of the (R)-product when the enzyme is 74 hours old. Fig. 3. nantioselective Switch Triggered by Co-solvents Co-solvents have been reported to have an impact on enantio- selectivity and there are indeed many examples in the literature. For instance, α-chymotrypsin exhibits a change of selectivity in anhydrous organic media, suggesting that the concentration of water molecules is critical to maintain the specific conformation of the enzyme active site, hence its specificity.[24] Klibanov et al. noted an inversion in enantiopreference when the protease from Aspergillus oryzae (AoP) catalyses the transesterification between g Currently, it is not fully understood whether this trend is due to the polarity or the bulkiness of solvent molecules interacting with the active site. Enantioselective Switch Triggered by Alternative Substrates In our example, the enantiopreference (ee (%)) of the catalyst improved considerably from yielding an almost racemic mixture in presence of 1.5 M DMSO, up to a significant predominance of the (S)-enantiomer when isopropanol was added to the reaction mix at the same concentration (Fig. 4). Enantioselective Switch Triggered by Co-solvents Other Parameters with a Key Role in the Behavior Changes of the enantiopreference (ee (%)) with different amino donors. In both cases the amino acceptor THF-ketone was 10 mM. The biotransformations were performed for 3 hours with 0.1 mM PLP in KPi buffer (100 mM at pH 8), at 37 °C. Fig. 4. Impact of co-solvent on the enantiopreference (ee (%)), and de- pendence on their logP. Each biotransformation contains purified HeWT (0.25 mg/mL),10 mM THF-ketone, 1 eq. S-MBA, 0.1 mM PLP and 1.5 M of co-solvent in KPi buffer (50 mM, pH 8), at 30 °C. Fig. 4. Impact of co-solvent on the enantiopreference (ee (%)), and de- pendence on their logP. Each biotransformation contains purified HeWT (0.25 mg/mL),10 mM THF-ketone, 1 eq. S-MBA, 0.1 mM PLP and 1.5 M of co-solvent in KPi buffer (50 mM, pH 8), at 30 °C. Fig. 4. Impact of co-solvent on the enantiopreference (ee (%)), and de- pendence on their logP. Each biotransformation contains purified HeWT (0.25 mg/mL),10 mM THF-ketone, 1 eq. S-MBA, 0.1 mM PLP and 1.5 M of co-solvent in KPi buffer (50 mM, pH 8), at 30 °C. Fig. 4. Impact of co-solvent on the enantiopreference (ee (%)), and de- pendence on their logP. Each biotransformation contains purified HeWT (0.25 mg/mL),10 mM THF-ketone, 1 eq. S-MBA, 0.1 mM PLP and 1.5 M of co-solvent in KPi buffer (50 mM, pH 8), at 30 °C. Fig. 3. Changes of the enantiopreference (ee (%)) with different amino donors. In both cases the amino acceptor THF-ketone was 10 mM. The biotransformations were performed for 3 hours with 0.1 mM PLP in KPi buffer (100 mM at pH 8), at 37 °C. Biocatalysis 393 CHIMIA 2023, 77, No. 6 Fig. 5. Enantiopreference shift in dependence of the ionic strength. Each biotransformation contained THF-ketone (10 mM), S-MBA (1 eq.), puri- fied HeWT (0.25 mg/mL), PLP (0.1 mM), and KPi-buffer (50 mM); pH 8, at increasing concentration of (A) NH4Cl (0.05–1.5 M) and (B) NaCl (0.01–3 M). vent. This principle is in line with what we reported, but in most cases, the investigated enzymes are lipases or esterases. There are a few reported cases of inversion under unusual conditions with other enzymes, but these are scattered and quite unique, not enough to envisage a general trend. Other Parameters with a Key Role in the Behavior With our work we postulated that the activity of HeWT is heavily influenced by the orientation of the substrate presented to the active site, with one orientation directing towards the carbonyl moiety and the other towards the C5 position, resulting in the transfer of the amino group to yield either the (R) or (S) configuration. Understanding this phenomenon implies further investigation, including single point mutations, molecular dynamic simulation and modelling. The ability to master the mechanism would certainly provide a valuable tool for the finetuning of biocatalysis, enabling the tai- lored synthesis of specific enantiomers. Acknowledgements The authors acknowledge the Swiss National Science Foundation (SNSF) (grant number200021_192274). Received: March 14, 2023 Received: March 14, 2023 [1] ‘Asymmetric Synthesis with Chemical and Biological Methods’, Eds D. Enders, K. E. Jaeger, 2007, https://doi.org/10.1002/9783527610648. [2] M. M. C. H. van Schie, J. D. Spöring, M. Bocola, P. Domínguez de María, D. Rother, Green Chem. 2021, 23, 3191, https://doi.org/10.1039/D1GC00561H. [3] J. McConathy, M. J. Owens, Prim. Care Companion CNS Disord. 2003, 5, https://doi.org/10.4088/PCC.v05n0202. [4] J. Yan, B. Xiang, D. Wang, S. Tang, M. Teng, S. Yan, Z. Zhou, W. Zhu, J. Agric. Food Chem. 2019, 67, 1784, https://doi.org/10.1021/acs.jafc.8b04536. g p g j [5] C. Caillet, L. Chauvelot-Moachon, J.-L. Montastruc, H. Bagheri, Br. J. Clin. Pharmacol. 2012, 74, 886, https://doi.org/10.1111/j.1365-2125.2012.04262.x. Fig. 5. Enantiopreference shift in dependence of the ionic strength. Each biotransformation contained THF-ketone (10 mM), S-MBA (1 eq.), puri- fied HeWT (0.25 mg/mL), PLP (0.1 mM), and KPi-buffer (50 mM); pH 8, at increasing concentration of (A) NH4Cl (0.05–1.5 M) and (B) NaCl (0.01–3 M). p g j [6] R. Bentley, Chem. Rev. 2006, 106, 4099, https://doi.org/10.1021/cr050049t. [6] R. Bentley, Chem. Rev. 2006, 106, 4099, https://doi.or [7] N. van Oosterwijk, S. Willies, J. Hekelaar, A. C. Terwisscha van Scheltinga, N. J. Turner, B. W. Dijkstra, Biochemistry 2016, 55, 4422, https://doi.org/10.1021/acs.biochem.6b00370. [8] M. D. Patil, G. Grogan, A. Bommarius, H. Yun, Catalysts 2018, 8, 254, https://doi.org/10.3390/catal8070254. p g [9] V. Tseliou, T. Knaus, M. F. Masman, M. L. Corrado, F. G. Mutti, Nat. Commun. 2019, 10, 1, https://doi.org/10.1038/s41467-019-11509-x. In previous experiments, the impact of ionic strength on the enantiopreference of HeWT was investigated. Remarkably, this type of dependence has not been extensively reported in the lit- erature. The screening involved different concentrations of NaCl (0.01–3 M) and NH4Cl (0.05–1.5 M) and the outcome presented a consistent trend where the (S)-selectivity changed in favor of the (R) as the salt concentration increased (Fig. 5). p g [10] E. Hegarty, F. Paradisi, CHIMIA 2020, 74, 890, https://doi.org/10.2533/chimia.2020.890. [11] L. Cerioli, M. Planchestainer, J. Cassidy, D.Tessaro, F. Paradisi, J. Mol. Catal. B Enzym. 2015, 120, 141, https://doi.org/10.1016/j.molcatb.2015.07.009. [12] C. M. Heckmann, L. Robustini, F. Paradisi, ChemBioChem 2022, 23, https://doi.org/10.1002/cbic.202200335. [13] A. Cipiciani, F. Bellezza, F. Fringuelli, M. G. Silvestrini, Tetrahedron Asymm. 2001, 12, 2277, https://doi.org/10.1016/S0957-4166(01)00416-5. [14] G. de Gonzalo, G. Ottolina, F. Zambianchi, M. W. Fraaije, G. Carrea, J. Mol. Catal. B Enzym. 2006, 39, 91, https://doi.org/10.1016/J.MOLCATB.2006.01.010. Considerations and Open Opportunities Despite significant efforts to elucidate the mechanism behind the stereopreference of HeWT, it is still unclear how the switch from (S) to (R) under different conditions occurs. It is not unlikely that various factors could contribute to how the substrate enters the active site, which we expect to be the key step that discriminates the conversion into one enantiomer over the other. The factors that appear to influence the enantioselectivity of HeWT include the presence of solvents (with varying degrees of influence), the ionic strength of the solution, and the ratio of amino donor over the amino acceptor. This was clear, especially when both cadaverine and IPA were used in excess compared to substrate THF-ketone. Other factors, such as temperature, pressure, and the ‘aging’ of the enzyme, have also been shown to affect the stereoselectivity in other enzymes, whereby these factors are not all well understood. These observations suggest that interactions involving H-bonds in the catalytic site, as well as differences in substrate solvation, are critical in determining the stereochemistry of the final product. The main examples reported in literature, with few exceptions, primarily attribute the inversion to the presence of an organic sol- p g [15] C. Zheng, V. T. Pham, R. S. Phillips, Catal. Today 1994, 22, 607, https://doi.org/10.1016/0920-5861(94)80126-6. [16] F. Hollmann, L. G. Otten, in ‘Wiley Encyclopedia of Chemical Biology’, John Wiley & Sons, Inc., Hoboken, NJ, USA, 2009, https://doi.org/10.1002/9780470048672.wecb150. p g [17] C. M. Heckmann, L. Robustini, F. 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Turner, ACS Catal. 2016, 6, 3880, https://doi.org/10.1021/acscatal.6b00782. License and Terms ce se a d e s This is an Open Access article under the terms of the Creative Commons Attribution License CC BY 4.0. The material may not be used for commercial purposes. The license is subject to the CHIMIA terms and conditions: (https://chimia.ch/chimia/about). The license is subject to the CHIMIA terms and conditions: (https://chimia.ch/chimia/about). The deﬁnitive version of this article is the electronic one that can be found at https://doi.org/10.2533/chimia.2023.390
https://openalex.org/W4285801510	https://www.researchsquare.com/article/rs-1837566/latest.pdf	English	null	Recent trends in anthropometric indices and lipoprotein indices as non-invasive biomarkers of atherosclerosis and coronary heart diseases in general population of Amritsar city	Research Square (Research Square)	2,022	cc-by	8,628	Monika Monu (  monikahg.gndu@gmail.com ) Monika Monu (  monikahg.gndu@gmail.com ) Guru Nanak Dev University Guru Nanak Dev University Recent trends in anthropometric indices and lipoprotein indices as non- invasive biomarkers of atherosclerosis and coronary heart diseases in general population of Amritsar city Recent trends in anthropometric indices and lipoprotein indices as non- invasive biomarkers of atherosclerosis and coronary heart diseases in general population of Amritsar city Monika Monu (  monikahg.gndu@gmail.com ) Introduction Above optimal levels of low density lipoprotein cholesterol (LDL-C) observed in the hypercholesterolemic condition can result in deposition of atherosclerotic plaques (Sharifi et al. 2019) in coronary arteries and in the proximal aorta at an early age and therefore increase the susceptibility to cardiovascular diseases (Ference et al. 2012; Trinder et al. 2020). As the condition does not cause symptoms it is often diagnosed on routine blood screening (Karr 2017). On the other hand optimal management strategies exist and have given considerable results (Grundy et al. 2019). Obesity has long been known to increase ratio of dyslipidemia (Gidding 1995) and as the Punjabis generally intake high fat diet which couple with modern lifestyle food habits and sedentary habits (Tripathy et al. 2016) may also be susceptible to dyslipidemia. In Amritsar city obesity was reported among residents in previous studies (Sidhu and Kumari 2000). Various studies have reported the association of dyslipidemia with body mass index (BMI) and waist circumference (Lokanath and Chandrashekariah 2014) considering these as measures of obesity (Bhardwaj et al 2011). Obesity has also been reported to be associated with serum LDL-C levels (Luo et al. 2014; Li et al. 2021). However, there appears to be sparse literature considering lipoprotein indices (total cholesterol/high density lipoprotein; triglyceride/high density lipoprotein; low density lipoprotein/high density lipoprotein) and no study from Amritsar has come to attention apparently. Although BMI and WC are positively associated with serum cholesterol, triglycerides and low density lipoproteins (Bhardwaj et al. 2011) and negatively with HDL-C levels (Shamai et al. 2011) yet there are some reservations as BMI does not distinguish between adipose tissue and lean body mass (Nevill et al. 2006). Similarly though waist circumference (WC), Waist Hip Ratio (WHR) are good predictors of abdominal fat body size however cannot be defined by these variables (Hsieh and Yashuaga 1999). Therefore, in the present study various anthropometric and lipoprotein indices as well as use of traditional anthropometric measurements were made for assessment of obesity and elucidate its association with Hypercholersterolemia. The anthropometric indices assessment includes Body fat percent (BF%), Total body fat mass (TBFM), A body shape index (ABSI), Body Roundness Index (BRI) and Body Adiposity Index (BAI); the lipoprotein indices considered are TG/HDL-C TC/HDL-C and LDL-C/HDL-C. Body fat percentage (BF%) is the total body fat both essential and stored body fat and it depends on BMI and the age of the person (Deurenberg et al. 1991). Research Article Keywords: low density lipoprotein cholesterol, cardiovascular diseases, atherosclerotic plaques, central obesity, body adiposity index, body roundness index, a body shape index, total body fat mass Posted Date: July 18th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1837566/v1 cense:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full Lic Page 1/14 Abstract Elevated low density lipoprotein cholesterol levels in circulation may get deposited in coronary arteries and result in atherosclerotic plaques at an early age and act as risk factor for cardiovascular diseases. More recent and detailed anthropometric indices in addition to traditional indices were assessed in this study along with lipid profile and lipoprotein indices in urban population (n=100) of Amritsar, Punjab. n=48 subjects comprised the case group and n=52 subjects the control group. Study was approved by the Institutional Ethics Committee of Guru Nanak Dev University and individual voluntary written informed consent was provided by the study group. 46% of cases were obese and lipids and lipoprotein indices were significantly higher in obese group while low density lipoprotein cholesterol and low density lipoprotein cholesterol/high density lipoprotein cholesterol also in overweight category. Factor analysis extracted the 7 components out of 23 variables with a cummulative variance of 81.45%. Females had higher central obesity, waist hip ratio, waist height ratio and body adiposity index. Pearson’s correlation analysis revealed a positive significant association of age (r=0.363, p=0.027), socio-economic status (r=0.328,p=0.023), waist hip ratio (r=0.298,p=0.042), waist height ratio (r=0.864,p=0 000) and body mass index (r=0.794,p=0.000) with low density lipoprotein cholesterol levels and of gender with atherogenic index(r=0.363,p=0.011). Also, a significant correlation of total body fat mass with atherogenic index(r=0.286,p=0.048) and and body roundness index with low density lipoprotein(r=0.345,p=0.016) was observed. Multivariate regression analysis revealed age (B=0.318,t=2.380,p=0.022) and gender (B=0.370,t=2.687,p=0.010) to be significant predictors of body roundness index as well as age (B=0.139,t=2.307,p=0.027) of low density lipoprotein cholesterol. The highest prediction accuracy was showed by waist circumference (AUC=0.597) and the least by a body shape index (AUC=0.458) as revealed by Receiver Operating Characteristics analysis. Introduction The TBFM is dependent of BF% as well as age (Deurenberg et al. 1991). The ABSI relies on WC, BMI and height and is associated with abdominal adipose tissue and is considered a risk factor for premature death (Krakauer and Krakauer 2012; Bertoli et al. 2017); it has been also used to evaluate health status of adolescents (Duncan et al. 2013). BRI predicts the percentage of body fat taking into consideration height and waist circumference (Thomas et al. 2013). The BRI can range from 1 to 16 with higher values in more rounded/obese person. The BAI is used to find the percent body fat from measurements of hip circumference of height (Bergman et al. 2011). Lipoprotein indices have shown more predictable considerations for heart diseases compared to values of lipoproteins per se (Calling et al. 2021; Hazem et al. 2021). Among these are TG/HDL TC/HDL and LDL-C/HDL-C ratio. High ratio of triglyceride to HDL-C (TG/HDL-C) is indicative of coronary disease development (da Luz et al. 2008). A simple index of Ischemi heart disease risk in TC/HDL-C (Lemieux et al. 2001; Calling et al. 2021) and for atherosclerosis (Patani et al. 2018; Quispe et al. 2020). Kunutsor et al. (2017) reported high LDL-C/HDL-C ratio as a risk factor for sudden cardiac death In this preliminary study healthy adults residing in an urban area of Amritsar were evaluated for these anthropometric and lipoproteins variables and indices to assess for presence of hypercholesterolemia. Results A total of 120 participants were contacted out of which (n=100; 20-50y of age) met the inclusion criteria and composed the study group. The general, demographic and clinical characteristics of the cases (males=27, females=21) and controls (males=20, females=32) have been presented in Table 1. Hypercholesterolemic and control groups were matched for age, gender, marital status and physical activity. All the study participants were normotensive, non alcoholic and non smokers while only 23% of cases were non vegetarian. On the basis of general obesity (body mass index) 43% of cases were overweight and 46% were obese. On the basis of waist circumference and waist hip ratio, 85% of cases were obese while on the basis of waist height ratio, 89.5% were obese. TG/HDL-C were above optimal in 16% of cases while TC/HDL-C and LDL-C/HDL-C in 8%. Among cases, 10.4% had very high, 27% high, 18.7% borderline high and 48 5% above optimal LDL-C levels while physical activity was not so common with only 8% of cases were moderately active. The control group had normal values for obesity and LDL-C levels and only 9% were moderately active. On comparing the cases and controls (Table 2) the hypercholesterolemic subjects had significantly higher values for weight(p=0.003), body mass index (p=0.016), waist circumference (p=0.017), total cholesterol (p=0.000), low density lipoprotein cholesterol(p=0.000), very low density lipoproteins (p=0.014) along with lipoprotein indices viz. TG/HDL-C (0.009), TC/HDL-C (0.001), LDL-C/HDL-C (P=0.000), while HDL-C levels were significantly higher in healthy group (p=0.025). Also, anthropometric indices including body fat% (p=0.016), total body fat mass (p=0.004) and a body shape index (p=0.012) were significantly higher in patients. On factor analysis (Table 3) out of 23 components 11 components were extracted with cummulative variance of 81.45%. Factor 1(eigen value- 4.766, % variance=19.8% ) was loaded with body fat% , body mass index, total body fat mass, waist circumference, body roundness index and hip circumference. Factor 2 was loaded with total cholesterol, LDL-/HDL-C, LDL-C, TC/HDL-C having eigen value of 4.411 and % variance of 18%. Anthropometric indices were calculated as - Body Mass Index (BMI) = Weight(kg)/Height2(m) (Keys et al 1972) Body Mass Index (BMI) = Weight(kg)/Height2(m) (Keys et al 1972) Body Fat Percentage (B F %) = (1 2 X BMI) + (0 23 X age) - 5.4 (Deurenberg et al 1991) Total Body Fat Mass (TBFM) = B F %/100 X Body weight (kg) (Deurenberg et al 1991) A Body Shape Index (ABSI) = Waist circumference/(BMI2/3 X Height1/2) (Krakauer and Krakauer 2012) Body Roundness Index (BRI) = 364 2 - 365 5 X √(1-[WC/(2π)2/(0 5 X Height)2] (Thomas et al 2013) Body Adiposity Index (BAI) = (Hip circumferece/height1.5) -18 (Bergman et al. 2011) Lipid profile - Latest and fasting blood screening reports of a maximum month old were referred to for noting levels of lipoproteins (TC, TG, HDL-C, LDL-C and VLDL-C). The atherogenic indices were calculated using TC/HDL-C, TC/HDL-C, LDL-C/HDL-C. NCEP ATP (III) guidelines were followed for classification purposes of lipids levels. LDL-C levels were calculated using Friedwald’s equation (Friedwald et al. 1972). Statistical analysis- The IBM/SPSS/21 version was used for analyses. Continuous variables are given as in means±Standard Deviation (SD) and categorical variables as numbers. Based on presence of Hypercholesterolemia as per ATPIII guidelines participants were categorized as cases; others were categorized as controls. Cases and control participants were stratified on the basis of gender, age, BMI and dietary pattern. Chi-square analysis was performed on categorical variables. Principal component analysis was performed for factor extraction. Student’s T-test, Pearson’s correlation analysis and mutlivariate linear regression analysis were performed. Multiple means were compared using one way ANOVA followed by post-hoc tukey’s t-test. A p-value of < 0 05 was considered statistically significant. Methodology The study was approved by the Institutional Ethics Committee of Guru Nanak Dev University and individual voluntary written informed consent was provided by the study group. Participants were contacted from the general population residing in Kabir Park Colony of Amritsar City. Inclusion criteria were residents of Amritsar City, unrelated adults with routine medical check-ups, no incidental/accidental/occupational exposure(s) and no history of chronic disease or recent in the past six months. Exclusion criteria were children, blood-relatives, non-residents of Amritsar, those on prescribed medication and those with health conditions. Using a face to face interview method a questionnaire was used to record personal and demographic information, lifestyle patterns, anthropometric measurements and medical details from routine blood screening reports of the participants. Random sampling study design was adopted and participants were contacted from every fifth house. Anthropometric measurements - using standard protocols (Weiner and Lourie 1981) measurements of height, hip circumference and waist circumference were made using non stretchable measuring tape. WC was measured at the centre of iliac crests and costal margins and hip circumference was measured at the greatest extrusion of buttock muscles Body weight (kg) was taken with minimal clothing on a standard analog weighing scale. Page 2/14 Page 2/14 Physiometric measurements (mmHg) were taken as average of three readings (Systolic blood pressure and Diastolic blood pressure) by using a sphygomanometer (cuff-size 12 5 -34 0 cm) and blood pressure measurements were classified as per Indian Hypertension guidelines (Chobanian et al. 2003; IGH IV 2019). The socioeconomic status (SES) assessed by considering income education and occupation modified Kuppuswamy and Uday Pareekh’s scale (Wani RT 2019). The scale indicates upper 26-29 (SES Class 1), upper middle 16-25 (SES Class 2), lower middle 11-15 (SES Class 3), upper lower 5-10 (SES Class 4) and C5 of lower (SES Class 5). Physical activity was categorized on the basis of duration of time spent in physical activity and in different activities as per Sullivan et al. 2011. Physical activity score of <140 - extremely inactive lifestyle, 140 - 169 - sedentary lifestyle, 170 -1 99 - moderately active person and of >2 00 vigorously active person. Anthropometric indices were calculated as - Discussion the present study is a first of its kind of association between obesity anthopometric variables indices lipids and lipoprotein indices parameters in this region. Lipid levels significantly differed between cases and healthy controls. General (22%) and central obesity (41%) were prevalent in cases and 12% males and 10% females of total participants were obese. Waist circumference, waist hip ratio, hip circumference and body adiposity index were higher in women. The prevalence of overweight and obesity was 28.6% and 12.8%, respectively as reported by Thakur et al. (2016) in Punjab. Central obesity was higher among women (Prasad et al. 2020 ; Deepa et al. 2009) and the present study’s gender differences are in concordance with the previous studies. Lipids (except HDL-C) and lipoprotein indices were higher in obese patients as well as LDL-C and LDL-C/HDL-C in overweight patients while HDL-C levels higher in patients with normal weight which is in agreement with the study carried out by Manawat et al. (2020). Among lipoprotein indices, TG/HDL-C proven to be highly significant predictor of myocardial infarction (Gaziano et al 1997; Bittner et al 2009) and TG/HDL-C >4 found to be powerful indicator of CVDs (Marotta et al 2009; Wan et al 2015). da Luz et al. in 2008 reported 170 subjects having higher TG/HDL-C levels and 72% among them had extensive coronary artery disease, while in present study 16% of cases had higher TG/HDL-C(>4) and an increase in this index may further contribute to progression of coronary artery disease in these 16% cases while 8% of cases had higher TC/HDL-C and LDL-C/HDL-C. A simple index of Ischemic heart disease is TC/HDL-C (Lemieux et al. 2001; Calling et al. 2021) and for atherosclerosis as well (Patani et al. 2018; Quispe et al. 2020); LDL-C/HDL-C high as a risk factor for sudden cardiac death (Kunutsor et al. 2017). In the present study all these indices were higher in cases than healthy controls as in agreement with these previous studies. Waist hip ratio, waist height ratio and body mass index were positively associated with LDL-C levels which may further contribute to CAD/CHD risk as according to studies (Chitra et al 2012; Wang et al 2018; Hazem et al 2021). Results Factor 3 (eigen value=3.018, % variance=12.5%) was loaded with a body shape index, waist hip ratio and waist height ratio VLDL and TG/HDL-C were loaded in factor 4 (eigen value=2.222, % variance=9.258) and body adiposity index and triglycerides in factor 5 (eigen value=1.889, % variance=7.871) Factor 6 (eigen value= 1.663, % variance=6.928) was loaded with socio-ecnomic status gender and age while factor 7 (eigen value=1 580, % variance=6 584) with alcohol intake dietary pattern and HDL-C. Within patients, (Table 4) total cholesterol triglycerides HDL-C VLDL LDL-C and lipoprotein indices were significantly higher (p≤0.001) in obese group while HDL-C levels were significantly elevated in normal BMI group as compared to other groups. Also, LDL-C levels and atherogenic index were significantly higher in overweight patients (p=0.000), while in healthy group no such differences were observed. As illustrated in table 5, Female patients had significantly increased waist circumference (p=0.045), hip circumference (p=0 025), waist hip ratio (p=0 045) and body adiposity index (p=0 000) as compared to male patients while TG/HDL-C(p=0.020) were significantly elevated in male patients. However, no significant differences were observed as a function of diet (Table 6) 6) Page 3/14 Page 3/14 Pearson’s correlation analysis(Table 7) revealed a significant positive correlation of age with TG/HDL-C (r=0.363, p=0.027), body roundness index (r=0.367, p=0.010), body fat% (r=0.307,p=0.034) and BMI (r=0.307, p=0 034) of gender with TG/HDL-C (r=0.363, p=0.011), body roundness index (r=0.422,p=0.003) and body adiposity index (r=0.541,p=0.000), of socioeconomic status with LDL-C levels (r=0.328, p=0 023) and body roundness index(r=0.330,p=0.022), of waist hip ratio with LDL-C (r=0.298, p=0.042) and total cholesterol(r=0.443,p=0.002), of waist height ratio (r=0.864,p=0.000) and BMI (0.794,p=0.000) with LDL-C levels. Furthermore, total body fat mass and body roundness index were significantly correlated with atherogenic index (r=0.286,p=0.048) and LDL-C levels (r=0.345,p=0.016). Multivariate linear regression analysis revealed age to be a significant predictor of TG/HDL-C (B=0.139, t=2.307,p=0.027) and body roundness index (B=0.318,t=2.380,p=0.022); gender of TG/HDL-C(B=0.353,t=2.342,p=0.024), body roundness index(r=0.370,t=2.687 p=0.010) and body adiposity index(B=0.570,t=4.228,p=0.000). Also waist height ratio was a significant predictor of LDL-C(B=0.347,t=1.764,p=0.045). Receiver Operating Characteristics(ROC) (Table 8)analysis revealed that the highest prediction accuracy of risk factors was shown by waist circumference(AUC=0.597), followed by hip circumference(AUC=0.577), waist height ratio(AUC=0.550) and then by total body fat mass (AUC=0.549) and least by a body shape index(AUC=0.458). Declarations nts: Efforts of Dr. Gursatej Gandhi for guidance for planning of study and proof reading are highly acknowledged cknowledgements: Efforts of Dr. Gursatej Gandhi for guidance for planning of study and proof reading are highly Funding: Departmental grants COSIST/SAP/FIST for carrying out study are highly acknowledged. Financial assistance from University Grants Commission- University with Potential for Excellence for Ph.D is highly acknowledged. ors declare no potential conflicts of interest with respect to research, authorship and/or publication of this article Statements and Declarations- Mrs. Monika Monu and Dr. Gursatej Gandhi declare no potential conflict of interest . ments and Declarations- Mrs. Monika Monu and Dr. Gursatej Gandhi declare no potential conflict of interest . Conclusion apparently, the present study is one of its kind which observed differences in obesity based on anthopometric variables and anthropometric indices as well as cardiovascular risks on the basis of lipid levels and lipoprotein indices between hypercholesterolemic and healthy subjects alongwith age and gender differences in general population of urban Amritsar. Also obesity stratified lipid levels and lipoprotein indices and association analysis have been reported in this study. This study has taken into account the non-invasive method of predicting the risk factors for heart diseases so that preventive measures and effective management of the risks should be taken as soon as possible. Increase in risk of cardiovascular diseases and coronary heart diseases in such an early age is a matter of concern and routine check-ups are suggested. The present study concludes the association of age, gender, socio-economic status and obesity with LDL-C levels. Study has certain limitations as well such as it was conducted on a small sample size. For getting better knowledge of such association studies and indices’ behaviour among hypercholesterolemic subjects, hospital based studies with larger sample size must be carried out. Discussion Body mass index, waist circumference and waist height ratio are good non- invasive predictors of severity of coronary artery disease risk (Hazem et al 2021) while body adiposity index and body roundness index proved to be best indicators of estimating CHD risk (Wang et al. 2018). To the best of our knowledge, no studies have reported the association of total body fat mass and body roundness index with LDL-C and atherogenic index. Age and gender emerged as significant predictors of body roundness index and body adiposity index in the present study which is in concordance with Bergman et al. (2011) Also, age and gender were significant predictors of LDL-C and LDL-C/HDL-C, respectively as also reported in the previous study (Trapani and Pallotini 2010). Socio-economic status was reported to be associated with lipids in study conducted by Santo et al. (2019) while age was reported to be associated with body fat% according to Deurenberg et al 1991 and a similar trend of results were revealed in the present study. Fujita et al. (2015) reported that a body shape index was not a good predictor of CVD/CHD risks and the present study’s results are in accordance. Results Pearson’s correlation analysis(Table 7) revealed a significant positive correlation of age with TG/HDL-C (r=0.363, p=0.027), body roundness index (r=0.367, p=0.010), body fat% (r=0.307,p=0.034) and BMI (r=0.307, p=0 034) of gender with TG/HDL-C (r=0.363, p=0.011), body roundness index (r=0.422,p=0.003) and body adiposity index (r=0.541,p=0.000), of socioeconomic status with LDL-C levels (r=0.328, p=0 023) and body roundness index(r=0.330,p=0.022), of waist hip ratio with LDL-C (r=0.298, p=0.042) and total cholesterol(r=0.443,p=0.002), of waist height ratio (r=0.864,p=0.000) and BMI (0.794,p=0.000) with LDL-C levels. Furthermore, total body fat mass and body roundness index were significantly correlated with atherogenic index (r=0.286,p=0.048) and LDL-C levels (r=0.345,p=0.016). Multivariate linear regression analysis revealed age to be a significant predictor of TG/HDL-C (B=0.139, t=2.307,p=0.027) and body roundness index (B=0.318,t=2.380,p=0.022); gender of TG/HDL-C(B=0.353,t=2.342,p=0.024), body roundness index(r=0.370,t=2.687 p=0.010) and body adiposity index(B=0.570,t=4.228,p=0.000). Also waist height ratio was a significant predictor of LDL-C(B=0.347,t=1.764,p=0.045). Receiver Operating Characteristics(ROC) (Table 8)analysis revealed that the highest prediction accuracy of risk factors was shown by waist circumference(AUC=0.597), followed by hip circumference(AUC=0.577), waist height ratio(AUC=0.550) and then by total body fat mass (AUC=0.549) and least by a body shape index(AUC=0.458). 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Demographic and clinical characteristics of the study groups Page 8/14 Cases (n=48) Controls(n=52) Total Males (27) Females (21) c2 (p value) Males (20) Female (32) c2 (p value) c2 (p value) Age (y) 20-35 25 20 0.051(0.821) 15 25 3.220(0.072) 5.702(0.127) 35-50 2 1 5 7 Marital status M 27 21 0.521(0.470) 20 32 0.557(0.455) 2.497(0.114) UM - - - - Dietary pattern Veg 18 19 2.563(0.109) 20 32 0.557(0.455) 17.922(0.000) Non Veg 9 2 - - Alcoholic - - 0.521(0.470) - 0.557(0.455) 2.497(0.114) Non Alcoholic 27 21 20 32 Smoking - - 0.521(0.470) - - 0.557(0.455) Non Smoking 27 21 20 32 Socio-economic status U 12 10 8.227(0.041) 1 5 4.486(0.213) 2.497(0.114) UM 8 5 12 10 UL 2 7 5 12 L 5 - 2 5 BMI(kg/m2)a <18 underweight - - 0.061(0.969) 3 1 1.058(0.303) 25.163(0.002) 18-22.9 normal 3 2 17 31 23-24.9 overweight 12 9 - - >25 obese 12 10 - - Waist circumference(cm)b Normal <85(M),<80(F) 5 2 0.215(0.642) 20 32 0.557(0.455) 34.840(<0.0001) Obese >85(M).>80(F 22 19 - - Waist hip ratioc Normal <0.88(M), <0.81 (F) 6 1 1.659(0.197) 20 32 0.557(0.455) 75.678(<0.0001) Obese >0.88(M),>0.81(F) 21 20 - - Waist height ratiod Normal <0.5 4 1 0.429(0.512) 20 32 0.557(0.455) 76.771(<0.0001) Obese ≥0.5 23 20 - - LDL-C levelse <100 (normal) - - 0.682(0.877) 20 32 0.557(0.455) 82.212(<0.0001) 100-129(above optimal) 12 10 - - 130-159(borderline high) 5 4 - - 160-189(high) 8 5 - - >190(very high) 2 3 - - Physical activityf Inactive lifestyle 12 10 0.623(0.732) 15 19 1.505(0.471) 40.510(<0.0001) Sedentary lifestyle 12 10 4 9 Moderately active person 3 1 1 4 Vigorously active person - - - - TG/HDL-Ce (≤2)normal 21 19 0.610(0.435) 20 32 0.557(0.455) 21.734(<0.0001) (>2) above optimal 6 2 - - TC/HDL-Ce (<5)normal 25 19 0.069(0.792) 20 32 0.557(0.455) 30.229(<0.0001) (≥5) above optimal 2 2 - - LDL-C/HDL-Ce (≤2)normal 23 20 0.091(0.762) 20 32 0.557(0.455) 30.229(<0.0001) (>2) above optimal 3 1 - - al. (2003); dHsieh and Muto (2004); eNCEP ATPIII, eSullivan et al., 2011. Tables TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/h Cases (n=48) Controls(n=52) Total Males (27) Females (21) c2 (p value) Males (20) Female (32) c2 (p value) c2 (p value) Age (y) 20-35 25 20 0.051(0.821) 15 25 3.220(0.072) 5.702(0.127) 35-50 2 1 5 7 Marital status M 27 21 0.521(0.470) 20 32 0.557(0.455) 2.497(0.114) UM - - - - Dietary pattern Veg 18 19 2.563(0.109) 20 32 0.557(0.455) 17.922(0.000) Non Veg 9 2 - - Alcoholic - - 0.521(0.470) - 0.557(0.455) 2.497(0.114) Non Alcoholic 27 21 20 32 Smoking - - 0.521(0.470) - - 0.557(0.455) Non Smoking 27 21 20 32 Socio-economic status U 12 10 8.227(0.041) 1 5 4.486(0.213) 2.497(0.114) UM 8 5 12 10 UL 2 7 5 12 L 5 - 2 5 BMI(kg/m2)a <18 underweight - - 0.061(0.969) 3 1 1.058(0.303) 25.163(0.002) 18-22.9 normal 3 2 17 31 23-24.9 overweight 12 9 - - >25 obese 12 10 - - Waist circumference(cm)b Normal <85(M),<80(F) 5 2 0.215(0.642) 20 32 0.557(0.455) 34.840(<0.0001) Obese >85(M).>80(F 22 19 - - Waist hip ratioc Normal <0.88(M), <0.81 (F) 6 1 1.659(0.197) 20 32 0.557(0.455) 75.678(<0.0001) Obese >0.88(M),>0.81(F) 21 20 - - Waist height ratiod Normal <0.5 4 1 0.429(0.512) 20 32 0.557(0.455) 76.771(<0.0001) Obese ≥0.5 23 20 - - LDL-C levelse <100 (normal) - - 0.682(0.877) 20 32 0.557(0.455) 82.212(<0.0001) 100-129(above optimal) 12 10 - - 130-159(borderline high) 5 4 - - 160-189(high) 8 5 - - >190(very high) 2 3 - - Physical activityf Inactive lifestyle 12 10 0.623(0.732) 15 19 1.505(0.471) 40.510(<0.0001) Sedentary lifestyle 12 10 4 9 Moderately active person 3 1 1 4 Vigorously active person - - - - TG/HDL-Ce (≤2)normal 21 19 0.610(0.435) 20 32 0.557(0.455) 21.734(<0.0001) (>2) above optimal 6 2 - - TC/HDL-Ce (<5)normal 25 19 0.069(0.792) 20 32 0.557(0.455) 30.229(<0.0001) (≥5) above optimal 2 2 - - LDL-C/HDL-Ce (≤2)normal 23 20 0.091(0.762) 20 32 0.557(0.455) 30.229(<0.0001) (>2) above optimal 3 1 - - 04); cSnehalatha et al. (2003); dHsieh and Muto (2004); eNCEP ATPIII, eSullivan et al., 2011. TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- sity lipoprotein aMisra et al. (2009); bWHO, (2004); cSnehalatha et al. (2003); dHsieh and Muto (2004); eNCEP ATPIII, eSullivan et al., 2011. TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- aMisra et al. (2009); bWHO, (2004); cSnehalatha et al. (2003); dHsieh and Muto (2004); eNCEP ATPIII, eSullivan et al., 2011. Tables TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein is (PCA) of studied variables Variables Communalities Components 1 2 3 4 5 6 7 Body fat % 0.950 .970 .033 -.021 .032 .041 .065 -.002 Body mass index 0.950 .970 .033 -.021 .032 .041 .065 -.002 Total body fat mass 0.927 .913 -.017 -.241 .158 -.096 .018 .003 Waist circumference 0.835 .819 -.039 .302 .146 .211 .079 -.008 Body roundness index 0.722 -.648 -.073 -.243 .213 -.410 -.132 .085 Hip circumference 0.779 .637 .018 .078 -.040 .599 -.040 .072 Low density lipoprotein cholesterol 0.956 -.067 .950 .081 -.033 .165 .120 .003 LDL-C/HDL-C 0.907 .135 .913 .044 -.073 -.217 -.018 -.004 TC/HDL-C 0.891 .160 .889 .053 .114 -.239 -.034 -.018 Total cholesterol 0.884 -.094 .833 -.095 .245 .318 .102 -.016 ABSI 0.951 -.146 .023 .958 .010 .038 .091 -.034 WHR 0.948 .023 -.021 .932 .123 -.185 .160 -.058 WHtR 0.996 .259 .030 .931 -.024 .202 .131 -.053 VLDL 0.928 .038 .003 -.040 .948 .130 -.048 -.084 TG/HDL-C 0.870 .132 .123 .182 .878 -.155 -.074 -.073 BAI 0.833 .541 .100 .083 -.288 .663 .027 -.012 Triglycerides 0.422 .076 .049 -.034 .187 .527 -.316 .022 Socioeconomic status 0.706 .206 .118 .120 .008 -.139 .783 -.044 Gender 0.536 .124 -.015 .120 -.199 .092 .675 -.049 Age 0.437 -.151 .198 .148 .193 -.244 .493 -.114 Alcohol intake 0.726 .063 -.124 .054 .002 -.115 .130 -.821 Dietary pattern 0.669 .051 -.083 -.025 -.174 -.125 -.062 .781 HDL-C 0.769 -.022 -.403 -.167 .370 .309 .315 .496 Eigen value 4.766 4.411 3.018 2.222 1.889 1.663 1.580 % of variance 19.857 18.381 12.577 9.258 7.871 6.928 6.584 Cummulative % 19.857 38.237 50.814 60.072 67.943 74.871 81.456 Page 9/14 Page 9/14 TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein, BF%- body fat %, ABSI- a body shape index, WHR- waist hip ratio, WHtR- waist height ratio, VLDL- very low density lipoprotein, BAI- body adiposity index, HDL-C high density lipoprotein cholesterol TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein, BF%- body fat %, ABSI- a body shape index, WHR- waist hip ratio, WHtR- waist height ratio, VLDL- very low density lipoprotein, BAI- body adiposity index, HDL-C high density lipoprotein cholesterol cases and controls Characteristics Case Group (n=48) Control Group (n=52) p- value Mean±S.D. Mean±S.D. Tables Lipid profile of healthy controls as a function of BMI categories Page 10/14 Variables Categories Total cholesterol(mg/dL) Triglycerides (mg/dL) HDL-C (mg/dL) NON-HDL-C (mg/dL) LDL-C (mg/dL) VLDL (mg/dL) TG/HDL-C TC/HDL-C LDL-C/HDL- C Body mass index (kg/m2) Underweight (<18)(n=4) 166.250±17.769 129.625±48.845 55.182±1.232 111.067±18.510 70.272±6.473 40.800±15.403 3.792±1.446 3.051±0.363 1.292±0.142 Normal(18-22.9)(n=32) 143.236±5.086 123.787±19.641 50.380±1.545 92.856±5.109 68.095±3.056 24.760±3.927 2.592±0.419 2.932±0.139 1.414±0.094 Overweight (23-24.9)(n=16) 142.804±5.655 117.095±10.236 51.495±2.515 91.309±5.742 66.929±4.736 24.376±2.382 2.590±0.313 2.902±0.178 1.384±0.132 Obese (>25)(n=48) 155.547±5.294 164.805±15.437 50.610±1.430 104.937±5.360 66.415±3.389 38.519±4.470 3.984±0.506 3.160±0.146 1.363±0.090 (One way ANOVA followed by Post Hoc Tukey t test)#P value 0.129 0.221 0.586 0.199 0.957 0.057 0.104 0.632 0.947 HDL-C high density lipoprotein-cholesterol, LDL-C low density lipoprotein cholesterol, VLDL-very low density lipoprotein, TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein HDL-C high density lipoprotein-cholesterol, LDL-C low density lipoprotein cholesterol, VLDL-very low density lipoprotein, TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein Table 5a. Comparison of continuous variables between males and females within case group Characteristics Males(n=5) Females(n=7) p- value Mean±S.D. Mean±S.D. Age (y) 44.750±6.849 40.750±11.295 0.561 Weight (kg) 78.250±9.878 67.000±11.015 0.179 Body Mass Index (kg/m2) 25.785±3.574 25.281±3.147 0.839 Systolic Blood Pressure (mmHg) 124.500±9.712 123.000±7.000 0.873 Diastolic Blood Pressure (mmHg) 83.250±5.377 80.000±0.000 0.272 Mean Arterial Pressure 97.000±5.873 94.497±2.335 0.459 Pulse Pressure 41.250±8.539 43.500±7.000 0.698 Waist Circumference (cm) 104.000±11.343 107.000±11.604 0.045 Hip Circumference (cm) 105.750±10.045 115.500±8.386 0.025 Waist Hip Ratio 0.924±0.039 0.983±0.051 0.045 Waist Height Ratio 0.597±0.068 0.658±0.062 0.237 Total cholesterol(TC) (mg/dL) 234.300±45.870 214.850±42.858 0.558 Triglycerides(TG) (mg/dL) 198.250±80.446 213.250±55.721 0.770 h density lipoprotein(HDL) (mg/dL) 49.562±6.851 43.655±9.774 0.360 w Density lipoprotein (LDL) (mg/dL) 140.122±37.325 136.092±33.388 0.877 ow density lipoprotein(VLDL) (mg/dL) 39.600±16.036 22.625±12.846 0.150 TG/HDL 2.731±0.224 1.980±0.143 0.020 TC/HDL 4.415±0.958 3.852±0.675 0.375 LDL/HDL 2.667±0.825 2.462±0.682 0.715 BF% 20.385±3.574 19.881±3.147 0.839 Total Body Fat Mass(TBFM) 16.142±4.628 13.561±4.361 0.448 A Body Shape Index(ABSI) 0.038±0.006 0.051±0.012 0.150 Body Roundness Index(BRI) 1.299±0.000 1.299±0.000 0.056 Body Adiposity Index(BAI) 28.141±6.282 37.762±3.448 0.000 p values in bold are significant Table 5a. Comparison of continuous variables between males and females within case group Characteristics Males(n=5) Females(n=7) p- value Mean±S.D. Mean±S.D. TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein, BF%- body fat % p values in bold are significant Tables Age (y) 40.812±14.842 33.000±3.529 0.605 Height (cm) 170.250±8.354 165.562±9.770 0.155 Weight (kg) 74.187±9.724 63.125±9.878 0.003 Body Mass Index (kg/m2) 25.726±3.297 22.981±2.754 0.016 Systolic Blood Pressure (mmHg) 125.375±8.539 126.437±6.439 0.694 Diastolic Blood Pressure (mmHg) 80.187±5.344 78.550±6.345 0.422 Mean Arterial Pressure 95.249±5.389 94.479±4.727 0.671 Pulse Pressure 45.187±8.026 47.937±9.080 0.371 Waist Circumference (cm) 101.687±9.328 91.687±12.809 0.017 Hip Circumference (cm) 107.250±12.315 100.062±11.755 0.102 Waist Hip Ratio 0.953±0.077 0.914±0.041 0.075 Waist Height Ratio 0.600±0.065 0.555±0.084 0.104 Total cholesterol(TC) (mg/dL) 218.743±32.240 154.375±25.061 0.000 Triglycerides(TG) (mg/dL) 185.500±55.860 175.875±11.590 0.058 High density lipoprotein(HDL) (mg/dL) 47.808±8.389 52.523±4.538 0.025 Low Density lipoprotein (LDL) (mg/dL) 137.336±29.006 64.303±21.957 0.000 Very low density lipoprotein(VLDL) (mg/dL) 41.706±34.196 29.225±13.386 0.014 TG/HDL 2.821±1.228 4.146±3.493 0.009 TC/HDL 4.345±1.135 3.052±0.613 0.001 LDL/HDL 2.781±1.097 1.223±0.578 0.000 BF% 20.326±3.297 17.581±2.754 0.016 Total Body Fat Mass(TBFM) 15.294±4.081 11.262±3.159 0.004 A Body Shape Index(ABSI) 0.416±0.010 0.052±0.014 0.012 Body Roundness Index(BRI) 1.299±0.000 1.299±0.000 0.397 Body Adiposity Index(BAI) 30.786±7.747 29.300±7.301 0.581 p values in bold are significant Table 4a. Lipid profile of case group as a function of BMI categories Variables Categories Total cholesterol(mg/dL) Triglycerides (mg/dL) HDL-C (mg/dL) Non hdl-c (mg/dL) LDL-C (mg/dL) VLDL (mg/dL) TG/HDL-C TC/HDL-C LDL-C/HDL-C Body mass index (kg/m2) Obese (>25) (n=48) 284.133±21.530a 248.666±39.176b* 19.626±0.923cd 264.506±20.683e*f 214.620±28.473h*I 49.866±7.802j* 12.936±2.529k* 14.446±0.520l*m 10.856±0.957n*o Overweight (23-24.9) (n=16) 265.575±11.378 225.718±20.056 31.928±2.269 233.646±13.013 196.813±10.075g* 42.359±4.717 8.396±1.286 10.046±1.067 7.442±0.856n* Normal(18- 22.9)(n=32) 237.950±13.075 177.875±7.828 39.823±3.464c 198.126±15.364f 176.463±12.071i 29.400±2.486j 4.330±0.697k 6.966±1.025m 5.117±0.893o Underweight (<18)(n=4) 221.983±5.210a 189.893±9.192b 40.240±1.53d 181.743±5.952e 155.897±4.584gh 31.866±2.382 4.543±0.520 6.005±0.469l 4.209±0.384n (One way ANOVA followed by Post Hoc Tukey t test)#P value 0.001 0.103 0.001 0.000 0.000 0.039 0.001 0.000 0.000 Table 4a. Lipid profile of case group as a function of BMI categories Table 4b. HDL-C high density lipoprotein-cholesterol, LDL-C low density lipoprotein cholesterol, VLDL-very low density lipoprotein, TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein erides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein, BF%- body fat % Table 5b. Comparison of continuous variables between males and females within control group Table 6. Comparison of continuous variables as a function of diet Tables Age (y) 44.750±6.849 40.750±11.295 0.561 Weight (kg) 78.250±9.878 67.000±11.015 0.179 Body Mass Index (kg/m2) 25.785±3.574 25.281±3.147 0.839 Systolic Blood Pressure (mmHg) 124.500±9.712 123.000±7.000 0.873 Diastolic Blood Pressure (mmHg) 83.250±5.377 80.000±0.000 0.272 Mean Arterial Pressure 97.000±5.873 94.497±2.335 0.459 Pulse Pressure 41.250±8.539 43.500±7.000 0.698 Waist Circumference (cm) 104.000±11.343 107.000±11.604 0.045 Hip Circumference (cm) 105.750±10.045 115.500±8.386 0.025 Waist Hip Ratio 0.924±0.039 0.983±0.051 0.045 Waist Height Ratio 0.597±0.068 0.658±0.062 0.237 Total cholesterol(TC) (mg/dL) 234.300±45.870 214.850±42.858 0.558 Triglycerides(TG) (mg/dL) 198.250±80.446 213.250±55.721 0.770 h density lipoprotein(HDL) (mg/dL) 49.562±6.851 43.655±9.774 0.360 w Density lipoprotein (LDL) (mg/dL) 140.122±37.325 136.092±33.388 0.877 ow density lipoprotein(VLDL) (mg/dL) 39.600±16.036 22.625±12.846 0.150 TG/HDL 2.731±0.224 1.980±0.143 0.020 TC/HDL 4.415±0.958 3.852±0.675 0.375 LDL/HDL 2.667±0.825 2.462±0.682 0.715 BF% 20.385±3.574 19.881±3.147 0.839 Total Body Fat Mass(TBFM) 16.142±4.628 13.561±4.361 0.448 A Body Shape Index(ABSI) 0.038±0.006 0.051±0.012 0.150 Body Roundness Index(BRI) 1.299±0.000 1.299±0.000 0.056 Body Adiposity Index(BAI) 28.141±6.282 37.762±3.448 0.000 Table 5a. Comparison of continuous variables between males and females within case group *p values in bold are significant Table 5b. Comparison of continuous variables between males and females within control group Page 11/14 Characteristics Sample Group (n=12) Control Group (n=88) p- value Mean±S.D. Mean±S.D. Age (y) 28.500±11.090 26.750±9.776 0.821 Height (cm) 178.250±7.410 160.00±5.416 0.451 Weight (kg) 71.500±8.698 58.500±10.115 0.099 Body Mass Index (kg/m2) 22.440±1.486 22.742±2.796 0.855 Systolic Blood Pressure (mmHg) 130.000±0.000 125.500±6.806 0.234 Diastolic Blood Pressure (mmHg) 75.000±10.000 77.500±5.000 0.670 Mean Arterial Pressure 93.335±6.670 93.497±1.916 0.964 Pulse Pressure 55.000±10.000 48.000±11.313 0.390 Waist Circumference (cm) 92.250±15.370 92.000±7.118 0.977 Hip Circumference (cm) 100.000±14.142 100.500±7.000 0.952 Waist Hip Ratio 0.919±0.030 0.915±0.019 0.811 Waist Height Ratio 0.516±0.075 0.574±0.035 0.210 Total cholesterol(TC) (mg/dL) 155.350±18.406 161.675±14.766 0.611 Triglycerides(TG) (mg/dL) 173.500±28.524 134.250±76.421 0.373 h density lipoprotein(HDL) (mg/dL) 55.730±6.115 51.185±2.507 0.218 w Density lipoprotein (LDL) (mg/dL) 66.435±14.126 83.620±9.425 0.089 ow density lipoprotein(VLDL) (mg/dL) 34.675±5.625 26.850±15.347 0.375 TG/HDL 3.242±0.618 2.622±1.470 0.467 TC/HDL 2.892±0.329 3.157±0.201 0.219 LDL/HDL 1.245±0.295 1.632±0.135 0.072 BF% 17.040±1.486 17.342±2.796 0.370 Total Body Fat Mass(TBFM) 12.257±2.286 10.345±3.220 0.201 A Body Shape Index(ABSI) 0.043±0.012 0.059±0.018 0.089 Body Roundness Index(BRI) 1.299±0.000 1.299±0.000 0.533 Body Adiposity Index(BAI) 23.927±4.664 31.675±3.263 0.533 Table 6. Comparison of continuous variables as a function of diet Page 12/14 Page 12/14 Page 12/14 Characteristics Vegetarian Non-vegetarian p- value Mean±S.D. Mean±S.D. Tables Body Mass Index (kg/m2) 25.420±0.726 27.533±1.399 0.175 Systolic Blood Pressure (mmHg) 125.375±8.539 126.437±6.439 0.694 Diastolic Blood Pressure (mmHg) 80.187±5.344 78.550±6.345 0.422 Mean Arterial Pressure 95.249±5.389 94.479±4.727 0.671 Pulse Pressure 45.187±8.026 47.937±9.080 0.371 Waist Circumference (cm) 99.486±12.273 102.454±9.114 0.462 Hip Circumference (cm) 101.297±10.434 107.909±12.397 0.084 Waist Hip Ratio 1.009±0.186 0.955±0.081 0.358 Waist Height Ratio 0.626±0.178 0.621±0.067 0.926 Total cholesterol(TC) (mg/dL) 210.627±34.645 225.609±37.170 0.222 Triglycerides(TG) (mg/dL) 173.189±43.374 167.545±35.688 0.696 h density lipoprotein(HDL) (mg/dL) 43.478±7.807 42.940±9.440 0.849 w Density lipoprotein (LDL) (mg/dL) 140.354±35.214 151.457±27.220 0.332 ow density lipoprotein(VLDL) (mg/dL) 23.064±9.833 22.100±10.348 0.787 TG/HDL 2.490±1.089 2.107±0.816 0.287 TC/HDL 4.479±0.945 4.461±1.189 0.959 LDL/HDL 2.987±0.896 3.040±1.119 0.869 BF% 20.020±4.419 22.133±4.642 0.175 Total Body Fat Mass(TBFM) 14.248±6.783 17.152±5.863 0.206 A Body Shape Index(ABSI) 0.621±3.497 0.041±0.017 0.588 Body Roundness Index(BRI) 1.299±0.000 1.299±0.000 0.567 Body Adiposity Index(BAI) 29.795±6.682 32.954±7.098 0.181 TG/HDL-C triglycerides/high density lipoprotein-cholesterol, TC/HDL-C- total cholesterol/high density lipoprotein-cholesterol, LDL/HDL- Low density lipoprotein/high density lipoprotein, Table 7. Pearson’s correlation analysis and multivariate linear regression analysis Study group Predictors Source of variation correlation Multivariate linear Regression r p B value(95%CI) t value p value Sample Group Age LDL-C 0.363 0.027 0.139 2.307 0.027 BRI 0.367 0.010 0.318 2.380 0.022 BF% 0.307 0.034 0.297 1.970 0.055 BMI 0.307 0.034 0.297 1.970 0.055 Gender LDL/HDL 0.363 0.011 0.353 2.342 0.024 BRI 0.422 0.003 0.370 2.687 0.010 BAI 0.541 0.000 0.570 4.228 0.000 Socio-economic status LDL-C 0.328 0.023 0.197 1.240 0.221 BRI 0.330 0.022 0.082 0.563 0.576 WHR LDL-C 0.298 0.042 0.176 0.140 0.889 TC 0.443 0.002 0.035 0.074 0.941 WHtR LDL-C 0.864 0.000 0.347 1.764 0.045 BMI LDL-C 0.794 0.000 0.051 0.345 0.731 TBFM LDL-C/HDL-C 0.286 0.048 0.140 0.736 0.466 BRI LDL-C 0.345 0.016 0.276 1.510 0.139 variate linear regression analysis Study group Predictors Source of variation correlation Multivariate linear Regression r p B value(95%CI) t value p value Sample Group Age LDL-C 0.363 0.027 0.139 2.307 0.027 BRI 0.367 0.010 0.318 2.380 0.022 BF% 0.307 0.034 0.297 1.970 0.055 BMI 0.307 0.034 0.297 1.970 0.055 Gender LDL/HDL 0.363 0.011 0.353 2.342 0.024 BRI 0.422 0.003 0.370 2.687 0.010 BAI 0.541 0.000 0.570 4.228 0.000 Socio-economic status LDL-C 0.328 0.023 0.197 1.240 0.221 BRI 0.330 0.022 0.082 0.563 0.576 WHR LDL-C 0.298 0.042 0.176 0.140 0.889 TC 0.443 0.002 0.035 0.074 0.941 WHtR LDL-C 0.864 0.000 0.347 1.764 0.045 BMI LDL-C 0.794 0.000 0.051 0.345 0.731 TBFM LDL-C/HDL-C 0.286 0.048 0.140 0.736 0.466 BRI LDL-C 0.345 0.016 0.276 1.510 0.139 Table 8. Tables Receiver Operating Charateristics analysis of the studied variables Page 13/14 Test Result Variables Area Under Curve Standard Error Asymptotic Significance Asymptotic 95% Confidence Interval Lower Bound Upper Bound HC .577 .057 .183 .465 .690 WC .597 .057 .094 .486 .708 WHR .529 .058 .622 .414 .643 WHTR .550 .058 .385 .437 .664 BMI .533 .058 .565 .420 .647 BF% .533 .058 .565 .420 .647 TBFM .549 .058 .394 .436 .663 ABSI .458 .058 .465 .343 .572 BRI .498 .058 .975 .384 .613 BAI .494 .058 .918 .380 .608 HC- Hip circumference, WC- waist circumference, WHR- Waist hip ratio, WHTR- Waist height ratio, BMI- body mass index, BF%- Body fat%, TBFM- Total body fat mass, ABSI- a body shape index, BRI- body roundness index, BAI- body adiposity index HC- Hip circumference, WC- waist circumference, WHR- Waist hip ratio, WHTR- Waist height ratio, BMI- body mass index, BF%- Body fat%, TBFM- Total body fat mass, ABSI- a body shape index, BRI- body roundness index, BAI- body adiposity index Table 8. figure Receiver Operating Charateristics analysis of the studied variables Figures Figure 1 Receiver Operating Charateristics analysis of the studied variables HC- Hip circumference, WC- waist circumference, WHR- Waist hip ratio, WHTR- Waist height ratio, BMI- body mass index, BF%- Body fat%, TBFM- Total body fat mass, ABSI- a body shape index, BRI- body roundness index, BAI- body adiposity index Figure 1 Receiver Operating Charateristics analysis of the studied variables HC- Hip circumference, WC- waist circumference, WHR- Waist hip ratio, WHTR- Waist height ratio, BMI- body mass index, BF%- Body fat%, TBFM- Total body fat mass, ABSI- a body shape index, BRI- body roundness index, BAI- body adiposity index Page 14/14 Page 14/14 Page 14/14
W4230830693.txt	https://link.springer.com/content/pdf/bfm%3A978-3-662-36445-1%2F1	de		Methoden der Mathematischen Physik	Springer eBooks	1,937	public-domain	3,170	DIE GRUNDLEHREN DER MATHEMATISCHEN WISSENSCHAFTEN IN EINZELDARSTELLUNGEN MIT BESONDERER BERÜCKSICHTIGUNG DER ANWENDUNGSGEBIETE GEMEINSAM MIT W. BLASCHKE HAMBURG M. BORN C. RUNGE GÖTTINGEN GÖTTINGEN HERAUSGEGEBEN VON R. COURANT GÖTTING E N BAND XII METHODEN DER MATHEMATISCHEN PHYSIK I VON R. COURANT UND D. HILBERT SPRINGER-VERLAG BERLIN HEIDELBERG GMBH 1924 METHODEN DER MATHEMATISCHEN PHYSIK VON R. COURANT ORD. PROFESSOR DER MATHEMATIK AN DER UNIVERSITÄT GÖTTINGEN UND D. HILBERT GEH. REG.-RAT • ORD. PROFESSOR DE R MATHEMATIK AN DER UNIVERSITÄT GÖTTINGEN ERST ER BAND MIT 29 ABBILDUNGEN SPRINGER-VERLAG BERLIN HEIDELBERG GMBH 1924 ISBN 978-3-662-35615-9 ISBN 978-3-662-36445-1 (eBook) DOI 10.1007/978-3-662-36445-1 ALLE RECHTE, INSBESONDERE DAS DER ÜBERSETZUNG IN FREMDE SPRACHEN, VORBEHALTEN. COPYRIGHT 1924 BY SPRINGER-VERlAG BERLIN HEIDELBERG URSPRUNGLICH ERSCHIENEN BEI]ULIUS SPRINGER IN BERLIN 1924 SOFTCOVER REPRINT OF THE HARDCOVER 1ST EDITION 1924 Vorwort. Von jeher hat die Mathematik mächtige Antriebe aus den engen Beziehungen gewonnen, welche zwischen den Problemen und Methoden der Analysis und den anschaulichen Vorstellungen der Physik bestehen. Erst die letzten Jahrzehnte brachten eine Lockerung dieses Zusammenhanges, indem sich die mathematische Forschung vielfach von ihren anschaulichen Ausgangspunkten ablöste und insbesondere in der Analysis manchmal allzu ausschließlich um Verfeinerung ihrer Methoden und Zuspitzung ihrer Begriffe bemühte. So kommt es, daß viele Vertreter der Analysis das Bewußtsein der Zusammengehörigkeit ihrer vVissenschaft mit der Physik und anderen Gebieten verloren haben, während auf der anderen Seite oft den Physikern das Verständnis für die Probleme und Methoden der Mathematiker, ja sogar für deren ganze Interessensphäre und Sprache abhanden gekommen ist. Ohne Zweifel liegt in dieser Tendenz eine Bedrohung für die Wissenschaft überhaupt; der Strom der wissenschaftlichen Entwicklung ist in Gefahr, sich weiter und weiter zu verästeln, zu versickern und auszutrocknen. Soll er diesem Geschick entgehen, so müssen wir einen guten Teil unserer Kräfte darauf richten, Getrenntes wieder zu vereinigen, indem wir unter zusammenfassenden Gesichtspunkten die inneren Zusammenhänge der mannigfaltigen Tatsachen klarlegen. Nur so wird dem Lernenden eine wirkliche Beherrschung des Stoffes ermöglicht und dem Forscher der Boden für eine organische Weiterentwicklung bereitet. Diesem Ziele soll für das Gebiet der mathematischen Physik das vorliegende Buch dienen. Es entwickelt mathematische Methoden, die im Anschluß an klassische physikalische Fragestellungen des 18. und 19. Jahrhunderts ausgebildet worden sind, und sucht die gewonnenen Ergebnisse zu einheitlichen mathematischen Theorien auszugestalten. Vollständigkeit erstreben wir nicht, hoffen aber doch, durch unsere Darstellung den Zugang zu einem wichtigen und an den schönsten Zusammenhängen reichen Gebiete zu erleichtern, dessen weiterer Ausbau eine überaus lohnende und für Mathematik wie Physik gleich ersprießliche Aufgabe ist. Unserer Einstellung entsprechend haben wir uns überaU bemüht, für die Betrachtungen und Beweise die einfachste mögliche Form zu finden. Jede blinde Benutzung des Apparates der Rechnung ist nach Möglichkeit vermieden und durch Überlegungen begrifflicher Natur ersetzt. Abgesehen von dem rein Methodischen enthält der vorliegende Band viele Einzelheiten, die auch dem Kenner neu sein dürften. Ein geschlossenes Ganzes wird dieser Band erst mit dem in Vorbereitung befindlichen zweiten Bande bilden. Durchweg spielen die Gesichtspunkte der Variationsrechnung die beherrschende Rolle, d. h. das Bestreben, mathematische Größen uncl Funktionen durch Extremums- VI Vorwort. eigenschaften zu charakterisieren. Immer mehr erweist sich die Variationsrechnung, in diesem aUgemeinen Sinne verstanden, als mächtiger Hebel der mathematischen Analysis und wichtiges Prinzip der Vereinfachung und Vereinheitlichung. Für den vorliegenden Band im einzelnen muß ich die Verantwortung allein übernehmen, da Anlage und Einzelausführungen zum größten Teil in meiner Hand lagen. Auch habe ich mir die Freiheit genommen, an zahlreichen Stellen des Buches größere Abschnitte aus eigenen Abhandlungen mit geringen Veränderungen abzudrucken. Wenn ich trotzdem darauf bestanden habe, daß auch nach außen hin die Autorschaft meines hochverehrten Lehrers, Kollegen und Freundes Hilbert mit zum Ausdruck kommt, so geschieht dies nicht nur im Hinblick auf das vielfach benutzte Material aus Hilbertschen Abhandlungen und Vorlesungen, welches in erhöhtem Maße noch im zweiten Bande zur Geltung kommen soll; vor allem wünsche ich vielmehr damit zu betonen, daß die hier vertretenen wissenschaftlichen und pädagogischen Bestrebungen Kinder der mathematischen Geistesrichtung sind, welche für immer mit Hilberts Namen verbunden bleiben wird. Über die Einzelheiten des behandelten Stoffes unterrichtet das ausführliche Inhaltsverzeichnis. Für die Anordnung waren methodische. nicht stoffliche Gesichtspunkte maßgebend. Jedes einzelne Kapitel bildet in gewissem Grade eine selbständige Einheit und kann daher im wesentlichen auch ohne Kenntnis der übrigen gelesen werden. Ein ausführliches Register soll die Orientierung in dem Buche erleichtern. Die Literaturangaben, insbesondere die jedem Kapitel beigegebenen Literaturverzeichnisse, machen keinerlei Anspruch auf systematische Vollständigkeit. Für den zweiten Band haben wir uns neben der allgemeinen Behandlung der klassischen Differentialgleichungen der Physik eine ausführliche Erörterung der Existenzfragen und der numerischen Berechnung der Lösungen vorbehalten, wobei die direkten Methoden der Variationsrechnung im Vordergrunde stehen sollen. Ferner sollen im zweiten Bande eine Ergänzung der hier im vierten Kapitel gegebenen Darstellung der Variationsrechnung durch die Hamilton-Jacobische Theorie und Anwendungen auf Fragen der neueren Physik Platz finden. Vielen treuen Helfern bei der Herstellung des Manuskriptes und bei der saueren Arbeit der Korrektur schulde ich Dank: E. BesselHagen, K.Friedrichs, K.Grandfot, E.Hellinger, P.]ordan, H.Kneser, 0. Neugebauer, A. Ostrowski, C. L. Siegel, A. Walther. Ebenso ist es mir eine angenehme Pflicht, auch an dieser Stelle der Verlagsbuchhandlung, welche in ihrer gewohnten Großzügigkeit die Arbeit des Autors durch jedes erdenkliche Entgegenkommen erleichtert hat, meinen herzlichen Dank auszusprechen. Göttingen, am 11. Februar 1924. R. Courant. Inhaltsverzeichnis. Erstes Kapitel. Die Algebra der linearen Transformationen und quadratischen Formen. § 1. Lineare Gleichungen und lineare Transformationen . . . Vektoren. - Seite Lineare Gleichungen, Tensoren, Bilinearformen. § 2. Lineare Transformationen mit linearem Parameter . . . § 3. Die Hauptachsentransformation der quadratischen Formen 6 9 Orthogonale Transformationen. Das Hauptachsenproblem. Die Durchführung der Hauptachsentransformation auf Grund eines Maximumprinzips. - Charakteristische Zahlen und Eigenwerte. Resolvente einer Form. § 4. Die Minimum-Maximum-Eigenschaft der Eigenwerte . . . § 5. Anwendungen . . . . . . . . . . . . . . . . . . . . 16 18 Orthogonale Vektorensysteme. Vollständigkeit. Lineare Darstellung eines Vektors durch ein Vektorensystem. - Lineare Unabhängigkeit und Gramsehe Determinante. - Lösung des zu einer Form gehörigen linearen Gleichungssystems. § 6. Ergänzungen und Aufgaben zum ersten Kapitel . . . . . 24 Determinantenabschätzung von Hadamard. - Simultane Transformation zweier quadratischer Formen in kanonische Gestalt. Trägheitsgesetz der quadratischen Formen. - Bilinearformen und quadratische Formen von unendlich vielen Variablen. - Unendlich kleinelineare Transformationen. -Variierte Systeme. - Auferlegung einer Bindung. - Elementarteiler eines Tensors oder einer Bilinearform. - Literatur zum ersten Kapitel. Zweites Kapitel. Das Problem der Reihenentwicklung willkürlicher Funktionen. § I. Orthogonale Funktionensysteme 33 Definitionen.- Orthogonalisierungvon Funktionen.- Besselsche Ungleichung. Vollständigkeitsrelation. Approximation im Mittel. Orthogonale Transformationen in unendlich vielen Veränderlichen. Gültigkeit der Ergebnisse bei mehreren unabhängigen Veränderlichen. Erweiterung der Voraussetzungen. § z. Das Häufungsprinzip für Funktionen . . . . . . . . . . § 3. Unabhängigkeitsmaß und Dimensionenzahl . . . . . . . Unabhängigkeitsmaß. Funktionenfolge. Asymptotische Dimensionenzahl einer 39 42 VIII Inhaltsverzeichnis. Seite 46 § 4. Die F ouriersche Reihe Vollständigkeit des Systems der trigonometrischen Funktionen. - Die Fouriersehe Reihenentwicklung stetiger Funktionen mit stückweise stetiger Ableitung. - Ausdehnung des Resultates auf stückweise stetige Funktionen. - Die Größenordnung der Fouriersehen Entwicklungskoeffizienten. § 5. Beispiele und Anwendungen für die Fouriersehe Reihe . . . 54 Das Dirichletsche Integral. - Beispiele. - Streckung des Grundgebietes. - Funktionalgleichung der Thetafunktion. - Die Poissonsche Formel. - Mehrfache Fouriersehe Reihen. § 6. Das Fouriersehe Integral. . . . . . . . . . . . . . . . 61 Plausibilitätsbetrachtungen. -Beweis des Fouriersehen Integraltheorems. - Reziprozitätsformeln. § 7. Beispiele für das Fouriersehe Integral 65 § 8. Die Polynome von Legendre 66 . . . . Erzeugung durch Orthogonalisierung der Potenzen 1, x, x~, ... Differentialgleichung und erzeugende Funktion. - Vollständigkeit. § 9. Der Approximationssatz von Weierstraß . . . . . . 69 Erster Beweis. - Zweiter Beweis. - Ausdehnung des Ergebnisses auf Funktionen von mehreren Veränderlichen. § 10. Beispiele anderer Orthogonalsysteme 72 Verallgemeinerung der zu den Legendreschen Polynomen führenden Fragestellung. - Die Tschebyscheffschen Polynome. Die J acobischen Polynome. - Die Hermiteschen Polynome. - Die Laguerreschen Polynome. § 11. Die zu einem Orthogonalsystem gehörigen Integralgleichungen 79 § 12. Ergänzungen und Aufgaben zum zweiten Kapitel. . . . . 82 Die Burwitzsehe Lösung des isoperimetrischen Problems. Gauß' Anwendung der Kugelfunktionen zur numerischen Berechnung von bestimmten Integralen. - Reziprozitätsformeln. - Einiges über Bernoullische Polynome und Zahlen. - Beispiele Fourierscher Reihen von Funktionen mit Unendlichkeitsstellen. - Spektrale Zerlegung durch Fouriersehe Reihe und Fouriersches Integral. - Vollständigkeit des Systems der trigonometrischen Funktionen. - Erzeugung vollständiger Funktionensysteme in mehreren Variablen durch solche in einer Veränderlichen. - Die Mellinschen Umkehrformeln. - Das Gibbssche Phänomen. - Sätze über die Gramsehe Determinante. Anwendung des Lebesgueschen Integralbegriffes. - Literatur zum zweiten Kapitel. Drittes Kapitel. Theorie der linearen Integralgleichungen. § I. Vorbereitende" Betrachtungen . . . Bezeichnungen und Grundbegriffe. Quellenmäßig dargestellte Funktionen. 99 Ausgeartete Kerne. - § 2. Die Fredholmschen Sätze für ausgeartete Kerne . . . . . 102 Inhaltsverzeichnis. § 3. Die Fredholmschen Sätze für einen beliebigen Kern § 4. Die symmetrischen Kerne und ihre Eigenwerte. . . IX Seite 104 107 Existenz eines Eigenwertes bei einem symmetrischen Kern. Die Gesamtheit der Eigenfunktionen und Eigenwerte. - Die Maximum-Minimum-Eigenschaft der Eigenwerte. § 5. Der Entwicklungssatz und seine Anwendungen 117 Der Entwicklungssatz. - Auflösung der inhomogenen linearen Integralgleichung. - Die Bilinearformel für die iterierten Kerne. Der Mercersche Satz. § 6. Die Neumannsehe Reihe und der reziproke Kern 122 § 7. Die Fredholmschen Formeln 124 § 8. Neubegründung der Theorie 128 - Ein Hilfssatz. - Die Eigenfunktionen eines symmetrischen Kernes. Unsymmetrische Kerne. - Stetige Abhängigkeit vom Kern. § 9. Erweiterung der Gültigkeitsgrenzen der Theorie 132 § 10. Ergänzungen und Aufgaben zum dritten Kapitel 134 Beispiele zur allgemeinen Theorie. - Singuläre Integralgleichungen. - Methode von E. Schmidt zur Herleitung der Sätze von Fredholm. - Methode von Enskog zur Auflösung symmetrischer Integralgleichungen. - Methode von Kellogg zur Bestimmung von Eigenfunktionen. - Beispiel eines unsymmetrischen Kernes ohne Nulllösungen. - Valterrasehe Integralgleichungen. - Die zu einem unsymmetrischen Kerne gehörigen adjungierten Orthogonalsysteme. Integralgleichungen erster Art. - Anwendung des Integrals von Lebesgue. - Die Methode der unendlich vielen Variablen. - Minimumseigenschaften der Eigenfunktionen. -Polare Integralgleichungen. - Symmetrisierbare Kerne. - Bestimmung des lösenden Kernes durch Funktionalgleichungen. - Die Stetigkeit der definiten Kerne. - Satz von Hammerstein. - Literatur zum dritten Kapitel. Viertes Kapitel. Die Grundtatsachen der Variationsrechnung. § 1. Die Problemstellung der Variationsrechnung . 142 Maxima und Minima von Funktionen. - Funktionenfunktionen. - Die typischen Probleme der Variationsrechnung. - Die charakteristischen Schwierigkeiten der Variationsrechnung. § 2. Ansätze zur direkten Lösung. 155 Isoperimetrisches Problem. - Das Ritzsehe Verfahren. Minimalfolgen. - Weitere direkte Methoden. - Prinzipielles über die direkten Methoden der Variationsrechnung. § 3. Die Differentialgleichungen der Variationsrechnung . . . . 165 Das einfachste Problem der Variationsrechnung. - Mehrere gesuchte Funktionen. - Auftreten höherer Ableitungen. - Mehrere unabhängige Variable. - Identisches Verschwinden des Eulerschen Ausdruckes. - Homogene Form der Eulerschen Differentialgleichungen. - Die Legendresche Bedingung. Inhaltsverzeichnis. X Seite § 4. Bemerkungen und Beispiele zur Integration der Eutersehen Differentialgleichung. 177 § 5. Randbedingungen . . 179 Allgemeiner Ausdruck für die erste Variation eines Integrals. Freie Ränder, natürliche Randbedingungen und Transversalität. § 6. Variationsprobleme mit Nebenbedingungen . . . . . . . . 186 - Isoperimetrische Probleme. - Endliche Bedingungsgleichungen. Allgemeine Nebenbedingungen. § 7. Der invariante Charakter der Eutersehen Differentialgleichungen . . . . . . . . . . . . . . . . . . . . . . . 193 Allgemeine Formeln. - Transformation von Au. Polarkoordinaten. - Elliptische Koordinaten. § 8. Die Greensehen Formeln. . . . . . . . . . . . . . . . 199 Greensehe Formeln für gewöhnliche Differentialausdrücke zweiter Ordnung. Adjungierte Ausdrücke. - Gewöhnliche Differentialausdrücke höherer Ordnung. - Partielle Differentialausdrücke. - Normalformen im elliptischen, parabolischen und hyperbolischen Falle. Beispiele. § 9. Das Hamittonsehe Prinzip und die Differentialgleichungen der Physik . . . . . . . . . . . . . . . . . . . . . . 207 Das Hamiltonsche Prinzip in der Punktmechanik - Schwingende Saite und schwingender Stab. - Schwingende Membran und Platte. § 10. Ergänzungen und Aufgaben zum vierten Kapitel . . . . . 212 Geometrische Deutung des Multiplikators von Lagrange. ReziVariationsproblem zu gegebener Differentialgleichung. prozität bei isoperimetrischen Problemen. - Kreisförmige Lichtstrahlen. - Das Problem der Dido. - Beispiel eines räumlichen Problems. - Das isoperimetrische Problem auf einer krummen Fläche. - Beispiel eines allgemeinen Mayerschen Problems. - Die Indikatrix und ihre Anwendungen. - Gleichzeitige Variation der abhängigen und unabhängigen Variablen. -Die Sätze von E. Noether über invariante Variationsprobleme. Integrale in der· Punktmechanik. - Transversalität bei mehrfachen Integralen. - Eulersche Differentialausdrücke auf krummen Flächen. - Tetrazyklische und pentasphärische Koordinaten. - Literatur zum vierten Kapitel. Fünftes Kapitel. Die Schwingungs- und Eigenwertprobleme der mathematischen Physik. § 1. Allgemeine Bemerkungen über lineare Differentialgleichungen 221 Das Superpositionsprinzip. Rand bedingungen. - Homogene und unhomogene § 2. Schwingungen von Systemen mit einem Freiheitsgrad . . . Integration der Differentialgleichung. - Anwendung auf die Theorie der Resonanzerscheinungen und der Registrierapparate. Behandlung der unhomogenen Gleichung im allgemeinen Fall. 222 Inhaltsverzeichnis. XI Seite § 3. Systeme von endlich vielen Freiheitsgraden 228 Hauptschwingungen. - Allgemeine Eigenschaften der schwingenden Systeme. - Ubergang von einem System zu einem benachbarten System. § 4. Systeme von unendlich vielen Freiheitsgraden . . . . . . 232 Die Methoden des Grenzübergangs zu unendlich vielen Freiheitsgraden. - Die homogene Saite. - Äußere Kräfte. - Allgemeiner Gedankengang der folgenden Untersuchungen. § 5. Die unhomogene Saite . . . . . . . . . . . . . . . . . 237 Die allgemeine unhomogene Saite und das Sturm-Liouvillesche Eigenwertproblem. - Kleine Unhomogenitäten. § 6. Der schwingende Stab . . . . . . . . . . . . . . . . § 7. Die schwingende Membran. . . . . . . . . . . . . . 24 3 245 Das allgemeine Eigenwertproblem der homogenen Membran. Erzwungene Bewegungen. - Knotenlinien. - Rechteckige Membran. - Kreisförmige Membran. - Besselsche Funktionen. - Die unhomogene Membran. § 8. Die schwingende Platte . . Allgemeines. - . . . . . . . . 256 Kreisförmige Begrenzung. § 9. Andere Eigenwertprobleme . . . . . . . 257 Probleme vom Sturm-Liouvilleschen Typus. Besselsche Funktionen, Legendresche Funktionen beliebiger Ordnung. Jacobische, Hermitesche, Laguerresche, Tschebyscheffsche Polynome. - Wärmeleitung und Eigenwertprobleme. - Schwingungen dreidimensionaler Kontinua. - Eigenwertprobleme der Potentialtheorie. Kugelfunktionen. - Randwertaufgabe der Potentialtheorie und Eigenwertprobleme. Lamesches- Problem. § 10. Die Greensehe Funktion und die Lösung der Eigenwertprobleme mit Hilfe der Integralgleichungstheorie . 273 Die Greensehe Funktion für gewöhnliche Differentialgleichungen. - Zusammenhang mit der Integralgleichungstheoric. - Die Konstruktion der Greensehen Funktion und die Greensehe Funktion im erweiterten Sinne. - Gewöhnliche Differentialgleichungen höherer Ordnung. - Partielle Differentialgleichungen. § 11. Beispiele für Greensehe Funktionen . . . . . . . . . . . 291 Gewöhnliche Differentialgleichungen. - Greensehe Funktion von LI u für Kreis und Kugel. - Greensehe Funktion und konforme Abbildung. - Die Greensehe Funktion der Potentialgleichung für eine Kugeloberfläche. § 12. Ergänzungen und Aufgaben zum fünften Kapitel . Systeme von zwei Freiheitsgraden. - Kegistrierung von Kurven durch Registrierapparate. - Poincares Deutung des Eigenwertproblems bei endlich vielen :Freiheitsgraden. - Dämpfung und Anfachung. - Beispiele zur schwingenden Saite. - Ein Satz über die Schwingungsgleichung. - Schwingungen des frei herabhängenden Seils und Besselsche Funktionen. - Die Greensehe Funktion der Gleichung LI u = 0 für ein Recht flach. - Die Greensehe Funktion für das Innere eines Rechtecks. - Die Greensehe Funktion für einen 299 XII Inhaltsverzeichnis. Kreisring. - Weitere Beispiele für explizit lösbare Fälle der Schwingungsgleichung. - Parameter in den Randbedingungen bei partiellen Differentialgleichungen. - Greensehe Tensoren für Differentialgleichungssysteme. - Belastete Orthogonalität und belastete Integralgleichungen. - Streckenspektrum und Integraldarstellungen. Verallgemeinerung der Entwicklungssätze für schwingende Saite und schwingenden Stab. - Analytische Fortsetzung der Lösungen der Gleichung Llu +Au = 0. - Ein Satz über die Knotenlinien der Lösungen von Alt+ Alt = o. - Beispiel für einen Eigenwert unendlich hoher Ordnung. - Grenzen für die Gültigkeit der Entwicklungssätze. - Literatur zum fünften Kapitel. Sechstes Kapitel. Anwendung der Variationsrechnung auf die Eigenwertprobleme. Seite § I. Die Extremumseigenschaften der Eigenwerte 322 Die klassischen Extremumseigenschaften. - Die Maximum-Minimum-Eigenschaft der Eigenwerte. -Bemerkungen über das Auftreten negativer Eigenwerte. § 2. Allgemeine Folgerungen aus den Extremumseigenschaften der Eigenwerte . . . . . . . . . . . . . . . . . . . . 329 Allgemeine Sätze. - Die Größenordnung der Eigenwerte. - Die Vollständigkeitseigenschaft der Eigenfunktionen. Stetigkeitseigenschaften der Eigenfunktionen. § 3. Der Entwicklungssatz . § 4. Die asymptotische Verteilung der Eigenwerte. Die Differentialgleichung Lllt +Alt = 0 bei Gebieten, welche aus endlich vielen Quadraten oder Würfeln,bestehen. - Ausdehnung des Resultates auf die allgemeine Differentialgleichung L[u] + Aeu = o. - Die Gesetze der asymptotischen Eigenwertverteilung für einen beliebigen Bereich. - Die Gesetze der asymptotischen Eigenwertverteilung für die Differentialgleichung LI u 2 u = 0 in verschärfter Form. -Vektorielle Randwertaufgaben. Das Gesetz der asymptotischen Eigenwertverteilung für die elektrischen Eigenschwingungen eines Hohlraumes. 342 344 + § 5. Die Knoten der Eigenfunktionen . . . . . . . . . . . . 363 § 6. Das asymptotische Verhalten der Sturm- Liouvilleschen Eigenfunktionen und die Erweiterung der Entwicklungssätze 367 Asymptotische Entwicklung der Eigenfunktionen. - Anwendung zur Verschärfung der Entwicklungssätze. - Übertragung der Resultate auf die Besselschen und Legendreschen Funktionen. § 7. Ergänzungen und Aufgaben zum sechsten Kapitel . . . . 375 Ableitung der Minimumseigenschaften der Eigenwerte aus ihrer Vollständigkeit.- Andere Minimumseigenschaften der Eigenwerte.Direkte Ableitung der Eigenwertgleichung. - Stetigkeitseigenschaft der Eigenwerte bei unendlich werdendem a. - Asymptotische Eigenwertverteilungbei der schwingenden Platte. -Aufgaben.- Parameter in den Randbedingungen. - Minimumsätze für Membran und Platte. - Minimumprobleme bei variabler Massenverteilung. - Literatur zum sechsten Kapitel. Inhaltsverzeichnis. XIII Siebentes Kapitel. Spezielle durch Eigenwertprobleme definierte Funktionen. Seite § I. Vorbemerkungen über lineare Differentialgleichungen zweiter Ordnung . . . . . . . . . . 381 § 2. Die Sesselsehen Funktionen . . . . . . . . . . . . . . 382 Anwendung der Laplaceschen Transformation. - Diskussion des Integrationsweges. Hankeische Funktionen. - Besselsche und Neumannsehe Funktionen. Integraldarstellung von ];.,(:>:). - Potenzreihendarstellung für h. (x). - Abhängigkeit vom Parameter),. Eine andere Integraldarstellung der Hankeischen und Besselschen Funktionen. - Charakterisierung der Hankeischen Funktionen durch ihr Verhalten im Unendlichen. - Explizite Darstellung der Neumannscheu Funktion. - Relationen zwischen den Besselschen Funktionen. - Die Nullstellen der Besselschen Funktionen. § 3. Die Kugelfunktionen von Legendre . 416 Das Schläflische Integral. - Die Integraldarstellungen von Laplace. - Die Legendreschen Funktionen zweiter Art. - Zugeordnete Kugelfunktionen. § 4. Die Kugelfunktionen von Laplace . . . . . . . . . . . . 420 + Aufstellung von 2n 1 Kugelfunktionen nter Dimension. Vollständigkeit des gewonnenen Funktionensystems. - Der Entwicklungssatz. - Das Poissonsche Integral. - Die Maxwell-Sylvestersehe Darstellung der Kugelfunktionen. § 5. Asymptotische Entwicklungen 430 Die Methode von Laplace. - Anwendung zum Beweis der Stirlingschen Formel. - Anwendung zur asymptotischen Berechnung der Hankeischen und Besselschen Funktionen für große Argumente. Sattelpunktmethode. - Anwendung der Sattelpunktmethode zur Berechnung der Besselschen Funktionen bei großem Parameter und großem Argument. - Allgemeine Bemerkungen über die Sattelpunktmethode. - Methode von Darboux. - Anwendung der Darbouxschen Methode zur asymptotischen Entwicklung der Legendreschen Polynome. Schlußbemerkung 444 Sachverzeichnis . 445 Druckfehlerberichtigung. Auf S. 40, Zeile 13 von unten, sind hinter die Worte "gleichmäßig stetigen" die Worte "und gleichmäßig beschränkten" einzuschalten.
https://openalex.org/W2916618829	http://eprints.whiterose.ac.uk/132546/1/13722_2017_Article_87.pdf	English	null	Proceedings of the 14th annual conference of INEBRIA	Addiction science & clinical practice	2,017	cc-by	35,606	his is a repository copy of Proceedings of the 14th annual conference of INEBRI White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/132546/ Version: Published Version Version: Published Version Article: Holloway, A.S., Ferguson, J., Landale, S. et al. (330 more authors) (2017) Proceedings of the 14th annual conference of INEBRIA. Addiction science & clinical practice, 12 (Suppl 1). ISSN 1940-0632 https://doi.org/10.1186/s13722-017-0087-8 https://doi.org/10.1186/s13722-017-0087-8 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? Amy Flynn, John R. Knight, Lon Sherritt, Sion K. Harris Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Center for Adolescent Substance Abuse Research, Boston Children’s Hospital, Boston, MA, USA g Correspondence: Aisha Holloway - aisha.holloway@ed.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A1 g Correspondence: Aisha Holloway - aisha.holloway@ed.ac.uk Correspondence: Aisha Holloway - aisha.holloway@ed.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A1 y y Addiction Science & Clinical Practice 2017, 12(Suppl 1): A1 p Correspondence: Amy Flynn - amy.ﬂynn@childrens.harvard.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A2 Background: Addressing alcohol harm in prisons can potentially reduce the risk of re-ofending, and costs to society, whilst tackling health inequalities. Health savings of £4.3 m and crime savings of £100 m per year can be a result of appropriate alcohol interventions. Prison therefore ofers an opportunity for the identiication, response and/or referral to treatment for those male remand prisoners who are consuming alcohol above recommended levels. There is however, limited evidence for the efectiveness, optimum timing of delivery, recommended length, content, implementation and economic beneit of Alcohol Brief Interventions (ABI) in the prison setting for male remand prisoners. As part of the PRISM-A study, we aimed to explore the ‘elements’ of an acceptable ABI for delivery, experiences of engagement with services/health professionals about alcohol use, alongside barriers and facilitators to implementation within the prison setting for male remand prisoners. Background: A previous study found adolescents receiving brief com- puter-facilitated screening and clinician advice (cSBA) in primary care reported lower rates of alcohol use at follow-up compared to usual care. One intervention component was provision of science-based information about alcohol risks for adolescents’ developing brain and health. A hypothesized intervention mechanism was enhancing per- ceived risk of harm of use, and this study examined whether perceived risk mediated the intervention efect. Materials and methods: We analyzed data from a quasi-experimen- tal trial of cSBA among 2096 12–18 year-old patients recruited from 9 New England practices. The study used a before-after design with practices being their own control. An 18-month Treatment as Usual (TAU) phase was followed by clinician training and an 18-month cSBA phase with computer-administered screening, individualized feed- back, health risk information, and clinician brief advice. We stratiied analyses by baseline past-12-month alcohol use and used mediated logistic regression modeling to examine any past-3-month drinking at 3-months. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. MEETING ABSTRACTS MEETING ABSTRACTS Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Addict Sci Clin Pract 2017, 12(Suppl 1):25 DOI 10.1186/s13722-017-0087-8 Addiction Science & Clinical Practice Open Access Published: 14 September 2017 Published: 14 September 2017 Conclusions: The importance of interpersonal trust indicated that intervention delivery by external organizations and nurses were favored in comparison to intervention delivery by prison staf and peer-prisoners. Proceedings of the 14th annual conference of INEBRIA New York, NY, USA, September 14–15, 2017 New York, NY, USA, September 14–15, 2017 Published: 14 September 2017 A1 What does an ABI look like when delivered in the prison setting: perspectives and experiences of male remand prisoners and relevant stakeholders Aisha S. Holloway1, Jennifer Ferguson2, Sarah Landale3, Laura Cariola3, Dorothy Newbury-Birch2 A2 y y 1Nursing Studies, School of Health in Social Science, The University of Edinburgh, Edinburgh, UK; 2Health and Social Care Institute, School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 3School of Health in Social Science, The University of Edinburgh, Edinburgh, UK Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? Amy Flynn, John R. Knight, Lon Sherritt, Sion K. Harris Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Center for Adolescent Substance Abuse Research, Boston Children’s Hospital, Boston, MA, USA Correspondence: Amy Flynn - amyﬂynn@childrens harvard edu Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? Amy Flynn, John R. Knight, Lon Sherritt, Sion K. Harris Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Center for Adolescent Substance Abuse Research, Boston Children’s Hospital, Boston, MA, USA Correspondence: Amy Flynn - amy.ﬂynn@childrens.harvard.edu Add S & Cl l P (S l ) A Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? Amy Flynn, John R. Knight, Lon Sherritt, Sion K. Harris Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Center for Adolescent Substance Abuse Research, Boston Children’s Hospital, Boston, MA, USA Correspondence: Amy Flynn - amyﬂynn@childrens harvard edu Exploring patients’ experiences of alcohol screening and brief intervention delivery in English primary care using Normalization Process Theory Amy J. O’Donnell, Eileen Kaner, Barbara Hanratty Institute of Health and Society, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne, UK Materials and methods: The present investigation used data col- lected as part of a multi-factorial randomized clinical trial (N = 439) comparing SBIRT, eSBIRT, and enhanced usual care for childbearing aged women receiving care in a reproductive health clinic. Partici- pants were pregnant and non-pregnant women presenting for out- patient visits at an urban reproductive healthcare clinic who were at least 18 years of age. Participants in the SBIRT and eSBIRT groups completed satisfaction and alliance ratings following a single-session motivational intervention targeting substance use (items rated on a Likert scale, ranging from 1 strongly disagree to 7 strongly agree). Trained raters independently rated audio recorded SBIRT sessions for the presence of six major intervention components. Raters also rated the occurrence of these components in the eSBIRT program. Descrip- tive analyses and t-tests were used to examine diferences between groups. g y Correspondence: Amy J. O’Donnell - amy.odonnell@newcastle.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A3 Background: Various policy measures have been introduced to encourage alcohol screening and brief intervention (ASBI) implemen- tation in English primary care, including clinical guidelines, inancial incentives, and the incorporation of consumption questions in routine health checks. Whilst there is some evidence of the impact of such measures on GPs and other clinicians, we have little knowledge of the views of patients themselves. We used Normalization Process Theory (NPT) informed interviews to explore patients’ experiences of, and per- spectives on, ASBI delivery in primary care. Materials and methods: Semi-structured interviews with 22 patients who had discussed alcohol consumption in primary care. NPT-based topic guide focused discussions, with interviews audio-recorded and transcribed verbatim. Two-phase analysis: (1) framework analysis to identify emergent themes; (2) thematic mapping against core NPT constructs (coherence, cognitive participation, collective action, relexive monitoring). Results: Participants in both groups were very satisied (M = 6.61 (0.57) for SBIRT; M = 6.36 (0.72) for eSBIRT) and felt allied (M = 6.79 (0.42) for SBIRT; M = 6.47 (0.62) for eSBIRT) with the intervention; though SBIRT participants were signiicantly higher in a few categories of each domain. Motivational intervention components received by each group were similar. g g Results: We found mixed understanding of the adverse health con- sequences of excessive drinking amongst patients (coherence), with particularly limited appreciation of longer-term risks. A3 Exploring patients’ experiences of alcohol screening and brief intervention delivery in English primary care using Normalization Process Theory Amy J. O’Donnell, Eileen Kaner, Barbara Hanratty Institute of Health and Society, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne, UK Correspondence: Amy J. O’Donnell - amy.odonnell@newcastle.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A3 Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? We tested 2 mediator variables representing trajecto- ries from baseline to 3-months in perceived risk of harm (PROH) of trying alcohol, and of binge drinking on weekends. Each trajectory variable had three categories: 2 = stayed high (moderate/great risk), 1 = increased to moderate/great risk, or 0 = stayed at, or declined to, no/low risk. We examined mediation by PROH of trying alcohol among baseline non-users, and PROH of binge drinking among base- line users. Materials and methods: Twenty-four in-depth interviews were conducted with adult male remand prisoners at one Scottish prison (n = 12) and one English prison (n = 12). A focus group at each of the prison sites was held with key stakeholders (e.g. prison nurses, prison oicers, voluntary alcohol/addiction services, health service managers and commissioners). Thematic analysis techniques utilizing NViVo 10 were employed. p y Results: A thematic content analysis of the interviews consistently highlighted that the majority of prisoners relected about the connection between alcohol consumption and criminal ofending, particularly in relation to ofenses involving physical assaults. They also expressed motivation to change their alcohol consumption. Both prisoner interviews and focus groups with stakeholders (N = 2), indicated the value of continuous follow-up support outside of the prison system and also the need to address the lack of stable social environments, which is often associated with alcohol and drug consumption. Stakeholders further identiied organizational barriers to the delivery of ABI, such as limited funding and manageable workloads. Results: Among baseline non-users (n = 1449), the cSBA efect was partially mediated by PROH (trying alcohol) (total efect beta [95% CI] = −0.773 [−1.481, −0.064]; indirect efect −0.066 [−0.206, −0.007]), with cSBA associated with higher PROH over time com- pared to TAU (0.140 [0.026, 0.255]), and higher PROH decreasing odds of reporting past-3-month drinking at follow-up (−0.482 [−0.925, −0.038]). Similarly, among baseline users (n = 647), PROH (binge Page 2 of 24 Page 2 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 drinking) partially mediated the cSBA efect (total efect −0.474 [−0.890, −0.058]; indirect efect −0.096, [−0.245, −0.016]), with cSBA enhancing PROH (0.204 [0.030, 0.378]) and higher PROH reducing odds of reporting drinking (−0.470 [−0.733, −0.207]). Conclusion: A brief computer-facilitated primary care intervention can enhance adolescents’ perceived risk of harm from alcohol, which in turn contributes to reduction in short-term drinking rates. Correspondence: Correspondence: Angeline Adam - angeline.adam@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A5 Correspondence: Angeline Adam - angeline.adam@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A5 Background: The TAPS Tool was developed as a brief substance use screening and assessment instrument for primary care. As part of a validation study of the TAPS Tool, we evaluated its acceptability to patients and feasibility of administration. Materials and methods: Participants (N = 2000) recruited from ive primary care clinics completed interviewer-administered (IA-TAPS) and computer self-administered (SA-TAPS) versions of the TAPS Tool. Time required and requests for assistance were recorded, and participants completed a 10-item questionnaire addressing user-friendliness, com- fort, and format preference. We examined results for all participants and for subgroups: elderly (>65 years), lower education (<high school), alcohol/drug use, sex, race, and ethnicity. Background: The TAPS Tool was developed as a brief substance use screening and assessment instrument for primary care. As part of a validation study of the TAPS Tool, we evaluated its acceptability to patients and feasibility of administration. Acceptability and feasibility of the tobacco, alcohol, prescription medication, and other substance use (TAPS) tool in U.S. primary care patients 1 2 3 Angeline Adam1, Robert P. Schwartz2, Li-Tzy Wu3, Geetha Subramaniam4, Gaurav Sharma5, Jennifer McNeely1 Angeline Adam1, Robert P. Schwartz2, Li-Tzy Wu3, Geetha Subramaniam4, Gaurav Sharma5, Jennifer McNeely1 1Department of Population Health, NYU School of Medicine, New York, NY, USA; 2Friends Research Institute, Inc., Baltimore, MD, USA; 3Department of Psychiatry and Behavioral Sciences, Department of Medicine and Duke Clinical Research Institute, Duke University Medical Center, Durham, NC, USA; 4Center for the Clinical Trials Network, National Institute on Drug Abuse, National Institutes of Health, Bethesda, MD, USA; 5The EMMES Corporation, Rockville, MD, USA Conclusions: This study provides novel patient insights into alcohol prevention practice in England. We conclude there is strong accept- ance of the screening role played by primary care clinicians but patients have less conidence in the efectiveness of alcohol interven- tions themselves. Alongside work to promote the beneits of struc- tured alcohol lifestyle advice, there is a need to better communicate the advantages of drinking within lower risk limits. Correspondence: Exploring patients’ experiences of alcohol screening and brief intervention delivery in English primary care using Normalization Process Theory There was some awareness of current alcohol guidelines but these were viewed lexibly, e.g. to accommodate increased consumption at special events. Screening usually took place within wider lifestyle discussions or new patient registrations; most described the experi- ence as routine and not especially sensitive. Screen-positive patients were unaware of receiving an intervention for their drinking. Whilst patients enacted a range of strategies to limit alcohol consumption (collective action), often involving family or friends (relexive moni- toring), they viewed such strategies as learned through experience rather than based on expert clinician advice. However, despite skep- ticism around clinicians’ ability to elicit truthful information from patients, or stimulate positive change (especially with heavy drink- ers, and against powerful socio-cultural inluencers), there was sup- port for primary care as a legitimate ASBI delivery setting (cognitive participation). Conclusions: Findings suggest that participant satisfaction and alli- ance with SBIRT and eSBIRT are comparable, and that participants are exposed to similar intervention elements regardless of delivery method. Correspondence: Amy M. Loree - amy.loree@yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A4 Conclusion: A brief computer-facilitated primary care intervention can enhance adolescents’ perceived risk of harm from alcohol, which in turn contributes to reduction in short-term drinking rates. Background: Screening, brief intervention, and referral to treatment (SBIRT) delivered in person or electronically (eSBIRT) is recommended for identifying women who use substances and helping them reduce or discontinue their use. However, it is not known how eSBIRT com- pares to SBIRT with respect to patients’ experience of satisfaction and therapeutic alliance with brief interventions. It is also unknown if SBIRT and eSBIRT deliver similar components of a brief intervention. Our aim was to compare satisfaction and alliance ratings following receipt of SBIRT and eSBIRT and to compare intervention components received in both SBIRT groups. Does perceived risk of harm mediate the efects of a primary care alcohol screening and brief advice intervention for adolescents? of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI, USA of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI, USA drinking) partially mediated the cSBA efect (total efect −0.474 [−0.890, −0.058]; indirect efect −0.096, [−0.245, −0.016]), with cSBA enhancing PROH (0.204 [0.030, 0.378]) and higher PROH reducing odds of reporting drinking (−0.470 [−0.733, −0.207]). Correspondence: Amy M. Loree - amy.loree@yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A4 Clinician experiences of healthy lifestyle promotion and perceptions of digital interventions as complementary tools for lifestyle behavior change in primary care 1 1 2 1 3 Anne H. Berman1, Karoline Kolaas1,2, Elisabeth Petersén1,3, Preben Bendtsen4, Erik Hedman1,2, Catharina Linderoth4, Ulrika Müssener4, Kristina Sinadinovic1, Fredrik Spak5, Ida Gremyr6, Anna Thurang1,3 1Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden; 2Gustavsberg Primary Care Clinic, Odelbergs väg 19, Gustavsberg, Sweden; 3Stockholm Center for Dependency Disorders, Box 17914, Stockholm, Sweden; 4Department of Medicine and Health Sciences, Linköping University, Linköping, Sweden; 5Department of Social Medicine, Gothenburg University, Gothenburg, Sweden; 6Division of Service Management and Logistics, Chalmers Technological University, Sweden Background: Due to the need for nurses to have competency in the delivery of aSBI, it is important to address gaps in nursing curricula. The University of Pittsburgh School of Nursing has been infusing SBIRT education into its undergraduate and graduate curricula for years. Most recently, the CDC publication on planning and implementing alcohol screening and brief intervention provided the framework for the WIP program for nurse leaders including: clinical nurse leaders, nursing administrators, and nursing informaticists. The Johns Hopkins School of Nursing (01-117-WIPFA 14-JU) and the Pitt School of Nursing (02-117-WIPFA 14-UPITT) worked together to develop a 13-module, on-line aSBI curricula. Correspondence: Anne H. Berman - anne.h.berman@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A6 Correspondence: Anne H. Berman - anne.h.berman@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A6 Materials and methods: The undergraduate curriculum includes 3-hour didactic, face-to-face SBIRT instruction, 15-weeks of clinical application, and infusion into the junior and senior years. The gradu- ate curriculum includes didactic, face-to-face, and video instruction embedded throughout DNP courses including: history and physical exam, management of acute and chronic conditions, as well as adoles- cent, women, and older adult health courses; followed by clinical infu- sion through the program. Lastly, the curriculum for the nurse leaders builds on JHU and UPITT’s previous successes, enabling the two teams to collaborate on the development of a single product using Articulate Storyline, the premier e-learning development platform hosted on a learning management system. Background: Evidence-based healthy lifestyle promotion in primary health care has been supported internationally by national policies and guidelines but implementation in routine primary health care has been slow. Referral to digital interventions could lead to a larger proportion of patients accessing structured interventions for healthy lifestyle changes, but such referral might have unknown implications for clinicians with patients accessing such interventions. A6 Clinician experiences of healthy lifestyle promotion and perceptions of digital interventions as complementary tools for lifestyle behavior change in primary care 1 1 2 1 3 The integration of alcohol screening and brief intervention (aSBI) into nursing curricula: CDC/American Association of Colleges of Nursing ‑ Workforce Improvement Projects (AACN‑WIP) and other initiatives 1 2 3 The integration of alcohol screening and brief intervention (aSBI) into nursing curricula: CDC/American Association of Colleges of Nursing ‑ Workforce Improvement Projects (AACN‑WIP) and other initiatives 1 2 3 Conclusions: Both formats of the TAPS Tool were well accepted. The SA-TAPS was preferred by subpopulations who may experience more stigma related to substance use, while the IA-TAPS was preferred by those who may have more diiculty using a computer. The time required for the TAPS would be feasible in most primary care settings, but patients who are older or less educated may need assistance with the self-administered version. Ann M. Mitchell1, Deborah Finnell2, Christine L. Savage3, Khadejah F. Mahmoud4 Ann M. Mitchell1, Deborah Finnell2, Christine L. Savage3, Khadejah F. Mahmoud4 1Department of Health and Community Systems, University of Pittsburgh School of Nursing, Pittsburgh, PA, USA; 2Department of Acute and Chronic Care, Johns Hopkins University, Baltimore, MD, USA; 3School of Nursing, Johns Hopkins University, Baltimore, MD, USA; 4School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA Correspondence: Ann M. Mitchell - ammi@pitt.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A7 A4 The mean time to complete the SA-TAPS was 4.5 min: 90% of the participants completed in <7 min. The SA- TAPS was completed more slowly by participants who were elderly (mean 6.1 min) or lower education (mean 6.0 min). Assistance was requested by 8% for the IA-TAPS, and by 25% for the SA-TAPS. SA-TAPS assistance was most frequently requested by those who were elderly (48%), lower education (38%), or used prescription drugs (31%). meaning not being able to do what is perceived as best for the patient due to lack of time and resources; and following one’s perception, meaning working from a gut feeling, which for our participants also meant deviating from clinical routines. Conclusions: In eforts to increase evidence-based practice and lighten the burden of clinicians in primary care, decision- and policy- makers planning the introduction of digital tools for healthy lifestyle promotion will need to explicitly deine their role as complements to face-to-face encounters. Our overriding hope is that this study will contribute to maintaining meaningfulness in the patient-clinician encounter, when digital tools are added to facilitate patient behavior change of unhealthy lifestyle behaviors. The mean time to complete IA-TAPS was 2.4 min; 90% of the partici- pants completed in <3 min. The mean time to complete the SA-TAPS was 4.5 min: 90% of the participants completed in <7 min. The SA- TAPS was completed more slowly by participants who were elderly (mean 6.1 min) or lower education (mean 6.0 min). Assistance was requested by 8% for the IA-TAPS, and by 25% for the SA-TAPS. SA-TAPS assistance was most frequently requested by those who were elderly (48%), lower education (38%), or used prescription drugs (31%). A7 The integration of alcohol screening and brief intervention (aSBI) into nursing curricula: CDC/American Association of Colleges of Nursing ‑ Workforce Improvement Projects (AACN‑WIP) and other initiatives 1 2 3 A4 Satisfaction, alliance and intervention experience: comparing provider‑ versus computer‑delivered brief motivational interventions for substance use among childbearing aged women Amy M. Loree1,2, Kimberly A. Yonkers3, Steven J. Ondersma4, Kate Gilstead-Hayden3, Steve Martino1,2 1VA Connecticut Healthcare System, West Haven, CT, USA; 2Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA; 3Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA; 4Merrill Palmer Skillman Institute and Department Satisfaction, alliance and intervention experience: comparing provider‑ versus computer‑delivered brief motivational interventions for substance use among childbearing aged women Amy M. Loree1,2, Kimberly A. Yonkers3, Steven J. Ondersma4, Kate Gilstead-Hayden3, Steve Martino1,2 Satisfaction, alliance and intervention experience: comparing provider‑ versus computer‑delivered brief motivational interventions for substance use among childbearing aged women 1 2 3 4 Materials and methods: Participants (N = 2000) recruited from ive primary care clinics completed interviewer-administered (IA-TAPS) and computer self-administered (SA-TAPS) versions of the TAPS Tool. Time required and requests for assistance were recorded, and participants completed a 10-item questionnaire addressing user-friendliness, com- fort, and format preference. We examined results for all participants and for subgroups: elderly (>65 years), lower education (<high school), alcohol/drug use, sex, race, and ethnicity. Page 3 of 24 Page 3 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Results: Almost all participants found the TAPS Tool easy to under- stand (99%), and said they would share results with their doctor (95%). 31% preferred the IA-TAPS, 38% the SA-TAPS, and 45% had no prefer- ence. The IA format was more frequently preferred by participants who were elderly (36 vs. 30%); less educated (49 vs. 26%); or used prescrip- tion drugs (34 vs. 30%). The SA format was preferred by participants who were African-American (40 vs. 35%); or used drugs (43 vs. 37%). Results: Almost all participants found the TAPS Tool easy to under- stand (99%), and said they would share results with their doctor (95%). 31% preferred the IA-TAPS, 38% the SA-TAPS, and 45% had no prefer- ence. The IA format was more frequently preferred by participants who were elderly (36 vs. 30%); less educated (49 vs. 26%); or used prescrip- tion drugs (34 vs. 30%). The SA format was preferred by participants who were African-American (40 vs. 35%); or used drugs (43 vs. 37%). The mean time to complete IA-TAPS was 2.4 min; 90% of the partici- pants completed in <3 min. A8 Materials and methods: Screening data from the three models, 19 Contracted Specialists (HEs), 16 In-house Specialists, and 37 In-house Generalists over the course of the 5-year CT SBIRT program (2011– 2016) were used to examine implementation model performance. Outcomes include the percentage of positive cases identiied and sub- stance use outcomes at 6-months following brief intervention. Materials and methods: Screening data from the three models, 19 Contracted Specialists (HEs), 16 In-house Specialists, and 37 In-house Generalists over the course of the 5-year CT SBIRT program (2011– 2016) were used to examine implementation model performance. Outcomes include the percentage of positive cases identiied and sub- stance use outcomes at 6-months following brief intervention. Results: The Contracted Specialist Model (HEs) identiied signii- cantly more positive cases (16.7%) than did the In-house Specialist model (11.1%) or the In-house Generalist model (3.7%). For past 30-day substance use, in a subset of patients followed at 6 months, there were signiicant changes in “days of alcohol binge use,” “days of alcohol use,” or “days of marijuana use” compared to baseline days of use; however, there were no signiicant patient outcome difer- ences across models. A8 Intervening during the event‑speciic pregame: a text message intervention to reduce new students’ alcohol use during Orientation Week Benjamin C. Riordan, Tamlin S. Conner, Jayde A. M. Flett, Damian Scarf Department of Psychology, University of Otago, Dunedin, New Zealand Correspondence: Benjamin C. Riordan - ben.riordan@postgrad.otago. ac.nz Addiction Science & Clinical Practice 2017, 12(Suppl 1): A8 Addiction Science & Clinical Practice 2017, 12(Suppl 1): A8 Addiction Science & Clinical Practice 2017, 12(Suppl 1): A8 Background: University students drink more during events than at any other time. One factor that may underlie the higher amount of alcohol consumed during events is pre-gaming (i.e., drinking before an event). In Study 1, we aimed to quantify the extent to which stu- dents’ pre-gamed before Orientation Week (O’Week) events using intercept surveys. In Study 2, we piloted a text message intervention targeting O’Week pre-gaming sessions, to determine whether we could reduce new students’ drinking during O’Week. Conclusions: The examination of the three models has implications for health policy and clinical practice. Dedicated HEs provide higher quality screening services by identifying at-risk patients at rates more consistent with those deined in the literature. Their intervention out- comes are similar to services provided by higher level, more costly staf. Provider reluctance to implement SBIRT services continues to be a major challenge. The need to explore alternative solutions is needed. Materials and methods: In Study 1 we administered breathalysers and surveys to 335 students who were entering three O’Week events. Participants self-reported the number of drinks they had consumed and their Blood Alcohol Concentration (BAC) was recorded. In Study 2 we trialled a text message intervention with new students residing in two residential colleges (Dorm 1: n = 100, Dorm 2: n = 241) who were assigned to either a control or intervention condition. All students reported their O’Week drinking. Students in the intervention condition also received text messages before four O’Week events at 7:30 p.m. and 9 p.m. that pointed out the social harms of drinking using collo- quial language. Clinician experiences of healthy lifestyle promotion and perceptions of digital interventions as complementary tools for lifestyle behavior change in primary care 1 1 2 1 3 This qualita- tive study aimed to explore the perceptions of clinicians in primary care on healthy lifestyle promotion with or without digital screening and intervention. Results: Education and clinical training had the most pronounced efect on indicators of Role Security including role adequacy (p < .05), role legitimacy (p < .05), and role support (p < .05) and Therapeutic Commitment including role speciic-self esteem (p < .05), and work satisfaction (p < .05) for people who use alcohol in the undergradu- ate student nurses. It had signiicant positive results on role legitimacy (p < .05), motivation (p < .05), and work satisfaction (p < .05) for people who use alcohol in the graduate nurse practitioner students. Lastly, there was also a positive efect on education and attitudes for the nurse leader group. Materials and methods: Focus group interviews were conducted at 10 primary care clinics in Sweden with clinicians from diferent health professions. Transcribed interviews were analyzed using a phenome- nological-hermeneutic method involving naïve understanding, struc- tural analysis and comprehensive understanding. Results: Two major themes captured clinicians’ perceptions on healthy lifestyle promotion: (1) the need for structured professional practice and (2) deicient professional practice as a hindrance to implemen- tation. Sub-themes in theme 1 were striving towards professional- ism, which for participants meant working in a standardized fashion, with replicable routines regardless of clinic, as well as being able to monitor statistics on individual patient and group levels; and embrac- ing the future with critical optimism, meaning expecting to develop professionally but also being concerned about the consequences of integrating digital tools into primary care, particularly regarding the importance of personal interaction between patient and provider. For theme 2, sub-themes were being in an unmanageable situation, Conclusions: These curricula infusion models provide easily accessible education and clinical training, bringing evidence-based aSBI/SBIRT to our current and future nurses. Education infused into multiple courses and levels of curricula can have positive efects on nurses’ Role Secu- rity and Therapeutic Commitment for providing care to individuals with alcohol use problems. Page 4 of 24 Page 4 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Correspondence: Add S & C Correspondence: Brendan J. Clark - brendan.clark@ucdenver.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A10 Background: Alcohol misuse is common in survivors of critical illness and is associated with an increased risk of morbidity and mortality. In this study, we adapted screening, brief intervention and referral to treatment (SBIRT) for survivors of critical illness to include motiva- tional interviewing (MI) and shared decision making techniques. This adaptation was designed to address the challenges in building thera- peutic alliance and autonomy and to address the need for referral to treatment in this population. We created a recovery navigator role to deliver this adapted intervention. We conducted a pilot study to assess acceptability and to ensure the feasibility and idelity of the recovery navigator role. Conclusions: Study 1 demonstrated that pre-gaming makes a sub- stantial contribution to the amount of alcohol students consume during events. Study 2 revealed a text message intervention target- ing pre-gaming reduced O’Week drinking in one dormitory but not another. One explanation for this inding is that the intervention was successful with the generally lighter drinkers in Dorm 1 but not the markedly heavier drinkers in Dorm 2. Materials and methods: This non-randomized pilot study was conducted in two urban medical intensive care units. Men with an AUDIT-C score of 4 or greater or women with an AUDIT-C score of 3 or greater who were admitted to the medical ICU and who provided informed consent were enrolled. We assessed baseline acceptability using the Client Satisfaction Questionnaire-8 (CSQ-8), MI fidelity using the Motivational Interviewing Skill Code version 2.1 (MISC), and feasibility using the percentage of subjects who received at least one session with the recovery navigator and the percentage of patients who received at least one session after hos- pital discharge. A10 A critical illness recovery navigator for alcohol: a pilot study Brendan J. Clark1, Jacqueline Jones2, Kathryne D. Reed1, Rachel M. Hodapp1, Ivor Douglas1, Ellen L. Burnham1, Laura Aagaard2, Paul F. Cook2 1Division of Pulmonary Sciences and Critical Care Medicine, School of Medicine, University of Colorado, Aurora, CO, USA; 2College of Nursing, University of Colorado, Aurora, CO, USA Results: In Study 1, students consumed on average 5.7 drinks before the events with a mean BAC of 0.062. In Study 2, students in Dorm 1 receiving the intervention reported consuming signiicantly fewer drinks during O’Week than those in the control condition (interven- tion = 9.7 vs. control = 15.5; t(98) = 2.138, p = .018) despite report- ing a similar amount of pre-university drinking (intervention = 5.8 vs. control = 6.4). However, there was no diference in drinking by Dorm 2 students at any time (O’Week: intervention = 36.7, control = 37.7, p = .768; pre-university: intervention = 13.4 vs. control = 16.0, p = .178). A8 Intervening during the event‑speciic pregame: a text message intervention to reduce new students’ alcohol use during Orientation Week Benjamin C. Riordan, Tamlin S. Conner, Jayde A. M. Flett, Damian Scarf Department of Psychology, University of Otago, Dunedin, New Zealand Correspondence: Benjamin C. Riordan - ben.riordan@postgrad.otago. ac.nz Addiction Science & Clinical Practice 2017, 12(Suppl 1): A8 Results: The Contracted Specialist Model (HEs) identiied signii- cantly more positive cases (16.7%) than did the In-house Specialist model (11.1%) or the In-house Generalist model (3.7%). For past 30-day substance use, in a subset of patients followed at 6 months, there were signiicant changes in “days of alcohol binge use,” “days of alcohol use,” or “days of marijuana use” compared to baseline days of use; however, there were no signiicant patient outcome difer- ences across models. A11 the implementation of adolescent SBI for substance use within a pri- mary care setting in the US. Two diferent implementation models for conducting brief interventions (BIs) were compared: the Generalist Model (GM), with BIs provided by a primary care provider (PCP), and the Specialist Model (SM), with BIs provided by behavioral health per- sonnel after hand-of by the PCP. Understanding how costs and out- comes vary by model is important for providers to plan for services, and for decision makers considering widespread implementation of SBI. We estimate the cost and cost-efectiveness of the two models of implementation. Optimizing the impact of alcohol and drug screening and brief intervention among a high‑risk population receiving services in New York City sexually transmitted disease clinics: a process and outcome evaluation of Project Renew Brett R. Harris1, Jiang Yu2, Margaret Wolﬀ3, Meighan Rogers4 1Department of Health Policy, Management, and Behavior, University at Albany School of Public Health, Rensselaer, NY, USA; 2Center on Addictions Research, University at Albany School of Social Welfare, Albany, NY, USA; 3Mount Sinai Icahn School of Medicine, New York, NY, USA; 4Bureau of STD Control, NYC Department of Health and Mental Hygiene, Long Island City, USA Correspondence: Brett R. Harris - brh24@nycap.rr.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A11 Materials and methods: The cost of SBI services was calculated using an activity-based costing methodology. Cost collection instruments retrieved staf time spent in each delivery activity and quantity and type of non-labor resources. Efectiveness was measured as the per- centage of patients receiving BI amongst those who needed it and retrieved from electronic medical records. Sensitivity analyses were conducted on the time and unit cost of hand-ofs. Correspondence: Brett R. Harris - brh24@nycap.rr.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A11 Correspondence: Brett R. Harris - brh24@nycap.rr.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A11 Background: Unhealthy substance use is associated with increased rates of sexually transmitted diseases (STDs), including HIV. In a high- risk STD clinic population in New York City (NYC), 30.5 and 16.5% reported a lifetime or current substance use disorder, respectively, yet only 1.4% were in treatment and 13.2% had ever been in treatment. Screening, brief intervention and referral to treatment (SBIRT) was irst implemented in STD clinics in 2005, piloted in one NYC clinic, with the goal of reducing substance use and the associated risky behaviors which increase the risk STD acquisition. A9 The examination of three SBIRT implementation models Bonnie McRee, Janice Vendetti, Karen Steinberg Gallucci, Kate Robaina Department of Community Medicine and Health Care, University of Connecticut School of Medicine, Farmington, CT, USA Correspondence: Bonnie McRee - mcree@uchc.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A9 The examination of three SBIRT implementation models Bonnie McRee, Janice Vendetti, Karen Steinberg Gallucci, Kate Robaina Department of Community Medicine and Health Care, University of Connecticut School of Medicine, Farmington, CT, USA Correspondence: Bonnie McRee - mcree@uchc.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A9 Background: The CT Screening Brief Intervention and Referral to Treatment (SBIRT) Program has employed three diferent models to implement services in 13 Federally Qualiied Health Centers. Initially, the program utilized the Contracted Specialist or Health Educator (HE) model. HEs were supervised by an outside agency to remove the time constraints identiied by the health care providers. Approximately two years later, the model shifted so that HEs became employees of and were supervised by the health centers. This model, the In-house Specialist, was intended to promote cohesion among the HE and health care team and to help sustain the program. To further address post-grant sustainability, a third model, the In-house Generalist, was utilized. In this model, in-house medical assistants were trained to administer the pre-screening tool and nurses or behavioral health staf conducted the full screen and provided the brief interventions, brief treatments or referrals to those screening positive for at-risk use. Results: We screened 37 and enrolled 8 patients over the course of 8 weeks. The median age of patients was 49 (range 31–66), 75% were male, and the median AUDIT score was 23 (range 12–33). Seven patients had an alcohol use disorder. The median CSQ-8 score was 32 (range 28–32), consistent with a high level of satisfaction. Each patient received at least one session with the recovery navigator (range 1–8) and six had at least one follow-up session after hospital discharge. Median MISC global ratings for acceptance, empathy, MI spirit and client self-explora- tion were 7 (range 5–7), 6 (range 4–7), 5 (range 4–6), and 6 (range 4–7), respectively; 98% of therapist utterances were MI consistent. Conclusions: These preliminary results demonstrate that a critical illness recovery navigator can deliver an adapted, MI-based SBIRT intervention to patients starting in the hospital and continuing after hospital discharge. A9 Page 5 of 24 Page 5 of 24 Page 5 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Would universal delivery of Screening and Brief Intervention in primary care improve or exacerbate alcohol‑related inequalities in health? A modelling study in England Colin Angus1, Sidney Sherborne1, Duncan Gillespie1, Amy O’Donnell2, Petra Meier1, Alan Brennan1 1School of Health and Related Research, University of Sheﬃeld, Sheﬃeld, Would universal delivery of Screening and Brief Intervention in primary care improve or exacerbate alcohol‑related inequalities in health? A modelling study in England Colin Angus1, Sidney Sherborne1, Duncan Gillespie1, Amy O’Donnell2, Petra Meier1, Alan Brennan1 1S h l f H l h d R l d R h U i i f Sh ﬃld Sh ﬃld Results: 130,597 pre-screenings for risky substance use were con- ducted, 66,989 (51%) of which were positive leading to 17,474 brief interventions and 1138 referrals. Between baseline and follow-up, there was a 19.7 and 43.2% decrease in days of alcohol and drug use, respectively (p < .05). Greater decreases in use were seen among patients ofered EBI (36.0 and 55.9% decrease in days of alcohol and drug use, respectively). Patients also self-reported reductions in num- ber of sexual contacts and experienced fewer days of depression and anxiety (p < .05). 1School of Health and Related Research, University of Sheﬃeld, Sheﬃeld, UK; 2Institute for Health and Society, University of Newcastle, Newcastle upon Tyne, UK Correspondence: Colin Angus - c.r.angus@sheﬃeld.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A13 Correspondence: Colin Angus - c.r.angus@sheﬃeld.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A13 Background: National public health bodies in the UK have repeatedly advocated for a universal program of Screening and Brief Interven- tions (SBIs) in primary care. Alcohol is a major driver of socioeconomic inequalities in health and population-level interventions are com- monly found to worsen inequalities unless speciically designed to avoid this. We aimed to quantify the likely impact of universal SBI delivery on health inequalities. Conclusions: Project Renew successfully expanded the reach of ser- vices from previous project iterations and led to reductions in sub- stance use, sexual risk behavior, and poor mental health which may help to prevent acquisition of HIV or other STDs. Based on positive results, services have been sustained under the ThriveNYC initiative, ensuring essential care to a large population of high-risk New Yorkers. Materials and methods: We analyzed national survey data on alco- hol consumption, primary care usage, AUDIT scores and self-reported SBI receipt, alongside hospital admissions and mortality records, to quantify the socioeconomic gradients in these outcomes. Results were combined using the Sheield Alcohol Policy Model to estimate the long-term health impacts of universal screening and their distribution across the socioeconomic spectrum. ost‑efectiveness of SBI implementation for adolescents l b 1 d d h 2 l 2 h Cost‑efectiveness of SBI implementation for adolescents Carolina Barbosa1, Brendan J. Wedehase2, Laura J. Dunlap2, Shannon G. Mitchell3, Kristi A. Dusek3, Robert P. Schwartz3, Jan Gryczynski3, Arethusa S. Kirk4, Marla T. Oros5, Colleen Hosler5, Kevin E. O’Grady6, Barry S. Brown7 1Behavioral Health Economics Program, RTI International, Chicago, IL, USA; 2Behavioral Health Economics Program, RTI International, Research Triangle Park, NC, USA; 3Social Research Center, Friends Research Institute, Baltimore, MD, USA; 4Total Health Care, Baltimore, MD, USA; 5Mosaic Group, Towson, MD, USA; 6Department of Psychology, University of Maryland, College Park, MD, USA; 7Department of Psychology, University of North Carolina Wilmington, Wilmington, NC, USA Correspondence: Carolina Barbosa - cbarbosa@rti.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A12 p Results: Individuals in the most deprived social group drank 16.3% less on average, yet were 14.5% more likely to attend primary care and 84.0% more likely to screen positive on AUDIT than those in the least deprived group, after adjusting for sociodemographic factors. Rates of hospitalization and mortality due to alcohol were 4.2 and 3.0 times greater respectively in the most deprived groups. Combining these social gradients, we estimate that the most deprived group would receive 27.2% fewer interventions than the least deprived, but experi- ence a 16.4% greater reduction in alcohol-related deaths. A11 Building upon lessons learned, Project Renew represents the third iteration of SBIRT implementation in NYC STD clinics with the goal of expanding the reach of SBIRT ser- vices within and across STD clinics citywide and decreasing substance use, poor mental health, and risky behavior. Results: The average cost of SBI per screen positive visit was $4 (SD $0.38) and $5 (SD $0.8) in generalist and specialist sites, respectively. The average time of a BI in the SM was more than triple that in the GM (18 vs. 5 min), and only 7% of patients needing a BI received one in the SM compared to 38% in the GM. In the SM, the hand-of process repre- sented a signiicant use of resources that was not required in the GM. The GM economically dominated the SM as it was both more efective and less costly. In sensitivity analyses, there were situations where the GM was more efective but at an additional cost, making the GM more cost-efective for willingness-to-pay thresholds below $1 per one addi- tional percentage increase in BI delivery. Materials and methods: SBIRT services were delivered February 2012-January 2015. Patients screening positive for substance mis- use on the AUDIT and/or DAST-10 were provided a brief intervention and interviewed using the Substance Abuse and Mental health Ser- vices Administration Government Performance and Results Act data collection tool at baseline and six-month follow-up. Patients scoring in Zones 3–4 (AUDIT > 15 or DAST-10 > 2) were ofered additional sessions of extended brief intervention (EBI). Service delivery was assessed using electronic medical records. Conclusion: The integration of behavioral health personnel into the BI delivery process in a primary-care setting providing general medi- cal and behavioral health services to adolescents might not be a cost- efective approach. A14 Efectiveness of a brief intervention group performed by nurses in addressing hazardous and harmful alcohol use Divane de Vargas, Janaina Soares Department of Maternal-Infant and Psychiatric Nursing, University of São Paulo, School of Nursing, São Paulo, Brazil Correspondence: Divane de Vargas - vargas@usp.br Addiction Science & Clinical Practice 2017, 12(Suppl 1): A14 A14 Efectiveness of a brief intervention group performed by nurses in addressing hazardous and harmful alcohol use Divane de Vargas, Janaina Soares Department of Maternal-Infant and Psychiatric Nursing, University of São Paulo, School of Nursing, São Paulo, Brazil Correspondence: Divane de Vargas - vargas@usp.br Addiction Science & Clinical Practice 2017, 12(Suppl 1): A14 Results: 782 patients participated. One-third (31.6%) screened posi- tive for unhealthy alcohol use and 19.7% for any drug use. Rates of being unemployed or unable to work were 44.8% overall, 46.9% for those with unhealthy alcohol use (χ2 p = 0.43 for diference between those who did vs. did not screen positive), and 62.8% for those with drug use (p < .01). Homelessness rates (including living “doubled up”) in the past year were 21.2% overall, 30.0% for those with unhealthy alcohol use (p < .01), and 43.4% for those with drug use (p < .01). Ina- bility to meet essential expenses was 42.7% overall, 43.0% for those with unhealthy alcohol use (p = 0.88), and 59.6% for those with drug use (p < .01). Telephone service was disconnected for 21.7% overall, 25.6% for those with unhealthy alcohol use (p = 0.11), and 29.8% for those with drug use (p = 0.02). Food insecurity rates were 52.9% over- all, 57.5% for those with unhealthy alcohol use (p = 0.09), and 65.6% for those with drug use (p < .01). Background: In Brazil, 20% of those who consume alcohol fulill cri- teria for hazardous/harmful use. This phenomenon contributes to a high prevalence of individuals who meet these criteria in the country’s health services, including in Primary Health Care (PHC) facilities. The objective of this study was to verify the efectiveness of a Brief Inter- vention Group (BIG) performed by nurses, in reducing the risky use of alcohol among PHC users. Materials and methods: A randomized controlled trial was con- ducted at a PHC facility in Sao Paulo City, Brazil. The sample consisted of 180 individuals. All participants completed the Alcohol Use Disor- ders Identiication Test (AUDIT); those with AUDIT scores between 8 and 19 were enrolled in the study; 44 participants completed all study phases. Individuals randomized to the experimental group were enrolled in the Brief Intervention Group (BIG), while individuals allocated to the control group received a lyer containing information about problems related to harmful alcohol consumption. Both groups participated in a follow-up review after 90 days. Integrated stepped care to address moderate alcohol use among HIV‑positive patients with liver disease: results from a randomized clinical trial 1 2 3 2 4 E. Jennifer Edelman1,2, Stephen A. Maisto3, Nathan B. Hansen2,4, Christopher J. Cutter1, Yanhong Deng5, James Dziura5, Lynn E. Fiellin1,2, Patrick G. O’Connor1, Roger Bedimo6, Cynthia Gibert7, Vincent C. Marconi8, David Rimland8, Maria C. Rodriguez-Barradas9, Michael S. Simberkoﬀ10, Amy C. Justice1,11, Kendall J. Bryant12, David A. Fiellin1,2 1Yale University School of Medicine, New Haven, CT, USA; 2Center for Interdisciplinary Research on AIDS, Yale University School of Public Health, New Haven, CT, USA; 3Syracuse University, Syracuse, NY, USA; 4College of Public Health, University of Georgia, Athens, GA, USA; 5Yale Center for Analytic Sciences, Yale University School of Public Health, New Haven, CT, USA; 6Veterans Aﬀairs North Texas Health Care System and UT Southwestern, Dallas, TX, USA; 7D.C. Veterans Aﬀairs Medical Center and George Washington University School of Medicine and Health Sciences, Washington, D.C., USA; 8Atlanta Veterans Aﬀairs Medical Center and Emory University School of Medicine, Decatur, GA, USA; 9Michael E. DeBakey Veterans Aﬀairs Medical Center and Baylor College of Medicine, Houston, Texas, Houston, TX, USA; 10VA NY Harbor Healthcare System and New York University School of Medicine, New York, NY, USA; 11VA Connecticut Healthcare System, Veterans Aging Cohort Study, West Haven, CT, USA; 12National Institute on Alcohol Abuse and Alcoholism HIV/AIDS Program, Bethesda, MD, USA E. Jennifer Edelman1,2, Stephen A. Maisto3, Nathan B. Hansen2,4, Christopher J. Cutter1, Yanhong Deng5, James Dziura5, Lynn E. Fiellin1,2, Patrick G. O’Connor1, Roger Bedimo6, Cynthia Gibert7, Vincent C. Marconi8, David Rimland8, Maria C. Rodriguez-Barradas9, Michael S. Simberkoﬀ10, Amy C. Justice1,11, Kendall J. Bryant12, David A. Fiellin1,2 Conclusion: The results suggest that brief intervention performed by nurses in-group in the context of the PHC was efective to reduce alco- hol consumption among individuals with hazardous/harmful alcohol use. A14 Efectiveness of a brief intervention group performed by nurses in addressing hazardous and harmful alcohol use Divane de Vargas, Janaina Soares Department of Maternal-Infant and Psychiatric Nursing, University of São Paulo, School of Nursing, São Paulo, Brazil Correspondence: Divane de Vargas - vargas@usp.br Addiction Science & Clinical Practice 2017, 12(Suppl 1): A14 The BIG intervention consisted of four group sessions, with weekly meetings and a follow- up session after 90 days. A mixed linear model was used to evaluate the efectiveness of the BIG in reducing alcohol consumption. Conclusions: ED patients have high rates of signiicant social needs, with higher rates found among patients with drug use. Unhealthy alcohol users had rates more similar to ED patients overall, with the exception of being more likely to experience homelessness. These indings suggest that ED SBIRT programs must recognize patients’ con- current social needs, which might impact the efectiveness of inter- ventions to address their substance use. Results: The experimental group had a statistically signiicant reduc- tion (p ≤ 0.01) of about 10 points in the mean AUDIT score after BIG (before BIG = 15.89 ± 6.62-corresponding to risky use; after BIG = 6.40 ± 5.05-corresponding to low-risk use), while maintaining low-risk use (6.69 ± 6.38–low-risk use). The control group had a sta- tistically signiicant reduction (p ≤ 0.01) of about 3 points in the mean AUDIT score after screening and feedback (baseline 13.11 ± 4.54-cor- responding to risky use; 30-day follow-up score 9.83 ± 5.54-cor- responding to risky use), and returned to the baseline pattern of alcohol use (13.00 ± 5.70-corresponding to risky use). The diferences between the two groups were statistically signiicant (p ≤ 0.01). Correspondence: Background: The cost-efectiveness of diferent implementation models of screening and brief intervention (SBI) delivery to adoles- cents has never been studied. A cluster randomized trial examined Conclusions: Whilst a program of universal SBI delivery in primary care in England would lead to higher levels of intervention delivery in less deprived groups, the greatest health beneits would be seen in Page 6 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 the most deprived groups. Universal screening would therefore nar- row, rather than widen existing socioeconomic inequalities in health. success of ED SBIRT programs, yet little research has examined SDOH among substance using ED patients. Materials and methods: We surveyed a random sample of ED patients at an urban, public hospital from November 2016–March 2017. Eligible patients were: ≥18 years old, medically/psychiatrically stable, not in police/prison custody, spoke English or Spanish, and had not already participated. RAs administered a 20–40 min survey. We used validated single-item screeners for current unhealthy alco- hol and drug use (Smith et al. 2009, 2010). Questions on self-reported past 12 month social needs were taken from national surveys or prior studies. Social determinants of health among emergency department patients who screen positive for unhealthy alcohol or drug use: implications for ED SBIRT 1 2 2 3 Kelly M. Doran1, Donna Castelblanco2, Ian Wittman2, Donna Shelley3, John Rotrosen4, Lillian Gelberg5 Kelly M. Doran1, Donna Castelblanco2, Ian Wittman2, Donna Shelley3, John Rotrosen4, Lillian Gelberg5 1 1Departments of Emergency Medicine and Population Health, NYU School of Medicine, New York, NY, USA; 2Department of Emergency Medicine, NYU School of Medicine, New York, NY, USA; 3Departments of Medicine and Population Health, NYU School of Medicine, New York, NY, USA; 4Department of Psychiatry, NYU School of Medicine, New York, NY, USA; 5Department of Family Medicine, David Geﬀen School of Medicine at UCLA, Department of Health Policy and Management, UCLA Fielding School of Public Health, Oﬃce of Healthcare Transformation and Innovation, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA Correspondence: E. Jennifer Edelman - ejennifer.edelman@yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A16 Correspondence: E. Jennifer Edelman - ejennifer.edelman@yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A16 Correspondence: Background: Despite recent decreases in the number of children and young people (CYP) who drink alcohol, the North East (NE) of England has one of the highest youth drinking rates in the country with 10% of CYP drinking regularly. This research aims to identify factors which might impact on alcohol consumption: (1) intergenerational inlu- ences; (2) mimicking positive behaviors; (3) alcohol accessibility; and (4) social media and friendship. 1School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 2Centre for Health Services Research, University of Kent, Canterbury, Kent, UK; 3Addictions Department, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK; 4Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK; 5School of Education, Communication and Language Sciences, Newcastle University, Newcastle upon Tyne, UK; 6Health Economics Group, Institute of Health and Society, Newcastle University, Richardson Road, Newcastle upon Tyne, UK; 7Centre for Public Health, Liverpool John Moores University, Liverpool, UK; 8Faculty of Education, Health and Community, Liverpool John Moores University, Liverpool, UK; 9Northumberland, Tyne and Wear NHS Foundation Trust, Newcastle upon Tyne, UK; 10Newcastle Clinical Trials Unit, Newcastle University, Newcastle upon Tyne, UK Materials and methods: An online survey was undertaken by CYP at seven schools in the North East of England. The survey was emailed by schools to all of their CYP in Years 7–10 (aged 11–15 years) and was completed in early 2017, with a target sample size of 1200. It explored alcohol use in CYP, why they do and do not drink, and explored their wider environment which may impact on their alcohol intake. Follow- ing completion of the survey, semi-structured interviews targeting 30–50 CYP were undertaken to explore in more detail the issues raised in the survey. y Correspondence: Emma Giles - e.giles@tees.ac.uk Correspondence: Emma Giles - e.giles@tees.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A17 Correspondence: Emma Giles - e.giles@tees.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A17 Addiction Science & Clinical Practice 2017, 12(Suppl 1): A17 Results: As of early March 2017, 760 CYP had completed the online survey, representing an average within school completion rate of 30%. In total, 32 in-depth interviews had also been undertaken with CYP from three schools. Final results for this study are expected at the end of April 2017. Preliminary results suggest that the majority of the sample do not currently drink alcohol. Reducing risky drinking in children and young people: alcohol perceptions and attitudes interview and survey indings G l 1 Cl h 2 S C l 3 f Reducing risky drinking in children and young people: alcohol perceptions and attitudes interview and survey indings Emma L. Giles1, Justine Clephane2, Simon Coulton3, Jennifer Ferguson1, David Gardiner4, John Holmes5, Neil Martin6, Grant J. McGeechan1, Colin Shevills6, Melanie Soutar7, Dorothy Newbury-Birch1 1School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 2Education Service, Wellbeing and Community Health Services Group, Brunel Building, 64 Regent Street, Blyth, UK; 3Centre for Health Services Research, University of Kent, Canterbury, Kent, UK; 4Public Health England North East, Waterfront 4, Goldcrest Way, Newburn Riverside, Newcastle upon Tyne, Tyne and Wear, UK; 5Section of Public Health ScHARR, University of Sheﬃeld, Regent Court, 30 Regent Street, Sheﬃeld, UK; 6Balance North East, Bede House, Durham, UK; 7Matrix Young People’s Service, 7 Burrow Street, South Shields, Tyne and Wear, UK Correspondence: Emma Giles - e.giles@tees.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A18 Conclusions: Non-treatment seeking HIV-positive patients with moderate alcohol consumption and liver disease rarely enter trials to address their drinking. ISC holds promise as an intervention to pro- mote abstinence in this population. Emma L. Giles1, Justine Clephane2, Simon Coulton3, Jennifer Ferguson1, David Gardiner4, John Holmes5, Neil Martin6, Grant J. McGeechan1, Colin Shevills6, Melanie Soutar7, Dorothy Newbury-Birch1 1School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 2Education Service, Wellbeing and Community Health Services Group, Brunel Building, 64 Regent Street, Blyth, UK; 3Centre for Health Services Research, University of Kent, Canterbury, Kent, UK; 4Public Health England North East, Waterfront 4, Goldcrest Way, Newburn Riverside, Newcastle upon Tyne, Tyne and Wear, UK; 5Section of Public Health ScHARR, University of Sheﬃeld, Regent Court, 30 Regent Street, Sheﬃeld, UK; 6Balance North East, Bede House, Durham, UK; 7Matrix Young People’s Service, 7 Burrow Street, South Shields, Tyne and Wear, UK Correspondence: Emma Giles - e.giles@tees.ac.uk Addiction Science & Clinical Practice 2017 12(Suppl 1) A18 Correspondence: Background: Among HIV-positive patients with liver disease, absti- nence from alcohol consumption is recommended, yet is often not systematically addressed in HIV treatment settings. We sought to eval- uate the efectiveness of integrated stepped care (ISC) versus treat- ment as usual (TAU) on alcohol abstinence. Materials and methods: From January 2013 through July 2016, we conducted a 24-week randomized controlled trial at ive Veterans Afairs Infectious Disease Clinics. Eligibility criteria included (1) mod- erate alcohol use—any alcohol consumption in the prior 30 days, but Background: Among HIV-positive patients with liver disease, absti- nence from alcohol consumption is recommended, yet is often not systematically addressed in HIV treatment settings. We sought to eval- uate the efectiveness of integrated stepped care (ISC) versus treat- ment as usual (TAU) on alcohol abstinence. Correspondence: Kelly M. Doran - Kelly.Doran@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A15 Correspondence: Kelly M. Doran - Kelly.Doran@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A15 Background: Emergency department (ED) patients have high levels of substance use as well as high levels of social needs that could impact health. Such social determinants of health (SDOH) may afect the Materials and methods: From January 2013 through July 2016, we conducted a 24-week randomized controlled trial at ive Veterans Afairs Infectious Disease Clinics. Eligibility criteria included (1) mod- erate alcohol use—any alcohol consumption in the prior 30 days, but Page 7 of 24 Page 7 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Timeline Follow Back (TLFB). Qualitative interviews with young people, parents, and school staf supplement the main trial, by exploring the barriers and facilitators to the efectiveness and implementation of the intervention. not meeting criteria for at-risk drinking or alcohol use disorder, and (2) liver disease—liver ibrosis (FIB-4 score >1.45) or detectable hepatitis C virus (HCV). Participants were randomized to ISC or TAU. ISC included: Step 1—Social Worker administered Brief Intervention with telephone booster; Step 2—Psychologist administered Motivational Enhance- ment Therapy over four sessions; and Step 3—Addiction Physician management with consideration of pharmacotherapy. Participants were “stepped up” to Step 2 and Step 3 if they reported any alcohol use in the prior 14 days at weeks 4 and 12, respectively. Generalized mixed efects models adjusted for baseline alcohol use and HIV dis- ease severity, were used to examine past 28 day abstinence at week 24 assessed by Timeline Followback. Correspondence: Target enrollment was 228. Results: In total, 4587 young people were surveyed at baseline, with 586 randomized. So far, thirty-one (n = 109) percent of the target (n = 352) follow-up sample have completed a survey at their 12 month time point, with ongoing follow-ups continuing until June 2017. In total, 27 qualitative interviews with school staf were conducted in 2016, and interviews with young people and parents are currently being conducted. Conclusions: Of 4587 young people surveyed, 1044 young peo- ple scored positive for risky drinking, and 586 are taking part in the trial. Qualitative interviews with school staf have indicated that they see the beneit of the intervention to schools, that there is staf sup- port for the intervention, and that is easy to undertake by school staf and to roll-out in schools should it be shown to be efective and cost-efective. Results: Among 6391 AUDIT-C screening tests performed, 3265 exceeded zero. Of these, we enrolled 95 participants before stopping the trial due to under-enrollment. Ninety-nine percent were men, 85% black, 12% white, 4% Hispanic, with a mean age of 61 years. At base- line, 85% had a FIB-4 >1.45, 56% were HCV-infected. The median CD4 cell count was 853 cells/mm3 and 24% had a detectable HIV viral load. The mean (standard deviation) drinks per day was 0.49 (0.59). Com- pared to TAU, ISC was associated with non-signiicant increased odds of past 28 day abstinence at week 24 (adjusted odds ratio [95% coni- dence interval] = 3.20 [0.88, 11.67]). A multi‑center individual‑randomized controlled trial of screening and brief alcohol intervention to prevent risky drinking in young people aged 14–15 in a high school setting 1 1 2 3 Emma L. Giles1, Dorothy Newbury-Birch1, Simon Coulton2, Paolo Deluca3, Colin Drummond3, Denise Howel4, Eileen Kaner4, Elaine McColl4, Ruth McGovern4, Stephanie Scott4, Elaine Stamp4, Harry Sumnall5, Luke Vale6, Viviana Alabani6, Amanda Atkinson7, Sadie Boniface3, Jennifer Ferguson1, Jo Frankham8, Eilish Gilvarry9, Nadine Hendrie2, Nicola Howe10, Grant J. McGeechan1, Amy Ramsey3, Grant Stanley7 Racial/ethnic disparities in alcohol Screening, Brief Intervention and Referral to Treatment among adult hypertensive patients in primary care settings 1 1 2 1 3 1Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA; 2Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA; 3Aurora Public Schools Division of Accountability and Research, Educational Services Center 1, Aurora, CO, USA Materials and Methods: An ongoing systematic review and narrative synthesis of eicacy and efectiveness trials of alcohol brief interven- tions from 10 databases from Jan 2000–Sept 2016 (including EMBASE, MEDLINE, PsycINFO, CINAHL, Web of Science) and grey literature sources (including databases and trial registries). Alcohol brief inter- vention deinitions are informed by National Institute of Clinical Excel- lence Public Health Guideline 24: Alcohol use disorders: prevention. The review was conducted in accordance with the Centre for Reviews and Dissemination (CRD) guidance and pre-registered on PROSPERO (CRD42016047185). Correspondence: Felicia W. Chi - felicia.w.chi@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A19 Correspondence: Felicia W. Chi - felicia.w.chi@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A19 Background: The high burden of unhealthy drinking and alcohol use disorders among patients with chronic conditions is well recognized. Alcohol Screening, Brief Intervention and Referral to Treatment (SBIRT) in adult primary care has been found eicacious in reducing hazard- ous drinking, and limited literature suggests positive efects of alcohol BI on blood pressure (BP) outcomes among hypertensive patients. This study examines whether there are racial/ethnic disparities in receipt of alcohol SBIRT among adult primary care hypertensive patients, by ana- lyzing data of a clustered, randomized controlled trial on SBIRT imple- mentation by primary care physicians (PCP arm) and non-physician providers (NPP and MA arm) in a large, integrated health care delivery system. Results: From the initial search of databases around 320 stud- ies were identiied, with the grey literature search ongoing. In the irst 95 studies included, there were seven diferent overarching domains used to summarize the outcomes; biomarkers, alcohol con- sumption (includes problems, hazardous, harmful, or risky drinking), economic factors and resource use, health measures, life impact, intervention factors, and psychological factors. Preliminary indings suggest the most commonly reported were consumption outcomes such as frequency of drinking (9.3% of 486 outcomes), frequency of heavy drinking (8.6%), typical drinks on occasion (10%) and num- ber of drinks in a week (8.4%). Fewest reported included economic factors and resource use, life factors (such as quality of life), and biomarkers. Correspondence: For those who do, they tend to consume alcohol on special occasions, such as birthdays, usually with parental approval. For those CYP that do not drink alcohol, they indi- cate that they perceive themselves to be too young to drink, that it is dangerous for health reasons, and that they wish to concentrate on their school performance. Also, they request more targeted informa- tion on alcohol. Background: Young people are a greater risk of problem drinking and are vulnerable to the efects of alcohol consumption, linked to early drinking and also the physiological and social consequences of alcohol use. The SIPS JR-HIGH study is a multicenter Randomized Controlled Trial (RCT) aiming to assess the efectiveness and cost-efectiveness of alcohol screening and brief intervention to reduce risky drinking in those aged 14–15 years in the English high school setting. Materials and methods: Thirty schools in England were recruited into the trial: 4 in London; 6 in the North West, 7 in Kent, and 13 in the North East. A baseline survey was undertaken between December 2015 and June 2016 by young people in school. Young people who screened positive for risky drinking using a single item screen (ASAQ) were allocated with equal probability to either a control arm consisting of usual school-based education on alcohol issues, or to the interven- tion arm augmenting usual education with a 30 min brief intervention, both delivered by school pastoral staf. At 12-month follow-up the pri- mary outcome of total alcohol consumed is being measured using the Conclusions: The results have implications for policy and practice partners on this research, in ensuring current drug and alcohol cam- paigns are appropriately ‘pitched’ to CYP, and that CYP are receiving messages as intended. Reinforcing the factors that CYP identify as rea- sons for not drinking when targeting alcohol screening and brief inter- ventions with CYP is a key consideration. Page 8 of 24 Page 8 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Correspondence: Addi i S i & Conclusions: Findings suggested comparable or higher SBIRT rates in non-White racial/ethnic groups compared to Whites among adult pri- mary care hypertensive patients, although certain patient factors may play an important role for diferent SBIRT delivery models. Background: Substance use in adolescence is linked to a range of negative life consequences. Consequently, there is a great need for social workers, nurses, and other health professionals to be trained in prevention and early intervention approaches to adolescent alco- hol and marijuana use. To this end, NORC at the University of Chicago along with leading professional education associations and experts partnered to develop and test an adolescent screening, brief interven- tion, and referral to treatment (SBIRT) curriculum for use in nursing and social work education. The study aimed to evaluate the impact of the education implemented with more than 4000 students in 32 nurs- ing and social work programs on students’ attitudes towards working with people who drink alcohol; perceived readiness, conidence, and competence; and skills. Racial/ethnic disparities in alcohol Screening, Brief Intervention and Referral to Treatment among adult hypertensive patients in primary care settings 1 1 2 1 3 Materials and Methods: Electronic health record (EHR) data on 139,179 adult hypertensive patients who had a primary care visit at 36 clinics during the irst year of the study were analyzed. We examined in each intervention arm, diferences in demographic and clinical charac- teristics, screening rates, and BI/RT rates among those who screened positive, across racial/ethnic groups. Multilevel Logistic regressions further assessed the associations between race/ethnicity and receipt of alcohol SBIRT in each intervention arm while accounting for cluster- ing of patients within physicians and clinics and adjusting for demo- graphics, baseline severity, anti-hypertensive medication adherence and comorbidity. Conclusions: These preliminary indings indicate, as expected, consid- erable variability in the outcomes reported in alcohol brief interven- tion trials. Whilst it is perhaps unsurprising that consumption is the most common domain measured, there was considerable variability in how this is described as an outcome, and even more variability in how it is measured in practice. This illustrates the challenges in synthesiz- ing the literature, and the need for a core outcome set to help alcohol brief interventionists to determine the minimum measurement stand- ard for research and evaluation. Results: We found diferences in demographic and clinical character- istics across racial/ethnic groups. Multilevel Logistic regressions found that compared to Whites, Asian/Paciic Islanders and Hispanics were more likely to receive screening in the PCP arm (adjusted Odds Ratios [95% conidence intervals] = 1.18 [1.07–1.30] and 1.12 [1.01–1.25], respectively), and African Americans and Hispanics were more likely to receive screening in the NPP and MA arm (adjusted Odds Ratios [95% conidence intervals] = 1.23 [1.01–1.50] and 1.19 [1.01–1.40], respectively). In both intervention arms, having uncontrolled BP was negatively associated with receiving screening for unhealthy drink- ing. However, no signiicant diferences were found in receiving BI/ RT when screened positive in either intervention arm. Interactions of race/ethnicity and gender were also examined. Research to practice: an evaluation of adolescent SBIRT training on student perceptions of conidence and attitudes toward implementing in the ield 1 2 3 Research to practice: an evaluation of adolescent SBIRT training on student perceptions of conidence and attitudes toward implementing in the ield 1 2 3 Correspondence: Gillian W. Shorter - gillianwshorter@gmail.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A20 A21 Research to practice: an evaluation of adolescent SBIRT training on student perceptions of conidence and attitudes toward implementing in the ield Hildie A. Cohen1, Tracy L. McPherson2, Cyrille Adam3 1NORC at the University of Chicago, Chicago, IL, USA; 2Public Health, NORC at the University of Chicago, Bethesda, MD, USA; 3Research and Development, Kognito, New York, NY, USA Correspondence: Hildie A. Cohen - cohen-hildie@norc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A21 A19 Background: Evidence to evaluate the eicacy and efectiveness of alcohol brief interventions is weakened by variability in measured outcomes and inconsistent reporting. This ongoing systematic review forms part of the larger Outcome Reporting in Brief Intervention Tri- als: Alcohol (ORBITAL) project aligned with the INEBRIA special interest group of the same name. The review aims to identify outcomes and wider domains used in eicacy and efectiveness trials of alcohol brief interventions. A19 Racial/ethnic disparities in alcohol Screening, Brief Intervention and Referral to Treatment among adult hypertensive patients in primary care settings Felicia W. Chi1, Constance Weisner1,2, Thekla B. Ross1, Jennifer Mertens3, Stacy Sterling1 1Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA; 2Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA; 3Aurora Public Schools Division of Accountability and Research, Educational Services Center 1, Aurora, CO, USA Correspondence: Felicia W. Chi - felicia.w.chi@kp.org Addiction Science & Clinical Practice 2017 12(Suppl 1): A19 Developing a brief motivational intervention for young adults intoxicated in the ED: results from an iterative qualitative design Jacques Gaume, Véronique Grazioli, Cristiana Fortini, Sophie Paroz, Nicolas Bertholet, Jean-Bernard Daeppen 1Alcohol Treatment Center, Lausanne University Hospital, Lausanne, Switzerland Correspondence: Jacques Gaume - jacques.gaume@chuv.ch Addiction Science & Clinical Practice 2017, 12(Suppl 1): A24 Correspondence: Jacques Gaume - jacques.gaume@chuv.ch Addiction Science & Clinical Practice 2017, 12(Suppl 1): A24 Materials and methods: A randomized controlled non-inferiority trial (e-BI versus face-to-face BI) of primary care-based facilitated access to an alcohol reduction website was conducted with 34 PCP’s of Catalo- nia (EFAR-Spain). A qualitative study on barriers and facilitators of e-BI was conducted with a subsample of the recruited GPs. Background: Harmful alcohol use among young adults is a major public health concern. In Switzerland, Emergency Department (ED) admissions for alcohol intoxication have increased substantially over the past decade, particularly among adolescents and young adults. Brief motivational interventions for young adults in the ED have shown promising but inconsistent results. Results: One-hundred ifteen GPs were trained for this project, but only eight GPs (7%) achieved the original recruitment goal (10 patients per GP), and recruitment required twenty-four months instead of the twelve months initially planned. The qualitative study (on-line survey) is therefore underway, focusing on the participant GPs in EFAR-project Spain. We expect to identify speciic barriers related to e-BI and the utility of e-BI for dealing with traditional barriers of BI (lack of training, time, specialized services to referred patients or incentives and risk of upsetting the patient). Materials and methods: Based on the literature on brief interven- tion and motivational interviewing eicacy and active ingredients, we developed a new motivational intervention model for young adults admitted in the ED with alcohol intoxication. Using an iterative qualita- tive design, we irst pre-tested this model by conducting 4 experimen- tal sessions to evaluate interventionists’ and patients’ experience, then conducted a consultation with 9 international experts using nominal group technique, then re-tested the model by conducting 6 experi- mental sessions to evaluate interventionists’ and patients’ experience. At each round, data collected were analyzed and discussed, and the intervention model updated accordingly. Conclusion: Our hypothesis is that facilitated access to e-BI does not sort out all the barriers to providing BI in primary care practice and adds new barriers that we should take into account in order to prop- erly implement facilitated access. The results of the qualitative study should help to reframe our facilitated access strategies. Results: Based on the literature, we found 6 axes for developing a new model: High level of relational factors (e.g. The variability of outcomes used in eicacy and efectiveness trials of alcohol brief interventions: a systematic review 1 2 1 3 Gillian W. Shorter1, Nick Heather2, Emma L. Giles1, Aisha Holloway3, Jeremy Bray4, Anne H. Berman5, Amy J. O’Donnell6, Dorothy Newbury-Birch1 1Alcohol and Public Health Team, School of Health and Social Care, Middlesbrough, Teesside, UK; 2Faculty of Health and Life Sciences, Northumbria University, Newcastle, Northumberland, UK; 3School of Health and Social Sciences, University of Edinburgh, Edinburgh, Midlothian, UK; 4Bryan School of Business and Economics, University of North Carolina at Greensboro, Greensboro, NC, USA; 5Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden; 6Institute of Health and Society, Newcastle University, Newcastle upon Tyne, Northumberland, UK Materials and methods: Students completed a pre-training evalua- tion survey, received adolescent SBIRT education including an online simulation training, and completed a post-training evaluation survey. A pretest–posttest within-subjects design was used to investigate the efects on student attitudes; conidence, competence, and readi- ness; and skills. Diferences between groups were also explored for Correspondence: Gillian W. Shorter - gillianwshorter@gmail.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A20 Page 9 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 were screened for eligibility using the AUDIT (n = 181). Those with an AUDIT score ≥6 completed a baseline survey (n = 121) and were ran- domized into an intervention (n = 71) or control group (n = 30). Par- ticipants in the intervention group received a brief intervention from a promotor at a day labor worker center. Personalized feedback was provided by promotores, using a tablet screen to display the partici- pants’ quantity of daily and weekly drinking. We conducted follow-up surveys at eight weeks following the baseline to assess changes in AUDIT scores, daily and weekly drinking. program-level variables. Through review of implementation progress reports and implementation teleconference calls, qualitative data was gathered to assess perceptions of how the education it into curricu- lum at diferent program levels (e.g., bachelor, masters, doctoral). Results: The adolescent SBIRT education was efective in improving a number of student outcomes assessed using the pretest–posttest evaluation survey of attitudes, conidence, competence, readiness, and skills. Diferences by program level were also observed. Conclusions: The implications of these indings suggest that ado- lescent SBIRT education including simulation-based training can positively afect student outcomes as they prepare to implement adolescent SBIRT in the ield. The indings can also inform educators on the diferences in outcomes among groups and inform curriculum infusion. Don’t take it for granted H Ló P l 1 A Hugo López-Pelayo1, Antoni Gual1, Lidia Segura2, Joan Colom2 1Grup de Recerca Addicions Clínic, Hospital Clínic de Barcelona, IDIBAPS. University of Barcelona, Barcelona, Catalonia, Spain; 2Program on Substance Abuse, Public Health Agency, Government of Catalonia, Barcelona, Catalonia, Spain Hugo López-Pelayo1, Antoni Gual1, Lidia Segura2, Joan Colom2 1Grup de Recerca Addicions Clínic, Hospital Clínic de Barcelona, IDIBAPS. University of Barcelona, Barcelona, Catalonia, Spain; 2Program on Substance Abuse, Public Health Agency, Government of Catalonia, Barcelona, Catalonia, Spain Conclusions: Given that there were no signiicant diferences across groups, discussing alcohol use with a promotor during the survey may have been enough to initiate changes in drinking behaviors among participants. Future research should assess appropriate interventions for reducing unhealthy alcohol use in this population. Correspondence: Hugo López-Pelayo - hlopez@clinic.cat Addiction Science & Clinical Practice 2017, 12(Suppl 1): A22 Correspondence: Hugo López-Pelayo - hlopez@clinic.cat Addiction Science & Clinical Practice 2017, 12(Suppl 1): A22 Developing a brief motivational intervention for young adults intoxicated in the ED: results from an iterative qualitative design Jacques Gaume, Véronique Grazioli, Cristiana Fortini, Sophie Paroz, Nicolas Bertholet, Jean-Bernard Daeppen 1Alcohol Treatment Center, Lausanne University Hospital, Lausanne, Switzerland empathy, alliance, avoid- ance of confrontation); Personalized feedback; Enhance discrepancy; Evoke change talk while softening sustain talk, strengthen ability and commitment to change; Completion of a change plan; Devote more time: longer sessions and follow-up options (face-to-face, tel- ephone, or electronic boosters; referral to treatment). Qualitative analysis of experimental sessions gave important insights regard- ing acceptability and feasibility of the model. Reinement comprised which feedback and information to provide and how, as well as how to deal with change planning with patients having vague change objectives. Experts’ consultation addressed numerous points, includ- ing relections on information and advice giving, as well as follow-up interventions. The variability of outcomes used in eicacy and efectiveness trials of alcohol brief interventions: a systematic review 1 2 1 3 y y g Results: At baseline, mean AUDIT scores were 19.1 for men in the intervention group (n = 71) and 21.5 for those in the control group (n = 30). Both groups had decreased their AUDIT scores at eight weeks (intervention, 15.6; control, 18.2) with no signiicant diferences between groups. Both groups also decreased their average number of drinks per drinking day from baseline to eight weeks (intervention, 2.9–1.8; control, 4.5–3.8, p < .05). Number of drinking days in the past two weeks also decreased in both groups from baseline to eight weeks (intervention, 5.7–4.2; control, 7.1–6.5, p < .05). A24 Background: Facilitated access to digital brief intervention (e-BI) has been proposed as a strategy for overcoming well-known barriers of BI in primary care practice (e.g. lack of time or fear of patient reactions). However, the ODHIN study showed that facilitated access to e-BI has no impact on BI implementation. The lack of success of facilitated access to e-BI in this study might be explained by the time required for delivery of e-BI, (which may take as much time as brief oral advice) and by the insuicient training of general practitioners (GPs) in e-BI. Developing a brief motivational intervention for young adults intoxicated in the ED: results from an iterative qualitative design Jacques Gaume, Véronique Grazioli, Cristiana Fortini, Sophie Paroz, Nicolas Bertholet, Jean-Bernard Daeppen 1Alcohol Treatment Center, Lausanne University Hospital, Lausanne, Switzerland A27 Materials and methods: PCPs from 3 clinics (HIV, safety net, and academic) were randomized into the CF-5A’s intervention or to usual care (UC). Adult patients who smoke were recruited in waiting rooms and assigned to their provider’s condition. Intervention patients completed the CF-5A’s and two tailored clinical summaries were gen- erated—one for the provider and one for the patient. UC patients completed an eligibility survey and consent only. Within 72 h of the appointment, patients completed a post-visit survey about their receipt of the 5A’s during their PCP encounter. Patients could partici- pate up to three times within the yearlong study period. Alcohol screening and brief interventions with male remand prisoners: a cross sectional survey of prevalence, feasibility and acceptability Jennifer Ferguson1, Dorothy Newbury-Birch1, Aisha S. Holloway2 1Health and Social Care Institute, School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 2Nursing Studies, School of Health in Social Science, The University of Edinburgh, Edinburgh, UK Alcohol screening and brief interventions with male remand prisoners: a cross sectional survey of prevalence, feasibility and acceptability Jennifer Ferguson1, Dorothy Newbury-Birch1, Aisha S. Holloway2 1Health and Social Care Institute, School of Health and Social Care, Teesside University, Middlesbrough, Tees Valley, UK; 2Nursing Studies, School of Health in Social Science, The University of Edinburgh, Edinburgh, UK Correspondence: Jennifer Ferguson - jennifer.ferguson@tees.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A27 Correspondence: Jennifer Ferguson - jennifer.ferguson@tees.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A27 Results: N = 221 providers saw n = 961 patients (n = 412 interven- tion; n = 549 UC) in n = 1340 total encounters with n = 1011 com- pleted post surveys (75.4% response). After accounting for 4-level nesting efects, GEE models showed intervention PCPs 32% more likely to “Assess” (OR 1.32; 95% CI, 1.01–1.72), 45% more likely to “Assist” (OR 1.45; 95% CI, 1.08–1.93), and 72% more likely to “Arrange” in the irst visit only (OR 1.72; 95% CI, 1.23–2.40), and 104% more likely to com- plete all 5A’s during the irst visit (OR 2.04; 95% CI, 1.35–3.07). Background: In the UK, a signiicant proportion of male remand pris- oners have alcohol problems. Alcohol Brief Interventions (ABIs) are an efective component of a population-level approach to harmful and hazardous drinking. ABI’s have been shown to reduce the aggregate level of alcohol consumed and therefore to reduce harm to the indi- vidual and to others. Vida PURA: results from a pilot randomized trial of screening and brief intervention with Latino day laborers 1 2 2 India J. Ornelas1, Suzanne Doyle2, Dennis Donovan2, Bonnie Duran3, Vanessa Torres1 1 1Department of Health Services, University of Washington, Seattle, WA, USA; 2Alcohol and Drug Abuse Institute, University of Washington, Seattle, WA, USA; 3School of Social Work, University of Washington, Seattle, WA, USA Correspondence: India J. Ornelas - iornelas@uw.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A23 Correspondence: India J. Ornelas - iornelas@uw.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A23 Background: Vida PURA is a culturally adapted intervention that con- sists of promotores providing screening and brief intervention at a day labor worker center to reduce unhealthy alcohol use among Latino day laborers. Conclusions: This iterative, multi-component design allowed develop- ing an intervention model embedded in recent research indings and theory advances, as well as feasible in a complex environment. Next step is a randomized controlled trial testing the eicacy of this model. Materials and methods: We conducted a pilot randomized control trial to test the eicacy of the Vida PURA intervention. Participants Addict Sci Clin Pract 2017, 12(Suppl 1):25 Page 10 of 24 Page 10 of 24 A26 Results: 502 surveys across the two sites were completed by remand and convicted prisoners. Of these, 79% scored positive on the AUDIT (8+), with 44% of prisoners scoring as probably dependent (20+). Of all prisoners, 37% thought 5 min of advice would be useful, and 51% thought 20 min of advice would be useful. Eighty-six percent said they would be willing to take part in a future ABI efectiveness study to test and to be followed-up to collect post-test data. Forty-seven percent said they did not feel pressurized to take part in a research study whilst detained in prison. A tablet‑based device for substance use and physical activity screening: spontaneous use in primary care waiting rooms 1 2 3 Jean-Bernard Daeppen1, Angéline Adam2, John A. Cunningham3, Nicolas Bertholet1 1 1Department of Community Medicine, Lausanne University Lausanne, Switzerland; 2Alcohol Treatment Center, Lausanne University Hospital, Lausanne Switzerland; 3Centre for Addiction and Mental Health, Toronto, Canada Correspondence: Jean-Bernard Daeppen - jean-bernard.daeppen@chuv.ch Addiction Science & Clinical Practice 2017, 12(Suppl 1): A26 Conclusions: Prevalence rates of risky drinking are higher in the crimi- nal justice system compared to the general population. Prisoners were generally accepting of screening and brief interventions in this setting. Background: Screening and brief intervention for unhealthy sub- stance use in primary care is challenging. Electronic devices may help clinicians to deliver screening and brief interventions to their patients, but spontaneous use in waiting rooms may be limited. A25 and screening results. On random half-days, a research assistant was present to ofer patients to use the device, allowing for comparison between spontaneous and assisted use of the device. The other days, a poster in the waiting room invited the patients to use the device. Results: Out of 1781 patients attending the 4 practices, 342 (19.2%) used the device. Spontaneous use was lower (243 completed screen out of 1501 patients, 16.2%), compared to use assisted by a research assistant (99 completed screen out of 280 patients, 35.4%). Data indi- cated a proile of heavier severity for patients with spontaneous use, compared to counterparts, being younger (44.5 [17.1 vs. 49.9 [16.9], p = .009), more likely to smoke cigarettes (41.3 vs. 29.6%, p = .04), and use drugs (11.5 vs. 4.1%, p = .04), respectively. No statistically signii- cant group diferences were observed regarding proportion of patients with unhealthy alcohol use (58.4 vs. 49.0%), prescription drug use (26.3 vs. 16.7%) and reporting insuicient physical activity (52.3 vs. 47.9%). Spontaneous use was associated with a 54.5% completion of an elec- tronic alcohol brief intervention (45.8% completion with assisted use). Conclusions: Spontaneous use was lower compared to assisted use and appeared to self-select patients with heavier tobacco and drug use who appear more likely to use the device. A25 Computer‑facilitated 5A’s for tobacco use disorders: using technology to improve screening and brief interventions Jason M. Satterﬁeld1, Steven Gregorich1, Nicholas J. Alvarado1, Ricardo Muñoz2, Gozel Kulieva1, Maya Vijayaraghavan3 1Department of General Internal Medicine, University of California San Francisco, San Francisco, CA, USA; 2PhD Program in Clinical Psychology, Palo Alto University, Palo Alto, CA, USA; 3Department of Medicine, University of California San Francisco, San Francisco, CA, USA Correspondence: Jason M. Satterﬁeld - Jason.Satterﬁeld@ucsf.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A25 Background: Clinical practice guidelines recommend that primary care providers (PCPs) deliver the 5A’s (ask, advise, assess, assist, and arrange) at every clinical encounter for the treatment of tobacco use disorders. Unfortunately, while most clinicians “ask” and “advise,” adherence to the “assist” and “arrange” steps remains low due to time and skill limitations. Innovative service delivery models are needed to improve 5A’s adherence. Objective: To evaluate efectiveness of a computer-facilitated 5A’s (CF- 5A’s) intervention to improve PCP 5A’s adherence. Primary outcomes include adherence to each “A” and to the 5A’s as a whole. A27 However, in relation to remand prisoners, there is no evidence as to how efective ABI’s could be. The aims of this study were therefore to explore the feasibility and acceptability of an ABI for adult male remand prisoners, and to develop an ABI for this group to be piloted in a future trial. This presentation presents the indings from the cross-sectional survey part of the study. Conclusion: The CF-5A’s model improved PCP’s 5A’s adherence. Efec- tiveness was attenuated by clinic site and afected by the number of visits with earlier visits showing stronger results. While this low cost intervention has great potential for improving the implementation and delivery of tobacco cessation and other services, future studies should identify ways to promote and sustain technology implementa- tion and integration with clinic procedures. Materials and methods: A cross-sectional survey of adult male remand and convicted prisoners (n = 502) was carried out at one Scot- tish prison and one English prison to assess prevalence of alcohol use disorders. The questionnaire also included questions related to pris- oners’ views on the acceptability and feasibility of ABIs in the prison system. A28 A28 Qualitative evaluation of the drink‑less project Joan Colom1, Lidia Segura-Garcia1, Estela Díaz1, Jorge Palacio-Vieira1, Antoni Gual2 1Program on Substance Abuse, Public Health Agency of Catalonia, Barcelona, Spain; 2Addiction Unit, Hospital Clínic, Barcelona, Spain Correspondence: Joan Colom - joan.colom@gencat.cat Addiction Science & Clinical Practice 2017, 12(Suppl 1): A28 Qualitative evaluation of the drink‑less project Joan Colom1, Lidia Segura-Garcia1, Estela Díaz1, Jorge Palacio-Vieira1, Antoni Gual2 1Program on Substance Abuse, Public Health Agency of Catalonia, Barcelona, Spain; 2Addiction Unit, Hospital Clínic, Barcelona, Spain Correspondence: Joan Colom - joan.colom@gencat.cat Addiction Science & Clinical Practice 2017, 12(Suppl 1): A28 A29 Can Amazon’s mechanical turk be used to recruit participants for Internet intervention trials? A pilot study involving a randomized controlled trial of a brief online intervention for hazardous alcohol use John A. Cunningham, Alexandra Godinho, Vladyslav Kushir Centre for Addiction and Mental Health, Toronto, Ontario, Canada Correspondence: John A. Cunningham - john.cunningham@camh.ca Addiction Science & Clinical Practice 2017, 12(Suppl 1): A29 A brief intervention for alcohol use with suicidal adolescents in inpatient psychiatric treatment 1 2 3 Kimberly H. M. O’Brien1, Laika D. Aguinaldo2, Christina M. Sellers3, Anthony Spirito4 1 Materials and methods: A cross-sectional observational study simul- taneously combining quantitative (systematic data of EIBI imple- mentation rates from the PHC monitoring system) and qualitative methodologies (semi-structure interviews, survey, etc.) was under- taken. A convenience sample was used. 1Department of Health and Human Development, Education Development Center, Waltham, MA, USA; 2Department of Social Work, The Ethelyn R. Strong School of Social Work at Norfolk State University, Norfolk, VA, USA; 3Department of Social Work, Boston College School of Social Work, Chestnut Hill, MA, USA; 4Department of Psychiatry and Human Behavior, Brown University, Providence, RI, USA Correspondence: Kimberly H. M. O’Brien - kobrien@edc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A30 Results: A total of 33 professionals were invited to participate (60% family doctors, 25% nurses and 15% health technicians). The major- ity were alcohol referents and 6 rejected. 84% of the professionals believe that the program has clearly contributed to the increase in the detection of risky drinkers, as conirmed by the clear increase of more than 35% reported by the monitoring system. 20% believe that it has contributed to facilitating and improving the relationship between PHC and Addiction specialist centers. Among the main weaknesses found: low professional motivation, lack of time, alcohol not a prior- ity, low incentives, high rotation, prejudices from professionals in front of patients with alcohol problems, and poor monitoring and feedback to professionals. Among the main strengths found: quality of train- ing, comprehensive program, program sustainability, computerized screening tools in the medical record, strong alliances with societies, strong alcohol referent network, high visibility of the program, and availability of referral to treatment. Background: Substance use assessment and counseling is frequently neglected in inpatient psychiatric units when the adolescent’s suicide risk is the primary focus of treatment. Given the potential role alco- hol could play in subsequent suicidal behaviors, greater attention to alcohol use in inpatient psychiatric treatment is critical. The purpose of this study was to test the feasibility and acceptability of a brief alcohol intervention with suicidal adolescent inpatients reporting past month drinking, and assess preliminary efects on alcohol use. Can Amazon’s mechanical turk be used to recruit participants for Internet intervention trials? A pilot study involving a randomized controlled trial of a brief online intervention for hazardous alcohol use Of those who agreed, a randomized half were asked to access an online brief intervention for drinking (CheckYourDrinking.net) and the other half were assigned to a no intervention control group (i.e., thanked and told that they would be re-contacted in 3 months). A brief intervention for alcohol use with suicidal adolescents in inpatient psychiatric treatment 1 2 3 Materials and methods: In the trial, 39 adolescents (Mage = 15.63; 80% female, 65% white) and their families were recruited from an urban inpatient psychiatric hospital and randomly assigned to the experimental intervention (EXP) or treatment as usual (TAU), stratify- ing by gender and frequency of alcohol use. At baseline and 3 month follow-up, alcohol use was measured by the Timeline Follow-back Interview. Adolescents randomized to EXP received an individual session to explore alcohol use as a risk factor for continued suicidal behaviors and create a change plan, and a subsequent family ses- sion to discuss the change plan and strengthen the adolescent’s commitment and self-eicacy as well as the parent’s ability to sup- port the adolescent. Adolescents in EXP were given an exit interview and session evaluation form to assess the intervention feasibility and acceptability. Conclusions: Changes in PHC are rather slow and require continuous sustainability actions. Organizational changes such as more time for preventive activities, CME, and empowerment through incentives and accreditation of the alcohol referent igure, and more patient-targeted raising awareness campaigns are key in order to overcome barriers. Qualitative evaluation of the drink‑less project J C l 1 Lidi S G i1 E l Dí 1 J Materials and methods: We developed a tablet-based device spe- ciically designed for primary care practices waiting rooms. The device ofers screening for tobacco, illicit drugs, prescription drugs, and physi- cal activity. Those screening positive for unhealthy alcohol use have the option of completing an electronic brief intervention. In Febru- ary 2017, we recorded the number of patients attending 4 primary care practices, the number of patients completing the screening, Page 11 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Background: After more than 20 years of research, evidence still shows that BI is efective in reducing alcohol quantity consumed in primary health care (PHC) (Platt et al., 2016). In Catalonia, in the framework of PHASE IV of the WHO Collaborative Project on Detec- tion and Management of Alcohol-related problems in PHC (Heather et al., 2004), we developed a country-wide strategy and have been implementing it under the principles of action research over the last 15 years. Here we will describe the core implementation strategy com- ponents at all levels, and present the results of the evaluation under- taken and the resulting decisions made to achieve an enduring and routine implementation. MTurk, there is potential for conducting trials employing this popula- tion (particularly if methods are employed to make sure that partici- pants receive the intervention). This potential is important as it could allow for the rapid conduct of multiple trials during the development stages of online interventions. A30 b A30 A brief intervention for alcohol use with suicidal adolescents in inpatient psychiatric treatment Kimberly H. M. O’Brien1, Laika D. Aguinaldo2, Christina M. Sellers3, Anthony Spirito4 1Department of Health and Human Development, Education Development Center, Waltham, MA, USA; 2Department of Social Work, The Ethelyn R. Strong School of Social Work at Norfolk State University, Norfolk, VA, USA; 3Department of Social Work, Boston College School of Social Work, Chestnut Hill, MA, USA; 4Department of Psychiatry and Human Behavior, Brown University, Providence, RI, USA Correspondence: Kimberly H. M. O’Brien - kobrien@edc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A30 A brief intervention for alcohol use with suicidal adolescents in inpatient psychiatric treatment Kimberly H. M. O’Brien1, Laika D. Aguinaldo2, Christina M. Sellers3, Anthony Spirito4 1Department of Health and Human Development, Education Development Center, Waltham, MA, USA; 2Department of Social Work, The Ethelyn R. Strong School of Social Work at Norfolk State University, Norfolk, VA, USA; 3Department of Social Work, Boston College School of Social Work, Chestnut Hill, MA, USA; 4Department of Psychiatry and Human Behavior, Brown University, Providence, RI, USA Correspondence: Kimberly H. M. O’Brien - kobrien@edc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A30 Can Amazon’s mechanical turk be used to recruit participants for Internet intervention trials? A pilot study involving a randomized controlled trial of a brief online intervention for hazardous alcohol use Results: Of the adolescents, 19 were randomized to EXP and 20 to TAU. All 19 in EXP completed the individual intervention (M = 75 min) and family intervention (M = 20 min) during their inpatient hospitaliza- tion. All 19 expressed satisfaction with the intervention, and all 19 cre- ated a change plan. Adolescents in EXP drank less alcohol at 3 month follow-up (M = 11.69, SD = 28.47) relative to baseline (M = 41.56, SD = 43.40), Z = −2.90, p < .05. Adolescents in TAU also drank less at follow-up (M = 6.37, SD = 14.58) compared to baseline (M = 30.90, SD = 53.75), Z = −3.46, p < .05. John A. Cunningham, Alexandra Godinho, Vladyslav Kushir Centre for Addiction and Mental Health, Toronto, Ontario, Canada Correspondence: John A. Cunningham - john.cunningham@camh.ca Addiction Science & Clinical Practice 2017, 12(Suppl 1): A29 Background: Amazon’s Mechanical Turk is a crowdsourcing platform that has been used extensively to collect psychological survey data. This pilot study sought to evaluate whether MTurk might also be a via- ble means of recruiting participants for online intervention research. Background: Amazon’s Mechanical Turk is a crowdsourcing platform that has been used extensively to collect psychological survey data. This pilot study sought to evaluate whether MTurk might also be a via- ble means of recruiting participants for online intervention research. Materials and methods: Participants were recruited to complete an online survey about their alcohol use through the MTurk platform. Those who met eligibility criterion for problem drinking were invited to complete a 3-month follow-up. Of those who agreed, a randomized half were asked to access an online brief intervention for drinking (CheckYourDrinking.net) and the other half were assigned to a no intervention control group (i.e., thanked and told that they would be re-contacted in 3 months). Conclusions: Results indicated that a brief alcohol intervention is feasible and acceptable to psychiatrically hospitalized suicidal adoles- cents, and may help to reduce their amount of alcohol use at 3 month follow-up. A larger fully powered study with a longer follow-up period is needed to test intervention efects and potential moderators. Materials and methods: Participants were recruited to complete an online survey about their alcohol use through the MTurk platform. Those who met eligibility criterion for problem drinking were invited to complete a 3-month follow-up. A pilot replication of QUIT, a randomized controlled trial of a brief intervention for reducing risky drug use, among Latino primary care patients 1 2 3 Lillian Gelberg1, Ronald M. Andersen2, Guillermina Natera Rey3, Mani Vahidi1, Melvin W. Rico1, Sebastian E. Baumeister4 1Department of Family Medicine, David Geﬀen School of Medicine at UCLA, Los Angeles, CA, USA; 2Fielding School of Public Health, University of California, Los Angeles, Los Angeles, CA, USA; 3National Institute of Psychiatry Ramón de la Fuente Muñiz, Mexico City, Mexico; 4Department of Epidemiology and Preventive Medicine, University of Regensburg, Regensburg, Germany Results: The cohort included 1198 pregnant women, 38% of whom were African-American and 15.5% of whom were Latina; 35% of partic- ipants had a Bachelor’s degree or higher, and 188 (15.9%) had a urine test positive for drugs or alcohol. Assessments were evenly distrib- uted across trimesters. The overall accuracy in identiication of a posi- tive urine screen was 83.8% for the NIDA Quick Screen, 73.9% for the WIDUS, 70.5% for the SURP-P, 68.7% for the CRAFFT, and 42.5% for the 5Ps. Sensitivity, speciicity, and positive and negative predictive values for the NIDA Quick Screen were 49.1, 90.0, 47.0, and 90.8, respectively. Conclusions: The parsimonious NIDA Quick Screen showed good accuracy but poor sensitivity in predicting urine drug screen results. Future analyses will consider potential new screening tools made from existing items, multi-step screening, and gold standards taking calen- dar-based recall and/or diagnostic status into account. Correspondence: Lillian Gelberg - lgelberg@mednet.ucla.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A33 Background: QUIT is the only primary care-based brief intervention that has previously shown eicacy for reducing risky drug use in the US (Gelberg et al., 2015). This pilot study replicated the QUIT rand- omized controlled trial in one of the ive original QUIT clinics that pri- marily serves Latinos. A34 A34 Web‑based intervention for people with harmful use or dependence of alcohol Magnus Johansson1, Christina Sinadinovic2, Anne H. Berman2, Ulric Hermansson2, Sven Andreasson1 1Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden; 2Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden Correspondence: Magnus Johansson - magnus.johansson.1@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A34 Web‑based intervention for people with harmful use or dependence of alcohol Magnus Johansson1, Christina Sinadinovic2, Anne H. Berman2, Ulric Hermansson2, Sven Andreasson1 1Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden; 2Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden Correspondence: Magnus Johansson - magnus.johansson.1@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A34 Materials and Methods: Design: Single-blind two-arm randomized controlled trial of patients enrolled from March–October 2013 with 3-month follow-up. Setting: Primary care waiting room of a FQHC in East Los Angeles. Participants: Adult primary care patients with risky drug use range 4–26 on the WHO ASSIST self-administered on tablet computers: 65 patients (32 intervention, 33 control); 51 (78%) completed follow-up; mean age 30.8 years; 59% male; 94% Latino. Interventions and measures: Inter- vention patients received: (1) brief (typically 3–4 min) clinician advice to quit/reduce their risky drug use, (2) video doctor message reinforc- ing the clinician’s advice, (3) health education booklet, and (4) up to two 20–30 min follow-up telephone drug use reduction coaching ses- sions. Control patients received usual care and cancer screening infor- mation. Primary outcome was reduction in the number of days of drug use in the past 30 days of the highest scoring drug (HSD) measured on the ASSIST, from baseline to 3-month follow-up. An evaluation of screening measures to detect substance use in pregnancy 1 2 3 Practices reported developing relationships with other service providers as an outcome of this work. Challenges included diiculties collecting data from EHRs, and incon- sistent insurance reimbursement policies for screening and BI, and follow-up with youth referred for behavioral health treatment. behavioral health treatment, fewer than one-third received referrals from their providers, either because they were already in treatment, or because parents or the patient refused. Practices reported developing relationships with other service providers as an outcome of this work. Challenges included diiculties collecting data from EHRs, and incon- sistent insurance reimbursement policies for screening and BI, and follow-up with youth referred for behavioral health treatment. Conclusions: Pediatric practices can readily incorporate SBIRT into routine screening protocols. Providers reported that SBIRT helped them to normalize conversations about drugs and alcohol as part of routine pediatric practice, and that patients were surprisingly open to further conversation. Closing the loop with youth referred for treat- ment remains a challenge. Materials and Methods: Four existing screening tools with pub- lished evidence regarding detection of drug use in pregnancy (SURP-P, CRAFFT, WIDUS, and 5Ps), plus the NIDA Quick Screen, were adminis- tered to pregnant women age 18 or over who were recruited from one of three sites. Screening tools were administered in counterbalanced order and were followed by collection of a urine sample that was tested for illicit and licit substances. An evaluation of screening measures to detect substance use in pregnancy 1 2 3 Results: A total of 423 participants were recruited, of which 85% were followed-up at 3-months. Only 1/3 of participants asked to access the online brief intervention did so. There was no signiicant diference between groups on the primary outcome variable—number of drinks in a typical week. One of three secondary outcome variables (AUDIT- C, highest number on one occasion, number of consequences expe- rienced) displayed a signiicant diference between condition, with those being asked to access the online intervention reporting signii- cantly greater reductions in AUDIT-C scores at follow-up compared to participants in the control condition (p = .004). Kimberly A. Yonkers1, Steven J. Ondersma2, Grace Chang3, Tiﬀany Blake-Lamb4 1 Kimberly A. Yonkers1, Steven J. Ondersma2, Grace Chang3, Tiﬀany Blake-Lamb4 1 1Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA; 2Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Detroit, MI, USA; 3Department of Psychiatry, VA Boston Healthcare and Harvard University, Boston, MA, USA; 4Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA Correspondence: Kimberly A. Yonkers - Kimberly.Yonkers@Yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A31 Correspondence: Kimberly A. Yonkers - Kimberly.Yonkers@Yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A31 Conclusions: Despite the current pilot showing only limited evidence of impact of the intervention among participants recruited through Page 12 of 24 Page 12 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Background: In 2016, there were over 3.9 million births in the US. The 2013 National Survey on Drug Use and Health suggests that from this group about 5% used illicit substances, 9% consumed alcohol and 15% smoked nicotine cigarettes. A ive-fold increase in antepartum maternal opiate use, coincident with an “epidemic” of opiate prescrip- tion abuse between 2000 and 2009 is also reported. These substances are harmful to mother and potentially her ofspring, rendering the identiication of women who use hazardous substances in pregnancy a public health priority. Unfortunately, only a few screening tools have been evaluated in pregnant women and none were tested against bio- chemical assays of substance use such as a urine toxicology test. The goal of this project was to compare the performance of ive screening tools for detection of substance use in women to a urine toxicology screen. behavioral health treatment, fewer than one-third received referrals from their providers, either because they were already in treatment, or because parents or the patient refused. Implementing SBIRT for youth and young adults in primary care: the New Hampshire Youth SBIRT initiative 1 2 1 Implementing SBIRT for youth and young adults in primary care: the New Hampshire Youth SBIRT initiative 1 2 1 Lea R. Ayers LaFave1, Kathleen M. Thies2, Amy L. Pepin1, Kara E. Sprangers1 Martha Bradley1, Shasta Jorgensen1, Nico A. Catano1, Adelaide R. Murray1, Deborah Schachter3 Lea R. Ayers LaFave1, Kathleen M. Thies2, Amy L. Pepin1, Kara E. Sprangers1, Martha Bradley1, Shasta Jorgensen1, Nico A. Catano1, Adelaide R. Murray1, Deborah Schachter3 1 2 1JSI Research and Training Institute, Inc., Bow, NH, USA; 2TLQ Associates Healthcare Consulting LLC, Bedford, NH, USA; 3New Hampshire Charitable Foundation, Concord, NH, USA 1JSI Research and Training Institute, Inc., Bow, NH, USA; 2TLQ Associates Healthcare Consulting LLC, Bedford, NH, USA; 3New Hampshire Charitable Foundation, Concord, NH, USA Correspondence: Lea R. Ayers LaFave - llafave@jsi.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A32 Correspondence: Lea R. Ayers LaFave - llafave@jsi.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A32 Background: Screen-Brief Intervention-Referral to Treatment (SBIRT) in pediatric practices normalizes conversations between youth and health care providers about alcohol and substance use, and supports guidance about healthy behaviors. SBIRT also identiies youth ages 12–22 whose current use of addictive substances places them at risk for developing substance use disorders, prompting providers’ brief intervention and referral for further assessment or treatment before substance use disorders develop. Results: Intervention patients reduced their past month HSD use by 4.5 more days than controls (p < .042, 95% CI 0.2, 8.7) by 3-month follow- up in an intent-to-treat linear regression analysis. Similar signiicant results were found using a complete sample regression analysis: 5.2 days (p < .03, 95% CI 0.5, 9.9). Conclusions: Findings further support the eicacy of QUIT in reducing risky drug use. Materials and methods: From May 2014 to June 2017, SBIRT was implemented as a standard of care in 23 pediatric practices in three cohorts across 10 organizations in New Hampshire—including aca- demic medical centers and FQHCs—serving over 25,000 youth. Sites adapted either the CRAFFT or S2BI screening tools for their electronic health record (EHR). Materials and methods included developing a playbook for implementing SBIRT, training in Brief Intervention (BI) for 174 providers, and technical assistance provided on site, by phone, and email, and during scheduled meetings and video conferences, to support changes in oice worklow, and integration of screening tools into EHRs for billing, documentation and data collection. Examination of referral to treatment as part of the brief intervention model 1 2 1 Megan A. O’Grady1, Sandeep Kapoor2, Cherine Akkari1, Camila Bernal1, Kristen Pappacena1, Jeanne Morley2, Mark Auerbach3, Charles J. Neighbors1, Nancy Kwon3, Joseph Conigliaro2, Jon Morgenstern4 1Division of Health Services Research, The National Center on Addiction and Substance Abuse, New York, NY, USA; 2Department of Internal Medicine, Emergency Medicine, and Psychiatry, Northwell Health, Great Neck, NY, USA; 3Departent of Emergency Medicine, Northwell Health, Great Neck, NY, USA; 4Department of Psychiatry, Northwell Health, Great Neck, NY, USA Materials and methods: A systematic review identiied k = 58 reports, describing 36 primary studies and 40 efect sizes (N = 3025 participants). Statistical methods calculated the inverse variance- weighted pooled correlation coeicient for the therapist to client and the client to outcome paths across multiple target behaviors (i.e., alco- hol use, other drug use, other behavior change). Results: Therapist MI-consistent skills were correlated with more cli- ent change talk (r = .55, p < .001) as well as more sustain talk (r = .40, p < .001). MI-inconsistent skills were correlated with more sustain talk (r = .16, p < .001), but not less change talk. When these measures were combined, as recommended in the Motivational Interviewing Skill Code, the overall technical hypothesis was supported. Speciically, proportion MI consistency was related with higher proportion change talk (r = .11, p = .004) and higher proportion change talk was related with reductions in risk behavior at follow up (r = −.18, p < .001). When tested as two independent efects, however, client change talk was not signiicant, but sustain talk was positively associated with worse outcomes (r = .20, p < .001). Finally, the relational hypothesis was not supported, but heterogeneity in technical hypothesis paths was par- tially explained by the a priori moderators of interest. Correspondence: Megan A. O’Grady - mogrady@centeronaddiction.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A35 Correspondence: Megan A. O’Grady - mogrady@centeronaddiction.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A35 Background: Screening, brief intervention, and referral to treatment (SBIRT) models in medical settings often rely on referrals to commu- nity treatment programs when patients with potential substance use disorder are identiied. Referrals have been studied much less than the screening and brief intervention components of the SBIRT model. In the available referral research there are mixed indings, with some studies showing that SBIRT increases treatment entry; others inding no relationship. Web‑based intervention for people with harmful use or dependence of alcohol h 1 h d 2 2 Web‑based intervention for people with harmful use or dependence of alcohol Magnus Johansson1, Christina Sinadinovic2, Anne H. Berman2, Ulric Hermansson2, Sven Andreasson1 1Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden; 2Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden Correspondence: Magnus Johansson - magnus.johansson.1@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A34 Results: While data collection will be completed in June 2017, the goal of 10,000 youth screened was exceeded in December 2016, with most sites sustaining screening rates above 85%. About 18% of youth were identiied as at risk. Although 29% of them needed referral to Background: Few harmful or dependent drinkers ever seek profes- sional help. This is largely due to stigma. Harmful drinkers are ashamed Page 13 of 24 Page 13 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 drug and alcohol use patterns, and health and psychosocial informa- tion. State data included date of treatment entry and level of care entered. of their problem and of going to a clinic. Many people prefer to use the internet to get information about alcohol over asking a doctor or friend. Web-based interventions for alcohol-problems reach individu- als who to a lesser extent come into contact with traditional addiction services. The interventions have shown small to moderate efects in reducing alcohol consumption. Few studies have focused on alcohol dependent users or the additional efect of guidance. Results: Fifty-ive percent of participants drank daily. During the past 30 days, 21% used marijuana and cocaine and 16% used opi- ates. Twenty-ive percent (107) of participants (99% from ED) entered treatment within 90 days of SBIRT (30% same day; 20% 1–7 days; 25% 8–30 days; 25% 31–90 days). The majority entered detox (79%); the remaining entered inpatient (12%) or outpatient (9%) programs. Mul- tivariate logistic regression analyses showed that having employment and trauma history decreased the likelihood of entering treatment within 90 days. Materials and Methods: A 3-arm randomized controlled trial was conducted at a well-established Swedish website aimed at the general public. New users from March 2015 to March 2017, with alcohol harm- ful use or dependence, were ofered to participate. After completing baseline questionnaires, all participants answered a survey about rea- sons for and preferences regarding web-based interventions. Correspondence: Correspondence: Molly Magill - molly_magill@brown.edu Addiction Science & Clinical Practice 2017 12(Suppl 1): A36 Background: In the present meta-analysis, we test the technical and relational hypotheses of Motivational Interviewing (MI) eicacy. We also propose an a priori conditional process model where heterogene- ity of technical path efect sizes should be explained by interpersonal/ relational (i.e., empathy, MI Spirit) and intrapersonal (i.e., client treat- ment seeking status) moderators. Web‑based intervention for people with harmful use or dependence of alcohol h 1 h d 2 2 After submitting the survey they were randomized to one of three forms of support: (1) information, (2) program as self-help or (3) program with on-line contact with a therapist. Participants were blinded to what kind of support the other groups received. The 8 module program consists of information-texts, videos and exercises based on Cognitive Behavioral Therapy and Motivational Interviewing. Conclusions: We found that a quarter of patients receiving referral as part of SBIRT entered treatment and identiied important patient-level referral predictors. SBIRT programs should continue to examine ways to increase treatment entry among those most in need. Patients with trauma history may beneit from trauma-informed SBIRT models. Examination of referral to treatment as part of the brief intervention model 1 2 1 Previous research also suggests that patient charac- teristics may be important in understanding the SBIRT referral process. As part of an evaluation of a large-scale SBIRT program, we examined the proportion of referred patients who entered treatment, as well as patient-level predictors of treatment entry. Conclusions: This meta-analysis provides additional support for the technical hypothesis of MI eicacy. Materials and methods: SBIRT was implemented in four emergency departments (ED) and 4 primary care practices in a large health sys- tem. Over 6000 patients screened positive on the AUDIT or DAST-10 during the irst 3.5 years of the program; 1091 (mostly ED patients [90%]) received a referral. About 40% consented to participation in the referral evaluation by allowing us to link program evaluation data with the New York State Client Data System treatment registry (n = 407). Program evaluation data included demographics, screening scores, The long view of meta‑analysis: testing technical, relational, d d l d l b f l The long view of meta analysis: testing technical, relational, and conditional process models in brief motivational intervention Molly Magill1, Timothy R. Apodaca2, Brian Borsari3, Jacques Gaume4, Ariel Hoadley5, J. Scott Tonigan6, Theresa Moyers6 y y 1Center for Alcohol and Addiction Studies, Brown University, Providence, RI, USA; 2Children’s Mercy Kansas City, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA; 3San Francisco Veterans Aﬀairs Health System and Department of Psychiatry, University of San Francisco, San Francisco, CA, USA; 4Lausanne University Hospital, Lausanne, 5 Discussion: Anonymity and access might be important reasons for choosing web-based treatment. The results from the randomized study will add knowledge of the efectiveness of web-based interven- tions, with or without guidance. Correspondence: Molly Magill - molly_magill@brown.edu A36 A36 The long view of meta‑analysis: testing technical, relational, and conditional process models in brief motivational intervention Molly Magill1, Timothy R. Apodaca2, Brian Borsari3, Jacques Gaume4, Ariel Hoadley5, J. Scott Tonigan6, Theresa Moyers6 1Center for Alcohol and Addiction Studies, Brown University, Providence, RI, USA; 2Children’s Mercy Kansas City, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA; 3San Francisco Veterans Aﬀairs Health System and Department of Psychiatry, University of San Francisco, San Francisco, CA, USA; 4Lausanne University Hospital, Lausanne, Switzerland; 5School of Public Health, Brown University, Providence, RI, USA; 6Center on Alcoholism, Substance Abuse, and Addictions, University of New Mexico, Albuquerque, NM, USA Correspondence: Molly Magill - molly_magill@brown.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A36 Results: Of 1175 participants with a mean age 45 years (SD = 13), 56% were women and 89% alcohol dependent (ICD). In the 7 days prior to study inclusion, participants consumed an average of 26 (sd = 17) drinks and their mean AUDIT score was 22 (SD = 6). Further, 37% showed symptoms of generalized anxiety (GAD-7) and 43% of depres- sion (MADRS-S). Participants were more ready to reduce their drink- ing (m = 8.4; SD = 1.9, on a 0–10 VAS-scale) than to stop (m = 8.4; SD = 1.9). The most endorsed reasons for using web-based interven- tion were anonymity and having access to intervention at any time. The most endorsed features were assessment feedback and online contact with a therapist. Data collection from the 3-month follow-up will be completed in July, 2017. The long view of meta‑analysis: testing technical, relational, and conditional process models in brief motivational intervention Molly Magill1, Timothy R. Apodaca2, Brian Borsari3, Jacques Gaume4, Ariel Hoadley5, J. Scott Tonigan6, Theresa Moyers6 1Center for Alcohol and Addiction Studies, Brown University, Providence, RI, USA; 2Children’s Mercy Kansas City, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA; 3San Francisco Veterans Aﬀairs Health System and Department of Psychiatry, University of San Francisco, San Francisco, CA, USA; 4Lausanne University Hospital, Lausanne, Switzerland; 5School of Public Health, Brown University, Providence, RI, USA; 6Center on Alcoholism, Substance Abuse, and Addictions, University of New Mexico, Albuquerque, NM, USA Correspondence: Molly Magill - molly_magill@brown.edu Addiction Science & Clinical Practice 2017 12(Suppl 1): A36 A37 Financial incentives for alcohol brief interventions in primary care in Scotland 1 2 3 Niamh M. Fitzgerald1, Lisa Schölin2, Amy J. O’Donnell3 1Institute for Social Marketing, UK Centre for Tobacco and Alcohol Studies, University of Stirling, Stirling, Scotland, UK; 2World Health Organization Regional Oﬃce for Europe; formerly Institute for Social Page 14 of 24 Page 14 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Marketing, University of Stirling, Stirling, Scotland, UK; 3Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK Marketing, University of Stirling, Stirling, Scotland, UK; 3Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK Correspondence: Niamh M. Fitzgerald - niamh.ﬁtzgerald@stir.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A37 Marketing, University of Stirling, Stirling, Scotland, UK; 3Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK Correspondence: Niamh M. Fitzgerald - niamh.ﬁtzgerald@stir.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A37 Marketing, University of Stirling, Stirling, Scotland, UK; 3Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK On random half-days, patients in the waiting room were encouraged by a research assistant to use the device and asked to complete a satis- faction questionnaire. Correspondence: Niamh M. Fitzgerald - niamh.ﬁtzgerald@stir.ac.uk Addiction Science & Clinical Practice 2017 12(Suppl 1): A37 Correspondence: Niamh M. Fitzgerald - niamh.ﬁtzgerald@stir.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A37 Correspondence: Niamh M. Fitzgerald - niamh.ﬁtzgerald@stir.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A37 Results: During the evaluation period and while the research assis- tant was present, 280 patients attended the practices and 99 (35.4%) used the device. Of them, 82 (82.3%) completed the satisfaction questionnaire. Mean (SD) age was 49.9 (16.9), 54.5% were female; 94% considered the device easy to use (“agree” or “strongly agree”), 93% considered the questions easy to understand, 79% considered their friends would be willing to use the device, while 8% reported that answering the questions made them uncomfortable, and 12% that they would prefer if the primary care physician asked the ques- tions. Most considered “useful” or “very useful” to be asked about their tobacco (92%), alcohol (92%), drug (99%), prescription drug use (91%) and physical activity (87%). Background: This study aimed to examine the evidence for inancial incentives for screening and brief intervention for alcohol (SBI) in pri- mary care and to explore the remuneration systems established in Scotland under the Scottish Government’s national SBI programme established in 2008. A37 Financial incentives for alcohol brief interventions in primary care in Scotland 1 2 3 Materials and methods: A rapid systematic literature review on the design and impact of inancial incentives on the delivery of SBI in pri- mary care, using PubMed; analysis of documentation and data regard- ing remuneration systems in three local areas in Scotland; in-depth semi-structured interviews with 5 key local and national stakeholders on the design and impact of remuneration models in Scotland. Conclusions: Among patients who used the device, its acceptability and usability were good. The developed device appears easy to use and patients generally perceived being asked about substance use and physical activity as useful. Nevertheless, the proportion of patients accessing the device remained limited. g p Results: From the 235 titles identiied in the systematic search, ten underwent full text review and four met inclusion criteria. Evidence in this area: is scarce, particularly in relation to systems implemented in routine practice; provides mixed evidence of impact on patient and provider outcomes; and does not indicate an optimum level of incentive. The three local remuneration models varied considerably in structure and rates of payment over time and in diferent areas: one provided core funding for community nursing; one made a single pay- ment for SBI (only where patients screened as needing intervention); and two paid separate incentives for screening and for brief inter- ventions following screening. No irm conclusions could be drawn about optimal models or levels of payment or the impact of changes over time. The ratio of brief interventions to screenings delivered also varied widely. Interviewees disagreed on whether incentives led to increased SBI delivery but suggested that the remuneration contracts enabled training and monitoring of delivery to be mandated. Distor- tions such as misrepresentation or gaming by claimants were not thought to be widespread. Correspondence: dd Correspondence: Nicolas Barticevic - nicolas.barticevic@gmail.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A39 Correspondence: Nicolas Barticevic - nicolas.barticevic@gmail.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A39 Conclusions: The establishment of 14 diferent local SBI remunera- tion systems in Scotland provided several opportunities for evaluation of natural experiments which have largely been missed. This study raises the possibility that inancial incentives operate as interventions in a complex system, rather than just incentivizing SBI delivery. Their structure, targets, and value are likely to have important implications, which ought to be studied carefully as part of, and to inform, future implementation initiatives. Background: In Chile, alcohol use is the leading cause of years lost due to disability. To address this issue, the Health Ministry has imple- mented a national program on Brief Interventions (BI) for Risky Alcohol use, in which Paramedics are the main BI providers. The objective of this work is to study the efectiveness of non-professional delivered BIs as they occur in the real world in Chilean primary care. Materials and Methods: A multi-center randomized open-label controlled trial was conducted in ive primary care centers in San- tiago de Chile. A total of 3247 people aged 18–45 were screened for moderate-risk alcohol use according to AUDIT (Alcohol Use Disorders Identiication Test), and 343 participants were randomized. The para- medic-delivered BI (n = 174) was compared to an informative pam- phlet (n = 169). The outcome measure was the AUDIT score at baseline and six months follow-up. A38 Acceptability and usability of a tablet‑based device for substance use and physical activity screening in primary care Nicolas Bertholet1, Angéline Adam1, John A. Cunningham2, Jean-Bernard Daeppen1 1 AUDIT‑linked Brief Intervention delivered by paramedics in Chilean primary care: a pragmatic randomized controlled trial Nicolas Barticevic1, Soledad Zuzulich2, Fernando Poblete3, Pablo Norambuena4 1 AUDIT‑linked Brief Intervention delivered by paramedics in Chilean primary care: a pragmatic randomized controlled trial Nicolas Barticevic1, Soledad Zuzulich2, Fernando Poblete3, Pablo Norambuena4 1Department of Family Medicina, Pontiﬁcia Universidad Católica de Chile, Santiago de Chile, Región Metropolitana, Chile; 2Nursing School, Pontiﬁcia Universidad Católica de Chile, Santiago de Chile, Región Metropolitana, Chile; 3Public Health Department, Pontiﬁcia Universidad Católica de Chile, Santiago de Chile, Región Metropolitana, Chile; 4Mental Health Department, Ministry of Health, Chile The integration of SBIRT into social work curricula at a university setting P l S 1 L T 2 M h l B l 1 Paul Sacco1, Laura Ting2, Michele Beaulieu1 Paul Sacco , Laura Ting , Michele Beaulieu 1School of Social Work, University of Maryland-Baltimore, Baltimore, MD, USA; 2University of Maryland-Baltimore County, Baltimore, MD, USA Correspondence: Paul Sacco - PSACCO@ssw.umaryland.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A40 Paul Sacco , Laura Ting , Michele Beaulieu 1School of Social Work, University of Maryland-Baltimore, Baltimore, MD, USA; 2University of Maryland-Baltimore County, Baltimore, MD, USA Correspondence: Paul Sacco - PSACCO@ssw.umaryland.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A40 Background: Presenters will review their SAMHSA-developed SBIRT curriculum focused on at-risk drinking, tobacco use and other drug use in social work (MSW) education. The curriculum was implemented through two methods: a standalone SBIRT (15 h, 1 credit) course and a hybrid foundation course infusion model. Results: Overall 45/406 (11.1%) accessed the website within 4 days of text (8th–12th March), most on the irst day. 43 respondents provided their gender—47% were women and 53% were men. Of the 38 who provided data, 25 (66%) were managers or professionals. 45 started the AUDIT, 41 (10.1%) completed the AUDIT-C, and 35 (8.6%) the full AUDIT. Of those who completed AUDIT-C, 36 (87.8%) scored positive (5 +). 3 registered for drink diary. Of the 35 completing the AUDIT, 10 were low risk (28.6%), 16 were increasing risk (45.7%), 5 were high risk (14.3%) and 4 (11.4%) were possibly dependent. Materials and methods: Standalone course students (n = 83) received in-person didactic instruction using an adapted version of the SAMHSA-SBIRT curriculum with role-plays, demonstration vid- eos, and values clariication exercises. All foundation irst year MSW students (n = 728) completed the hybrid infusion model combining 6 h of online and in-class training, including role-play exercises, vid- eos and didactic instruction. All trainees completed satisfaction sur- veys and self-report scales assessing SBIRT knowledge, attitudes, and behaviors pre-intervention, post-intervention (30 day) and 6-months post-intervention. Trainees in the standalone course were also evalu- ated through videotaped standardized patient interviews (pre- and immediate post-test) assessing their SBIRT skills and coded by trained staf using a standardized rating scale. Qualitative interviews focused on students’ perceptions of SBIRT and their use of SBIRT in practice. Conclusions: This study suggests that it may well be feasible to use SMS messaging as a means to facilitate patient access to on line screening and brief intervention packages. A42 Results: Overall, the social work SBIRT project has reached compa- rable or greater numbers of health professionals than 2013–2016 SAMHSA grantees; levels of trainee satisfaction were comparable with similar programs. Data from standalone coursework suggests coni- dence, knowledge and behavior change increased due to training, but leveled of at 6-months post-training (Sacco et al., in press). Qualitative interviews indicate trainees have diiculties implementing SBIRT due to lack of clarity about whether it was their role (or any social worker’s job) to conduct screening and brief intervention. Trainees also felt uncomfortable making recommendations to agency administrators to implement SBIRT given their student role. Additional structural barriers prevent SBIRT implementation as agency funding mandates require the use of other, speciic assessment tools, or limit substance use screening to addiction counselors. Use of SMS texts for facilitating access to online alcohol interventions: a feasibility study 1 2 3 Paul George Wallace1, Matthew Andrews2, Kate Daley3, Don Shenker4, Louise Gallagher5, Rod Watson6, Tim Weaver7, Amy O’Donnell8 1South London Health Innovation Network, London, UK; 2Safe Sociable London Partnership, London, UK; 3South London Health Innovation Network, London, UK; 4Alcohol Health Network, London, UK; 5Department Public Health, Royal Borough of Kingston, London, UK; 6South London Health Innovation Network, London, UK; 7Middlesex University, London, UK; 8University of Newcastle, Newcastle, UK Correspondence: Paul George Wallace - paul.wallace@nihr.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A41 Materials and methods: We conducted a randomized controlled trial to study the feasibility and eicacy of an SBIRT programme for haz- ardous drinkers presenting in an ED. All patients older than 18 years attending the ED were potentially eligible. Cognitively impaired or medically unstable patients were excluded, as were patients seeking treatment for alcohol use. Patients were randomized to two groups, with the control group receiving two lealets—one regarding alcohol use, and the other giving information about the study protocol. The intervention group received the same lealets as well as a brief motiva- tional intervention on alcohol use and, where appropriate, a referral to specialized treatment. The primary outcomes were the proportion of hazardous drinkers measured by AUDIT-C scale and the proportion of patients attending specialized treatment at 1.5 and 4.5 months. Results: Of 3027 patients attending the ED, 2044 (67%) were potentially eligible to participate, 247 (12%) screened positive for hazardous drinking, and 200 agreed to participate. 72% of the par- ticipating sample were men, and the mean age was 43 years. Fol- low-up rate was 78%. At 1.5 months, the intervention group showed Background: Facilitated access to online alcohol intervention can have a signiicant impact on reducing alcohol misuse and ill-health and ofers a potentially cost efective alternative to face to face inter- vention. SMS text messaging ofers a novel means to implement facili- tated access from general practice and this project was designed to test its feasibility and potential utility. p g p Results: Of 3027 patients attending the ED, 2044 (67%) were potentially eligible to participate, 247 (12%) screened positive for hazardous drinking, and 200 agreed to participate. 72% of the par- ticipating sample were men, and the mean age was 43 years. Fol- low-up rate was 78%. A40 previously completed the AUDIT-C. The texts included a short mes- sage—“Healthier drinking choices? Take the alcohol health check http://e-drink-check.kingston.gov.uk/ref/p1c1”. The online package accessed via the link enabled the patient to respond online to the AUDIT questions and to be categorized as low risk, increasing risk, high risk or possibly dependent. Respondents were then provided with brief advice and health-risk feedback on their score, based on motivational interviewing techniques, designed to increase self- awareness and provide techniques to address the issue and motivate health improvement. Feasibility and efectiveness of a specialized brief intervention for hazardous drinkers in an emergency department l 1 Cl Ol 2 C l 3 bl 3 l Pol Bruguera1, Clara Oliveras2, Carolina Gavotti3, Pablo Barrio3, Fleur Braddick3, Hugo López-Pelayo3, Laia Miquel3, Montse Suárez4, Carla Bruguera5, Lídia Segura5, Joan Colom5, Antoni Gual3 1 Pol Bruguera , Clara Oliveras , Carolina Gavotti , Pablo Barrio , Fleur Braddick3, Hugo López-Pelayo3, Laia Miquel3, Montse Suárez4, Carla Bruguera5, Lídia Segura5, Joan Colom5, Antoni Gual3 1Grup de Recerca, Addiccions Clínic (GRAC), Addictive Behaviours Unit, Psychiatry Department, Hospital Clínic de Barcelona, Barcelona, Spain; 2Psychiatry Department, Hospital Clínic de Barcelona, Barcelona, Spain; 3Grup de Recerca Addiccions Clínic (GRAC), Addictive Behaviours Unit, Psychiatry department, Hospital Clínic de Barcelona, Barcelona, Spain; 4Emergency Department, Hospital Clínic de Barcelona, Barcelona, Spain; 5Program on Substance Use, Public Health Agency, Government of Catalonia, Barcelona, Spain Correspondence: Antoni Gual and Pol Bruguera - tgual@clinic.cat and pbruguer@clinic.cat Correspondence: Antoni Gual and Pol Bruguera - tgual@clinic.cat and pbruguer@clinic.cat Addiction Science & Clinical Practice 2017, 12(Suppl 1): A42 Conclusions: Our SAMHSA project has been successful in training signiicant numbers of social work students. Student satisfaction has been high and training outcomes suggest that the training is efec- tive. Uptake of SBIRT by social work interns in practice has been lim- ited. System-level factors and social worker perceptions about scope of practice may be barriers to implementation. Addiction Science & Clinical Practice 2017, 12(Suppl 1): A42 Addiction Science & Clinical Practice 2017, 12(Suppl 1): A42 Introduction: Screening, brief intervention, and referral to treatment (SBIRT) programs have been developed, evaluated and shown to be efective, particularly in primary care and general practice. Neverthe- less, efectiveness of SBIRT in emergency departments (EDs) has not been clearly established. We aimed to evaluate the feasibility and ei- cacy of an SBIRT program conducted by psychiatrists specialized in addictive disorders and motivational interviewing techniques in the ED of a tertiary hospital. The integration of SBIRT into social work curricula at a university setting P l S 1 L T 2 M h l B l 1 Such an approach ofers real promise for substantially increasing the delivery of screening and brief intervention in primary care, but larger studies will be required to establish the cost efectiveness of this approach compared with tradi- tional face to face delivery. Acceptability and usability of a tablet‑based device for substance use and physical activity screening in primary care 1 1 2 Nicolas Bertholet1, Angéline Adam1, John A. Cunningham2, Jean-Bernard Daeppen1 1 1Alcohol Treatment Center, Department of Community Medicine and Health, Lausanne University Hospital, Lausanne, Switzerland; 2Centre for Addiction and Mental Health, Toronto, Ontario, Canada Correspondence: Nicolas Bertholet - Nicolas.Bertholet@chuv.ch Addiction Science & Clinical Practice 2017, 12(Suppl 1): A38 Results: From the total people screened, 11% were at moderate risk for alcohol use (AUDIT 8–15), and 2% at high risk (AUDIT over 15). Recruited participants had a mean age of 29 years, 57% were male, and the average AUDIT score was 10.5 (SD 2.6), which did not difer between groups. Changes to the “real practice” conditions had to be made to permit full protocol implementation. Lack of time, competi- tion with other tasks, and lack of space were some of the problems for screening procedures and delivery of BI. 58 paramedics were trained on AUDIT and BI delivery, and 32 reached the standards to participate in the study. Additional training was needed to ensure proper AUDIT administration and BI structure. Only 10 paramedics inally partici- pated in the study, mainly due to administrative constraints. To date, 120 participants have completed follow-up. The mean AUDIT score at follow-up is 6.2 for all participants, with no signiicant diferences observable between groups so far. Background: In primary care practices, electronic screening can potentially overcome implementation barriers and help clini- cians. Acceptability and usability are key elements in successful implementation. Materials and methods: With the help of product designers, we developed a tablet-based device for primary care waiting rooms. The device was designed to inspire ease and comfort and the tablet pro- gram was carefully designed to maximize ergonomics. It comprises screening for tobacco, alcohol, illicit drugs, prescription drug use and physical activity. A summary of the screening results with emoticons is then presented to patients. Patients who screen positive have the option to answer additional questions on alcohol use and to receive an electronic brief intervention. In February 2017, the device was piloted in 4 diferent primary care practices in suburban and rural Swit- zerland. The aim was to assess acceptability and usability of the device. Conclusions: Paramedics can implement AUDIT linked BIs, but they need special training and accommodation to integrate it into their practice and tasks. Both groups had lower AUDIT scores (by 4 points), but completion of follow-up is necessary to determinate if there is an efect attributable to BI. Acceptability and usability of a tablet‑based device for substance use and physical activity screening in primary care 1 1 2 Page 15 of 24 Page 15 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 A43 A di A dissemination model for delivering SBI in high schools Richard L. Brown1, Julie Whelan Capell2, D. Paul Moberg3, Julie Maslowsky4, Laura A. Saunders1 A dissemination model for delivering SBI in high schools Richard L. Brown1, Julie Whelan Capell2, D. Paul Moberg3, Julie Maslowsky4, Laura A. Saunders1 1 1Department of Family Medicine and Community Health, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2JulieINK, LLC, Milwaukee, WI, USA; 3Population Health Institute, University of Wisconsin-Madison, Madison, WI, USA; 4Department of Kinesiology and Health Education, University of Texas-Austin, Austin TX, USA Correspondence: Richard L. Brown - rlbrown@wisc.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A43 1Department of Family Medicine and Community Health, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 2JulieINK, LLC, Milwaukee, WI, USA; 3Population Health Institute, University of Wisconsin-Madison, Madison, WI, USA; 4Department of Kinesiology and Health Education, University of Texas-Austin, Austin TX, USA Correspondence: Richard L. Brown - rlbrown@wisc.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A43 Results: Among 1220 patients who screened positive for unhealthy alcohol use via the SISQ, 882 (72.3%) had an AUDIT score of 0–15 and 338 (27.7%) had a score >15. The sensitivity and speciicity of the SISQ in detecting an AUDIT score of >15 is 89.3% and 74.0%, respectively, when the SISQ frequency of heavy drinking is dichotomized at less than or equal to monthly versus weekly or more. Background: Alcohol and drug screening and brief intervention (SBI) prolongs abstinence and reduces substance use for adolescents. Numerous barriers to delivering SBI in healthcare settings have been delineated. Dissemination models are needed so that all adolescents can receive SBI. Conclusion: In this sample of ED patients with unhealthy alcohol use, the reported SISQ frequency of heavy drinking can be used as an indi- cator of severity to diferentiate between patients who should receive brief advice versus a more substantive intervention. Materials and methods: Ten high schools in Southeastern Wisconsin, USA, were recruited as sites for universal SBI administration to all ninth- or tenth-graders. School administrators agreed to use opt-out recruit- ing with students and parents. They expressed a preference for SBI administration by non-school personnel, as school staf did not have time, and students would reveal more accurate information. In Janu- ary, 2016, eight college seniors were trained to serve as health coaches and administer SBI. Preparing the future workforce: development and ield test evaluation of adolescent SBI simulation training 1 h 1 b h1 ll d 2 Weiwei Liu1, Tracy L. McPherson1, Sabrina Bauroth1, Cyrille Adam2, Dawn L. Lindsay3, Piper Lincoln3, Holly Hagle4 1Public Health, NORC at the University of Chicago, Bethesda, MD, USA; 2Research and Development, Kognito, New York, NY, USA; 3Research and Evaluation, Institute for Research, Education and Training in Addiction, Pittsburgh, PA, USA; 4Education and Training, Institute for Research, Education and Training in Addiction, Pittsburgh, PA, USA Correspondence: Tracy McPherson - McPherson-Tracy@norc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A45 Results: Over 95% of students in the participating grades received SBI. About two-thirds of the 2525 students who received SBI were freshmen, and one-third were sophomores. Self-reported prevalence of past-year substance use was 18% for alcohol, 10% for marijuana, and 3% for other drugs. Over 95% of the students reported comfort talking with their health coach and trusted that the information they revealed would remain con- idential, as coaches promised. After the SBI session, 87% of the past-year drinkers, 75% of the past-year marijuana users, and 80% of the past-year users of other drugs reported stronger intention to reduce their substance use in the next month. Over 94% of the abstinent students reported stronger intention to continue abstinence over the next year. Background: Nursing and social work programs from across the U.S. were recruited to participate in a learning collaborative to integrate adolescent SBI training into curriculum. NORC partnered with the American Association of Colleges of Nursing, Council on Social Work Education, Center for Clinical Social Work, IRETA, Kog- nito, and subject matter experts (SMEs) to develop and evaluate an adolescent alcohol and marijuana SBI online simulation training program. The program aims to prepare students, educators, and practitioners to conduct Brief Negotiated Interviews using Motiva- tional Interviewing (MI) strategies. This study aimed to evaluate the effectiveness of the training on key outcomes including students’ knowledge; attitudes toward working with patients/clients who use alcohol; readiness, confidence, and competence; and adoles- cent SBI skills. Conclusions: SBI administration by non-school staf holds promise as a method of delivering universal SBI to high school students. Use of SMS texts for facilitating access to online alcohol interventions: a feasibility study 1 2 3 At 1.5 months, the intervention group showed Materials and methods: A London practice used iPLATO software to send campaign and reminder SMS texts to 406 patients who had Page 16 of 24 Page 16 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 The single-item screening question (SISQ) to identify unhealthy alco- hol use may also provide information on severity to inform the brief intervention (Saitz et al. 2009, 2010, 2014). We assessed the SISQ accu- racy for identifying which ED patients with unhealthy alcohol use should receive a brief intervention versus brief advice. greater reductions in alcohol consumption, and fewer patients con- tinuing with hazardous alcohol use (26.4 vs 48.1%, p = 0.0053). The SBIRT program also increased the probability of attending special- ized treatment, compared to the control condition (19.4 vs 6.1%, p = 0.0119). Materials and methods: Non-clinician health coaches conducted brief health surveys on a non-targeted, random sample of English- or Spanish-speaking adult patients at an urban, public hospital during the hours of 8 a.m. and 12 a.m. on all days of the week. Patients were asked the SISQ, “How many times in the past 12 months have you had [X] or more alcoholic drinks in a day?” (where X is 5 for men and 4 for women, and any use is considered positive). Patients with positive responses were asked to use the following frequency options: “Never,” “Less than monthly,” “Monthly,” “Weekly,” “Daily or almost daily.” These options were preferred to numerical frequencies during piloting. All patients who screened positive (only) were asked the 10-item Alcohol Use Identiication Test (AUDIT). We compared the SISQ responses to the AUDIT dichotomized at 15; AUDIT scores of 0–15 receive education and advice; scores of >15 receive brief intervention and possible referral. Conclusion: The SBIRT program in the ED was found to be feasible and efective in identifying hazardous drinkers, reducing hazardous alco- hol use and increasing treatment for alcohol problems. A43 A di Between January and May, 2016, they delivered SBI at the high schools. Most sessions lasted about 15 min. Each student initially completed screening and brief assessment questionnaires via computer. The computer immediately printed out a summary of the student’s responses. The coach reviewed the computer printout and administered motivational interventions aimed to prolong abstinence or reduce use. Each student returned to the computer at the end of the session and anonymously answered several questions about their experience with the health coach and its impacts. A44 Materials and methods: The SBI with Adolescents simulation train- ing program was developed in collaboration with partners and SMEs, then beta tested and usability tested. Following development and testing, schools of nursing and social work (n = 11) were recruited from the learning collaborative to participate in the ield test evalu- ation. Among the 1390 nursing and social students who participated, 593 (42.7%) accessed the training; 797 (57.3%) did not, facilitating the comparison between trained and untrained students on key outcomes. Students completed a pretest and two posttest surveys online, approxi- mately 30 days apart. OLS regression was used to compare trained and untrained students at the two post-tests, while controlling for pretest. Longitudinal growth modeling was used to assess the efect of the sim- ulation training on the starting point and change of a given outcome. A44 Using the alcohol single‑item screening question in the emergency department for screening and risk‑stratiication Ryan P. McCormack1, Joy Scheidell2, Mirelis Gonzalez1 1Department of Emergency Medicine, NYU School of Medicine, New York, NY, USA; 2Department of Population Health, NYU School of Medicine, New York, NY, USA Correspondence: Ryan P. McCormack - Ryan.McCormack@nyumc.org Treatment of alcohol dependence in primary care compared to specialist care: a randomised controlled trial 1 2 1 Sara Wallhed Finn1, Anders Hammarberg2, Sven Andréasson1 1Department of Public Health Sciences, Karolinska Institutet, Centre for Psychiatry Research, Stockholm Health Care Services, Stockholm, Sweden; 2Department of Clinical neurosciences, Karolinska Institutet, Centre for Psychiatry Research, Stockholm Health Care Services, Stockholm, Sweden Results: Across study activities, barriers and facilitators of curriculum implementation appeared in several key stakeholder groups: students, educators (including faculty, ield supervisors, nurse preceptors), and program administrators. A number of key elements speciic to the CFIR components of Inner Setting, Characteristics of Individuals, and Process emerged such as stakeholder and leadership buy-in; previous exposure to SBIRT; comfortability with substance use/mental health issues; familiarity with technology; utilization of program liaisons/ champions; curriculum and course adaptability; and training of ield supervisors and preceptors. Correspondence: Sara Wallhed Finn - sara.wallhed-ﬁnn@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A46 Background: A minority of all individuals with alcohol depend- ence seek treatment. A possible way to reduce this treatment gap is to ofer treatment in primary care. Most treatment studies in primary care have included individuals with hazardous consumption, and alco- hol dependence has been studied to a lesser extent. There is a need to develop brief and efective treatment models that are feasible to implement. We have developed a stepped care model for treatment of hazardous drinking and alcohol dependence in primary care, “the 15-method”. The model consists of three steps: (1) identiication of problem drinking and brief advice, (2) assessment, with feedback, and (3) four brief sessions based on CBT and motivational interviewing. These sessions can be combined with pharmacological treatment. In this trial steps (2) and (3) are studied. Objective: To investigate if treat- ment for alcohol dependence, using a stepped care model, in primary care is as efective as specialist addiction care. Conclusions: Pre-service education is a critical component to pre- paring the medical and behavioral workforce to implement SBIRT in a range of settings. The CFIR model identiied barriers to and facili- tators of curriculum integration found in nursing, social work, and inter-professional education. Findings from this study can help inform approaches to the integration of SBIRT education taken by educa- tors and program leaders in academic institutions around the globe. Moreover, the indings can be used by professional associations who provide guidance on educational standards and curriculum infusion. Materials and methods: Randomized controlled non-inferiority trial, between groups parallel design, not blinded. Correspondence: Background: Despite the support for screening and brief intervention to identify alcohol and related problems in medical and behavioral health settings, this skill is rarely integrated into the professional edu- cation of future practitioners. NORC, in partnership with the Council on Social Work Education and the American Association of Colleges of Nursing, led a multi-year learning collaborative to infuse adolescent SBIRT curriculum in schools of social work and nursing. Conclusions: The indings suggest that online simulation training can positively impact student outcomes and shows great promise as a method for preparing students to conduct brief interventions using MI skills with adolescents using alcohol and marijuana. g Materials and methods: NORC partnered with IRETA on secondary analysis utilizing the Consolidated Framework for Implementation Research (CFIR) to conceptualize barriers and facilitators to the imple- mentation of adolescent SBIRT curriculum into undergraduate and graduate nursing and social work programs. Data from implementa- tion progress reports, learning collaborative calls, and implementation calls with individual schools working on infusing SBIRT curricula were reviewed by two raters and categorized according to the CFIR model. Results: Across study activities, barriers and facilitators of curriculum implementation appeared in several key stakeholder groups: students, educators (including faculty, ield supervisors, nurse preceptors), and program administrators. A number of key elements speciic to the CFIR components of Inner Setting, Characteristics of Individuals, and Process emerged such as stakeholder and leadership buy-in; previous exposure to SBIRT; comfortability with substance use/mental health issues; familiarity with technology; utilization of program liaisons/ champions; curriculum and course adaptability; and training of ield supervisors and preceptors. Materials and methods: NORC partnered with IRETA on secondary analysis utilizing the Consolidated Framework for Implementation Research (CFIR) to conceptualize barriers and facilitators to the imple- mentation of adolescent SBIRT curriculum into undergraduate and graduate nursing and social work programs. Data from implementa- tion progress reports, learning collaborative calls, and implementation calls with individual schools working on infusing SBIRT curricula were reviewed by two raters and categorized according to the CFIR model. SBIRT in an interprofessional context for healthcare students and professionals 1 2 2 Shauna P. Acquavita1, Ruth Anne Van Loon2, Rachel Smith2, Bonnie J. Brehm3, Tiﬃny Diers4, Karissa Kim5, Andrea Barker2 1School of Social Work, College of Allied Health Sciences, University of Cincinnati, Cincinnati, OH, USA; 2University of Cincinnati, School of Social Work, Cincinnati, OH, USA; 3College of Nursing, University of Cincinnati, Cincinnati, OH, USA; 4College of Medicine, University of Cincinnati, Cincinnati, OH, USA; 5James L. Winkle College of Pharma University of Cincinnati, Cincinnati, OH, USA Results: Preliminary results of the intention-to-treat analysis (n = 231) conirms non-inferiority for the primary outcome at twelve months fol- low up. Weekly alcohol consumption in primary care (n = 111) was 9.7 grams higher compared to specialist care (n = 120), (95% CI −30.4 to 49.7), p = 0.64. Correspondence: Shauna P. Acquavita - acquavsa@ucmail.uc.e Addiction Science & Clinical Practice 2017, 12(Suppl 1): A48 Background: Students across the healthcare professions need SBIRT knowledge and skills to be prepared to work with patients in a variety of settings. An interprofessional SBIRT course for students in medicine, nursing, pharmacy, and social work was developed to provide this education and to prepare students for collaborative practice. Conclusions: A stepped care model is a promising approach for treat- ment of alcohol dependence in primary care. This may be a way to broaden the base of treatment for alcohol dependence, reducing the current treatment gap. Materials and methods: SBIRT education was presented in a hybrid course. In the irst half, students completed online modules on substance use disorders (SUD) and had SBIRT virtual simulations. The mid-term exam was a standardized patient experience where interprofessional teams administered SBIRT. Finally, clinical experiences in community agencies and the university’s medical center allowed interprofessional teams to implement SBIRT with patients/clients. Quantitative and quali- tative data provided feedback on course content and experiences. Treatment of alcohol dependence in primary care compared to specialist care: a randomised controlled trial 1 2 1 The non-inferiority limit was set to 50 g of alcohol per week. 288 adults fulilling ICD-10 crite- ria for alcohol dependence were randomized to treatment in primary care (n = 144) or specialist care (n = 144). General practitioners at 12 primary care centers received one day of training in the model. Pri- mary outcome was change in weekly alcohol consumption at twelve months follow up compared to baseline, as measured with Time Line Follow Back. Secondary outcomes were heavy drinking days, severity of dependence, consequences of drinking, psychological health, qual- ity of life, satisfaction with treatment and biomarkers. Using the alcohol single‑item screening question in the emergency department for screening and risk‑stratiication k1 h d ll2 l l 1 Ryan P. McCormack1, Joy Scheidell2, Mirelis Gonzalez1 1Department of Emergency Medicine, NYU School of Medicine, New York, NY, USA; 2Department of Population Health, NYU School of Medicine, New York, NY, USA Correspondence: Ryan P. McCormack - Ryan.McCormack@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A44 Correspondence: Ryan P. McCormack - Ryan.McCormack@nyumc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A44 Background: The adoption of screening and intervention for unhealthy alcohol use in emergency departments (EDs) remains extremely limited. Streamlining the assessment would be more acceptable to staf, who cite time and competing priorities as barriers. Page 17 of 24 Page 17 of 24 Page 17 of 24 Page 17 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Pittsburgh, PA, USA; 4Training and Education, Institute for Research, Education and Training in Addiction, Pittsburgh, PA, USA Correspondence: Sarah E. King - king-sarah@norc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A47 Pittsburgh, PA, USA; 4Training and Education, Institute for Research, Education and Training in Addiction, Pittsburgh, PA, USA Correspondence: Sarah E. King - king-sarah@norc.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A47 Results: Comparisons of demographic characteristics between trained and untrained students showed no diferences by gender and age. The simulation training was efective in improving a number of key outcomes. Results showed a small to medium standardized efect size, and students who received the training showed sharper growth in their attitudes, conidence, competency, and readiness to implement SBI in the ield at the posttest. A49 B i Barriers and facilitators of implementation of screening, brief intervention and referral to treatment (SBIRT) for behavioral health problems in pediatric primary care: a qualitative interview study Stacy A. Sterling1, Ashley L. Jones1, Asheley C. Skinner2, Agatha Hinman1 1Division of Research, Kaiser Permanente Northern California Division of Research, Oakland, CA, USA; 2Department of Medicine, Clinical Research Institute, Duke University, Durham, NC, USA Correspondence: Stacy A. Sterling - stacy.a.sterling@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A49 Barriers and facilitators of implementation of screening, brief intervention and referral to treatment (SBIRT) for behavioral health problems in pediatric primary care: a qualitative interview study Stacy A. Sterling1, Ashley L. Jones1, Asheley C. Skinner2, Agatha Hinman1 1Division of Research, Kaiser Permanente Northern California Division of Research, Oakland, CA, USA; 2Department of Medicine, Clinical Research Institute, Duke University, Durham, NC, USA Correspondence: Stacy A. Sterling - stacy.a.sterling@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A49 Background: Pediatric primary care is an opportune setting for Screen- ing, Brief Intervention and Referral to Treatment (SBIRT), but it has not been widely implemented, and there is little research on factors con- tributing to its implementation. This study used data from qualitative interviews (n = 20) with Kaiser Permanente Northern California (KPNC) and community-based pediatric primary care, specialty mental health and substance abuse treatment clinicians, policymakers and staf to examine factors which may inhibit or facilitate implementation of SBIRT in pediatric primary care. We used the Consolidated Framework for Implementation Research (CFIR) to inform our analysis, which used an inductive approach to build an understanding of the complexities involved in SBIRT implementation in pediatric primary care. Materials and methods: Randomized clinical trial with 500 WIDUS- positive postpartum women. Participants were randomly assigned to either a time control condition or a single-session, tailored brief inter- vention. The primary outcome was days of drug use over the 6-month follow-up period; secondary outcomes included urine and hair analy- sis results at 3- and 6-month follow-up. Results: 36.1% of participants acknowledged drug use in the 3 months prior to pregnancy, but 89% tested positive for drugs at the 6-month follow-up. Participants rated the intervention as easy to use (4.9/5) and helpful (4.4/5). Analyses revealed no between-group diferences in drug use. Exploratory analyses also showed that intervention efects were not moderated by baseline severity, WIDUS score, or readiness to change. A50 interviewing (MI) (95%), or experience using MI with clients (58%). There were signiicant improvement in scores for competency in SBIRT (Measured by University of Pittsburgh’s SBIRT Medical and Residency Training Survey), from pre to post course (p < .0005). Perceived con- idence and preparedness (p < .0005) for conducting a SBI improved after completing a SBI virtual simulation. Qualitative data included “The experiences gave me an excellent opportunity to apply SBIRT in a real setting… and allowed me to work collaboratively with other pro- fessions that may play a role in the impact alcohol and drugs has on one’s health. (Social Work)”; “If other members of the healthcare team are also trained in SBRT skills then the chances of a patient receiving the proper screening and intervention would be greatly increased…In the future, I plan to share my SBRT training…” (Medicine).” Computer‑delivered indirect screening and brief intervention for drug use in the perinatal period: a randomized trial Steven J. Ondersma1, Dace S. Svikis2, Casey L. Thacker3, Ken Resnicow3, Jessica R. Beatty4, James Janisse5, Karoline Puder6 1Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Wayne State University, Detroit, MI, USA; 2VCU Institute for Women’s Health and Departments of Psychology, Psychiatry, and Obstetrics/Gynecology, Virginia Commonwealth University, Richmond, VA, USA; 3School of Public Health, University of Michigan, Ann Arbor, MI, USA; 4Merrill-Palmer Skillman Institute, Wayne State University, Detroit, MI, USA; 5Department of Family Medicine and Public Health Sciences, Wayne State University, Detroit, MI, USA; 6Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA Computer‑delivered indirect screening and brief intervention for drug use in the perinatal period: a randomized trial Steven J. Ondersma1, Dace S. Svikis2, Casey L. Thacker3, Ken Resnicow3, Jessica R. Beatty4, James Janisse5, Karoline Puder6 1Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Wayne State University, Detroit, MI, USA; 2VCU Institute for Women’s Health and Departments of Psychology, Psychiatry, and Obstetrics/Gynecology, Virginia Commonwealth University, Richmond, VA, USA; 3School of Public Health, University of Michigan, Ann Arbor, MI, USA; 4Merrill-Palmer Skillman Institute, Wayne State University, Detroit, MI, USA; 5Department of Family Medicine and Public Health Sciences, Wayne State University, Detroit, MI, USA; 6Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA Conclusions: An interprofessional hybrid course is an efective method for providing education about SUD and interventions and for teaching SBIRT skills, topics often missing from disciplinary curricula. A49 B i Materials and methods: Audiotapes of interviews were transcribed, and using NVivo software, were double-coded, independently, by coders blind to each other’s coding. Themes were created based on the broad constructs of the CFIR model: outer setting, inner setting, characteristics of the intervention, characteristics of the individuals involved, and process of implementation. Within those overarching constructs, SBIRT-speciic sub-themes were developed, based on par- ticipant responses and informed by the extant literature. Percentage coder agreement and a Kappa Coeicient were calculated to measure inter-rater reliability, by interview, node, and across the sample. Conclusions: The present trial showed no evidence of eicacy for an indirect, single-session, computer-delivered brief interven- tion designed as a complement to indirect screening. More direct approaches that still do not presume active drug use may be possible and appropriate. Barriers to and facilitators of integrating adolescent SBIRT training in nursing, social work and inter‑professional education 1 2 1 2 Sarah E. King1, Dawn L. Lindsay2, Tracy L. McPherson1, Rachael Vargo2, Brayden N. Kameg3, Holly Hagle4 1 1Public Health Department, NORC at the University of Chicago, Bethesda, MD, USA; 2Research and Evaluation, Institute for Research, Education and Training in Addiction, Pittsburgh, PA, USA; 3Department of Health and Community Systems, University of Pittsburgh School of Nursing, Results: Prior to the course, most students (n = 43) had ten hours or less of training in SUD (81%), course content on motivational Page 18 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Correspondence: Steven J. Ondersma - s.ondersma@wayne.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A50 Correspondence: Steven J. Ondersma - s.ondersma@wayne.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A50 Correspondence: Steven J. Ondersma - s.ondersma@wayne.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A50 Background: Under-reporting of drug use in the perinatal period is well-documented, and signiicantly limits brief intervention reach. The Wayne Indirect Drug Use Screener (WIDUS) is a validated screener focusing on correlates of drug use rather than drug use itself. The present trial tested the eicacy of a single-session computer-deliv- ered screening and brief intervention designed for use with indirect screen-positive cases; this intervention sought to motivate reduc- tions in substance use without presuming its presence. It did so by engaging participants in a tailored review of “parenting strengths” that are associated with positive child outcomes, one of which was “a healthy home” (deined as absence of substance abuse); other speci- ied strengths, such as safety and emotional health, were selected in part because of their negative association with substance use. Partici- pants were invited to consider whether they could enhance their or “their home’s” strengths in any of those areas. Participants who indi- cated interest in change were helped to set speciic goals and identify resources for facilitating change (such as treatment). Innovative strategies for brief interventions targeting alcohol dependence: program development and implementation for treatment in primary care d ﬁ k h Results: Inner setting factors, such as time, screening instruments and weak linkages between department and organizations were most fre- quently discussed as barriers to SBIRT implementation. Outer setting factors such as conidentiality laws and societal attitudes about sub- stance use were also frequently cited as inluencing implementation, as were patient characteristics, such as co-occurring mental health concerns and linguistic needs. Many respondents also discussed the role of clinician training and SBIRT skills. The single most frequently discussed implementation facilitator was adopting an integrated, embedded-behavioral health clinician model of SBIRT. Inter-rater reli- ability was high: Kappa coeicients ranged from 0.78 to 0.85, and per- cent agreement between coders was >98% across the constructs. Sven Andréasson, Ann-Soﬁe Bakshi Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden, Stockholm, Sweden Correspondence: Sven Andréasson - sven.andreasson@gmail.com Addiction Science & Clinical Practice 2017, 12(Suppl 1): A51 Correspondence: A50 The hybrid model meets the challenges of scheduling, geographical location, professional curriculum requirements, and administrative buy-in. Practicing SBI skills in clinical sites with preceptors present is a unique experience that is highly valued by students. Estimating efectiveness of components of a smartphone app‑ Drink Less—to reduce excessive alcohol consumption: a factorial randomised control trial Estimating efectiveness of components of a smartphone app‑ Drink Less—to reduce excessive alcohol consumption: a factorial randomised control trial Materials and methods: Data were collected through semi-struc- tured interviews with general practitioners, working in primary care units participating in TAP. Ten informants were interviewed at base- line after they had attended a one-day training in TAP and before program implementation. Follow-up interviews were conducted after 6 months. The interviews were recorded, transcribed and analyzed through qualitative thematic content analysis. Claire V. Garnett1, David Crane1, Jamie Brown2, Robert West2, Susan Michie1 Claire V. Garnett1, David Crane1, Jamie Brown2, Robert West2, Susan Michie1 1Department of Clinical, Educational and Health Psychology, University College London, London, England; 2Department of Behavioural Science and Health, University College London, London, England Correspondence: Claire V. Garnett - c.garnett.12@ucl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A53 1Department of Clinical, Educational and Health Psychology, University College London, London, England; 2Department of Behavioural Science and Health, University College London, London, England Correspondence: Claire V. Garnett - c.garnett.12@ucl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A53 Results: At baseline, informants expressed a need for increased knowl- edge on how to treat patients with alcohol disorders. They observed that several patients seemed to have problematic alcohol consump- tion but lacked a systematic method for interventions. In the follow- up study, the informants stated that TAP had provided a systematic treatment tool, which functioned well in the primary care setting. The program was perceived as potentially efective in reducing alcohol consumption and as resource efective. Furthermore it was considered uncomplicated and easy to use. Also, the informants were motivated to integrate it into the regular practice of their primary care units. Background: Smartphone apps have the potential to help drinkers reduce hazardous and harmful alcohol consumption. However, there have been few evaluations of the efectiveness of these apps and none to our knowledge that estimates the efects of individual intervention components. This study aimed to evaluate the efectiveness of inter- vention components of an alcohol reduction app, Drink Less. Materials and methods: Drink Less is a freely available app to any individual in the UK making an attempt to reduce their drinking. The app was structured around goal setting with information on the UK drinking guidelines, units and alcohol-related harms. Optimizing engagement with eSBI: The BRANCH app targeting harmful drinking in young adults Results: Of 672 study participants, 27% responded to follow- up. At baseline, the mean past week consumption was 39.9 units (SD = 27.34) and mean AUDIT score was 19.1 (SD = 6.56). There were no signiicant main efects of the intervention modules on either measure. There were two-way interactions between enhanced Self-monitoring and Feedback and Action Planning on AUDIT score (F = 5.818, p = 0.016) and between enhanced Normative Feedback and Cognitive Bias Re-training on past week consumption (F = 4.676, p = 0.031). Enhanced Self-monitoring and Feedback was used more often and rated more positively for helpfulness, satisfaction and rec- ommendation than the minimal version. Background: Electronic screening and brief intervention (eSBI) smartphone apps demonstrate potential to reduce harmful drink- ing. However, low user engagement rates with eSBI reduce overall efectiveness. The Alcohol Theme of the Collaboration for Leadership in applied Health Research and Care (CLAHRC) South London, has developed an eSBI app targeting harmful drinking in young adults which included strategies for optimising engagement. The app, called ‘BRANCH’, was evaluated with a mixed-methods design, includ- ing a randomised controlled trial (RCT), which compared a basic and enhanced version of the app, as well qualitative interviews with par- ticipants from the RCT. The qualitative component, exploring partici- pants’ engagement with the app is presented here. Conclusions: Individual enhanced modules were not more efective compared with their minimal condition. The combinations of Self- monitoring and Feedback with Action Planning, and Normative Feed- back with Cognitive Bias Re-training resulted in signiicant reductions in alcohol-related outcomes when both modules were enhanced. Users rated the Self-monitoring and Feedback module signiicantly more positively when it was enhanced. Materials and methods: Qualitative 1:1 interviews were conducted with participants recruited from the basic and enhanced arms of the BRANCH RCT. Half of the participants were high engagers (logged in more than twice), the other half low engagers (logged in less than twice). Interviews explored participants’ experiences of using the app, including barriers and facilitators to engagement. A detailed thematic analysis was undertaken. Estimating efectiveness of components of a smartphone app‑ Drink Less—to reduce excessive alcohol consumption: a factorial randomised control trial The app ofered access to ive additional intervention modules—Normative Feedback, Cognitive Bias Re-training, Self-monitoring and Feedback, Action Plan- ning and Identity Change—to help them achieve their goal. Exces- sive drinkers (AUDIT ≥ 8) who were aged 18+ were orthogonally randomised to receive ‘enhanced’ or ‘minimal’ versions of each of the ive modules (to a total of 25 experimental conditions). The primary outcome measure was change in past week consumption at one- month follow-up. Secondary measures were change in AUDIT score, usage data and usability ratings. A factorial between-subjects ANOVA assessed main and interactive efects of the app modules using an intention-to-treat analysis. Conclusion: Knowledge gained from the TAP study can be used to further develop and implement innovative treatment strategies for alcohol problems. Importantly, the stigma problem, which to a large extent generates a threshold for seeking help, can be reduced. TAP is tailored to primary care and the results of the study indicate that pri- mary care practitioners can be efectively engaged to deliver treat- ment for alcohol dependence. A53 A53 was implemented in 12 primary care units in Stockholm, Sweden. The aim of the present study is to investigate the functionality of the TAP- program, as experienced by the participating practitioners. was implemented in 12 primary care units in Stockholm, Sweden. The aim of the present study is to investigate the functionality of the TAP- program, as experienced by the participating practitioners. Optimizing engagement with eSBI: The BRANCH app targeting harmful drinking in young adults Joanna M. Milward, Paolo Deluca, Andreas Kimergard, Colin Drummond Addictions Department, King’s College London, London, UK Correspondence: Joanna M. Milward - joanna.milward@kcl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A52 Optimizing engagement with eSBI: The BRANCH app targeting harmful drinking in young adults Joanna M. Milward, Paolo Deluca, Andreas Kimergard, Colin Drummond Addictions Department, King’s College London, London, UK Correspondence: Joanna M. Milward - joanna.milward@kcl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A52 Correspondence: Background: Alcohol use disorders are common, although a majority of afected individuals are reluctant to seek and undergo treatment in addiction care units. A major reason for this is the stigma attached to alcohol problems and treatment, generating a need to develop and implement stigma reducing interventions. Consequently, we devel- oped the program’ treatment for alcohol dependence in primary care (TAP)’ for brief intervention tailored to the primary care setting. TAP Conclusions: Participants identiied many challenges to SBIRT imple- mentation, but responses suggested a number of pragmatic policy and care delivery changes which could be taken to encourage its greater adoption. Page 19 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Smartphone apps targeting risky and excessive drinking patterns among university students show difering subgroup efects over 20 weeks 1 2 1 3 Materials and methods: 1640 adolescents (aged 14–17) attending ten EDs across three regions of England were screened for alcohol consumption and randomized into the trials. 73% across the two trials were follow up at 12 months. g p Materials and Methods: Students from six campuses were invited to a three-armed trial (A). Those with hazardous alcohol use (n = 2166) were randomly assigned to one of two smartphone apps ofer- ing feedback on real-time estimated blood alcohol concentration (eBAC) levels, or to a control group, with three follow-ups at 6, 12 and 20 weeks. At 6 weeks, participants in the app groups with excessive weekly alcohol consumption of >9 (women) or >14 (men) drinks per week (n = 257), were ofered participation in a second trial (B); con- senters (n = 186) were randomly assigned to a skills-based app or a waitlist group, and compared with an assessment-only control group. Results: Six-week analyses (n = 2166) replicated our earlier trial from 2014, re-conirming earlier results: the Promillekoll app was associated with higher quantity and frequency of drinking compared to controls, and a higher risk for excessive drinking; the PartyPlanner group did not difer from controls. Lower-risk drinkers from trial A (n = 1177) up to 20 weeks did not difer from controls on main outcomes. However, sub-analyses showed that individuals with higher consumption had higher motivation to reduce intake. In both intervention groups, con- sumption was lower for more highly motivated participants compared to controls at 6- and 20-week follow-ups. Latent class analysis of par- ticipants in both trials (n = 2166) revealed a class (n = 146) that drank several days a week and that difered signiicantly from the remain- ing cohort in gender, age, and alcohol consumption. For this class, access to the Promillekoll app appeared marginally associated with lower quantity over time; access to the skills-based TeleCoach app was clearly associated with fewer drinking days up to 20 weeks. Results: In both trials no signiicant diferences in outcome were found between groups on either primary or secondary outcome meas- ures. This supported the null hypothesis that PFBA and eBI are no more efective nor cost efective in reducing alcohol consumption in low-risk drinkers than screening alone. For those allocated to eBI, 34% actually engaged with the intervention after leaving the ED. No rela- tionship was identiied between engagement with the intervention and alcohol consumption at month 12. Efectiveness and cost efectiveness of a smartphone based electronic alcohol intervention for adolescents: indings from the SIPS jr trials 1 2 1 1 SIPS jr trials Paolo Deluca1, Simon Coulton2, Sadie Boniface1, Kim Donoghue1, Eilish Gilvarry3, Eileen Kaner4, Ellen Lynch4, Ian Maconochie5, Ruth McGovern4, Dorothy Newbury-Birch6, Ceri Phillips7, Rhys Pockett7, Tom Phillips1, R. Patton8, Ian Russell9, John Strang1, Colin Drummond1 1Department of Addictions, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK; 2Centre for Health Services Studies, University of Kent, Canterbury, Kent, UK; 3Northumberland, Tyne and Wear NHS Foundation Trust, UK; 4Institute of Health and Society, Newcastle University, Newcastle, UK; 5Paediatric Emergency Medicine, Imperial College London, London, UK; 6School of Health and Social Care, Teesside University, Middlesbrough, UK; 7Swansea Centre for Health Economics, College of Human and Health Sciences, Swansea University, Swansea, Wales, UK; 8School of Psychology, University of Surrey, Guildford, UK; 9Swansea University Medical School, Swansea, Wales, UK Paolo Deluca1, Simon Coulton2, Sadie Boniface1, Kim Donoghue1, Eilish Gilvarry3, Eileen Kaner4, Ellen Lynch4, Ian Maconochie5, Ruth McGovern4, Dorothy Newbury-Birch6, Ceri Phillips7, Rhys Pockett7, Tom Phillips1, R. Patton8, Ian Russell9, John Strang1, Colin Drummond1 Materials and methods: First, results from a national survey of health plan activities related to SBI were synthesized with literature review and analysis. Next, a public forum was convened and a policy brief was presented to provide education and motivation regarding the impor- tance of SBI. Finally, Massachusetts health care stakeholders, repre- senting health plans, provider groups, state and federal government, and researchers came together to discuss how to advance alcohol SBI eforts in the state and qualitative analyses were conducted. 1Department of Addictions, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK; 2Centre for Health Services Studies, University of Kent, Canterbury, Kent, UK; 3Northumberland, Tyne and Wear NHS Foundation Trust, UK; 4Institute of Health and Society, Newcastle University, Newcastle, UK; 5Paediatric Emergency Medicine, Imperial College London, London, UK; 6School of Health and Social Care, Teesside University, Middlesbrough, UK; 7Swansea Centre for Health Economics, College of Human and Health Sciences, Swansea University, Swansea, Wales, UK; 8School of Psychology, University of Surrey, Guildford, UK; 9Swansea University Medical School, Swansea, Wales, UK Results: A number of key issues and next steps were identiied, which fell into strategies for providers and those for health plans. For pro- viders, the importance of utilizing non-physician staf for SBI deliv- ery, improving medical education, and ofering a toolbox for primary care providers to use were noted. Smartphone apps targeting risky and excessive drinking patterns among university students show difering subgroup efects over 20 weeks 1 2 1 3 y Results: Twenty participants were recruited from January to March 2017. Findings suggest that eSBI for young people is successful for speciic user-types who are motivated to monitor their health and enjoy entering physical health data. Ease-of-access and low data-entry burden costs directly afected users’ engagement with the app. Conclusions: This is the irst study to explore participant engagement with an eSBI speciically targeting harmful drinking in young adults. ESBI may be appropriate for speciic user groups of digital technology. Young people are proicient technology users who want simple and fast interactions with eSBI apps. If eSBI apps do not meet these expec- tations then young people quickly cease use altogether. Implications of the indings to improve engagement are discussed in the context of future app development. Results: Twenty participants were recruited from January to March 2017. Findings suggest that eSBI for young people is successful for speciic user-types who are motivated to monitor their health and enjoy entering physical health data. Ease-of-access and low data-entry burden costs directly afected users’ engagement with the app. Anne H. Berman1,2, Ingvar Rosendahl1, Claes Andersson3, Mikael Gajecki1,2, Kristina Sinadinovic1, Matthijs Blankers4,5,6 1Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden; 2Stockholm Center for Dependency Disorders, Stockholm, Sweden; 3Malmö University, Department of Criminology, Malmö, Sweden; 4Trimbos Institute—The Netherlands Institute of Mental Health and Addiction, Utrecht, The Netherlands; 5Arkin Mental Health Care, Amsterdam, The Netherlands; 6Department of Psychiatry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Conclusions: This is the irst study to explore participant engagement with an eSBI speciically targeting harmful drinking in young adults. ESBI may be appropriate for speciic user groups of digital technology. Young people are proicient technology users who want simple and fast interactions with eSBI apps. If eSBI apps do not meet these expec- tations then young people quickly cease use altogether. Implications of the indings to improve engagement are discussed in the context of future app development. Correspondence: Anne H. Berman - anne.h.berman@ki.se Addiction Science & Clinical Practice 2017, 12(Suppl 1): A54 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Page 20 of 24 Page 20 of 24 Background and Aims: University students with risky drinking are a clear target group for intervention via smartphone apps. This study compared three diferent apps over a 20-week period, for university students with hazardous and excessive drinking patterns. A56 A56 Reducing risky drinking: what health care systems can do Maureen T. Stewart1, Amity E. Quinn2, Mary Brolin1, Brooke Evans1, Constance Horgan1 1Institute for Behavioral Health, The Heller School for Social Policy and Management, Brandeis University, Waltham, MA, USA; 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada Correspondence: Maureen T. Stewart - mstewart@brandeis.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A56 Reducing risky drinking: what health care systems can do Maureen T. Stewart1, Amity E. Quinn2, Mary Brolin1, Brooke Evans1, Constance Horgan1 1 1Institute for Behavioral Health, The Heller School for Social Policy and Management, Brandeis University, Waltham, MA, USA; 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada Correspondence: Maureen T. Stewart - mstewart@brandeis.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A56 Conclusions: Smartphone apps targeting eBAC can inluence drink- ing levels up to 20 weeks for university students with hazardous use and higher motivation to reduce their drinking. A skills-based app that reduces intake among students with excessive weekly consumption can be particularly efective for students with daily drinking habits. Background: Risky, non-dependent alcohol use is prevalent in the United States, and Massachusetts has higher rates of alcohol use and binge drinking than other states. Screening and brief intervention (SBI) is an evidence-based practice to address risky alcohol use, but numerous barriers are slowing the rate of SBI uptake. While healthcare providers play a critical role in increasing the use of alcohol SBI, health care systems have a role encouraging further expansion. The goal of this project was to bring health care system stakeholders together to identify actionable steps to advance alcohol SBI and improve popula- tion health in Massachusetts. Smartphone apps targeting risky and excessive drinking patterns among university students show difering subgroup efects over 20 weeks 1 2 1 3 However, females were more likely to download the app than males (p < 0.001). Conclusions: In both trials we found that engagement with the eBI intervention was low in participants randomized to eBI. Only a third of participants engaged with the eBI platform after leaving ED. This may have limited the impact of the eBI intervention compared to control intervention. However, as these were pragmatic trials, this is likely to be the level of engagement expected in the typical patient recruited for ED. Low screening and follow‑up for unhealthy alcohol use in Medicaid health plans 1 1 1 1 Junqing Liu1, Fern McCree1, Doug Kanovsky1, Tyler Oberlander1, Huan Zhang1, Ben Hamlin1, Robert Saunders1, Mary B. Barton1, Sarah H. Scholle1, Patricia Santora2, Chirag Bhatt3, Kazi Ahmed3 1 2 1National Committee for Quality Assurance, Washington, DC, USA; 2The Substance Abuse and Mental Health Services Administration, Rockville, MD, USA; 3FEi systems, Columbia, MD, USA Materials and methods: This study used descriptive and multivari- able analyses of VA administrative data for patients eligible for screen- ing (N = 16,118). The study assessed irst-year rates and predictors of screening and of positive screens, both for drug use and for unhealthy alcohol use, for which screening was already required. Background: National studies report that about 20% of adults engage in hazardous drinking. The US Preventive Services Task Force (USPSTF) recommends that providers screen adults for alcohol misuse. Many state Medicaid programs cover screening, but little is known about Medicaid plan performance. This pilot study examines the rate of screening and follow-up for unhealthy alcohol use among Medicaid enrollees. Results: During the irst year, 70% of patients were screened for drug use, and 84% were screened for unhealthy alcohol use. In multivariable analyses, screening for drug use was more likely for patients who had 8 or more days with VA visits or were aged 60 or over. Patients with a prior drug use disorder diagnosis were much less likely to be screened. Strong predictors of a positive drug use screen included a prior diag- nosis of drug use disorder, prior mental health visits, younger age or being unmarried. Materials and methods: We used 2015 data submitted by two Med- icaid plans. We adapted the American Medical Association’s measure, Unhealthy Alcohol Use: Screening and Brief Counseling, for health plan reporting. Additional testing of this measure was undertaken to determine whether it met requirements for inclusion in the Health- care Efectiveness Data and Information Set (HEDIS). We examined the percentage of adult members who received unhealthy alcohol use screening using a recommended standardized tool (e.g., AUDIT, AUDIT-C) and received follow-up care if they screened positive. We studied annual screening and investigated follow-up care within three months after a positive screening, using a random sample of 108 (Plan A) and 120 (Plan B) adult members. Plans provided de-identiied, member-level data based on case management records, claims and manual chart review of electronic health records (EHR). A57 Background: Unhealthy drug use is a concern in many settings, including military and veteran populations. In 2013, the Veterans Administration (VA) medical center in Bedford, Massachusetts, started requiring routine annual screening for unhealthy drug use in outpa- tient primary care and mental health settings, using a validated sin- gle question. This new policy builds on prior experience with annual screening for unhealthy alcohol use, which the VA has required of phy- sicians since 2004. Efectiveness and cost efectiveness of a smartphone based electronic alcohol intervention for adolescents: indings from the SIPS jr trials 1 2 1 1 Strategies for health plans included the plans themselves conducting screening, improving performance measurement—plans were particularly interested in a possible HEDIS alcohol screening measure, payment reform, and support for referral to treatment. Finally, strategies for other stakeholders to advance SBI included partnerships with state and federal organizations, researcher support and assistance, public education campaigns, and working with consumer groups to harness community support. Correspondence: Paolo Deluca - Paolo.Deluca@kcl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A55 Correspondence: Paolo Deluca - Paolo.Deluca@kcl.ac.uk Addiction Science & Clinical Practice 2017, 12(Suppl 1): A55 Background: This paper reports two linked randomized controlled trials which aimed to evaluate the efectiveness and cost-efective- ness of two BI intervention strategies among adolescents attending Emergency Departments (EDs), compared with screening alone. One trial focused on high-risk adolescent drinkers and the other focused on those identiied as low-risk or abstinent from alcohol. In both tri- als our primary outcome measure was quantity of alcohol consumed at 12 months after randomization. Our primary (null) hypothesis was: Personalized Feedback and Brief Advice (PFBA) and Personalized Feed- back plus electronic Brief Intervention (eBI) are no more efective than screening alone in reducing alcohol consumed at 12 months after ran- domization measured by the AUDIT-C. Conclusions: Providers, delivery systems, and payers all have roles to play in improving how risky drinking is addressed in the state. Next steps identiied by stakeholders included: scheduling follow-up meet- ings, developing and disseminating SBI and referral implementation and practice guidelines, ofering training and support for providers and health care systems in the state, and improving performance measurement. Page 21 of 24 Page 21 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Low screening and follow‑up for unhealthy alcohol use in Medicaid health plans 1 1 1 1 Conclusions: The drug screening initiative was relatively successful in its irst-year implementation, having screened 70% of eligible subjects. This proportion was somewhat lower than the 84% achieved for the longer-established alcohol screening program. Importantly, physicians failed to screen for drug use many of those patients who were most likely to screen positive, thereby missing many opportunities to treat and refer patients with drug use disorders. Future reinements should include better training clinicians in how to ask sensitive questions and how to deal with positive screens. Universal versus targeted screening for tobacco and drug use in adult primary care Universal versus targeted screening for tobacco and drug use in adult primary care Constance M. Weisner, Kelly C. Young-Wolﬀ, Wendy Y. Lu, Felicia W. Chi, Stacy A. Sterling Division of Research, Kaiser Permanente, Oakland, CA, USA Correspondence: Constance M. Weisner - Constance.Weisner@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A59 Results: 40% of Plan A members were screened in 2015 for alcohol use; 29% screened positive. 33% of members who screened positive received follow-up care, generally within two months after screen- ing. 38% of Plan B members were screened in 2015; one member had documented positive results and received follow-up care within two months. Screening rates were based on non-standardized screen- ing tools because standardized tools were rarely used. Screening and follow-up care were rarely documented in structured EHR ields. Claims or case management data alone would ind only half the care provided. Constance M. Weisner, Kelly C. Young-Wolﬀ, Wendy Y. Lu, Felicia W. Chi, Stacy A. Sterling Division of Research, Kaiser Permanente, Oakland, CA, USA Correspondence: Constance M. Weisner - Constance.Weisner@kp.org Addiction Science & Clinical Practice 2017, 12(Suppl 1): A59 Background: The optimal strategy for accomplishing screening and intervention for all substances in primary care has not been found. One question has to do with the comprehensiveness of using haz- ardous drinking as the foundation, with further screening for other substances of those who meet hazardous drinking criteria. Aims: To explore the relative eiciency of targeted versus universal screening of tobacco use disorders, drug use disorders and opioid-at-risk use in adult primary care. Conclusions: Despite national recommendations, rates of screening and follow-up for unhealthy alcohol use are low in Medicaid popu- lations. USPSTF-recommended standardized tools are rarely used; results are seldom documented in structured electronic data. Struc- tured EHR ields for standardized screening tools and follow-up care will allow easier monitoring of care quality. Provider education, greater access to treatment and quality measure reporting can encourage bet- ter care for alcohol misuse. Materials and methods: Kaiser Permanente Northern California has incorporated alcohol SBIRT into adult primary care. A total of; 2312,922 adults were screened for unhealthy drinking during 2014– 2015. Of those, 253,821 (11%) screened positive (i.e., exceeding daily or weekly limits). We compared rates of diagnosis-based tobacco use and drug use disorders, and opiate-at-risk use (prescription patterns) among all patients screened for unhealthy drinking (universal) versus those screened positive (targeted). Correspondence: Dominic Hodgkin - hodgkin@brandeis.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A58 A60 Receipt of alcohol screening and brief intervention among transgender and non‑transgender adults in the U.S.: exploratory indings from a national health survey John R. Blosnich1, Keren Lehavot2, Joseph E. Glass3, Emily C. Williams2 1Department of Veterans Aﬀairs, VA Pittsburgh Healthcare System, Center for Health Equity Research and Promotion, Pittsburgh, PA, USA; 2Denver-Seattle Center of Innovation for Veteran-Centered Value-Driven Care, VA Puget Sound Healthcare System, Seattle, WA, USA; 3Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA Correspondence: John R. Blosnich - john.blosnich@va.gov Addiction Science & Clinical Practice 2017, 12(Suppl 1): A60 Materials and methods: Behavioral Risk Factor Surveillance System 2014 data from eight US states were used to estimate patterns of alco- hol use and receipt of alcohol screening and brief intervention among persons reporting sexual orientation and a checkup in the last two years (N = 47,800). Analyses were conducted in 2016–2017. Results: Gay men had a higher age-adjusted prevalence of any alcohol use compared to heterosexual men in the past 30 days (79.5 vs. 60.1%), and bisexual women trended toward signiicance in having a higher prevalence of risky drinking (14.4 vs. 4.9%), heavy episodic drinking (21.1 vs. 10.8%), and any unhealthy alcohol use (22.8 vs. 12.0%) com- pared to heterosexual women in the past 30 days. We found few difer- ences in receipt of alcohol screening and brief intervention by sexual orientation. Speciically, lesbian women were more likely to report being asked about heavy episodic drinking than heterosexual women during a checkup, and among those reporting unhealthy alcohol use, gay men were less likely, and bisexual men were more likely, to report receiving brief intervention compared to heterosexual men. Background: Prior research shows that many transgender individu- als (persons whose current gender identity is diferent from their sex assigned at birth) must teach their healthcare providers about transgender healthcare delivery, and transgender individuals experi- ence discrimination and harassment within the healthcare system. Consequently, it is unclear whether transgender individuals are less likely to receive alcohol screening and brief intervention during clini- cal visits in comparison with their non-transgender peers. Materials and methods: Data are from the 2014 Behavioral Risk Fac- tor Surveillance System surveys of eight US states that administered both the Alcohol Screening and Brief Intervention (ASBI) module and the Sexual Orientation and Gender Identity module. A62 A62 Racial/ethnic and gender diferences in receipt of brief intervention among patients with unhealthy alcohol use in the U.S. Veterans Health Administration Emily C. Williams1, Joseph E. Glass2 1Department of Health Services, University of Washington School of Public Health, Seattle, WA, USA; 2Kaiser Permanente Washington Research Institute, Seattle, WA, USA Correspondence: Emily C. Williams - Emily.Williams3@va.gov Addiction Science & Clinical Practice 2017, 12(Suppl 1): A62 Implementing single‑item screening for drug use in a Veterans Administration outpatient setting 1 2 1 The proportion of those meeting opioid-at-risk use criteria was lower among those who screened positive for hazardous drinking (3.8%) than among all patients screened (5.4%); indings were similar across demographic groups. We will conduct multivariate analyses focusing on each sub- stance, examining patient characteristics. Background: Sexual minorities, including lesbian, gay, and bisexual women and men, experience a wide array of health disparities and have recently been designated as a health disparity population by the National Institutes of Health. Despite evidence of alcohol disparities between sexual minority and heterosexual individuals in the general population, research has not examined whether there are disparities in receipt of alcohol screening and brief intervention—together consid- ered one of the highest prevention priorities for US adults. This study examined diferences in alcohol use and receipt of alcohol screening and brief intervention across sexual minority status. Conclusion: A limitation is that we measured “disorders” rather than “at-risk” use, except for opioids. Aside from tobacco, targeted screen- ing was not comprehensive; strategies are needed for more complete screening. Correspondence: dd Correspondence: Emily C. Williams - Emily.Williams3@va.gov Addiction Science & Clinical Practice 2017, 12(Suppl 1): A62 Correspondence: Emily C. Williams - Emily.Williams3@va.gov Addiction Science & Clinical Practice 2017, 12(Suppl 1): A62 Background: Intersectionality theory posits that individuals with multiple marginalized social statuses are likely to be at magniied risk for poor health and health care than those with one or no marginal- ized social statuses. While previous studies have reported gender and racial/ethnic diferences in receipt of brief intervention for unhealthy alcohol use, no study has assessed the intersection of race and gender in association with receipt of brief intervention. In a national sample of patients with unhealthy alcohol use, we examined receipt of brief intervention across race/ethnicity and gender. Conclusions: This is the irst study on provision of alcohol-related care for transgender individuals among a population-based sample with a direct non-transgender comparison group. Results did not identify signiicant diferences between groups, and in the rare instances dif- ferences were observed, indings suggested transgender individuals received more brief interventions than their non-transgender peers. Diferences notwithstanding, alcohol screening and brief intervention should be increased among both groups. Materials and methods: VA outpatients who had one or more posi- tive screens for unhealthy alcohol use (AUDIT-C ≥ 5) documented in their medical records between 10/2009 and 5/2013 were eligible. Pois- son regression models with a race by gender interaction were it to identify the predicted prevalence and 95% conidence intervals (CI) of having brief intervention documented ≤14 days after a positive alco- hol screen for American Indian, Asian/Paciic Islander, black, Hispanic, and white men and women. Models included a robust sandwich esti- mator to account for correlation resulting from multiple screens. A60 The analytic sam- ple (n = 49,208) included all individuals who provided their gender identity and had a medical checkup in the last two years (a prerequi- site for the ASBI module). Analyses were conducted in 2016–2017. Conclusions: Overall similarities between sexual minorities and heter- osexuals in alcohol use and receipt of screening and brief intervention are encouraging. Nonetheless, a potential area of disparity emerged with respect to gay men’s lower likelihood of being ofered advice about drinking compared to heterosexual men among those for which brief interventions are indicated. Future research is needed to under- stand mechanisms underlying this disparity, as well as to assess varia- tion in quality of alcohol-related care across sexual minority status. Results: A weighted proportion of 0.6% (n = 283) of the sample self- identiied as transgender. No signiicant diferences in alcohol con- sumption (i.e., any alcohol use in the past 30 days, heavy alcohol use, heavy episodic drinking) were observed between transgender and non-transgender groups. Transgender and non-transgender groups did not difer in being asked by a healthcare provider if they drink alcohol, how much alcohol they drank, or if they had any heavy episodic drink- ing. Although a greater proportion of transgender than non-transgen- der individuals (31.7 vs. 19.9%) reported being ofered advice about unhealthy alcohol use, this diference was not statistically signiicant (p = .054). Compared with non-transgender individuals, transgender individuals who received alcohol use screening were more likely to report being advised to quit or reduce their drinking (20.7 vs. 7.8%; p = .012); but adjustment for socio-demographics attenuated this dif- ference (adjusted odds ratio = 2.31, 95% CI 0.91–5.86, p = .077). Implementing single‑item screening for drug use in a Veterans Administration outpatient setting 1 2 1 Dominic Hodgkin1, Wenwu Gao2, Elizabeth L. Merrick1, Charles E. Drebing3, Mary Jo Larson1, Constance M. Horgan1, Monica Sharma4, Nancy M. Petry5, Richard Saitz6 Dominic Hodgkin1, Wenwu Gao2, Elizabeth L. Merrick1, Charles E. Drebing3, Mary Jo Larson1, Constance M. Horgan1, Monica Sharma4, Nancy M. Petry5, Richard Saitz6 Results: The proportion of tobacco use disorders was 7.0% among all adults who were screened and 12.1% among those exceeding either daily or weekly limits. The prevalence ranged across age groups (12.7– 20.6%) and genders (females 17.7%, males 19.9%), and were much higher for those exceeding both daily and weekly limits. On the other hand, only 4% of those with tobacco disorders reported exceeding both daily and weekly drinking limits. 1Institute for Behavioral Health, Schneider Institutes for Health Policy, Heller School for Social Policy and Management, Brandeis University, Waltham, MA, USA; 2Psychology Service, Bedford Department of Veterans Aﬀairs Medical Center, Bedford, MA, USA; 3Psychology Service, Bedford Department of Veterans Aﬀairs Medical Center, Bedford, MA, USA; 4Primary Care Service, Bedford Department of Veterans Aﬀairs Medical Center, Bedford, MA, USA; 5University of Connecticut School of Medicine, CT, USA; 6Department of Community Health Sciences, Boston University School of Public Health, Boston, MA, USA Proportions of drug use disorders were similar using both approaches: 1.8% among all patients screened, and 2.3% among those exceeding daily or weekly limits. Proportions were higher among those exceed- ing weekly limits, especially those aged 18–25 and 26–44 for whom the proportions were twice as high as from universal screening. How- ever, the vast majority of those with drug disorders (86%) would have Correspondence: Dominic Hodgkin - hodgkin@brandeis.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A58 Page 22 of 24 Page 22 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 of Veterans Aﬀairs, VA Pittsburgh Healthcare System, Center for Health Equity Research and Promotion, Pittsburgh, PA, USA; 3Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA Correspondence: Keren Lehavot - keren.lehavot@va.gov Addiction Science & Clinical Practice 2017, 12(Suppl 1): A61 been missed by the targeted screening approach. The proportion of those meeting opioid-at-risk use criteria was lower among those who screened positive for hazardous drinking (3.8%) than among all patients screened (5.4%); indings were similar across demographic groups. We will conduct multivariate analyses focusing on each sub- stance, examining patient characteristics. been missed by the targeted screening approach. Racial/ethnic and gender diferences in receipt of brief intervention among patients with unhealthy alcohol use in the U.S. Veterans Health Administration Emily C. Williams1, Joseph E. Glass2 1Department of Health Services, University of Washington School of Public Health, Seattle, WA, USA; 2Kaiser Permanente Washington Research Institute, Seattle, WA, USA Emily C. Williams1, Joseph E. Glass2 1Department of Health Services, University of Washington School of Public Health, Seattle, WA, USA; 2Kaiser Permanente Washington Research Institute, Seattle, WA, USA Diferences in receipt of alcohol‑related care across race/ethnicity among VA patients living with HIV and unhealthy alcohol use 1 1 2 3 Diferences in receipt of alcohol‑related care across race/ethnicity among VA patients living with HIV and unhealthy alcohol use 1 1 2 3 Diferences in receipt of alcohol‑related care across race/ethnicity among VA patients living with HIV and unhealthy alcohol use Kara M. Bensley1, India Ornelas1, Gary Chan2, Julie Dombrowski3, John Fortney4, Anna D. Rubinsky5, Gwen T. Lapham6, Joseph E. Glass6, Emily C. Williams1 Background: Women are at the highest risk of misusing substances during their reproductive years. Given the fact that many women in this age group often use their reproductive health provider as their primary source of medical care, this setting may be an optimal place to screen and deliver a brief intervention to women who misuse substances. The goal of this project was to determine whether Screening, Brief Interven- tion, and Referral to Treatment delivered electronically (e-SBIRT) or by cli- nician (SBIRT) reduces substance misuse more than enhanced usual care (EUC) in women seeking routine care in a reproductive health setting. i l d h d ( ) f d Background: Women are at the highest risk of misusing substances during their reproductive years. Given the fact that many women in this age group often use their reproductive health provider as their primary source of medical care, this setting may be an optimal place to screen and deliver a brief intervention to women who misuse substances. The goal of this project was to determine whether Screening, Brief Interven- tion, and Referral to Treatment delivered electronically (e-SBIRT) or by cli- nician (SBIRT) reduces substance misuse more than enhanced usual care (EUC) in women seeking routine care in a reproductive health setting. Materials and Methods: Women (N = 439) from two reproduc- tive healthcare clinics who smoked cigarettes or misused alcohol, illicit drugs, or prescription medication were randomized to a 20-min e-SBIRT, 20-min SBIRT, or EUC (receipt of a pamphlet with informa- tion and referrals). Assessments occurred at baseline and one, three, and six months post-baseline. Co-primary outcomes were days/month of primary substance use and post-intervention service utilization for participant reported primary substance. Kara M. Bensley1, India Ornelas1, Gary Chan2, Julie Dombrowski3, John Fortney4, Anna D. Rubinsky5, Gwen T. Lapham6, Joseph E. Glass6, Emily C. Williams1 Kara M. Bensley1, India Ornelas1, Gary Chan2, Julie Dombrowski3, John Fortney4, Anna D. Rubinsky5, Gwen T. Lapham6, Joseph E. Glass6, Emily C. Diferences in receipt of alcohol‑related care across race/ethnicity among VA patients living with HIV and unhealthy alcohol use 1 1 2 3 Williams1 1Department of Health Services, University of Washington, Seattle, WA, USA; 2Department of Health Services and Department of Biostatistics, University of Washington, Seattle, WA, USA; 3Department of Medicine and Allergy and Infectious Diseases, University of Washington, WA, USA; 4Department of Psychiatry and Behavioral Sciences, University of Washington, and Health Services Research and Development, Veterans Health Administration Puget Sound, Seattle, WA, USA; 5Kidney Health and Research Collaborative, University of California San Francisco, San Francisco, CA, USA; 6Kaiser Permanente Washington Health Research Institute, Seattle, WA, USA Materials and Methods: Women (N = 439) from two reproduc- tive healthcare clinics who smoked cigarettes or misused alcohol, illicit drugs, or prescription medication were randomized to a 20-min e-SBIRT, 20-min SBIRT, or EUC (receipt of a pamphlet with informa- tion and referrals). Assessments occurred at baseline and one, three, and six months post-baseline. Co-primary outcomes were days/month of primary substance use and post-intervention service utilization for participant reported primary substance. Correspondence: Kara M. Bensley - kbensley@uw.edu Addiction Science & Clinical Practice 2017 12(Suppl 1): A63 Correspondence: Kara M. Bensley - kbensley@uw.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A63 Correspondence: Kara M. Bensley - kbensley@uw.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A63 Background: Unhealthy alcohol use (UAU) is particularly risky for patients living with HIV (PLWH) and associated with worse outcomes for racial/ethnic minorities. While evidence-based interventions for UAU are available, whether receipt of such interventions is equitable across racial/ethnic groups among PLWH is unknown. Results: The number of participants randomized included: 143 to e-SBIRT, 145 to SBIRT, and 151 to EUC, with retention >84% at all points. At baseline, the mean (SD) days per month of primary sub- stance use were 23.7 (7.7) for e-SBIRT, 23.2 (8.3) for SBIRT and 24.2 (7.7) for EUC, which respectively declined to 19.7 (11.2), 19.3 (11.2), and 22.8 (8.9) at one month, 17.8 (11.9), 18.0 (12.0), and 21.4 (10.6) at three months, and 16.2 (12.5), 17.0 (12.3), and 19.1 (11.8) at six months. Estimated declines were greater in e-SBIRT [β (SE) = −0.090 (0.034), p = 0.008; Cohen’s d = 0.19 at one month, 0.30 at three months, and 0.17 at six months] and SBIRT [β (SE) = −0.078 (0.037), p = 0.038; Cohen’s d = 0.17 at one month, 0.22 at three months, and 0.06 at six months] compared to EUC. Service utilization did not difer between groups. Electronic‑ and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: a randomized clinical trial 1 2 1 Electronic‑ and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: a randomized clinical trial Kimberly A. Yonkers1, Steven J. Ondersma2, Ariadna Forray1, Todd A. Olmstead3, Kathryn Gilstad-Hayden1, Trace Kershaw4, Steve Martino4 1Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA; 2Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Detroit, MI, USA; 3Lyndon B. Johnson School of Public Aﬀairs, University of Texas at Austin, Austin, TX, USA; 4School of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT, USA; 4Department of Psychiatry, Yale School of Medicine, VA Connecticut Healthcare, New Haven, CT, USA Correspondence: Kimberly A. Yonkers - Kimberly.Yonkers@Yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A64 Electronic‑ and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: a randomized clinical trial 2 Electronic‑ and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: a randomized clinical trial Kimberly A. Yonkers1, Steven J. Ondersma2, Ariadna Forray1, Todd A. Olmstead3, Kathryn Gilstad-Hayden1, Trace Kershaw4, Steve Martino4 1Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA; 2Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Detroit, MI, USA; 3Lyndon B. Johnson School of Public Aﬀairs, University of Texas at Austin, Austin, TX, USA; 4School of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT, USA; 4Department of Psychiatry, Yale School of Medicine, VA Connecticut Healthcare, New Haven, CT, USA Correspondence: Kimberly A. Yonkers - Kimberly.Yonkers@Yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A64 Kimberly A. Yonkers1, Steven J. Ondersma2, Ariadna Forray1, Todd A. Olmstead3, Kathryn Gilstad-Hayden1, Trace Kershaw4, Steve Martino4 1Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA; 2Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Detroit, MI, USA; 3Lyndon B. Johnson School of Public Aﬀairs, University of Texas at Austin, Austin, TX, USA; 4School of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT, USA; 4Department of Psychiatry, Yale School of Medicine, VA Connecticut Healthcare, New Haven, CT, USA Conclusions: Race and gender, individually and intersectionally, appear to inluence receipt of brief intervention among patients with unhealthy alcohol use. Black women with unhealthy alcohol use appear to be at the greatest risk of under-receipt of brief interven- tion. Future research should investigate mechanisms underlying these associations. Alcohol use and receipt of alcohol screening and brief intervention in a representative sample of sexual minority and heterosexual adults receiving health care 1 2 3 1 Keren Lehavot1, John R. Blosnich2, Joseph E. Glass3, Emily C. Williams1 1Denver-Seattle Center of Innovation for Veteran-Centered Value-Driven Care, VA Puget Sound Healthcare System, Seattle, WA, USA; 2Department Page 23 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Results: The sample included 830,825 outpatients (3% women, 25% non-white), relecting 1,172,606 positive screens (1–5 per patient); 74% had documented brief intervention. Both American Indian and black patients were less likely than white patients to receive brief inter- vention (p-values <0.001), as were women relative to men (p < 0.001), and a signiicant interaction between race and gender was identiied (p < 0.001). The predicted prevalence of receiving brief intervention ranged from 66.8% (95% CI 65.7–67.8) among black women to 74.7% among Asian/Paciic Islander (95% CI 74.6–74.8), Hispanic (95% CI 74.0–75.1), and white (95% CI 74.6–74.8) men. Identiied diferences are consistent with research in non-HIV speciic samples but mechanisms underlying them are unknown and should be further investigated. Diferences in receipt of alcohol‑related care across race/ethnicity among VA patients living with HIV and unhealthy alcohol use 1 1 2 3 Materials and methods: VA electronic health record (EHR) data were used to identify alcohol screens positive for UAU (AUDIT-C ≥ 5) between 10/1/09 and 5/30/13 among black, Hispanic, and white patients with a past-year diagnosis for HIV and a documented home zip code. We measured three domains of evidence-based care from the EHR: brief intervention (advice to reduce or abstain from drink- ing) within 14 days of the screen among all positive screens; and spe- cialty addictions treatment and pharmacotherapy (ills for naltrexone, disuliram, acamprosate or topiramate) within one year of screening among those with diagnosed alcohol use disorder (AUD). Poisson regression models and recycled predictions were used to estimate adjusted predicted prevalence of receiving alcohol-related care. Mod- els were adjusted for age, sex, rurality, VA eligibility status, marital sta- tus, and region and accounted for correlation within patients using a robust sandwich estimator. Conclusions: E-SBIRT and SBIRT signiicantly decreased days of pri- mary substance use among women in reproductive healthcare cent- ers; neither SBIRT resulted in more service utilization than EUC. A63 f Correspondence: Kimberly A. Yonkers - Kimberly.Yonkers@Yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A64 A RCT comparing computer and therapist‑delivered SBIRT for alcohol/drugs in an urban primary care setting D S S iki1 S J O d 2 P l Dill 3 Mi h l F W Dace S. Svikis1, Steven J. Ondersma2, Pamela Dillon3, Michael F. Weaver4 1Department of Psychology; Institute for Women’s Health, Virginia Commonwealth University, Richmond, VA, USA; 2Merrill-Palmer Skillman Institute and Department of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI, USA; 3Center for Clinical and Translational Research, Virginia Commonwealth University, Richmond, VA, USA; 4University of Texas Health Science Center, Houston, TX, USA Materials and methods: In study 1, 22 participants viewed high empathy only, high positive regard only, and education-only content to evaluate discriminability. Pilot participants viewed the content for these cells in counterbalanced order and ranked them for preferabil- ity, understanding, and supportiveness. In study 2, 100 heavy-drink- ing undergraduates were randomly assigned to either a high- or a low-empathy e-BI. Intentions to reduce drinking were assessed both before and after the intervention, yielding a state motivation change score. Correspondence: Dace S. Svikis - dssvikis@vcu.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A66 Correspondence: Dace S. Svikis - dssvikis@vcu.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A66 Background: The present study examined 3 and 6 month outcome data from a 4-arm randomized controlled trial comparing computer- directed and therapist-delivered brief interventions for heavy/prob- lem drinking and drug use in patients attending an urban primary care clinic. Results: In study 1, the high empathy e-BI was rated as most like- able (z = 3.01, p < .01), supportive (z = 3.01, p < .01), understanding (z = 3.47, p < .01), and airming (z = 2.56, p = .01). Participant ratings for the control and positive regard conditions did not difer. In study 2, participants in the high-empathy condition reported greater increases in intentions to reduce drinking over the course of the study (β = .24, p < .05). Materials and methods: Primary care patients with heavy/problem alcohol or drug use were identiied using an anonymous computer health survey. Those providing informed consent (N = 713) were randomized to one of 4 study groups: computer-directed interven- tion (CACI), therapist-delivered intervention (CATI), assessment only control (CA), and minimal screen-only control (SC). SC participants answered no substance-use related questions until 3 and 6 month follow-up, when Timeline Follow-back (TLFB) data on alcohol and other drug use were obtained. The sample was 61% female and 77% African American, with mean age of 45.3 years. Electronic and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: economic analyses 1 2 3 Results: Among 3310 PLWH with UAU (4376 screens), 1968 were black, 257 were Hispanic, and 1085 were white; 2233 (50%) had AUD. Among all with UAU, no racial/ethnic diferences in brief intervention were observed. Adjusted prevalences were 55.9% (95% CI 54.7–64.9%) among black, 59.8% (54.7–64.6%) among Hispanic, and 59.2% (56.4–61.9%) among white patients (p = 0.1223). Among those with AUD, adjusted prevalence of specialty addictions treatment difered across race/ethnic- ity with 52.1% (49.2%, 55.0%) for black patients, 39.2% (31.5%, 46.8%) for Hispanic patients, and 33.5% (29.3%, 37.7%) for white patients (p < 0.001). No diferences in pharmacotherapy were observed; adjusted prevalences were 6.0% (4.6%, 7.4%) among black; 7.1% (2.2–11.9%) among Hispanic, and 6.5% (4.4–8.5%) among white patients (p = 0.7857). Steve Martino1, Todd A. Olmstead2, Steven J. Ondersma3, Ariadna Forray4, Kathryn Gilstad-Hayden4, Trace Kershaw5, Kimberly A. Yonkers4 1Department of Psychiatry, Yale School of Medicine, VA Connecticut Healthcare System, West Haven, CT, USA; 2Lyndon B. Johnson School of Public Aﬀairs, University of Texas at Austin, Austin, TX, USA; 3Department of Psychiatry and Behavioral Neurosciences and Merrill-Palmer Skillman Institute, Detroit, MI, USA; 4Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA; 5School of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT, USA C d S l d Conclusion: Among patients with AUD, receipt of specialty addictions treatment appeared to be most common among black patients. No other racial/ethnic diferences in alcohol-related care were identiied. Correspondence: Steve Martino - steve.martino@yale.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A65 Page 24 of 24 Addict Sci Clin Pract 2017, 12(Suppl 1):25 Page 24 of 24 Background: The American College of Obstetricians and Gynecolo- gists recommends the use of Screening, Brief Intervention, and Refer- ral to Treatment (SBIRT) in reproductive healthcare centers to help women reduce or stop substance misuse. A recent trial showed both electronically-delivered SBIRT (e-SBIRT) and clinician-delivered SBIRT, when compared to enhanced usual care (EUC), resulted in a signiicant reduction in the days of primary substance use over a 6-month follow- up period. However, decision makers need guidance about the rela- tive cost-efectiveness of these two approaches. This study presents the cost and cost-efectiveness of e-SBIRT and SBIRT relative to EUC in reducing substance misuse among women seeking routine care in reproductive health settings. SC, respectively), but did not reach signiicance. A67 Common factors and ethopoeia in computer‑delivered brief intervention for alcohol use: a factorial trial Emily R. Grekin1, Jennifer D. Ellis1, Steven J. Ondersma2, Jessica R. Beatty3, Lucy McGoron3 1Department of Psychology, Wayne State University, Detroit, MI, USA; 2Merrill Palmer Skillman Institute and Department of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI, USA; 3Merrill Palmer Skillman Institute, Wayne State University, Detroit, MI, USA Correspondence: Emily R. Grekin - grekine@wayne.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A67 A RCT comparing computer and therapist‑delivered SBIRT for alcohol/drugs in an urban primary care setting D S S iki1 S J O d 2 P l Dill 3 Mi h l F W Analyses compared 7-day point prevalence rates of alcohol use, binge drinking and illicit drug use across the 4 study groups, stratiied by the primary substance identiied using a computer algorithm at baseline. Conclusions: Results suggest that empathy within the context of an e-BI may increase acceptability and motivation to change. Findings are consistent with the Media Equation Theory and suggest that life- like technology elicits social responses. The ongoing factorial trial (expected N = 352) will yield key data regarding diferences in drink- ing at follow-up as a function of common factors, with and without a spoken voice or presence of an animated narrator. Common factors and ethopoeia in computer‑delivered brief intervention for alcohol use: a factorial trial 1 1 2 Emily R. Grekin1, Jennifer D. Ellis1, Steven J. Ondersma2, Jessica R. Beatty3, Lucy McGoron3 1 Emily R. Grekin1, Jennifer D. Ellis1, Steven J. Ondersma2, Jessica R. Beatty3, Lucy McGoron3 1 1Department of Psychology, Wayne State University, Detroit, MI, USA; 2Merrill Palmer Skillman Institute and Department of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI, USA; 3Merrill Palmer Skillman Institute, Wayne State University, Detroit, MI, USA Correspondence: Emily R. Grekin - grekine@wayne.edu Addiction Science & Clinical Practice 2017, 12(Suppl 1): A67 Results: From the provider perspective, e-SBIRT dominated both EUC and SBIRT (i.e., on average, e-SBIRT both costs less and leads to more days of abstinence during the 6-month follow-up than EUC and SBIRT). From the societal perspective, (a) e-SBIRT dominates SBIRT, and (b) compared to EUC, the cost of using e-SBIRT to attain an additional day of abstinence is 14 cents. Background: Media Equation Theory implies that common relation- ship factors, such as empathy and positive regard, are active in elec- tronic brief interventions (e-BIs) just as they are in person-delivered interventions, particularly when lifelike characteristics are present. We will test this hypothesis by: (1) evaluating discriminability of common factors in an e-BI context; (2) measuring immediate post-e-BI changes in state motivation with and without common factors present; and (3) evaluating the efect of common factors, with and without the pres- ence of lifelike characteristics, on changes in alcohol use at follow-up. Data from steps 1 and 2 are presented below; step 3 is ongoing as an NIAAA-funded factorial trial. Conclusions: e-SBIRT is a promising cost-efective approach for use in reproductive healthcare centers to help women reduce or stop sub- stance misuse. Electronic and clinician‑delivered screening, brief intervention, and referral to treatment for women in reproductive healthcare centers: economic analyses 1 2 3 Similarly, 7-day point prevalence binge abstinence also showed non-signiicant advantages for CACI at 3-months which reached signiicance at the 6-month follow-up (87% abstinence for CACI vs. 75, 63, and 75% for CATI, CA, and SC; p < .02). No group diferences were found for illicit drug use at either 3 or 6 month follow-up. Conclusions: No group diferences were found on illicit drug use, with similar rates of use across the 2 intervention and 2 control groups, mirroring most other SBIRT trials. However, the computer-delivered intervention was associated with signiicantly less binge drinking at the six-month follow-up. Additional analyses will examine other out- come measures such as frequency (days) of use and urinalysis drug use assays. Materials and methods: Cost data were collected prospectively dur- ing the trial. Both variable and ixed costs for each condition were estimated from the provider and patient perspectives. Efectiveness was assessed in terms of the number of days per month of primary substance use during the 6-month follow-up period. Incremental cost-efectiveness ratios and cost-efectiveness acceptability curves were used to determine the relative cost-efectiveness of the three conditions. Publisher’s Note S Results: The algorithm placed N = 343 (48%) of participants into a heavy alcohol use subgroup and N = 370 (52%) were placed in an illicit drug use subgroup. Within the alcohol use subgroup, 7-day point prevalence abstinence trends for any use favored the CACI condition (e.g., 55% abstinence at 6 months vs. 44, 41 and 42% for CATI, CA, and Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional aﬃliations.
https://openalex.org/W2951812325	https://europepmc.org/articles/pmc6127909?pdf=render	English	null	Effective normalization for copy number variation in Hi-C data	BMC bioinformatics	2,018	cc-by	13,088	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Normalization is essential to ensure accurate analysis and proper interpretation of sequencing data, and chromosome conformation capture data such as Hi-C have particular challenges. Although several methods have been proposed, the most widely used type of normalization of Hi-C data usually casts estimation of unwanted effects as a matrix balancing problem, relying on the assumption that all genomic regions interact equally with each other. Results: In order to explore the effect of copy-number variations on Hi-C data normalization, we first propose a simulation model that predict the effects of large copy-number changes on a diploid Hi-C contact map. We then show that the standard approaches relying on equal visibility fail to correct for unwanted effects in the presence of copy-number variations. We thus propose a simple extension to matrix balancing methods that model these effects. Our approach can either retain the copy-number variation effects (LOIC) or remove them (CAIC). We show that this leads to better downstream analysis of the three-dimensional organization of rearranged genomes. Conclusions: Taken together, our results highlight the importance of using dedicated methods for the analysis of Hi-C cancer data. Both CAIC and LOIC methods perform well on simulated and real Hi-C data sets, each fulfilling different needs. Keywords: Normalization, Hi-C, Cancer, Copy-number Keywords: Normalization, Hi-C, Cancer, Copy-number Effective normalization for copy number variation in Hi-C data Nicolas Servant1,2,3† , Nelle Varoquaux4,5†, Edith Heard6, Emmanuel Barillot1,2,3 and Jean-Philippe Vert3,1,2,7 Nicolas Servant1,2,3† , Nelle Varoquaux4,5†, Edith Heard6, Emmanuel Barillot1,2,3 and Jean-Philippe Vert3,1,2,7 Servant et al. BMC Bioinformatics (2018) 19:313 https://doi.org/10.1186/s12859-018-2256-5 Servant et al. BMC Bioinformatics (2018) 19:313 https://doi.org/10.1186/s12859-018-2256-5 Background One of the exciting discoveries that has emerged from systematic sequencing of cancer genomes was the high frequency of mutations in genes known to regulate epigenetic processes such as chromatin associated proteins, DNA methylation, or his- tone variants and modifications [14]. The contribution of altered epigenomes in the process of tumorigenesis is thus at last being unraveled thanks to the combination of genomic and epigenomic interrogation. More recently, genetic and epigenetic alterations in the non-coding part of the genome, including distal regulatory elements such as enhancers or insulators, have been reported and found to impact gene expression in cancer [15]. This has led to intense interest in the spatial proximity and 3D organi- zation of cancer genomes. Losada et al. [16] reviews the effect of somatic mutations in cohesin complex proteins (which play a citical role in TADs organization and chro- mosome looping) in various types of cancer. Groeschel et al. [17] and Taberlay et al. [18] describe how disruptions in genome organization (respectively in leukemia and prostate cancer) lead to major epigenetic and transcrip- tional changes. Lastly, Hnisz et al. [19], Weischenfeldt et al. [20], and Beroukhim et al. [21] show how disruptions in long range DNA looping and genome rearrangements lead to enhancer hijacking and Flavahan et al. [22] link insulator dysfunctions to oncogene activation in cancer. Thus, changes in chromosome conformation at differ- ent scales are now considered as key potential players in cancer, as well as important potential biomarkers. In the context of cancer Hi-C data, an additional per- turbation related to chromosomal rearrangements must be considered. Amplified genomic regions have a greater chance of being pull-down during the library preparation, while genomic regions with lower copy numbers are more difficult to detect. To date, such copy number variants (CNVs) are usually ignored in cancer Hi-C data nor- malization, although they raise interesting and important questions both at the biological and methodological levels. The real impact of CNVs on contact frequencies remains difficult to assess. For instance, a tandem amplification has a very different impact on local chromatin organiza- tion compared to the gain of a complete chromosome. Similarly, a genomic duplication could lead to different changes in contact frequencies depending on whether the event occurs within a TADs or across/at a TAD boundary [10]. Background (TADs). These have been proposed as possible functional units of regulation, and are generally preserved across cell types, as well as being conserved between mammals [3, 4, 6, 7]. TAD boundaries are frequently associated with CTCF binding sites. CTCF is also involved in the estab- lishment of chromatin loops between convergent target sites [3]. These chromatin loops are believed to provide scaffold for promoter-enhancer contacts and can there- fore be implicated in gene activation (see Bouwman et al. [8] for a review). The spatial organization of the genome and the physical interactions occurring within and between chromosomes can play important roles in gene regulation and in genome function in general. The organization and folding of mam- malian chromosomes within the nucleus involve multiple hierarchical chromatin structures (see Bonev et al. [1] for a review). At the megabase-scale, the genome in the inter- phase nucleus is divided into compartments of open and closed chromatin, that are respectively associated with gene-rich, actively transcribed regions and gene-poor, silent regions [2, 3]. Compartment organization varies across physiological conditions and during cell differen- tiation [4, 5]. At the sub-megabase scale, chromosomes are also partitioned into topological associated domains Given the important recent insights that chromosome conformation techniques have provided into 3D genome organization in a normal context, the application of such approaches to a disease context offers great promises to explore the effect of perturbations in 3D genomic organi- zation on cell regulation (see Kirjger et al. [9] for a review). At a high enough resolution, such techniques can be used to characterize links between disease-associated sequence variants and the gene regulatory landscape. For example, structural variants can disrupt boundaries between TADs, Correspondence: nicolas.servant@curie.fr †Nicolas Servant and Nelle Varoquaux contributed equally to this work. 1Institut Curie, PSL Research University, F-75005 Paris, France 2INSERM, U900, F-75005 Paris, France Full list of author information is available at the end of the article Correspondence: nicolas.servant@curie.fr †Nicolas Servant and Nelle Varoquaux contributed equally to this work. 1Institut Curie, PSL Research University, F-75005 Paris, France 2INSERM, U900, F-75005 Paris, France Full list of author information is available at the end of the article Servant et al. BMC Bioinformatics (2018) 19:313 Page 2 of 16 et al. [23] for a review). In Hi-C experiments, the con- tact frequencies between two genomic loci are roughly proportional to the reads counts observed between two regions after sequencing [2]. Background However, as is the case for many high-throughput technologies, the raw contact fre- quencies are affected by systematic biases such as GC content, mappability, or restriction fragment size [24]. Estimating and correcting these biases is therefore an important step in ensuring accurate downstream analy- sis. In the past few years, several methods and packages have been developed to normalize Hi-C data (see Ay and Noble [25] for a review). These methods fall into two main categories: explicit factor correction methods or matrix balancing algorithms. Explicit-factor normaliza- tion methods require an a priori knowledge of the Hi-C systematic biases. Yaffe et al. [24] first proposed a non- parametric model to estimate the probability of observing a contact between two loci given these biases. The main limitation of this method is its computational cost. Sub- sequently, Hu et al. [26] proposed a much faster explicit correction method, based on Poisson or Negative Bino- mial regression which can be applied at the bin resolution, and gives similar performance compared to the original method. Unlike the explicit factor correction methods, the matrix balancing methods do not assume any spe- cific source of biases, and are in theory able to correct for all unwanted variances in the contact map [3, 27, 28]. Applying such methods leads to an optimization prob- lem that can be solved efficiently and precisely using the Sinkhorn and Knopp algorithm [28], or the Knight and Ruiz algorithm [3]. and consequently can act as driver events in the mis- regulation of associated gene expression [10, 11]. Over the past decade, major advances have been made in both high-throughput sequencing techniques and data availability from large patient cohorts across multiple can- cer types, enabling a comprehensive and systematic explo- ration of genomic and epigenomic landscapes of a wide variety of cancers. While cancer has been shown to have a genetic component, our appreciation of the inherent epi- genetic complexity is more recent and has dramatically increased over the last few years. At the genetic level, cancer is frequently associated with the sequential acqui- sition of somatic variants, both at the single nucleotide and at the copy number levels [12]. The different alter- ations that characterize tumors are usually caused by a few functional driver events, which occur among many non-functional passenger events, mainly located in the non-coding part of the genome [13]. Background Their linear proximity on the genome would therefore explain the massive increase of contact frequencies that we observed in real data, and which are not modeled by our simulation. We then sum- marized both data in one dimension (1D) by summing the contact frequencies over each row. Overall, the sim- ulated MCF7 profile is well correlated with the profile of real MCF7 Hi-C data (Spearman cor=0.877, Fig. 1f, Table S2). We can observe that the sum of interactions for a genomic window is proportional to the copy number. These observations lead us to believe that our simulation method appropriately models the effect of copy number variations in Hi-C data. as an unwanted effect, and to remove it during the nor- malization step [29]. This strategy indeed makes sense for the detection of a genome-wide list of significant contacts, or for the direct comparison of samples with different chromosomal rearrangement profiles. On the other hand, the signal from copy number alterations can also be con- sidered as important biological information, that can be of interest for 3D modeling, genome reconstruction of can- cer cells, or to simply further characterize the genomic landscape of a tumor [30]. Here, we propose to further explore the impact of CNVs on Hi-C data and provide tools that deal with its effects on data normalization. First, we develop a model simulating large copy number rearrangements on a diploid Hi-C con- tact map. Using such simulated data, we demonstrate that the naive matrix balancing algorithm which is commonly used to normalize Hi-C data, cannot be applied to can- cer Hi-C data. We then propose two methods that extend the ICE algorithm and correct the data from systematic biases, either considering the CNVs as a bias to remove or as an interesting signal to conserve in the data struc- ture. Finally, we apply these methods to several disease associated Hi-C data sets, demonstrating their relevance. The ICE normalization is not suitable for cancer Hi-C data Several methods have been proposed to remove unwanted technical and biological variations from Hi-C data. Among them, the matrix-balancing methods leverage a small number of hypotheses on the biases and on the properties of Hi-C data to formulate their normalization procedure: these do not assume any specific source of bias, and are (as long as the hypotheses are fulfilled) able to cor- rect for any factors affecting contact frequencies [27, 28]. Background In this context, the iterative correction method (ICE, [28]) has been successfully applied to many diploid Hi-C data sets. ICE relies on two assumptions: (1) the bias between two regions i and j can be represented as the product of individual biases of these regions : NICE ij = βiβjCij; (2) each bin should interact approximately the same number of times: i NICE ij = k, where C represents the raw count matrix, NICE the ICE normalized count matrix, β the bias vectors and k a constant. Background Addressing the question of CNVs during normaliza- tion is therefore an important challenge in the analysis of Hi-C data and their interpretation in the context of genetic and epigenetic mis-regulation in disease. Developing accurate and quantitative methods to ana- lyze the chromatin conformation derived from disease- associated cells/tissues is therefore of increasing interest to a wide community of researchers and pathologists. In addition to standard microscopy approaches, several 3C-based methods are now used: these rely on digestion and religation of fixed chromatin to estimate the proba- bility of contact between two genomic loci (see Ramani The question of how copy number signal should be treated depends mainly on the related biological ques- tions. One strategy is to consider the copy number effect Page 3 of 16 Page 3 of 16 Servant et al. BMC Bioinformatics (2018) 19:313 Both our simulations and the real data show blocks of higher/lower contact frequencies in regions affected by large copy number variants. Interestingly, for the high- est copy numbers, both profiles increase concurrently, but not at the same rate. One explanation would be that these regions of very high copy number correspond to com- plex rearrangements such as tandem focal amplifications in cis and translocation in trans. Their linear proximity on the genome would therefore explain the massive increase of contact frequencies that we observed in real data, and which are not modeled by our simulation. We then sum- marized both data in one dimension (1D) by summing the contact frequencies over each row. Overall, the sim- ulated MCF7 profile is well correlated with the profile of real MCF7 Hi-C data (Spearman cor=0.877, Fig. 1f, Table S2). We can observe that the sum of interactions for a genomic window is proportional to the copy number. These observations lead us to believe that our simulation method appropriately models the effect of copy number variations in Hi-C data. Both our simulations and the real data show blocks of higher/lower contact frequencies in regions affected by large copy number variants. Interestingly, for the high- est copy numbers, both profiles increase concurrently, but not at the same rate. One explanation would be that these regions of very high copy number correspond to com- plex rearrangements such as tandem focal amplifications in cis and translocation in trans. Simulating the effect of copy number variations on Hi-C data In addition, the contact frequency observed in trans between loci i and k is the sum of 4 interactions between non homologous chromosomes (red dashed lines). b. In the context of segmental rearrangement, these properties can be extended and generalized if loci i and j belong to the same DNA segment, or to different segments (see “Methods” section and Additional file 1: Figure S1). c. Simulation of cancer Hi-C data from normal diploid (C) data by calculating the scaling factor matrix (p). Colors in scaling factor matrix represent the level of gains (red) and loss (green) to simulate. For each interaction Csim ij , the simulated count is finally estimated using a binomial down-sampling method (see “Methods” section). d. Intra-chromosomal maps of chromosome 1 and 2 before (top) and after (bottom) simulation of copy number changes. Copy number effects are characterized by blocks of high/lower signal. Overall, the simulation conserves the structure and the counts/distance properties of the Hi-C maps. e. Validation of the simulation model using Hi-C data from MCF10A cell line from which we simulated the expected copy number of MCF7 cancer cell line (MCF7 simulated). The mean O/E (Observed/Expected) counts per block of copy number of intra (cis) and inter-chromosomal (trans) maps at 1 Mb resolution is represented. Looking at the intra-chromosomal maps of chr3 and 8 demonstrates that our model efficiently simulates large copy number events. f. 1D genome-wide profiles of near-diploid MCF10A, simulated MCF7 and real MCF7 Hi-C data. MCF7 gain and losses are represented in red and green Fig. 1 Simulation of cancer Hi-C data. a. In Hi-C data from diploid cells, the contact frequency measured between two loci i and j is equal to the sum of 2 cis interactions (black solid lines) occurring within an individual allele and of 2 trans interactions between homologous chromosomes (transH, black dashed lines). In addition, the contact frequency observed in trans between loci i and k is the sum of 4 interactions between non homologous chromosomes (red dashed lines). b. In the context of segmental rearrangement, these properties can be extended and generalized if loci i and j belong to the same DNA segment, or to different segments (see “Methods” section and Additional file 1: Figure S1). c. Simulation of cancer Hi-C data from normal diploid (C) data by calculating the scaling factor matrix (p). Simulating the effect of copy number variations on Hi-C data BMC Bioinformatics (2018) 19:313 a b d c e f a b c c c b d e f Fig. 1 Simulation of cancer Hi-C data. a. In Hi-C data from diploid cells, the contact frequency measured between two loci i and j is equal to the sum of 2 cis interactions (black solid lines) occurring within an individual allele and of 2 trans interactions between homologous chromosomes (transH, black dashed lines). In addition, the contact frequency observed in trans between loci i and k is the sum of 4 interactions between non homologous chromosomes (red dashed lines). b. In the context of segmental rearrangement, these properties can be extended and generalized if loci i and j belong to the same DNA segment, or to different segments (see “Methods” section and Additional file 1: Figure S1). c. Simulation of cancer Hi-C data from normal diploid (C) data by calculating the scaling factor matrix (p). Colors in scaling factor matrix represent the level of gains (red) and loss (green) to simulate. For each interaction Csim ij , the simulated count is finally estimated using a binomial down-sampling method (see “Methods” section). d. Intra-chromosomal maps of chromosome 1 and 2 before (top) and after (bottom) simulation of copy number changes. Copy number effects are characterized by blocks of high/lower signal. Overall, the simulation conserves the structure and the counts/distance properties of the Hi-C maps. e. Validation of the simulation model using Hi-C data from MCF10A cell line from which we simulated the expected copy number of MCF7 cancer cell line (MCF7 simulated). The mean O/E (Observed/Expected) counts per block of copy number of intra (cis) and inter-chromosomal (trans) maps at 1 Mb resolution is represented. Looking at the intra-chromosomal maps of chr3 and 8 demonstrates that our model efficiently simulates large copy number events. f. 1D genome-wide profiles of near-diploid MCF10A, simulated MCF7 and real MCF7 Hi-C data. MCF7 gain and losses are represented in red and green b b e b b d e f e e e d d f f Fig. 1 Simulation of cancer Hi-C data. a. In Hi-C data from diploid cells, the contact frequency measured between two loci i and j is equal to the sum of 2 cis interactions (black solid lines) occurring within an individual allele and of 2 trans interactions between homologous chromosomes (transH, black dashed lines). Simulating the effect of copy number variations on Hi-C data Due to the large number of genomic and epigenomic fac- tors possibly involved, predicting the true effect of copy- number variations on the 3D organization of the genome is challenging. We propose a simple mathematical model to simulate the effect of abnormal karyotypes on a diploid Hi-C data set by estimating the enrichment in interac- tions due to CNVs (see “Methods” section and Fig. 1). Our model is based on the assumptions that (1) copies of chro- mosomes are independent and have similar 3D structure and (2) that the impact of copy number changes is higher than 3D structure variations that occur across cell types. Therefore, for a given copy number profile, our model will estimate the expected Hi-C contact maps in the presence of CNVs (Fig. 1d). We therefore applied our simulation model to assess the ability of the ICE normalization method to correct for CNVs. We simulated two data sets with different proper- ties from the publicly available human IMR90 Hi-C data [3]; a highly rearranged data set with segmental gains, losses and a focal amplification up to 10 copies (Fig. 1c) and a case of aneuploidy with gain or loss of entire chro- mosomes (Additional file 1: Figure S2a). While the sim- ulations were performed genome-wide, we restricted the CNVs to the first chromosomes to ease the results inter- pretation and visualization. The ground-truth normalized data was found by applying ICE to the original diploid data. We were thus able to assess the performance of ICE to correct for unwanted sources of variation, including the In order to validate our simulation model, we exploited available Hi-C data from two epithelial cell lines: the MCF7 breast cancer cell line and the MCF10A near- diploid, non-tumorigenic cell line [5]. We extracted the copy number information of the MCF7 line from Affymetrix SNP6.0 array, filtering out any altered seg- ments that were lower than the MCF10A’s Hi-C map res- olution (1 Mb) and applied our simulation model on the normal-like data, thus obtaining a simulation of MCF7’s abnormal Hi-C data. We then compared our simulated results with the real MCF7 Hi-C data set. As expected, the contact counts (for both the simulated data and the real data) are correlated with the copy number (Fig. 1e). Page 4 of 16 Servant et al. Estimation of copy number from Hi-C data Analyzing cancer samples usually requires access to CNV profiles. External sources of data (such as whole-genome sequencing or microarray data) can be used to infer DNA breakpoints along the genome, and thus to define DNA segments of equal copy number. If such data is not avail- able, we propose to directly infer the copy number profile from the Hi-C contact maps (see “Methods” section). Although in theory, all sequencing reads are useful to esti- mate the copy number profile, we first validated that using the counts from the contact maps (i.e. the subset of valid interaction products) is sufficient. For this, we compared our estimated copy number profile from MCF7 and T47D Hi-C contact maps, with the results of the Control-FREEC software [31] that directly uses the aligned sequencing If the downstream analysis is restricted to intra- chromosomal interactions, one may ask whether apply- ing ICE independently to each intra-chromosomal maps could mitigate the introduction of biases. We therefore independently normalized by ICE all intra-chromosomal maps. Although the effects are less strong, we observed a b Fig. 2 Impact of matrix balancing normalization on simulated cancer Hi-C data. a. Simulated Hi-C contact maps (500 kb resolution) of the first four chromosomes and contact frequencies presented as the sum of genome-wide contacts per locus, using either all (inter and intra-chromosomal), cis (intra-chromosomal) or trans (inter-chromosomal) contacts. Rearranged regions are highlighted in red (gain) or green (loss). The 1D profile of ICE data is constant genome-wide as expected under the assumption of equal visibility. However, the iterative correction on simulated cancer data results in an shift of contacts between altered regions (see arrows for examples). b. Block-average error matrix of simulated raw and ICE cancer data (150 Kb resolution) (See Additional file 1: Method 1.4). The iterative correction does not allow to correct for segmental copy number bias a b b a Fig. 2 Impact of matrix balancing normalization on simulated cancer Hi-C data. a. Simulated Hi-C contact maps (500 kb resolution) of the first four chromosomes and contact frequencies presented as the sum of genome-wide contacts per locus, using either all (inter and intra-chromosomal), cis (intra-chromosomal) or trans (inter-chromosomal) contacts. Rearranged regions are highlighted in red (gain) or green (loss). The 1D profile of ICE data is constant genome-wide as expected under the assumption of equal visibility. Simulating the effect of copy number variations on Hi-C data Colors in scaling factor matrix represent the level of gains (red) and loss (green) to simulate. For each interaction Csim ij , the simulated count is finally estimated using a binomial down-sampling method (see “Methods” section). d. Intra-chromosomal maps of chromosome 1 and 2 before (top) and after (bottom) simulation of copy number changes. Copy number effects are characterized by blocks of high/lower signal. Overall, the simulation conserves the structure and the counts/distance properties of the Hi-C maps. e. Validation of the simulation model using Hi-C data from MCF10A cell line from which we simulated the expected copy number of MCF7 cancer cell line (MCF7 simulated). The mean O/E (Observed/Expected) counts per block of copy number of intra (cis) and inter-chromosomal (trans) maps at 1 Mb resolution is represented. Looking at the intra-chromosomal maps of chr3 and 8 demonstrates that our model efficiently simulates large copy number events. f. 1D genome-wide profiles of near-diploid MCF10A, simulated MCF7 and real MCF7 Hi-C data. MCF7 gain and losses are represented in red and green Servant et al. BMC Bioinformatics (2018) 19:313 Page 5 of 16 copy number, by comparing the obtained matrices to the ground-truth. the same phenomenon in complex rearrangements (Additional file 1: Figure S3). Before running the ICE normalization, we first rep- resented the data in 1D, by summing each row of the matrix. As previously mentioned, the sum of genome- wide interactions per bin is proportional to the copy number (Fig. 2a). After applying ICE, each genomic region now interacts the same number of times genome-wide, as expected. However, ICE leads to an imbalance between cis and trans contact counts; cis contact counts are now depleted for regions with high copy number, and trans contact counts are enriched (Fig. 2b). On the other hand, lost regions now present higher contact probabilities than regions of gain in cis. The same conclusions can be made in the context of aneuploidy (Additional file 1: Figure S2). However, we notice that in this case, ICE can yield to the expected results if the analysis is restricted to intra- chromosomal contacts. Altogether, these results demonstrate that ICE does not adequately normalize data from cells with abnormal kary- otypes. More importantly, using ICE on cancer Hi-C data can lead to a misinterpretation of the contact probabili- ties between rearranged regions. Its use is therefore not recommended in this context. Estimation of copy number from Hi-C data However, the iterative correction on simulated cancer data results in an shift of contacts between altered regions (see arrows for examples). b. Block-average error matrix of simulated raw and ICE cancer data (150 Kb resolution) (See Additional file 1: Method 1.4). The iterative correction does not allow to correct for segmental copy number bias Servant et al. BMC Bioinformatics (2018) 19:313 Page 6 of 16 reads to call the CNVs (Additional file 1: Figure S4). We observe a good correlation between both methods (MCF7 spearman cor=0.888, T47D spearman cor=0.855) therefore validating our approach. telomeric pattern is expected, even in a diploid sample, as the assumption of equal visibility in these regions is questionable. We then applied our segmentation strategy to our Hi-C simulated data (Fig. 3b). In order to assess the robust- ness of this approach, we further simulated 100 addi- tional data sets with distinct CNVs profiles. Results on this larger number of data sets show a 91% recall and a precision of 62.4%. In particular, we observe that the pre- cision of breakpoint detection can sometimes be lower in highly amplified regions. Finally, we also applied our seg- mentation procedure to the IMR90 diploid data set. As expected, we obtain a nearly uniform copy number profile (Additional file 1: Figure S6c). Interestingly, we frequently observe a decrease of contacts at telomeric regions which therefore results in a breakpoint in the segmentation. This LOIC: a novel normalization strategy for cancer Hi-C data As presented above, ICE relies on the assumption of equal visibility of each genomic bin. In the presence of copy number variations, this assumption does not hold: genomic bins with higher copy number variations will interact overall more frequently than genomic bins of lower CNVs. In addition, the copy number effect between loci i and j (Bij), cannot be decomposed into the prod- uct of an effect in locus i and an effect in locus j, thereby also violating the ICE hypothesis. Instead, we propose to extend the ICE model, through the assumption that equal visibility remains true across regions of identical copy number. In addition, biases associated to fragments (such a c b Fig. 3 Generalization of matrix balancing algorithms for cancer Hi-C data. a. Rationale of LOIC method versus standard ICE method. The LOIC method extends the ICE normalization by constraining the genome-wide Hi-C 1D profile to follow the copy number signal. b. Estimation of copy number from Hi-C data Segmentation of the Hi-C 1D genome-wide profile of simulated cancer data. The red line represents the smoothing line that estimate the copy number level. c. LOIC normalized Hi-C contact maps of simulated data on the first four chromosomes. The 1D profiles are represented by the sum of genome-wide contacts at each locus using either all (inter and intra-chromosomal), cis (intra-chromosomal) or trans (inter-chromosomal) contacts. As a results, we can see that the LOIC method allows to normalize cancer Hi-C data keeping into account the copy number information a c b b Fig. 3 Generalization of matrix balancing algorithms for cancer Hi-C data. a. Rationale of LOIC method versus standard ICE method. The LOIC method extends the ICE normalization by constraining the genome-wide Hi-C 1D profile to follow the copy number signal. b. Segmentation of the Hi-C 1D genome-wide profile of simulated cancer data. The red line represents the smoothing line that estimate the copy number level. c. LOIC normalized Hi-C contact maps of simulated data on the first four chromosomes. The 1D profiles are represented by the sum of genome-wide contacts at each locus using either all (inter and intra-chromosomal), cis (intra-chromosomal) or trans (inter-chromosomal) contacts. As a results, we can see that the LOIC method allows to normalize cancer Hi-C data keeping into account the copy number information Fig. 3 Generalization of matrix balancing algorithms for cancer Hi-C data. a. Rationale of LOIC method versus standard ICE method. The LOIC method extends the ICE normalization by constraining the genome-wide Hi-C 1D profile to follow the copy number signal. b. Segmentation of the Hi-C 1D genome-wide profile of simulated cancer data. The red line represents the smoothing line that estimate the copy number level. c. LOIC normalized Hi-C contact maps of simulated data on the first four chromosomes. The 1D profiles are represented by the sum of genome-wide contacts at each locus using either all (inter and intra-chromosomal), cis (intra-chromosomal) or trans (inter-chromosomal) contacts. As a results, we can see that the LOIC method allows to normalize cancer Hi-C data keeping into account the copy number information Servant et al. BMC Bioinformatics (2018) 19:313 Page 7 of 16 “Methods”section). We refer to this method as CNV- Adjusted Iterative Correction (CAIC). as fragment length, GC-content, or mappability) can still be decomposed into the product of two region-specific biases. j We applied CAIC normalization to the two simulated data sets. Estimation of copy number from Hi-C data Looking at the 1D signal of the CAIC nor- malized data using the cis and trans data validates that the method tends to remove the CNV effect (Fig. 4a). In addition, the unbalanced effect that we previously observed with the ICE normalized data disappeared. We then divided the normalized contact matrices by the expected count matrices, thus removing the structure due to genomic proximity. Taking the average per block, we observe that the expected CAIC matrices are much more uniform than the ICE normalized matrices (Additional file 1: Figure S7 and S8). On the aneuploid simulated data set, it is worth noting that ICE and CAIC yield very close results in cis. We then compared the normalized contact maps to the ground-truth by computing the error matrix as well as three additional error measures (see Fig. 4b and Additional file 1: Methods 1.4). We observe that the copy number effect is well removed both on the aneuploid and highly rearranged data set (Table S3). We thus first propose to extend ICE by assuming that the sum of contacts for a given genomic bin is constant across genomic bins of identical copy number (Fig. 3a): i CLOIC ij = kj, where kj is the interaction profile asso- ciated to the copy number of j (see “Methods” section). We refer to this method as a LOcal Iterative Correction (LOIC). When there is no copy number alteration, LOIC solves exactly the same problem as ICE. y p We applied the LOIC procedure to our highly rear- ranged simulated Hi-C data set using the breakpoint posi- tions estimated by our segmentation procedure (Fig. 3c). As expected, we observe that the genome-wide sum of contacts of each bin is proportional to the copy number, and that bins within a DNA segment are normalized to the same level of interactions. The previous effects on the cis and trans sum of contacts observed using the standard ICE strategy, is no longer found. In addition, we calcu- lated the effective fragment length, the GC content and the mappability features for each 500 Kb bin as already proposed [26], and then represented the average contact frequencies among those genomic features. Despite a few local enrichments due to CNVs, we observe that LOIC normalization is as effective as ICE normalization for correction of GC content, effective fragment length and mappability (Additional file 1: Figure S5). Application to breast cancer Hi-C data A number of studies performed Hi-C experiments on can- cer samples or cell lines [5, 18, 32]. We further explored the effect of our normalization procedures on two previ- ously published Hi-C data from breast cancer cell lines: T47D [32] and MCF7 [5]. We processed the T47D and MCF7 samples from raw data files to raw contact maps using the HiC-Pro pipeline [33]. As already seen in our simulation data (Fig. 2a), we observe a strong copy number effect on the raw contact maps with respec- tively higher/lower contact frequency on gained/lost DNA regions in both samples (Fig. 5, Additional file 1: Figure S9). Applying ICE on these data sets does not entirely remove the copy number effect, and tends to flip the coverage profile between gained/lost regions in cis, therefore validating our previous observations on simu- lated data (Fig. 5a, Additional file 1: Figure S9a). In conclusion, the LOIC strategy presented here can be seen as a generalization of the ICE method allowing to correct the Hi-C maps for systematic biases while keep- ing the copy number signal. In this sense, applying either methods to a diploid data set leads to identical results. Estimation of copy number from Hi-C data We then turned to the aneuploid simulated data set. In this case, LOIC enables conservation of the inter-chromosomal scaling factor due to CNVs. Differences in intra-chromosomal maps between ICE and LOIC remain negligible and are related to the segmentation profile (Additional file 1: Figure S6a, b). Altogether, these results demonstrate that the CAIC normalization procedure effectively removes copy-number effects from Hi-C contact maps. CAIC: estimating and removing the copy-number effect on cancer Hi-C data In order to estimate the copy number signal from these cell lines, we segmented the 1D Hi-C profile as previ- ously described. Interestingly, on both T47D and MCF7 data we observed a very good correlation between the copy number signal extracted directly from the Hi-C data and the copy number profile extracted from SNP6 Affymetrix array (Fig. 5b, Additional file 1: Figure S9b, Spearman cor=0.87 for both MCF7 and T47D data) We then applied the LOIC strategy presented above, so that the sum of each column/row follows the segmentation profile extracted from the data. As expected, we observe that the LOIC normalized contact maps conserves the We also set out to estimate and to correct the effect intro- duced by copy number changes. We assume that copy number effects can be represented as a block-constant matrix where each block is delimited by a copy number change (see “Methods” section). In addition, we assume that, on average, each pair of loci interacts the same way as any other pair of loci at the same genomic distance s. In summary, the raw interaction count Cij is roughly equal to the product of a CNV bias Bij and the expected contact count at genomic distance s: Cij ≃Bijes(i,j). We thus cast an optimization problem to find the CNV block biases B and the expected contact count at genomic distance s (see Servant et al. BMC Bioinformatics (2018) 19:313 Page 8 of 16 a b Fig. 4 CNV-adjusted normalization of cancer Hi-C data. a. Hi-C contact maps of the four first chromosomes of our highly rearranged simulated data, together with the 1D signal of all, cis and trans data. Regions in red and green correspond to simulated gains and losses. b. Block-average error matrix of simulated ICE and CAIC Hi-C data. The CAIC efficiently removed the CNV effect, whereas the ICE normalization does not allow to correct for its effect b a b a Fig. 4 CNV-adjusted normalization of cancer Hi-C data. a. Hi-C contact maps of the four first chromosomes of our highly rearranged simulated data, together with the 1D signal of all, cis and trans data. Regions in red and green correspond to simulated gains and losses. b. Block-average error matrix of simulated ICE and CAIC Hi-C data. CAIC: estimating and removing the copy-number effect on cancer Hi-C data The CAIC efficiently removed the CNV effect, whereas the ICE normalization does not allow to correct for its effect Interestingly, when we compared the WT sample and the samples with the duplication events normalized by the ICE method, we observed that the ICE normalized maps do not allow duplication effects to be observed clearly. This is in agreement with our previous observa- tions on cancer Hi-C data. We then applied our LOIC strategy following the observed 1D coverage profiles. As illustrated in Fig. 6, the LOIC normalization is able to remove systematic biases while keeping the copy number effect. The effects of both intra and inter-TAD dupli- cation can therefore be clearly observed, validating the interest of our method for the study of local structural rearrangements. copy number properties, and that the biases introduced by the ICE normalization no longer hold true. In addition, we also applied the CAIC normalization to correct for CNVs signal. Looking at the correlation between Hi-C counts and the copy number signal validates the efficiency of the methods (Fig. 5c, Additional file 1: Figure S10). In con- clusion, applying the LOIC and CAIC methods on both cancer data set allows us to correct for systematic bias while conserving or removing the copy number structure. Normalization of capture-Hi-C data with genomic duplication In addition to cancer data, we also investigated the rel- evance of LOIC for the normalization of Hi-C data in samples with local structural events. Recently, Franke et al. [10] investigated the effect of local duplications on chro- matin structure and, in particular, on the formation of new TADs. We processed the capture Hi-C data across the Sox9 locus and generated the raw and ICE contact maps at 10kb resolution. We focused our analysis on samples with inter-TAD (dup-S) and intra-TAD (dup-L) duplications. In the context of an inter-TAD duplication, Franke et al. [10] described the formation of a new domain in the duplicated region by comparing the raw contact maps of wild type (WT) and dup-L samples (Fig. 6c-d). Removing the CNVs signal avoids misinterpretation of the chromosome compartment calling of cancer Hi-C data We also explored the impact of CNVs and normal- ization on chromosome compartment calling. In intra- chromosomal contact maps, chromosome compartment profiles appear as checker-board-like interaction patterns, shifting from blocks with either high or low interac- tion frequency. Thus, chromosome compartments are usually detected using a Principal Component Analysis (PCA) on the correlation matrix of the distance-corrected intra-chromosomal contact maps. The first principal Servant et al. BMC Bioinformatics (2018) 19:313 Page 9 of 16 a b c Fig. 5 Normalization of T47D Hi-C data. a. Hi-C contact maps (250 Kb resolution) of the first four chromosomes of T47D cancer Hi-C sample. When looking at the 1D cis and trans profiles, we observed that ICE introduces a bias in the normalized data, therefore validating the observation made on the simulated data. We then applied the LOIC and CAIC normalizations in order to efficiently correct the data from systematic bias, while removing or keeping the CNVs effect. b. Estimatation of the copy number signal from the Hi-C data after correction and segmentation of the 1D profile. The inferred copy number signal from the Hi-C data are highly correlated with the copy number profile from Affymetrix SNP6.0 array. c. Correlation of raw and normalized contact frequencies with the copy number a a b b b c c Fig. 5 Normalization of T47D Hi-C data. a. Hi-C contact maps (250 Kb resolution) of the first four chromosomes of T47D cancer Hi-C sample. When looking at the 1D cis and trans profiles, we observed that ICE introduces a bias in the normalized data, therefore validating the observation made on the simulated data. We then applied the LOIC and CAIC normalizations in order to efficiently correct the data from systematic bias, while removing or keeping the CNVs effect. b. Estimatation of the copy number signal from the Hi-C data after correction and segmentation of the 1D profile. The inferred copy number signal from the Hi-C data are highly correlated with the copy number profile from Affymetrix SNP6.0 array. c. Correlation of raw and normalized contact frequencies with the copy number component then distinguishes the open (A) from closed (B) compartments [2] (see Additional file 1: Methods). H3K4me. Respectively, closed compartments are asso- ciated with repressive marks such as H3K27me3 or H3K9me3 (Fig. 7a). Removing the CNVs signal avoids misinterpretation of the chromosome compartment calling of cancer Hi-C data We performed compartment calling analysis on the MCF7 Hi-C data set normalized by ICE, LOIC, or CAIC methods and integrated the results with the histone marks data obtained from the ENCODE project [34]. Surpris- ingly, we observed that compartment calling is globally not affected by the CNVs on MCF7 data (Additional file 1: Figure S12) with around 8% of chromosome compart- ments switching from open to closed states (and vice- versa) according to the normalization method (Additional file 1: Figure S13a). We then assessed how the A/B com- partments correlated with the active and repressive his- tone marks genome-wide (see Additional file 1: Methods). As expected, open compartments are associated with open-chromatin marks such as H3K27ac, H3K36me3 and Interestingly, looking at each chromosome indepen- dantly shows that, on the MCF7 data, the chromosome 8 harbours distinct compartment patterns according to the normalization method (Additional file 1: Figure S13a). In this case, it is clear that the copy number affects the PCA analysis and the compartment calling (Fig. 7b, c). We therefore conclude that it is important to correct for the copy number effect before running such analysis, and therefore that, by definition, the LOIC normaliza- tion method is not appropriate to this task. Applying the CAIC normalization method outperforms the other methods, resulting in a compartment profile which is well correlated with active/inactive histone marks, and which Servant et al. BMC Bioinformatics (2018) 19:313 Page 10 of 16 a c d b Fig. 6 Duplication in capture-Hi-C and normalization. a. 1D profiles of capture-Hi-C wild-type sample (WT), with intra-TAD duplication (dup-S) or with inter-TAD (dup-L) duplication [10]. As expected, the duplication samples are characterized by twice more contacts at the duplicated sites. b. Raw and ICE normalized contact maps of WT sample. c. Normalization of the dup-S sample with the ICE and LOIC methods. The duplication effect is visualized by subtracting the normalized WT and dup-S contact maps. d. Same approach applied to the dup-L sample b a b a c d c d c d Fig. 6 Duplication in capture-Hi-C and normalization. a. 1D profiles of capture-Hi-C wild-type sample (WT), with intra-TAD duplication (dup-S) or with inter-TAD (dup-L) duplication [10]. As expected, the duplication samples are characterized by twice more contacts at the duplicated sites. b. Raw and ICE normalized contact maps of WT sample. c. Normalization of the dup-S sample with the ICE and LOIC methods. Removing the CNVs signal avoids misinterpretation of the chromosome compartment calling of cancer Hi-C data The duplication effect is visualized by subtracting the normalized WT and dup-S contact maps. d. Same approach applied to the dup-L sample Discussion h Histone marks enrichment on chromosome 8 of MCF7 sample. On this chromosome the copy number has a strong impact on the compartment calling c Results of the compartment calling for the chromosome 8 of Fig. 7 Detection of chromosome compartments. a. Genome-wide enrichment of ChIP-seq histone marks in open (A) or closed (B) compartments. Open compartments are enriched in open-chromatin marks, whereas closed compartments are enriched in repressive marks. The results are concordant genome-wide, whatever the normalization method applied. b. Histone marks enrichment on chromosome 8 of MCF7 sample. On this chromosome, the copy number has a strong impact on the compartment calling. c. Results of the compartment calling for the chromosome 8 of MCF7 sample (first principal component), together with normalized ChIP-seq tracks. Open chromatin domains are in red. Closed domains in blue strategy that we have applied here requires a high resolu- tion diploid Hi-C data set as an input. However, as Hi-C sequencing depth increases, this should no longer be a limitation. our model is able to predict the effects of large copy number changes on interaction patterns by estimating a subsampling coefficient that can be applied in the pres- ence of copy number variation. In addition to providing us with a theoretical framework to assess the enrich- ment and depletion of contact counts with respect to copy number variations, such a model can be used to assess how normalization methods and downstream analyses are affected by copy number variation. Our model is never- theless simplistic and can be elaborated upon. For exam- ple, we consider that duplicated regions are non-tandemly rearranged events, which, in some cases, certainly under- estimates the intra-chromosomal effect of copy number. In addition, the model does not integrate any biologi- cal knowledge and is therefore not designed to simulate changes due to the alteration of regulatory elements such as insulator regions. We also note that the subsampling In our study, we go on to demonstrate that applying ICE (the most commonly used normalization method on Hi- C data) to data sets with abnormal copy number profiles leads to unbalanced corrections between amplified and lost regions and between inter and intra-chromosomal contacts. This unwanted effect can then lead to prob- lematic results in downstream analysis (for example, in the identification of open and closed compartments). Discussion h is closer to the normal MCF10A chromosome 8 profile (Fig.7b, c). The compartments pattern of the chromosome 8 extracted from the ICE normalized data is concordant with our previous conclusion that ICE is not appropriate to correct for CNVs, potentially leading to a wrong inter- pretation of the compartment profile. However, we also noticed that, in this case, the A/B compartments can be rescued by looking at the second principal component of the PCA. Chromosome conformation techniques provide a means to investigate links between the 3D organization of the genome and biological processes such as the functional and phenotypic effects of genomic variation in disease. Unsurprisingly, structural and copy number variations have also been observed to affect the genome architecture of cancer cells, perturbating TADs as well as the cell’s reg- ulation [18, 35, 36], and as a result, the contact count maps of the genome. How do such genomic rearrangements impact on the performance of existing pipelines? Altogether, these results demonstrate that although compartment calling seems globally unaffected by copy number effects, applying the CAIC strategy to normal- ize the data improves the detection of A/B compartments, avoiding potential issues in their interpretation. In order to better understand how large copy number variations can affect Hi-C data, we first propose a sim- ple simulation model. From an existing diploid data set, Servant et al. BMC Bioinformatics (2018) 19:313 Page 11 of 16 a b c Fig. 7 Detection of chromosome compartments. a. Genome-wide enrichment of ChIP-seq histone marks in open (A) or closed (B) compartments. Open compartments are enriched in open-chromatin marks, whereas closed compartments are enriched in repressive marks. The results are concordant genome-wide, whatever the normalization method applied. b. Histone marks enrichment on chromosome 8 of MCF7 sample. On this chromosome, the copy number has a strong impact on the compartment calling. c. Results of the compartment calling for the chromosome 8 of MCF7 sample (first principal component), together with normalized ChIP-seq tracks. Open chromatin domains are in red. Closed domains in blue b a a b c b a a c c Fig. 7 Detection of chromosome compartments. a. Genome-wide enrichment of ChIP-seq histone marks in open (A) or closed (B) compartments. Open compartments are enriched in open-chromatin marks, whereas closed compartments are enriched in repressive marks. The results are concordant genome-wide, whatever the normalization method applied. b. Methods identification of breakpoints. We thus also propose a seg- mentation procedure to directly extract the copy number signal and the location of breakpoints from the Hi-C contact maps. Although this step is crucial for the nor- malization and can be challenging for noisy samples, our procedure performs well on all the data set used in this study, including the normal diploid IMR90 sample. It also confirms that the Hi-C technique could become in the near-future a powerful approach to infer CNV profile in tumors. identification of breakpoints. We thus also propose a seg- mentation procedure to directly extract the copy number signal and the location of breakpoints from the Hi-C contact maps. Although this step is crucial for the nor- malization and can be challenging for noisy samples, our procedure performs well on all the data set used in this study, including the normal diploid IMR90 sample. It also confirms that the Hi-C technique could become in the near-future a powerful approach to infer CNV profile in tumors. Let us first introduce some notations. Given a segmen- tation of the genome into n genomic windows (or bins), Hi-C data can be summarized by a n-by-n symmetric matrix C, in which each row and column corresponds to a specific genomic loci and each entry Cij the num- ber of times loci i and j have been observed in con- tact. Let K ∈Rn the copy number profile of the sample of interest, which we represent as a piecewise constant vector. We denote by s(i, j) the genomic distance between the loci, defined as the number of base pairs between the cen- ter of the two loci; if i and j are not part of the same chro- mosome, we extend this definition by setting s(i, j) = ∞. In this paper, we derive different ways to normalize the raw count matrix C: we denote by Ny the contact count matrix normalized with method y (e.g. NICE represents the ICE normalized count matrix). Both CAIC and LOIC methods perform well on sim- ulated and real Hi-C data sets, each fulfilling different needs. From a methodological point of view, we note that our assumption that the CNV effect is constant for each block delimited by a copy number change, cannot always be fulfilled, especially for very large genomic alterations. Discussion h We therefore propose two new normalization methods that can remove systematic biases: LOIC, which pre- serves the effect of copy number variation, and CAIC, which removes the effects of copy number. In order to achieve this, both normalization methods require the Page 12 of 16 Page 12 of 16 Servant et al. BMC Bioinformatics (2018) 19:313 Simulation of cancer Hi-C data Before we turn to how to appropriately model cancer Hi-C data, let us first review some terminology. In the literature, cis-contact counts refers to the contact counts between two loci of the same chromosome: this includes intra- chromosomal contact counts but also inter-chromosomal contact counts of homologous chromosomes. In this paper, we restrict the use of cis-contact counts to con- tact counts issued from the same DNA fragment, and we denote by “trans-homologous” (transH) interactions, the interactions between homologous chromosomes. Note that cis and transH contact counts are mostly indistin- guishable (with the exception of allele-specific Hi-C) in Hi-C data hence the simplification of terminology usually used. We now return to the problem at hand: how to sim- ulate a contact count matrix Csim of a cancer genome with abnormal copy number from a raw diploid contact count matrix C. In order to model the change in con- tact count abundances due to copy number variation, we first need to understand precisely which interactions are observed in the case of a simple diploid genome. For that purpose, we denote by Eij the expected contact count between loci i and j, and Ecis ij , EtransH ij and Etrans ij the expected cis, trans and transH-contact counts between i and j. Methods In such cases, modeling the CNV effect according to the distance between pairs of interacting loci could be an interesting extension to our current model. The choice of which normalization method to use in Hi- C data analysis, will depend on the specific context and biological question. As an example, we demonstrate here that although chromosome compartment calling and PCA analysis are not dramatically affected by the copy number changes in MCF7 data, inappropriate normalization of the data can lead to incorrect interpretation of the results of such analysis. This effect can be even greater with other cell lines, as it is dependent on the copy number profile of the tumor (data not shown). This first experiment shows that CAIC, which removes the copy number biases, can enable existing analysis pipelines to be applied to cell lines with abnormal karyotypes. Yet, in other contexts, keeping the CNV informations could also be pertinant. For exam- ple, it may be of interest to examine the precise effect of local structural rearrangements and CNVs at smaller scale such as TADs level. As illustrated here with capture Hi-C data, applying our LOIC normalization procedure could be useful to remove systematic biases appropriately, while keeping the copy number structure. Estimation of copy number from the contact count matrix In order to derive a scaling factor pij which incorporates the copy number effect, we need to estimate Ecis ij , Etrans ij t H In order to derive a scaling factor pij which incorporates the copy number effect, we need to estimate Ecis ij , Etrans ij and EtransH ij , which is impossible without further assump- tions. In practice, little is known about the probability of contact between homologous chromosomes, which is therefore difficult to estimate. However, we know that the chromosomes usually occupy their own space (chromo- some territories) within the nucleus. We therefore make the assumption that all chromosomes are independent, and that the contact probability between homologous chromosomes can be estimated using the trans interac- tion between non-homologous chromosomes. We thus consider that Etrans ij = EtransH ij = Etrans. Estimation of copy number from the contact count matrix The copy number signal can be directly inferred from the Hi-C data in two steps. We first calculate the one- dimensional (1D) signal as the sum of genome-wide con- tact per bin, assuming that this signal reflects the true contact frequencies including the systematic Hi-C biases and the CNVs signal. We further calculate the GC con- tent, the mappability and the effective fragment length of each bin end as already proposed [26]. The local genomic features of all chromosome bins are defined as the aver- age of the corresponding features among all overlapping fragment ends. We then apply a Poisson regression model to correct the signal from GC content, mappability and fragment length, using the model proposed by Hu et al. [26]. The corrected profile is obtained by subtracting the fitted values to the observed data, and rescaled to be cen- tered on 1. The normalized 1D data are then segmented using a pruned dynamic programming algorithm [38]. The segmented profile is smoothed with the GLAD pack- age [39] in order to optimize the breakpoint locations and to remove false positives events. The segmentation is an important step of the method which may need to be adjusted according to the signal-to-noise ratio of the data. In this study, we apply the same parameters to all data sets and we consider the smoothed line after the segmentation as the Hi-C derived copy number profile. Conclusions Taken together, the analyses covered here confirm that Hi-C can be a powerful technique to explore strutural variations on tumor samples and highlight the impor- tance of using dedicated methods for the analysis of such data. The two new methods we introduce here (LOIC and CAIC) perform well on both simulated and real cancer Hi- C data sets, each answering different biological questions. As the application of Hi-C techniques to cancer and other samples to explore the 3D architecture of their genomes will continue to grow in the coming years, the importance of using the right techniques and developing more accu- rate tools to characterize such data sets appropiately is critical. Taken together, the analyses covered here confirm that Hi-C can be a powerful technique to explore strutural variations on tumor samples and highlight the impor- tance of using dedicated methods for the analysis of such data. The two new methods we introduce here (LOIC and CAIC) perform well on both simulated and real cancer Hi- C data sets, each answering different biological questions. • if loci i and j belong to the same chromosome, the expected contact count Eij is the sum of (1) cis-counts from either of the homologous chromosomes; (2) the transH-counts between the two homologous chromosomes: • if loci i and j belong to the same chromosome, the expected contact count Eij is the sum of (1) cis-counts from either of the homologous chromosomes; (2) the transH-counts between the two homologous chromosomes: Eij = 2Ecis ij + 2EtransH ij Servant et al. BMC Bioinformatics (2018) 19:313 Page 13 of 16 • if loci i and j belong to different chromosomes, then the observed contact counts Eij is the sum of either of the four possible trans interactions: We thus obtain, for each entry of the contact count matrix Cij, a ratio pij corresponding to the expected fac- tor of enrichment or depletion of interactions for the loci i and j. In order to make the estimation of pij more robust, we estimate it constant per blocks of identical copy num- bers by taking the median of the empirical values in each block. (See Additional file 1: Figure S11). Thus, the fac- tor matrix p can be assumed to be block constant between regions of identical copy number variations. We thereby smooth p by computing the median scaling factor of block of similar copy number. • if loci i and j belong to different chromosomes, then • if loci i and j belong to different chromosomes, then Eij = KiKjEtrans ij Eij = KiKjEtrans ij Now that we have derived how contact counts are decomposed in terms of cis, transH and trans contact counts, we can leverage those relationships to simulate the effect of copy number variations on contact count matrices. Conclusions Eij = 4Etrans ij Eij = 4Etrans ij This can be generalized to polyploid genome or to the context of chromosomal abnormalities (Additional file 1: Figure S1). • if loci i and j belong to the same chromosome, let k be the number of cis interactions. If i and j belong to the same DNA segment, k = Ki = Kj. When i and j belong to different DNA segments, k could in theory take values between 0 and min(Ki, Kj). Here, we simulated the data with k = 2, or k = min(Ki, Kj) if Ki < 2 or Kj < 2. Then • if loci i and j belong to the same chromosome, let k be the number of cis interactions. If i and j belong to the same DNA segment, k = Ki = Kj. When i and j belong to different DNA segments, k could in theory take values between 0 and min(Ki, Kj). Here, we simulated the data with k = 2, or k = min(Ki, Kj) if Ki < 2 or Kj < 2. Then Finally, the simulated contact counts Csim ij are generated by a binomial subsampling strategy of Cij by a probability equal to pij max(pij) [37]: Csim ij ∼B(Cij, pij) (1) (1) Eij = kEcis ij + (KiKj −k)EtransH ij The reason for choosing a binomial subsampling as opposed to a simpler multiplication of the original Hi-C counts by a CNV-dependent factor, is that if Cij follows a Poisson or Negative Binomial distribution, then Csim ij fol- lows the same distribution with modified expectation [37]. One limitation of this model is that the simulated counts can only be smaller than the original counts, which may be problematic if we start from small counts. It thus requires a diploid Hi-C data set with a sufficient sequencing depth to apply the downsampling strategy. . • if loci i and j belong to different chromosomes, then Additional file Additional file 1: Supplementary Methods and Figures. This file contains supplemental methods, Tables S1-S3, and Figures S1-S13 (PDF 19,614 kb) In order to apply the proposed method, one needs to know a priori the set of bins with a given copy number or the copy number breakpoints. It can either be found via Estimation of copy number from the contact count matrix j j From these relationships, we then calculate the scal- ing factor pij as following (recall that Eij is the expected copy number between i and j on genome with abnormal chromosomal interactions): • we estimate Etrans as the median trans-contact count; • Ecis ij = Cij −Etrans; if loci i and j belong to the same chromosome, E • if loci i and j belong to the same chromosome, pij = Eij 2Ecis ij +2Etrans j if loci i and j belong to different chromosomes, j • if loci i and j belong to different chromosomes, pij = KiKj 4 Page 14 of 16 Servant et al. BMC Bioinformatics (2018) 19:313 To validate our estimation of copy number from Hi-C contact maps, we simulate an additional 100 data sets with varying copy number variations, as follows: probing the samples to estimate it using specific technolo- gies or through prior knowledge on the cell-line or sample studied. When none of these options are available, we can leverage the information provided by Hi-C data directly to estimate it. • draw a number of breakpoints along the genome; • remove all breakpoints that fall into non mappable genome; LOIC: Correcting technical biases of Hi-C cancer data To normalize the contact count matrix, we adapt the ICE method proposed by Imakaev et al. [28] to incorporate the copy number effect. In particular, we use similar assump- tions. First, the bias between two regions i and j can be decomposed as the product of two region-specific biases βij = βiβj. ∀i, j ∈[ 1, n] , Cij = βiβjNLOIC ij , (2) (2) In addition, we assume that, on average, each pair of loci interacts roughly the same way as any pair of loci at the same genomic distance s: where β ∈Rn is a vector of bin-specific biases, such as gc-content, fragment lengths, mappability, etc. where β ∈Rn is a vector of bin-specific biases, such as gc-content, fragment lengths, mappability, etc. Second, all copy-number identical regions interact as much: NCAIC l,m ≃e(s(l, m)) , (4) (4) where e(s) is the expected contact count at genomic dis- tance s. We leverage this assumption to cast an optimiza- tion problem: ∀i ∈[1, n] , n j=1 NLOIC ij = 1 \|{l \| Kl = Ki}\| l\|Kl=Ki n j=1 Clj . (3) (3) min e,B i,j NLOIC ij −Bije(s(i, j)) 2 subject to B is block-constant e decreasing min e,B i,j NLOIC ij −Bije(s(i, j)) 2 We refer to the second hypothesis as the “local equal- visibility assumption” to contrast it with ICE’s “equal- visibility assumption”: instead of enforcing an interaction profile constant across all the genome, we enforce an interaction profile constant for regions of identical copy number. We solve this optimization problem genome-wide by iter- atively estimating the block constant matrix B and the expected counts function e using an isotonic regression. Note that the trans estimation can be done jointly on the whole genome independently from the cis estimation. We solve this optimization problem genome-wide by iter- atively estimating the block constant matrix B and the expected counts function e using an isotonic regression. Note that the trans estimation can be done jointly on the whole genome independently from the cis estimation. Similarly to Imakaev et al. [28], this problem can be solved exactly using matrix-balancing algorithms (under the assumption that the matrix is full decomposable [40]). Note that if there is no copy number variations, this boils down to solving exactly the same problem as ICE. LOIC: Correcting technical biases of Hi-C cancer data On the other hand, in the presence of copy number variation, the resulting interaction profile will be a constant piecewise function, whose value depends on the copy number of the two loci. Additional file Additional file 1: Supplementary Methods and Figures. This file contains supplemental methods, Tables S1-S3, and Figures S1-S13 (PDF 19,614 kb) Abbreviations 1D: One dimension; CNVs: Copy number variants; CAIC: CNV-adjusted iterative correction; ICE: Iterative correction and eigenvector decomposition; CAIC: Removing the copy number effect • draw the copy number coefficient from a poisson distribution of size 1. add 1 to these values; The previous section describes how to normalize the raw contact counts matrix C to adjust for unwanted variations such as GC-content, mappability, fragment lengths, while keeping the copy number information. We now propose to estimate the effect of copy number variations on the contact count matrix to offer the possibility of removing it. We denote by NCAIC the normalized contact count matrix where the CNV effect has been removed. • redraw any copy number coefficient that is equal to its neighbour; • apply the simulation model presented above on these copy number profiles. The copy number profiles are available as supplementary data. We assume that the copy number effect for each pair of loci is identical for element with identical copy-number variations. This reflects that the copy-number effect between loci i and j is related to the amount of genetic material of those two regions, and thus identical between all pairs with similar copy-number variations. We can thus model the normalized contact count matrix as the product of a block-constant matrix B and corrected matrix NCAIC: NCAIC ij = BijNLOIC ij , where each block is a function of the copy number in i and in j. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 14. Plass C, Pfister SM, Lindroth AM, Bogatyrova O, Claus R, Lichter P. Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. 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https://openalex.org/W2973932540	https://europepmc.org/articles/pmc6801931?pdf=render	English	null	Elemental Contamination in Indoor Floor Dust and Its Correlation with PAHs, Fungi, and Gram+/− Bacteria	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	10,713	Received: 21 August 2019; Accepted: 20 September 2019; Published: 23 September 2019 Abstract: In this study, we performed elemental analysis for ﬂoor dust samples collected in Jordanian microenvironments (dwellings and educational building). We performed intercorrelation and cluster analysis between the elemental, polyaromatic hydrocarbon (PAH), and microorganism concentrations. In general, the educational building workshops had the highest elemental contamination. The age of the dwelling and its occupancy played a role on the elemental contamination level: older and more occupied dwellingshad greater contamination. The elemental contamination at a dwelling entrance was observed to be higher than in the living room. We found exceptionally high concentrations for Fe and Mn in the educational workshop and additionally, Hg, Cr, and Pb concentrations exceeded the limits set by the Canadian Council of Ministers of the Environment. According to the cluster analysis, we found three major groups based on location and contamination. According to the enrichment factor (EF) assessment, Al, Co, Mn, Ti, and Ba had EF < 2 (i.e., minimal enrichment) whereas P, S, Pb, Sb, Mo, Zn, Hg, and Cu had EF > 40 (i.e., extremely enriched). In contrast, Ca and P were geogenically enriched. Furthermore, signiﬁcant Spearman correlations indicated nine subgroups of elemental contamination combined with PAHs and microbes. Keywords: ICP-OES; cluster analysis; Spearman correlation; dwellings; educational building International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Elemental Contamination in Indoor Floor Dust and Its Correlation with PAHs, Fungi, and Gram+/−Bacteria Sharif Arar 1,* , Afnan Al-Hunaiti 1, Mohanad H. Masad 2 , Androniki Maragkidou 3, Darren Wraith 4 and Tareq Hussein 5,6,* 1 Department of Chemistry, School of Science, University of Jordan, Amman 11942, Jordan; a.alhunaiti@ju.edu.jo 1 Department of Chemistry, School of Science, University of Jordan, Amman 11942, Jordan; a.alhunaiti@ju.edu.jo 2 Water, Environment and Arid Region Research Center (WEARRC), Al al-Bayt University, Al-Mafraq 25113, Jordan; mohanad@aabu.edu.jo q j 3 Finnish Meteorological Institute, Atmospheric Dispersion Modelling, P.O. Box 503, FI-00101 Helsinki, Finland; androniki.maragkidou@fmi.ﬁ 4 School of Public Health and Social Work, Queensland University of Technology, Queensland 4000, Australia; d.wraith@qut.edu.au 4 School of Public Health and Social Work, Queensland University of Technology, Queensland 4000, Australia; d.wraith@qut.edu.au 5 Department of Physics, The University of Jordan, Amman 11942, Jordan Department of Physics, The University of Jordan, Amman 11942, Jordan 6 Institute for Atmospheric and Earth System Research (INAR), University of Helsinki, PL 64, FI-00014 Helsinki UHEL, Finland * Correspondence: s.arar@ju.edu.jo (S.A.); tareq.hussein@helsinki.ﬁ(T.H.) www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2019, 16, 3552; doi:10.3390/ijerph16193552 1. Introduction Pollution has been a general and common problem for a long time, aﬀecting both indoor and outdoor environments [1]. Indoor pollution has been a focus of research for many years as people spend most of their time (more than 80%) indoors (e.g., dwellings, workplaces, oﬃces, schools, etc.) [2–5]. Indoor ﬂoor dust has been reported in many studies as it acts a sink for various airborne pollutants including chemical pollution (e.g., heavy metals, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and a vast range of organic compounds) and biological contamination (e.g., microorganisms, fungi, bacteria, viruses, insects and their dry parts, dustmites, and cells from humans, plants, and animals). These mixtures of pollutants can come from common or diﬀerent sources but actually metabolize and act synchronically with each other in vivo and in vitro [6]. Int. J. Environ. Res. Public Health 2019, 16, 3552; doi:10.3390/ijerph16193552 2 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 Indoor airborne pollutants might originate from either indoor or outdoor sources. In general, indoor sources of air pollution are closely linked to occupants’ activities and the use of household electronic devices, chemical products, building materials, and combustion processes (solid, liquid, and biomass fuels, etc.) [7–15]. Regardless of their source origin, they settle and accumulate in ﬂoor dust indoors. , ) [ ] g g , y Heavy metals are non-degradable, toxic, mutagenicity, and carcinogenicity. As a common type of indoor ﬂoor dust pollution, they have adverse health eﬀects such as damage to the nervous system, cardiovascular deaths, slow growth development, and asthma [9,16–19]. Exposure to heavy metals can occur via three main pathways: dermal, inhalation, and ingestion [20,21]. Hand to mouth ingestion is a major pathway for small children and crawling infants. Therefore, assessment of heavy metal contamination in indoor ﬂoor dust is an important topic. However, risk assessment studies focused on a single type of pollution (e.g., organic, inorganic, or biological) and few studies presented combine investigations for heavy metals with other types of pollution and their sources and health risk assessments at the same time [22]. For instance, heavy metals in indoor ﬂoor dusts are considered an important indicator of urban microenvironments because they are linked to urban activities (e.g., tailpipe and none-tailpipe emissions as well as industrial and agricultural activities) and inhabitants activities besides degradation and use of furniture, appliances, building materials [13,23–33]. 2.2.1. Elemental Analysis The elemental analysis was performed by applying inductively coupled plasma optical emission spectroscopy (ICP-OES). We analyzed nineteen elements: P, S, Al, Fe, Pb, Ca, Ba, Cr, Sb, Mo, Ti, Zn, Cd, Hg, Ni, Mn, Cu. Co, and Sr. The dust samples were ﬁrst digested before applying the elemental analysis. The used glassware were soaked overnight with 10% nitic acid then rinsed with distilled water followed by ultra-high-quality deionized water. Floor dust samples were dried overnight in an oven at 105 ◦C. We transferred 0.2–0.25 g of each dust sample (weighed to the nearest ﬁfth digest with a Mettler Toledo 5-digit analytical balance AB 135S) into a glass (Duran) beaker for aqua regia digestion in an open system [45]. The digestion was made with 8 ml of 65% nitric acid (HNO3, analar, Fluka, Switzerland) and 3 mL of 35%–37% hydrochloric acid (HCl analar, Fluka, Reinach, Switzerland). The digested sampled in beakers were covered with watch glasses and heated to boiling (120–160 ◦C) for four hours. The watch glasses were then removed to allow solvent evaporation until the samples were dry. Then another 3 mL of 65% nitric acid was added and left to evaporate before reaching digested sample volume of about 0.5 mL. The solutions were diluted to 5 mL with ultra-high-quality deionized water (conductivity around 0.05501 µS/cm), then ﬁltered with What man ﬁlter paper no 42 (0.45 µm) into 25 mL volumetric ﬂask and tipped to the mark with ultra-high quality deionized water (Milli-Q puriﬁcation system). Elements determination with the ICP-OES was carried out in triplicate with 10% of the samples prepared in duplicate. A PerkinElmer instrument model optima 2000 DV was used operating with a Meinhard type c nebulizer and quartz torch. The following operating conditions were used: viewing height of 15 mm, replicate read time of 20 s per replicate, plasma gas ﬂow of 15 L/min, auxiliary gas ﬂow of 0.2 mL/min, sample aspiration rate of 0.8 mL/min, and pump ﬂow rate of 1.5 L/min. The detection wavelengths for each element are listed in Table S3 and the calibration procedure and lower detection limit [46] are described in the supplementary material (see also Table S4). All measured elements showed high linearity with coeﬃcients of determination (R2) ranging from 0.9706 for Al to 0.9997 for Sr. The method detection limits ranged from 0.004 µg/g for Sr to 122.88 µg/g for S as indicated in detail in Table S4. 1. Introduction While many reports and studies about trace elements and heavy metals in indoor ﬂoor dust (e.g., educational buildings and residential areas) can be found in the literature [28,34–36], there are few reports in Jordanian microenvironments. For example, Al-Momani and Shatnawi [37] reported heavy metals concentrations in selected household dust in Jordan. Jardat et al. [38] reported the inorganic constituents of ﬂoor dust inside oﬃces in an industrial area in Jordan. Al-Mdanatet al. [39] reported concentrations of heavy metals in indoor dust in Karak city, Jordan. Some of these studies were focused on the determination of a heavy metals content, health risk assessment, source origin, and spatial distribution employing multivariate analysis, enrichment factors, and contamination degree [40,41]. Organic pollutants may originate from open ﬁres, unsafe combustion of biomass fuels including coal, charcoal and kerosene, tobacco smoking, gas stoves, and wood burning units. They are major sources of carbon monoxide and polyromantic hydrocarbons (PAHs). In addition, solid waste management and chemical spills could produce polychlorinated dibenzo-dioxins/furans (PCDDS/Fs) and polychlorinated biphenyls (PCBs). In addition to residual pesticides, such as pyrethroides and diethyltoluamide from spraying farms, homes, and schools. Volatile organic compounds (VOCs) are released from diﬀerent sources including paints, glues, resins, polishing material, cleaning agents and household products. Microorganisms belong to biological pollutants that include animal dander, dust mites (fungi and bacteria), molds (Penicillium/Aspergillus), infectious and allergic agents (cat dander), and pollen. These organisms originate from indoor or outdoor environments. They proliferate in dirt, water damaged surfaces, air conditions, humidiﬁers, carpets, and furniture [6,7,11]. Recently, we also performed simple statistical correlations between the microbes and PAHs in selected indoor environments in Amman, Jordan [42]. In this study we extended the analysis to quantify elemental contamination in indoor ﬂoor dust In this study, we extended the analysis to quantify elemental contamination in indoor ﬂoor dust collected inside selected microenvironments (dwellings and an educational building) in Amman, Jordan. Dwellings and an educational building were studied due to the expected high concentrations of particulate matter, bio-aerosols, chemical pollutants based on area expansion and anthropogenic activities. We also investigated the relationship between heavy metals, microbes, and PAHs based on multivariate and cluster analysis. Understanding the correlations among pollutants could be an initial step for risk assessment of combined exposures to multiple associated chemicals of indoor ﬂoor dust contamination. According to the best of our knowledge, such analysis has not been done elsewhere. 3 of 15 Int. J. 1. Introduction Environ. Res. Public Health 2019, 16, 3552 2.1. Floor Dust Samples Floor dust samples were collected during 3–9 April 2015 from eight dwellings and an education building located in Amman, Jordan (Supplementary Material, Figures S1 and S2, and Tables S1 and S2). All these indoor microenvironments were naturally ventilated. The weather conditions during the sampling period are presented in Figure S3. Two samples were taken from each dwelling (living room and main entrance). To collect a reasonable amount of ﬂoor dust samples, the dwellings were not vacuum cleaned for 3–4 days before sample collection. The educational building was the Department of Physics (University of Jordan), where all samples were collected on the same day. Although smoking was prohibited inside the university buildings, sometimes this was violated. As described by Maragkidou et al. [43,44]. The ﬂoor dust samples were collected by using a regular vacuum cleaner equipped with vacuum dust bags (Allied Filter Fabrics Pty., Ltd., Berkeley Vale, Australia). The sample collection was for 3 min of vacuum cleaning. Immediately after dust collection, the dust bags were closed and put inside a zipped plastic bag. Each dust sample was then put in a glass vial, wrapped with aluminum foil and stored in the freezer (−20 ◦C) until the chemical and biological analysis was due. 2.2. Chemical and Biological Analysis 2.2.1. Elemental Analysis Recoveries ranged from 86.4% to 102% with an average recovery of 93%. 4 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 2.3. Enrichment Factor (EF) We sorted the elements sources as natural or anthropogenic (non-crustal)by using the enrichment factor (EF) Equation (1): EF = Cx/Cre f dust Cx/Cre f crust , (1) (1) where Cx is the concentration of the measured element and Cref is the concentration of the reference elements (here chosen to be Fe). The subscript dust refers todust samples whereas crust to crustal concentrations. Here ﬁve EF ranges can be assumed [47]: EF less than 2 as minimal enrichment, EF in the range 2–5 as moderate, EF in the range 5–20 as signiﬁcant, EF in the range 20–40 as high, and EF more than 40 as extremely high. 2.2.2. PAHs and DNA Analysis For the purpose of cluster analysis with other pollutants in the same samples, we recall the chemical and biological analysis of the same samples that was previously presented by Maragkidou et al. [43,44] and Al-Hunaiti et al. [42]. As mentioned, the main purpose of this manuscript was to focus on the heavy metals analysis and apply cluster analysis to group the chemical and biological contaminations against the indoor environments. As such, the PAHs and qPCR results (Tables S5–S8), which were previously discussed before by Maragkidou et al. [44] and Al-Hunaiti et al. [42], are not the main focus of this manuscript. As described by Maragkidou et al. [44], the previous chemical analysis consisted of polycyclic aromatic compounds (PAHs), which are described in the supplementary material (see also Tables S5 and S6); and was performed by applying gas chromatography mass spectrometry (GC-MS). As described by Al-Hunaiti et al. [42], the biological analysis was performed by applying quantitative PCR (qPCR) analyses and it included total fungal DNA, group of Aspergillus spp./Penicillium, fungi, and Grampositive and Gramnegative bacteria. 3.1. Elemental Concentrations and Enrichment Factor These concentrations were about 340 (Pb), 260 (Cr), and 80 (Hg) µg/g for Pb, Cr, and Hg, respectively, which exceeds the reported values in indoor dust reported in previous studies [13,16,29,37,38,49]. Here, the concentrations of Pb, Cr, and Hg exceeded the Canadian Council of Ministers of the Environment (CCME) limits [38,50]. The concentration of Hg was also high (about 130 µg/g) in a lecture room opposite to the ﬁrst-year educational laboratories, where they use mercury in an educational experiment. Inside the educational building (Figure 1, Table S10), the highest contaminated areaswere the WSA (workshop main room) and Wsb (metals cutting and welding area). The lecture room in the ground ﬂoor was higher in elemental contamination compared to oﬃces on the ﬁrst and second ﬂoors. The BC (main hall) located in the ground ﬂoor was the least contaminated. The results of total elemental concentration for the indoor environments were contrary or inversely proportional in general to the PAHs concentration results to the same environments reported previously by Maragkidou et al. [43,44]. p p y y g The educational workshop area exhibited the highest concentrations of Fe (~72,900 µg/g and ~33,100 µg/g in two locations). This was mainly due to machinery and welding activities inside the workshop area. The Fe concentration reported here in this study exceeded what was previously reported by Jaradat et al. [38] in an industrial area (13,300 µg/g). The workshop area also had the highest total combined content of Zn (6400 µg/g), Cu (11,500 µg/g), and Mn (1300 µg/g) compared to other investigated areas in this study; these are also attributed to welding activities [29,30,49]. These concentrations exceeded what was previously reported in industrial oﬃces [13,16,29,37,38]. Welding and machinery activities in the workshop area also lead to the highest concentrations of Pb, Cr and Hg among all samples. These concentrations were about 340 (Pb), 260 (Cr), and 80 (Hg) µg/g for Pb, Cr, and Hg, respectively, which exceeds the reported values in indoor dust reported in previous studies [13,16,29,37,38,49]. Here, the concentrations of Pb, Cr, and Hg exceeded the Canadian Council of Ministers of the Environment (CCME) limits [38,50]. The concentration of Hg was also high (about 130 µg/g) in a lecture room opposite to the ﬁrst-year educational laboratories, where they use mercury in an educational experiment. All dwellings’ ﬂoor dust samples showed varying concentrations of Pb, and Cr. 2.4. Cluster Analysis Hierarchical clustering was applied by running Cluster 3.0 program and using the average linkage method and Pearson correlation (centered correlation) as a similarity measure in order to decide on the appropriate number of clusters for our work as well as to investigate the association among PAHs, heavy metals and bio-contaminants inside the Jordanian dwellings and the university areas. This program combines, arranges, and analyzes to show relationships between data elements. During the hierarchical analysis, all the data (genes and arrays) were mean centered in log-space and normalized. The visualization of clustered data was performed by tree diagrams-dendrograms generated with Java Tree View 1.1.6r4. Vertical analysis was based on dissimilarities among areas and groups. The horizontal analysis was based on ranking the indoor environments in similar groups based on contamination levels. In this analysis, all variables (diﬀerent types of contaminants) are considered jointly (rather than separately as in Spearman’s correlation). Spearman’s correlation was also estimatedfor correlated pollutants regardless to the indoor environments, where the following codes were used: PA (Penicillium/Aspergillus), G+ (gram positive bacteria), G-(gramnegative bacteria), F(fungi), PAH1(phenanthrene), PAH2 (anthracene), PAH3 (ﬂuoranthene), PAH4 (pyrene), PAH5 (Benzo[a]anthracene), PAH6 (chrysene), PAH7 (benzo[b]ﬂuoranthene), PAH8 (benzo[k]ﬂuoranthene), PAH9 (benzo[j]ﬂuornathene), PAH10 (benzo[a]pyrene), PAH11 (indeno[1,2,3-CD]pyrene), PAH12 (dibenz[a,h]anthracene), PAH13 (benzo[g,h.i]perylene). 5 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 3.1. Elemental Concentrations and Enrichment Factor The elemental concentrations of ﬂoor dust samples (described in detail in Figure S1 and S2, Tables S1 and S2) are listed in Table S9 and shown in Figure 1. According to the mean value and regardless to the sample collection location, the highest elemental concentration was found for Ca (28,500 µg/g, range 700–67, 800 µg/g), P (9200 µg/g, as high as 19,200 µg/g), Fe (8400 µg/g, range 900–72,900 µg/g), Zn (4500 µg/g; as high as 8500 µg/g), S (2800 µg/g, range 700–7900 µg/g), Al (700 µg/g, range 30–5500 µg/g), and Cu (500 µg/g, as high as 11,500 µg/g). The mean concentration was in the range 100–500 µg/g for Ba, Ti, Mn, and Sr whereas for Cr, Pb, Ni, and Hg it was between 10–100 µg/g. The rest of the elements (Mo, Sb, Cd, and Co) had mean concentrations less than 10 µg/g. The percent relative standard deviations (RSDs) for all samples elemental replicate analysis were <20%, which is the acceptable limit for digestion methods for trace analysis [48]. p g y [ ] Inside the educational building (Figure 1, Table S10), the highest contaminated areaswere the WSA (workshop main room) and Wsb (metals cutting and welding area). The lecture room in the ground ﬂoor was higher in elemental contamination compared to oﬃces on the ﬁrst and second ﬂoors. The BC (main hall) located in the ground ﬂoor was the least contaminated. The results of total elemental concentration for the indoor environments were contrary or inversely proportional in general to the PAHs concentration results to the same environments reported previously by Maragkidou et al. [43,44]. The educational workshop area exhibited the highest concentrations of Fe (~72,900 µg/g and ~33,100 µg/g in two locations). This was mainly due to machinery and welding activities inside the workshop area. The Fe concentration reported here in this study exceeded what was previously reported by Jaradat et al. [38] in an industrial area (13,300 µg/g). The workshop area also had the highest total combined content of Zn (6400 µg/g), Cu (11,500 µg/g), and Mn (1300 µg/g) compared to other investigated areas in this study; these are also attributed to welding activities [29,30,49]. These concentrations exceeded what was previously reported in industrial oﬃces [13,16,29,37,38]. Welding and machinery activities in the workshop area also lead to the highest concentrations of Pb, Cr and Hg among all samples. 3.1. Elemental Concentrations and Enrichment Factor Dwellings samples also indicated the presence of P and S elements in appreciable concentrations where the average ratio of P:S was 4:1 indicating crustal sources enriched with anthropogenic sources such as high temperature process (e.g., metal smelting, oil combustion and vehicular emissions) [51]. The comparison of elemental concentrations inside dwellings (entrance vs. living room) are demonstrated in Figure 1 (see also Table S10). In general, elemental concentrations at the entrance were higher than that was in the living rooms for all dwellings except for dwellings H and A2. For dwellings DH2, DH3, A3, A4, and DH5, the total elemental concentration ratio between living room and entrance area was 0.9, 0.5, 0.6, 0.8, and 0.6; respectively. As for A2 and H, ratio was 3.1 and 1.7; respectively. This ratio provides an indication about source origin (as indoor or outdoor) for the elemental contamination. Non-crustal elements with EF < 2 are relocated by dust ﬁne particles as reported by Al-Momani and Shatnawi [37]; this conﬁrms that contamination in living room for dwellings A2 and H was an indoor activity. Int. J. Environ. Res. Public Health 2019, 16, 3552 Int. J. Environ. Res. Public Health 2019, 16, x 6 of 15 6 of 15 Figure 1 Elemental concentrations in the indoor floor dust 0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000 Entrance Living Room Entrance Living Room Entrance Living Room Entrance Living Room Entrance Living Room Entrance Living Room Entrance Living Room Entrance Living Room Corridor Office P200 Office P209 Office P300 Office P303 Lecture Room L102 Lecture Room L230 Workshop Office Workshop welding DH2 A2 DH3 DH4 A3 A4 DH5 H Dwellings Educational Builing Concentration [µg/g] P S Zn Ti Al Ba Mn Sr Cr Cu Pb Ni Mo Sb Cd Hg Co Fe Ca Figure 1. Elemental concentrations in the indoor ﬂoor dust. Fi 1 El t l t ti i th i d fl d t Dwellings Educationa Figure 1. Elemental concentrations in the indoor ﬂoor dust. g The EF values are shown in Figure 2 (see also Table S11). The elements Ba, Ti, Co, and Al had minimal enrichment EF (mean value <2) whereas Sr and Mn had moderate (mean value in the range 2–5).Elements (Al, Ti, Ba) coming from Khamaseen dust conssit partially of aluminum silicate minerals, and quartz; these elements reflect the natural lithological and mineralogical composition of the dust related material and source area [52]. 3.1. Elemental Concentrations and Enrichment Factor Calcium is widely distributed in other minerals such as feldspar, amphibole and pyroxene, and is often associated with clay minerals such as illite, chlorite and Ca-montmorillonite. Ca is geogenically (naturally) enriched element as calcite which dominates the limestone deposition in the North African and Eastern Mediterranean dust (EF = 10) [52]. In general, the entrance area (EF in the range 5–14) of dwellings showed Ca concentrations higher than that found in the living rooms area. The corridor, lecture rooms, and offices in the educational building also showed high Ca concentration (EF in the range 7–16). EF values up to 10 are considered coming from the geogenic enrichment and more than 10 involved contribution from anthropogenic sources like cement factories, fertilizers especially that there was a construction site beside the educational building [52]. As for Cr, which is a toxic element with many adverse effects to humans [19,20]; it showed higher concentrations (EF = 10–28) in the living rooms than that in the entrances of the dwellings. This element is typically a by-product of fossil fuel combustion inside dwelling and from wood preservatives, chrome pigments (e.g., lead chromate) used in paints, printing inks, and The EF values are shown in Figure 2 (see also Table S11). The elements Ba, Ti, Co, and Al had minimal enrichment EF (mean value <2) whereas Sr and Mn had moderate (mean value in the range 2–5). Elements (Al, Ti, Ba) coming from Khamaseen dust conssit partially of aluminum silicate minerals, and quartz; these elements reﬂect the natural lithological and mineralogical composition of the dust related material and source area [52]. Calcium is widely distributed in other minerals such as feldspar, amphibole and pyroxene, and is often associated with clay minerals such as illite, chlorite and Ca-montmorillonite. Ca is geogenically (naturally) enriched element as calcite which dominates the limestone deposition in the North African and Eastern Mediterranean dust (EF = 10) [52]. In general, the entrance area (EF in the range 5–14) of dwellings showed Ca concentrations higher than that found in the living rooms area. The corridor, lecture rooms, and oﬃces in the educational building also showed high Ca concentration (EF in the range 7–16). EF values up to 10 are considered coming from the geogenic enrichment and more than 10 involved contribution from anthropogenic sources like cement factories, fertilizers especially that there was a construction site beside the educational building [52]. 3.1. Elemental Concentrations and Enrichment Factor As for Cr, which is a toxic element with many adverse eﬀects to humans [19,20]; it showed higher concentrations (EF = 10–28) in the living rooms than that in the entrances of the dwellings. This element is typically a by-product of fossil fuel combustion inside dwelling and from wood preservatives, chrome pigments (e.g., lead chromate) used in paints, printing inks, and anti-corrosive materials [26,27]. anti-corrosive materials [26,27]. Mean EF >40 (i.e., extremely high) was obtained for Hg, Sb, Cd, Zn, S, P, Mo, and Pb with mean values of EF = 1190, 364, 284, 248, 156, 145, 82, and 51; respectively. High EF indicates the presence of anthropogenic sources such as burning fossil fuels, power plants, metallurgy industry, maintenance equipment, workshops, chemical factories, pains and pigments [13,14,26] whereas EF<5 indicates Saharan dust which contains minor amounts of phosphorite [52] Mean EF > 40 (i.e., extremely high) was obtained for Hg, Sb, Cd, Zn, S, P, Mo, and Pb with mean values of EF = 1190, 364, 284, 248, 156, 145, 82, and 51; respectively. High EF indicates the presence of anthropogenic sources such as burning fossil fuels, power plants, metallurgy industry, maintenance equipment, workshops, chemical factories, pains and pigments [13,14,26] whereas EF < 5 indicates Saharan dust, which contains minor amounts of phosphorite [52]. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants 3.2. Cluster analysis and Intercorrelation of Pollutants We examined Spearman’s correlation coefficients at insignificant estimates with p-value threshold of 0.10 among three types of pollutants concentrations: elemental, biological (fungi and bacteria), and PAHs. According to the cluster analysis, we identified three groups based onmicro environment and total contamination (Figure 3). The first group(Group 1) included the workshop area at the educational building, entrance areas of dwellings H and A4, and living rooms areas of d lli H A4 A2 d DH2 Th d (G 2) i l d d h d i l b ildi We examined Spearman’s correlation coeﬃcients at insigniﬁcant estimates with p-value threshold of 0.10 among three types of pollutants concentrations: elemental, biological (fungi and bacteria), and PAHs. According to the cluster analysis, we identiﬁed three groups based onmicro environment and total contamination (Figure 3). The ﬁrst group (Group 1) included the workshop area at the educational building, entrance areas of dwellings H and A4, and living rooms areas of dwellings H, A4, A2, and DH2. The second group (Group 2) included the educational building premises and the entrance areas Int. J. Environ. Res. Public Health 2019, 16, 3552 J , , i d th t f d premises and the entrance areas of dw 7 of 15 ed the ded the Environ. Res. Public Health 2019, 16, 3552 7 wellings DH3 and DH5. The third group(Group 3) included the entrance areas of dwellings D DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. mises and the entrance areas of dwellings DH3 and DH5. The third group(Group 3) included ance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings DH , and A3. Figure 2.Enrichment factor for each indoor environment. Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Entrance Entrance Entrance Entrance Entrance Entrance Entrance Entrance Corridor Office P209 Office P303 Lecture Room L230 Workshop welding DH2 A2 DH3 DH4 A3 A4 DH5 H Dwellings Educational Builing EF value Hg Sr Co Cu Mn Ni Cd Zn Ti Mo Sb Cr Ba ca Pb Al S P Figure 2. Enrichment factor for each indoor environment. mises and the entrance areas of dwellings DH3 and DH5. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants The third group(Group 3) included ance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings D 4, and A3. Figure 2.Enrichment factor for each indoor environment. Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Entrance Entrance Entrance Entrance Entrance Entrance Entrance Entrance Corridor Office P209 Office P303 Lecture Room L230 Workshop welding DH2 A2 DH3 DH4 A3 A4 DH5 H Dwellings Educational Builing EF value Hg Sr Co Cu Mn Ni Cd Zn Ti Mo Sb Cr Ba ca Pb Al S P Figure 3. Hierarchical clustering (dendrogram) showing three major clusters. of dwellings DH3 and DH5. The third group(Group 3) included the entrance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. entrance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. g , , , g g , DH4, and A3. of dwellings DH3 and DH5. The third group(Group 3) included the entrance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. entrance areas of dwellings DH2, A2, DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. g g g DH4, and A3. A2, DH4, and A3 as well as the living room areas of dwellings DH3, DH4, and A3. Fi 2 E i h t f t f h i d i t 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Entrance Entrance Entrance Entrance Entrance Entrance Entrance Entrance Corridor Office P209 Office P303 Lecture Room L230 Workshop welding DH2 A2 DH3 DH4 A3 A4 DH5 H Dwellings Educational Builing EF value Hg Sr Co Cu Mn Ni Cd Zn Ti Mo Sb Cr Ba ca Pb Al S P Figure 2. Enrichment factor for each indoor environment. Figure 2.Enrichment factor for each indoor environment. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants K-means clustering values of the three clusters with the diﬀerent pollutants. Figure 4. K-means clustering values of the three clusters with the diﬀerent pollutants. Figure 4.K-means clustering values of the three clusters with the different pollutants. As shown in Figure 4, bacteria (gram positive and negative), Penicillinum/Aspergillus, and fungi are strongly associated with P, S, Fe, and Ca and to less extent with PAH1 (phenanthrene) for Groups 1 and 2 as demonstrated by the K-means clustering values. The major source of sulfurin the ambient environment is the combustion of sulfur-containing fossil fuels such as coal and crude petroleum. In addition, P and S are extremely enriched with values exceeding the 100 indicating the involvement of anthropogenic activity (e.g., combustion and vehicular emissions and fine particulate matter [51]) related to the area or locations in Group 1 and Group 2. As for Group 3, it showed bacteria(gram positive and negative), Penicillinum/Aspergillus, and fungi group strongly with Fe, Ca, Ti, Zn, Mn, Cu, and to a lower extent PAH4 (pyrene), PAH3 (fluoranthene), and PAH1 (phenanthrene) based on their K-means clustering values. The presence of extremely enriched Cu and high concentrations of Fe is a strong indication for anthropogenic activities. The low K-means clustering valuesof P and S in Group 3 compared to Groups 1 and 2 could be due to different sources and role of biological contamination [43,51]. The Spearman correlations are presented in Figure 5 (see also Figure 6). PAH1 (phenanthrene), hi h i f f il b i d l ki i i i l l d i h i i The Spearman correlations are presented in Figure 5 (see also Figure 6). PAH1 (phenanthrene), which is from fossil burning and natural gas cooking, is positively correlated with gram positive bacteria (unable to metabolize the phenanthrene angular structure [42]) and negatively with the extremely enriched Cu and Zn, which could be coming from diﬀerent anthropogenic sources (e.g.,·paints, cosmetics [27–29,43]). Gram positive bacteria correlates positively with P (correlation factor ~0.38 and p = 0.09), which could be explained may be by the presence of some gram positive bacterium strains that are reported to be responsible for phosphate solubilization and uptake like Bacillus spp. and Actino bacteria respectively, and are considered to be strong competitors in relation to other organisms [53,54]. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 Entrance Entrance Entrance Entrance Entrance Entrance Entrance Entrance Corridor Office P209 Office P303 Lecture Room L230 Workshop welding DH2 A2 DH3 DH4 A3 A4 DH5 H Dwellings Educational Builing EF value Hg Sr Co Cu Mn Ni Cd Zn Ti Mo Sb Cr Ba ca Pb Al S P Figure 2.Enrichment factor for each indoor environment. Figure 2. Enrichment factor for each indoor environment. Figure 2.Enrichment factor for each indoor environment. igu e Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. Figure 3. Hierarchical clustering (dendrogram) showing three major clusters. Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. Figure 3. Hierarchical clustering (dendrogram) showing three major clusters. Figure 3 Hierarchical clustering (dendrogram) showing three major clusters Figure 3.Hierarchical clustering (dendrogram) showing three major clusters. Figure 3. Hierarchical clustering (dendrogram) showing three major clusters. 8 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 As shown in Figure 4, bacteria (gram positive and negative), Penicillinum/Aspergillus, and fungi are strongly associated with P, S, Fe, and Ca and to less extent with PAH1 (phenanthrene) for Groups 1 and 2 as demonstrated by the K-means clustering values. The major source of sulfurin the ambient environment is the combustion of sulfur-containing fossil fuels such as coal and crude petroleum. In addition, P and S are extremely enriched with values exceeding the 100 indicating the involvement of anthropogenic activity (e.g., combustion and vehicular emissions and ﬁne particulate matter [51]) related to the area or locations in Group 1 and Group 2. As for Group 3, it showed bacteria (gram positive and negative), Penicillinum/Aspergillus, and fungi group strongly with Fe, Ca, Ti, Zn, Mn, Cu, and to a lower extent PAH4 (pyrene), PAH3 (ﬂuoranthene), and PAH1 (phenanthrene) based on their K-means clustering values. The presence of extremely enriched Cu and high concentrations of Fe is a strong indication for anthropogenic activities. The low K-means clustering valuesof P and S in Group 3 compared to Groups 1 and 2 could be due to diﬀerent sources and role of biological contamination [43,51]. Int. J. Environ. Res. Public Health 2019, 16, x 8 of 15 0 1 2 3 4 5 6 K-means clustring values Group 1 Group 2 Group 3 Figure 4. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants Gram positive bacteria is negatively with Fe (correlation factor −0.48 and p = 0.02) and Zn (correlation factor −0.50 and p = 0.02), where these elements are consumed for essential and normal functioning of living organisms, extracellular and intracellular functions in addition to bio-sorption ability. Most microorganisms have surface antigens such as proteins, polysaccharides, teichoic acids, O-chain lipid oligo, and polysaccharides that have strong aﬃnity and selectivity to bind or complex with a certain metal cation [55,56]. In addition, almost all species of bacteria originated from metal-rich environments, where bacteria uses metal cofactors to facilitate key cellular processes, such as production of energy and replication [57]. However, these elements could become toxic and lethal for microorganisms at high concentrations because of forming radical compounds in the cell of the microorganism [55–57], as noticed from the negative correlation. which is from fossil burning and natural gas cooking, is positively correlated with gram positive bacteria (unable to metabolize the phenanthrene angular structure [42]) and negatively with the extremely enriched Cu and Zn, which could be coming from different anthropogenic sources (e.g., i t ti [27 29 43]) G iti b t i l t iti l ith P ( l ti f t g PAH2 (Anthracene) showed negative correlations with Ni and Cu. This could be due to the fact that both Ni (signiﬁcantly enriched) and Cu (extremely and highly enriched) come from diﬀerent anthropogenic sources, such as tires, oil and gasoline additives, and metal parts in addition to certain 9 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 types of pigments whereas anthracene could be coming from wood preservatives and diluents for pigments and colors [27,29,32,43]. This correlation could also indicate that Penicillinum/Aspergillus and other strains of fungi show high tolerance for toxic heavy metals, such as Ni and Cu [57,58]. PAH3 (ﬂuoranthene), PAH4 (pyrene), and PAH6 (chrysene) are positively associated with gramnegative bacteria and Ca, while PAH7 (benzo[b]ﬂuoranthene) is positively correlated with Gram negative bacteria and Ca and negatively associated with Gram positive bacteria and Penicillinum/Aspergillus. G+ bacteria is able to metabolite these PAHs in contrast to G- bacteria. The presence of strong association with Ca. PAH9 (benzo[j]ﬂuornathene) was moderately associated with Ca, and Ti. PAH10 (benzo[a] pyrene) had a moderate positive association with gramnegative bacteria, Pb, and Ti. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants PAH11 (Indeno[1,2,3-CD]pyrene) was associated with Pb, which could be related to common anthropogenic sources, such as heating with kerosene, charcoal, and oil, in addition to using petroleum chemicals [43]. PAH13 (benzo[g,h.i]perylene) was associated with Pb, Ca, and Ti and negatively correlated with gram positive bacteria. In general, many factors inﬂuence the association between microorganism strain type and its ability to metabolite certain structure of PAHs depending on its active structural regions (bay region, bay-like region, M-region, and K-region) that end with Diol-epoxide active metabolic intermediates. In addition, the associationis dependent on the heavy metal anthropogenic common source and the microorganism tolerance or consumption for these heavy metals or elements [56,59,60]. Furthermore, the major metal-metal associations and minor PAH–metal or metal–bioaerosol associations indicates that the three classes of pollutants correlating in Figure 5 exhibit in general diﬀerent emission resources, accumulation and metabolism pathways. As also shown in Figure 5, Cu correlated with Zn (correlation coeﬃcient 0.73 and p < 0.001), Ni (correlation factor 0.55 and p = 0.01), and Mn (correlation factor 0.75 and p < 0.001). In general, Cu and Zn are grouped as extremely enriched elements that can be related to wear parts of alloys, where Cu is used in diﬀerent machinery parts and in brass automotive radiators whereas Zn is used in diﬀerent parts of alloys and vehicles and as antioxidant and lubricant in oils. Also, Pb is associated positively with Zn and Cu; this indicates that these may be coming from the same sources [29,31,41]. Other metal–metal associations are indicated in Figure 5, where the signiﬁcant strong correlations of metals are supported by an earlier section (Section 3.1) by enrichment factor grouping levels. In addition, we used skewness values for pollutants which provide information about the symmetric distribution of pollutants including metals, where the degree of deviation from symmetric distribution involves the contribution of anthropogenic activities [24,32,41,49] and supports data from enrichment factors and Spearman correlation data and grouping of pollutants explained in the text. Where skewness value less than −1 and greater than 1 is considered highly skewed, while values (−1 to −0.5 and 1 to 0.5) are considered to be moderately skewed, and values (−0.5 to 0.5) is considered symmetrically distributed (no anthropogenic involvement). Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants After Investigation the relationship between pollutants regardless of the indoor environment for example: PA and F had skew values 3.6 and 4 (correlation 0.467), whereas PAH1, PAH3, and PAH4 have skew values 3.8,4.2, and 4.3 respectively which make them act as one group as in Figure 5 coming from the same source (correlation factors higher than 0.79. The same is applied for PAH11 and PAH13 with skew values 1.2, and 1.3 respectively and correlation coeﬃcient of 0.9344. Zn and Cu skew values where 1.4 and 1.6 respectively (correlation 0.727) as in Figure 5 and for Mn and Cu 2.6 and 1.6 respectively (correlation 0.75). For example (PAH1, G+, Zn, Cu) they had skew values 4, 2.2, 1.4, and 1.6 respectively indicating high involvement of anthropogenic activity where average EF values were 248 and 31 respectively (signiﬁcantly and extremely enriched) the four components had correlation around 0.4. These skew values for some extent were supportive for the data explanation mentioned above. 10 of 15 10 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 Int. J. Environ. Res. Public Health 2019, 16, x Figure 5.Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coefficient). Crosses refer to insignificant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coefficient (x-axis scale). Figure 5. Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coeﬃcient). Crosses refer to insigniﬁcant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insigniﬁcant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coeﬃcient (x-axis scale). Figure 5.Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coefficient). Crosses refer to insignificant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coefficient (x-axis scale). Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants e 5.Plot of the correlation (Spearman) estimates for the variables analyzed (the size of th spond to the absolute size of the correlation coefficient). Crosses refer to insignificant e l th h ld f 0 10 B i l i di t ti l ti bl i l i di t Figure 5.Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coefficient). Crosses refer to insignificant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coefficient (x axis scale) Figure 5. Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coeﬃcient). Crosses refer to insigniﬁcant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insigniﬁcant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coeﬃcient (x-axis scale). at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coefficient (x-axis scale). Figure 5.Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coefficient). Crosses refer to insignificant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coefficient (x axis scale) Figure 5. Plot of the correlation (Spearman) estimates for the variables analyzed (the size of the circles correspond to the absolute size of the correlation coeﬃcient). Crosses refer to insigniﬁcant estimates at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insigniﬁcant correlation is marked as (×). The intensity of color is an indication for the value of the correlation coeﬃcient (x-axis scale). at a p-value threshold of 0.10. Brown circles indicate negative correlation, blue circlesindicate positive correlation and insignificant correlation is marked as (×). 4. Conclusions In this study, we quantify elemental contaminations found in indoor ﬂoor dust samples that were collected inside some microenvironments (dwellings and an educational building) in Amman, Jordan. We also investigated the relationship between heavy metals, microbes, and PAHs based on multivariate and cluster analysis leading to the following major ﬁndings. The average concentration of elements in µg/g in samples varied form extremely high amounts of Ca (~29,000 µg/g), and Fe (8400 µg/g) to moderate amounts of Ni (40 µg/g), and Cr (~90 µg/g), and lower concentrations of Cd (~5 µg/g) and Co (~2 µg/g). In general, the educational building workshops (~49,400 µg/g) were the highest contaminated by elements followed by the living room for H dwelling (66,500 µg/g), While the lowest concentration in indoor environments was the main entrance area for A2 dwelling (2nd ﬂoor apartment) located in the north eastern part of the capital Amman. In addition, for dwellings, the elemental contamination in the entrance was found to be greater than the living room for all dwellings except for dwelling H (entrance < living room) and dwelling A2 (entrance < living room). Pollution assessment by mean values of EF of these elements indicated which elements are extremely enriched and which elements are with minimum enrichment as: Hg (~1110), Sb (~350), Cd (~300), Zn (~250), S (~160), P (~150), Mo (~80), Pb (~50), Cu (~30), Cr (~15), Ni (~7), Ca (~7), Sr (~4), Mn (~2), Ba (~1), Ti (~1), Co (~1), Al (~ 0).Where Al, Co, Mn, Ti, and Ba are considered with minimal enrichment <2.Sr is considered moderate enrichment whereas Ca, Ni, Cr are considered to be signiﬁcantly enriched. The other elements P, S, Pb, Sb, Mo, Zn, Hg, and Cu are considered to be extremely highly enriched in general. In contrast, Ca, and P were geogenically enriched. Investigation of the relationship between pollutants (regardless of the indoor environment) resulted in a potential grouping based on sources for pairs or a variety of pollutants which would be of high probability of common source of emissions including (PA and F), (phenanthrene, Anthracene, and ﬂuoranthene), (Indeno[1,2,3-CD]pyrene and benzo[g,h.i]perylene), (Zn and Cu), and (Mn and Ni). This could be very useful in health risk assessment based on combined exposures to multiple associated pollutants. The Spearman correlation analysis was at 0.1 signiﬁcance level and was conﬁrmed by skew values for each pollutant indicated major signiﬁcant correlations for PAH–PAH, metal–metal, microorganism–microorganism. Saharan dust, which contains minor amounts of ph 3 2 Cl t l i d I t l ti f P ll t t 3.2. Cluster Analysis and Intercorrelation of Pollutants The intensity of color is an indication for the value of the correlation coefficient (x-axis scale). ( ) -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 Pollutant correlation facor value S Pb Ca Ti Zn Ni Cu Gram+ bacteria Gram- Bacteria Figure 6.Bar presentation of the obtained correlation data of PAHs with gram positive bacteria, gramnegative bacteria, Penicillinum/Aspergillus, fungi, and elements. -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 Pollutant correlation facor value S Pb Ca Ti Zn Ni Cu Gram+ bacteria Gram- Bacteria Figure 6. Bar presentation of the obtained correlation data of PAHs with gram positive bacteria, gramnegative bacteria, Penicillinum/Aspergillus, fungi, and elements. Figure 6.Bar presentation of the obtained correlation data of PAHs with gram positive bacteria, gramnegative bacteria, Penicillinum/Aspergillus, fungi, and elements. Figure 6. Bar presentation of the obtained correlation data of PAHs with gram positive bacteria, gramnegative bacteria, Penicillinum/Aspergillus, fungi, and elements. Figure 6.Bar presentation of the obtained correlation data of PAHs with gram positive bacteria, gramnegative bacteria, Penicillinum/Aspergillus, fungi, and elements. In general, we concluded from Pearson correlation factors for individual pollutants and skew values that the higher skew value, the higher enrichment factor for an element. In addition, the more the matching of skew values among diﬀerent pollutants the more there will be the correlation to make the same group. 11 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 So in addition to PAH–PAH correlations, each PAH is correlated with metals and microorganisms to form its unique group (PAH–element–microorganism) as described in Figure 6 as in groups regardless if the correlation is positive or negative: (PAH1,G+,Zn, Cu), (PAH2, PA,Ni,Cu), (PAH3,Ca,G-), (PAH6,G-,Ca), (PAH7,G+,G-,PA,Ca), (PAH9,G+,Ca,Ti),(PAH10,G-,Ti,Pb), (PAH11,Pb), (PAH13,G+,G-,Ti, Pb,Ca). 4. Conclusions Furthermore, the important major ﬁnding in this study reveals a signiﬁcant number of tri-component correlations related to PAH–metal–microorganism including:(phenanthrene, G+, Zn, Cu), (Anthracene, PA, Ni, Cu), (ﬂuoranthene, Ca, G-), (chrysene, G-, Ca), (benzo[b]ﬂuoranthene, G+, G-, PA, Ca), (benzo[j]ﬂuornathene, G+, Ca, Ti), (benzo[a]pyrene, G-, Ti, Pb), (Indeno[1,2,3-CD]pyrene, Pb), (benzo[g,h.i]perylene, G+, G-, Ti, Pb, Ca). Many factors inﬂuence the association between microorganism strain type and its ability to metabolite certain structure of PAHs depending on its active structural regions (bay region, bay-like region, M-region, and K-region) that end with Diol-epoxide active metabolic intermediates. In addition, an association is dependent on heavy metal anthropogenic common source and the microorganism tolerance or consumption for these heavy metals or elements. Given the complexity of the data analysed, the major metal-metal associations and minor PAH–metal or metal–bioaerosol associations indicate that the three classes of pollutants exhibit in general diﬀerent emission resources, accumulation and metabolism pathways. 12 of 15 12 of 15 Int. J. Environ. Res. Public Health 2019, 16, 3552 Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/16/19/3552/s1, Figure S1: A map of Amman with site locations of the dwellings marked with yellow landmarks (abbreviations as listed in Table S1) and the campus of the University of Jordan (marked with red, see also Figure S2 and Table S2); Figure S2: A map of the University of Jordan campus and a schematic chart of the Department of Physics with indications for the rooms from where ﬂoor dust samples were collected; Table S1: Features of the dwellings and ﬂoor, where the dust samples were collected; Table S2: A summary about the rooms and ﬂoor surfaces from which the dust samples were collected at the Department of Physics, the University of Jordan. Samples were collected on April 29, 2015. 4. Conclusions The bare ﬂoor tiles used in the building are cement ﬁlled with small marble stones; Figure S3:Ambient temperature, relative humidity, and accumulated rain (since April 1st); Table S3: ICP-OES detection wavelengths (nm); Table S4: Calibration curve equation for measured elements; Table S5: Polycyclic aromatic hydrocarbons (PAHs [ng/g]) concentrations in the dust samples collected from the dwellings (Maragkidou et al., 2016); Table S6: Polycyclic aromatic hydrocarbons (PAHs [ng/g]) concentrations in the dust samples collected from the educational building (Maragkidou et al., 2017); Table S7: Microbe concentration [cell equivalent/mg] based on the qPCR-DNA analysis of the dust samples collected from the dwellings; Table S8: Microbe concentration [cell equivalent/mg] based on the qPCR-DNA analysis of the dust samples collected from the educational building; Table S9: Elemental concentrations [µg/g] of the ﬂoor dust samples; Table S10: Total measured elemental concentration; Table S11: Average enrichment factor (EF) and range for measured elemental concentrations. Author Contributions: Conceptualization, T.H., S.A., and A.A.-H.; methodology, T.H., A.M., S.A., M.H.M. and D.W.; formal analysis, S.A., A.A.-H., M.H.M., and D.W.; investigation, T.H., S.A., A.A.-H., A.M., and D.W.; resources, T.H., S.A., and M.H.M.; writing—original draft preparation, S.A. and T.H.; writing—review and editing, all authors; visualization, T.H., S.A., A.A., and D.W.; supervision, T.H., A.A., and S.A.; project administration, T.H. and S.A.; funding acquisition, T.H., A.M., and S.A. Funding: This research was funded by the Deanship of Academic Research at the University of Jordan; European Commission (FP7-PEOPLE-2012-ITN) Marie Curie ITN (HEXACOMM, project no. 315760), and Academy of Finland Center of Excellence (grant no. 272041). This manuscript was written and completed during the sabbatical leave of the last author’s (Tareq Hussein) that was spent at the University of Helsinki and supported by the University of Jordan during 2019. Acknowledgments: Open access funding provided by University of Helsinki. The authors would like to thank Stuart Harrad and Yuning Ma (University of Birmingham, School of Geography, Earth & Environmental Sciences, Division of Environmental Health & Risk Management) for their support in the PAHs analysis. The authors also thanks Marten Täubel and Anne Hyvärinen (National Institute for Health and Welfare (THL), Department of Health Protection Living Environment and Health Unit, Kuopio, Finland) for their support in the qPCR analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest. References The use of calcium carbonate-enriched clay minerals and diammonium phosphate as novel immobilization agents for mercury remediation: Spectral investigations and ﬁeld applications. Sci. Total Environ. 2019, 646, 1615–1623. [CrossRef] 11. Bruce, N.; Perez-Padilla, R.; Albalak, R. Indoor air pollution in developing countries: A majorenviron and public health challenge. Bull. 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https://openalex.org/W4221083253	https://0277.pubpub.org/pub/omssyof5/download/pdf	German	null	Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung	null	2,022	cc-by	6,397	027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Deborah Keller Published on: Mar 14, 2022 DOI: https://doi.org/10.21428/1bfadeb6.3bc2e7e1 License: Creative Commons Attribution 4.0 International License (CC-BY 4.0) 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Published on: Mar 14, 2022 DOI: https://doi.org/10.21428/1bfadeb6.3bc2e7e1 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung ABSTRACT Die Kinderbuchsammlungen und Archive des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM stellen ein wichtiges kulturelles Erbe der Schweiz dar. Sie enthalten auch internationale und historische Bestände von Rang und werden von der Forschungsabteilung des Instituts und im Rahmen von Kooperationen mit Universitäten und Pädagogischen Hochschulen beforscht. Der Beitrag stellt insbesondere die historischen Sammlungen Hürlimann, Keckeis und Waldmann sowie das Johanna Spyri-Archiv mit ihren aktuellen Impulsen für die Forschung vor. The children’s book collections and archives of the Swiss Institute for Children’s and Youth Media SIKJM (keine englische Übersetzunng auf der Webseite) represent an important cultural heritage of Switzerland. They also contain international and historical holdings of high standing and are researched by the Institute’s research department and within the framework of cooperation with universities and colleges of education. The article highlights in particular the historical collections Hürlimann, Keckeis and Waldmann as well as the Johanna Spyri Archive with their current impulses for research. 2. Beziehungsreicher Kern der Sammlung Am besten erschlossen unter den historischen Sammlungen ist diejenige von Bettina Hürlimann (1909-1983). Geboren in Weimar als Tochter des Verleger-Ehepaars Gustav Kiepenheuer und Irmgard Kiepenheuer-Funke, kam Bettina Kiepenheuer in ihrem offenen Elternhaus mit bedeutenden Literaten und Künstlern der Zeit in persönlichen Kontakt – darunter, um nur wenige zu nennen, Joseph Roth, Bertold Brecht, Ernst Toller, Mies van der Rohe, Jean Renoir, Lotte Reiniger, Wilhelm Furtwängler oder Oskar Kokoschka. Nachdem sich ihre ersten Pläne, nämlich die Kunstakademie zu besuchen oder ein Architekturstudium aufzunehmen, zerschlagen hatten, machte Bettina Kiepenheuer zunächst eine Ausbildung als Typographin. So war sie mehrfach prädestiniert für die Laufbahn, die sie später einschlug. Mit Martin Hürlimann verheiratet, dem Atlantis- Verleger und Gründer der gleichnamigen Zeitschrift (später Du), wurde sie selbst auch Verlegerin, zuerst in Berlin, ab 1939 in die Heimat ihres Ehemanns exiliert, in Zürich. Als junge Mutter auf der Suche nach gehaltvollen Kinderbüchern für ihre eigenen vier Kinder entdeckte sie schliesslich ihre Leidenschaft für das Kinderbuch. Bis heute lebt der Name Atlantis als Kinderbuch-Verlagszweig, heute unter dem Dach des Kampa Verlags, fort. Die Kollektion, die Bettina Hürlimann im Laufe der Jahre aufbauen sollte, ist stark von dieser Herkunft und Biografie geprägt. Einen lebendigen Einblick in ihr umtriebiges Leben gewährt die (auch ins Englische übersetzte) Autobiographie Sieben Häuser (1976, S. 185), die weitgreifende Zusammenhänge erhellt und zugleich einen Teil europäischer Kunst-, Kultur- und Geistesgeschichte der ersten Hälfte des 20. Jahrhunderts einfängt. Verena Rutschmann, die von 1977 bis 2007 am Institut als Forscherin wirkte und wohl die profundeste Kennerin der SIKJM-Sammlungen ist, hat diese einmal unter dem Titel „Weltoffenheit und Eigenart“ vorgestellt (2007) und damit zwei Pole benannt, welche auch die Hürlimann-Sammlung treffend charakterisieren. In der Tat ist vielleicht die Weltoffenheit, ja die Weltläufigkeit derjenige Pol, den die Hürlimann-Sammlung am meisten verkörpert. Gerade aus heutiger Sicht, wenn (vermeintliche) Sprachbarrieren, aber auch die schiere Masse publizierter Bücher die Spezialisierungen immer weiter vorantreiben, ist es bemerkenswert, mit welcher Leichtigkeit, noch ganz ohne World Wide Web, Bettina Hürlimann ihre Antennen in alle vier Himmelsrichtungen ausstreckte, Trouvaillen und Trophäen mit sicherem Instinkt aufspürte und oft genug in ihre Sammlung heimführte. Sie reiste nach England und Frankreich, in die Niederlande und nach Skandinavien, nach Russland, in die USA und nach Japan. Ihr an der Typographie geschultes Auge, ihr eminentes Flair für das Grafische und die Illustration erkannte dort mit feinem Gespür und auf anhieb Qualitäten und Innovationen. 1. Einleitung Das Schweizerische Institut für Kinder- und Jugendmedien SIKJM ist einer Fachwelt von Lehr- und Bibliothekspersonen und einer breiteren Öffentlichkeit bekannt für seine regen Aktivitäten im Bereich der Literaturvermittlung und der literalen Förderung, die in einer sich rasch wandelnden Medienlandschaft orientierende Wirkung entfalten. Dass es daneben auch ein Hort einzigartiger historischer wie auch zeitgenössischer Bücherschätze darstellt, ist vielleicht etwas weniger bekannt: Die SIKJM-Bibliothek beherbergt mit über 70’000 erschlossenen Titeln eine hochkarätige Sammlung internationaler Kinder- und Jugendliteratur sowie einschlägiger Fachliteratur, und sie dokumentiert umfassend das Kinderbuchschaffen aller vier Sprachregionen der Schweiz. Im geschichtsträchtigen Jahr 1968 wurde der Vorläufer des SIKJM, das Schweizerische Jugendbuchinstitut (SJI), als Forschungs- und Dokumentationsstelle für Kinder- und Jugendliteratur von Franz Caspar unter dem Dach der Johanna Spyri-Stiftung gegründet. 2002 schloss es sich mit dem Schweizerischen Bund für Jugendliteratur (SBJ) zum heutigen Institut zusammen. Den Kern der historischen Bestände bilden Schenkungen privater Kinderbuchsammlungen an das Institut, die wichtigsten darunter von Bettina Hürlimann, Elisabeth Waldmann und Peter Keckeis, die nachfolgend näher vorgestellt werden. Darüber hinaus erhält das SIKJM als Schweizer Sektion von IBBY (International Board on Books for Young People), das den Hans Christian Andersen Award verleiht, jeweils alle dafür nominierten Bücher sowie die auf der „IBBY Honour List“ ausgezeichneten Titel. Die Bibliothek bietet somit auch im Bereich der zeitgenössischen internationalen Kinder- und Jugendliteratur eine repräsentative Auswahl. 2 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 2. Beziehungsreicher Kern der Sammlung Das weite Netzwerk, das sie sich so aufbaute, nährte ihre Sammlung über viele Jahre hinweg und wurde durch ihr offenes Haus aufrechterhalten. Hürlimann war überzeugt, dass das Kinderbuch eine „internationale Angelegenheit“ (1976, S. 185) sei, und war bei der von Jella Lepman, unter Beteiligung von Lisa Tetzner und Kurt Kläber veranlassten, 1953 in Zürich erfolgten IBBY-Gründung dabei. 3 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung So gliedert sich denn der von Ruth Fassbind-Eigenheer erstellte und mit kenntnisreicher Einleitung versehene Gesamtkatalog Die Kinderbuchsammlung Bettina Hürlimann (1992) auch mehr nach Ländern und Kontinenten als nach Gattungen oder Themen. Die Vielsprachigkeit der Sammlung ist sowohl Chance als auch Herausforderung für das SIKJM. Sie bietet zahlreiche Ansatzpunkte für Kooperationen mit Einzelphilologien oder mit komparatistischen und interdisziplinären Projekten. Dabei kommt der Skandinavistik ein besonderer Stellenwert zu, während etwa die herausragende Sammlung japanischer Bilderbücher noch viel Potential für Forschungskooperationen bietet. So gliedert sich denn der von Ruth Fassbind-Eigenheer erstellte und mit kenntnisreicher Einleitung versehene Gesamtkatalog Die Kinderbuchsammlung Bettina Hürlimann (1992) auch mehr nach Ländern und Kontinenten als nach Gattungen oder Themen. Die Vielsprachigkeit der Sammlung ist sowohl Chance als auch Die thematische und literaturgeschichtliche Erschliessung der Sammlung wurde zuallererst jedoch von Bettina Hürlimann selbst betrieben. Die Arbeit an ihrem Buch Europäische Kinderbücher aus drei Jahrhunderten (Hürlimann, 1963), das auf grosses Echo stiess und auf Englisch, Spanisch und Japanisch übersetzt wurde, ging zentral von den eigenen Vorlieben aus und gab oft den Anstoss für weitere Erwerbungen, wie sie in ihrer Autobiographie betont: Die mir so zufallenden Bereicherungen meiner Bibliothek bestimmten meine etwas einseitige Arbeitsweise, in der ich bei Deutungen und Beschreibungen solche Werke ausführlicher behandelte, die ich durch eigene Anschauung kannte. Meine Leser spürten diese direkte Beziehung, und für mich hatte das Sammeln damit einen lebendigen Sinn, ja war eine Notwendigkeit geworden. (1976, S. 150) Die mir so zufallenden Bereicherungen meiner Bibliothek bestimmten meine etwas einseitige Arbeitsweise, in der ich bei Deutungen und Beschreibungen solche Werke ausführlicher behandelte, die ich durch eigene Anschauung kannte. 2. Beziehungsreicher Kern der Sammlung Meine Leser spürten diese direkte Beziehung, und für mich hatte das Sammeln damit einen lebendigen Sinn, ja war eine Notwendigkeit geworden. (1976, S. 150) Ein Kapitel dieses historischen Überblicks ist der Geschichte des Schweizer Kinderbuches gewidmet und geht auf die Zürcher Neujahrsblätter ein, deren ältestes auf das Jahr 1645 zurückreicht, also noch vor Comenius’ Orbis Pictus (1658). Hürlimann formuliert dabei die interessante These, diese Neujahrsblätter – in ihrer Sammlung mit einem stattlichen Bestand vor allem aus den Musikgesellschaften und aus der Stadt-Bibliothek Zürich vertreten –, die zuerst ja für Kinder ausgegeben wurden, stellten den eigentlichen Ursprung der Schweizer Kinderliteratur dar. (1963, S. 262) Kostbarkeiten und Rara der Hürlimann-Kollektion liessen sich lange aufzählen: Viele wertvolle Erstausgaben wie etwa jene von Des Knaben Wunderhorn (1806-1808) oder von Peter Pan (1906), die erste französischsprachige Robinson Crusoe-Ausgabe (1721) oder Bände kinderliterarischer Gründungswerke aus dem ausgehenden 18. Jahrhundert wie das Bilderbuch für Kinder von Bertuch oder die Kupfersammlung zu J.B. Basedows Elementarwerke für die Jugend und ihre Freunde gehören dazu. Als Höhepunkt ihres Sammlerdaseins bezeichnet Bettina Hürlimann den Moment, als Ehemann Martin ihr die Erstausgabe der Grimm’schen Kinder- und Hausmärchen (1812) schenkte. (vgl. Hürlimann 1976, S. 149) Einen der thematischen Schwerpunkte der Kollektion bilden die Robinsonaden: Ausgehend von Robinson Crusoe, jenem Schlüsseltext der europäischen Romanentwicklung, der hundertfach für Kinder und Jugendliche bearbeitet wurde und in der Form von Campes Robinson der Jüngere (1779) als „erster deutschsprachiger Text spezifischer Kinderliteratur“ (von Glasenapp, 2020, S. 2) gilt, beschäftigte sich Bettina Hürlimann nicht nur sammelnd, sondern auch forschend mit dieser weitverzweigten Textfamilie. Ihr zusammengetragenes Material 4 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung hat sie in einem Artikel der Zeitschrift Du vorgestellt (1966), hegte darüber hinaus jedoch einen nicht mehr verwirklichten Plan: 2: Exlibris der Sammlung Bettina Hürlimann Bettina Hürlimanns besonderes Interesse für die Illustration bescherte dem Institut rund 400 Bettina Hürlimanns besonderes Interesse für die Illustration bescherte dem Institut rund 400 Originalillustrationen – Skizzen, Zeichnungen, Drucke, Collagen, Scherenschnitte, Hefte und Buchmaquetten –, die aus der Buchherstellung im Atlantis-Verlag hervorgingen oder als Freundschaftsgeschenke von Bilderbuchschaffenden ihren Weg in die Sammlung fanden. Bis dato nur analog erschlossen, konnten sie 2019 in einem Projekt durch die SIKJM-assoziierte Forscherin Anna Lehninger neu geordnet und in den Online- Katalog aufgenommen werden, indem die Verbindungen zu den gedruckten Büchern hergestellt wurden. Dies stellt einen wichtigen Beitrag dar, um der Illustration einen gewichtigeren Platz in der Kinderbuchforschung einzuräumen (vgl. Lehninger, 2019). Schliesslich besitzt das SIKJM auch Bettina Hürlimanns Nachlass, der neben ihren Schriften und Vorträgen die umfangreiche Korrespondenz mit Autor:innen und Illustrator:innen aus aller Welt aufweist und für die Forschung immer wieder wertvolle Einsichten und Zusammenhänge bereithält. hat sie in einem Artikel der Zeitschrift Du vorgestellt (1966), hegte darüber hinaus jedoch einen nicht mehr verwirklichten Plan: hat sie in einem Artikel der Zeitschrift Du vorgestellt (1966), hegte darüber hinaus jedoch einen nicht mehr verwirklichten Plan: Ich wollte und will ein Buch darüber schreiben, aber je mehr ich darüber lerne, desto grösser, ja hoffnungslos gross erscheint mir das Thema. (1976, S. 153) Ich wollte und will ein Buch darüber schreiben, aber je mehr ich darüber lerne, desto grösser, ja hoffnungslos gross erscheint mir das Thema. (1976, S. 153) 2019, zum 300. Geburtstag von Defoes Original, hat sich neben vielen anderen Institutionen auch das SIKJM wieder neu mit Robinson beschäftigt. Der dort aufgegriffene Aspekt der Ikonographie Robinsons in den Illustrationen, in weitem diachronem Bogen betrachtet, wird durch die SIKJM-Forschung aktuell weiterverfolgt. Das Thema ist auch insofern sinnfällig, als die Hürlimann-Sammlung von der Robinson- Darstellung buchstäblich geprägt ist: Bettina Hürlimann erblickte nämlich in einem ikonisch gewordenen Requisit Robinsons, dem Sonnenschirm, ein Symbol menschlicher Würde (vgl. 1963, S. 81) und wählte Robinson deshalb als Exlibris für ihre gesamte Sammlung, sodass nun rund 4’000 Bücher des SIKJM- Bestandes mit einem Robinson-Stempel versehen sind. Die aktuelle Beschäftigung mit Robinson nimmt aber auch weitere Bestände, intern aus der Sammlung Keckeis, extern aus der in Rapperswil ansässigen privaten Robinson-Bibliothek in den Blick. Auch bestehen Kooperationen mit dem Englischen Seminar der Universität Zürich, das sich ebenfalls mit der Sammlung Rapperswil befasst, sodass in der Region Zürich in der Folge des Robinson-Jubiläums aktuell eine intensive Auseinandersetzung mit dem Thema stattfindet. 5 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Abb. 1: Defoe, Daniel. La Vie et les Avantures surprenantes de Robinson Crusoe. Illustrationen von Bernard Picard. Amsterdam, 1721. Frontispiz des 1. Bandes. Abb. 1: Defoe, Daniel. La Vie et les Avantures surprenantes de Robinson Crusoe. Illustrationen von Bernard Picard. Amsterdam, 1721. Frontispiz des 1. Bandes. 6 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Abb. 2: Exlibris der Sammlung Bettina Hürlimann Abb. 2: Exlibris der Sammlung Bettina Hürlimann Abb. 3. Historische Schweizer Kinderliteratur von 1750 bis 1900 Ebenfalls seit Gründungszeiten war der Verleger Peter Keckeis (1920-2007) an den Geschicken des Schweizerischen Jugendbuchinstituts beteiligt. Er prägte als wichtiger Akteur auch des allgemeinen Literaturbetriebs der 1960er und 1970er Jahre massgeblich die Verlage Benziger und Huber, war Förderer von Autoren wie Kuno Räber oder Walter Matthias Diggelmann und mit Berufskollegen wie Otto F. Walter oder eben dem Ehepaar Hürlimann befreundet (vgl. Frey, 2007). Im Rahmen der Zürcher Historischen 7 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Kinderbuchgesellschaft und am SJI in regem Austausch mit den beiden Sammlerinnen Hürlimann und Waldmann, hat er dem Institut die historischen Schweizer Bestände seiner Kinderbuchsammlung, rund 400 Titel, geschenkt (in der Folge „Sammlung Keckeis“ genannt). Wenn hier von „Schweizer Beständen“ die Rede ist, so muss präzisiert werden, dass Keckeis darin auch Bücher ausländischer Autor:innen einschloss, die in der Schweiz herausgegeben wurden. So finden sich etwa Franz Poccis Dichtungen (1843) aus dem Verlag Hurter, Schaffhausen. Kinderbuchgesellschaft und am SJI in regem Austausch mit den beiden Sammlerinnen Hürlimann und Waldmann, hat er dem Institut die historischen Schweizer Bestände seiner Kinderbuchsammlung, rund 400 Titel, geschenkt (in der Folge „Sammlung Keckeis“ genannt). Wenn hier von „Schweizer Beständen“ die Rede ist, so muss präzisiert werden, dass Keckeis darin auch Bücher ausländischer Autor:innen einschloss, die in der Schweiz herausgegeben wurden. So finden sich etwa Franz Poccis Dichtungen (1843) aus dem Verlag Hurter, Schaffhausen. Wenn auch kein Katalog zur Sammlung existiert, so hat Peter Keckeis sie teilweise mit eigenen Publikationen kommentiert. Und auch er hatte, wie Bettin Hürlimann, an einer Geschichte des europäischen Kinderbuchs gearbeitet, die allerdings unveröffentlicht geblieben ist (vgl. Frey, 2007, S. 220). Sein immenses Wissen ist schliesslich in die umfangreiche und für Forschungen zur historischen Schweizer Kinderliteratur unschätzbar wertvolle Annotierte Bibliographie der Schweizer Kinder- und Jugendliteratur von 1750-1900 (Weilenmann, 1993) eingeflossen. Die Sammlung Keckeis ergänzt sich mit den Helvetica in der Sammlung Hürlimann auf besonders glückliche Weise, etwa was die Neujahrsblätter – wie erwähnt bei Hürlimann sehr zahlreich, bei Keckeis jedoch kaum vorhanden – oder auch die Ausgaben des Schweizerischen Robinson von Johann David Wyss betrifft. 3. Historische Schweizer Kinderliteratur von 1750 bis 1900 Manche Werke bedeutender Autor:innen, die bei Hürlimann kaum vorkommen und auch in ihren Publikationen keine Erwähnung finden, hat Keckeis integral gesammelt, so etwa jene des Winterthurer Malers und Schriftstellers August Corrodi (1826-1885). Teile und Aspekte seines Werks konnte das SIKJM in jüngster Zeit unter anderem im Rahmen einer Kooperation mit dem Sinergia-Projekt „The Power of Wonder – Die Instrumentalisierung von Bewunderung, Erstaunen und Überraschungen in Wissens-, Macht- und Kunstdiskursen“ (unter Leitung von Mireille Schnyder, Universität Zürich, und Nicola Gess, Universität Basel) vorstellen. So konnte gezeigt werden, wie Corrodi auf innovative Weise stereotype aufklärerische Konzepte ebenso wie romantische Kindheitsbilder aufbricht und mit seinen humoristischen Inszenierungen neue, produktive Narrative des Staunens schafft (Keller, 2021). Die ebenfalls in diesem Projekt entstandene virtuelle Ausstellung Staunen im Kinderbuch (Konzept: Mireille Schnyder und Daniela Hahn), welche sich sowohl an Forschende wie auch an ein breites Publikum richtet und einige Perlen der SIKJM-Bibliothek präsentiert, „verfolgt anhand ausgewählter Beispiele aus Kinderbüchern von der Mitte des 17. bis Anfang des 20. Jahrhunderts, wie sich Formen, Objekte, Ziele und Wertungen des Staunens über die Jahrhunderte veränderten“ (Schnyder & Hahn, 2021). Wiederum ausgehend von Corrodi, stellt die Sammlung Keckeis ferner einen wichtigen Fundus für geplante Studien des SIKJM zur Komik in der historischen Schweizer Kinderliteratur dar. 8 8 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Abb. 3: Corrodi, August: Schloss Waldegg und seine Bewohner. Ein Sommerferienbuch für die Jugend. Stuttgart, 1860. Titelbild von August Corrodi. Abb. 3: Corrodi, August: Schloss Waldegg und seine Bewohner. Ein Sommerferienbuch für die Jugend. Stuttgart, 1860. Titelbild von August Corrodi. Der „digital turn“ (vgl. u.a. die Ausgabe Digital Humanities und wissenschaftliche Bibliotheken dieser Zeitschrift) macht(e) auch vor dem SIKJM nicht Halt. Bereits seit 2010 stehen rund 300 Titel, vorwiegend aus der Sammlung Keckeis, auf der Online-Plattform Schweizer Drucke e-rara zum Download zur Verfügung. Aktuell wird geprüft, wie diese auch für ein umfassendes Online-Portal deutschsprachiger historischer Kinderbücher nutzbar gemacht werden könnten, das in Kooperation verschiedener Bibliotheken in Deutschland im Entstehen begriffen ist. Zusätzliche Helvetica des SIKJM sowie auch des Spyri-Archivs (vgl. unten) werden identifiziert, um das Portal weiter zu ergänzen. Dieses wird die Grundlage für breite, interdisziplinäre und internationale Forschungsinitiativen im Bereich der deutschsprachigen historischen Kinderliteratur bilden. 4. Amerikanische Bilderbücher von der Zürcher Bahnhofstrasse Noch näher an den „Endverbraucher:innen“ als die Verleger:innen Keckeis und Hürlimann war die Buchhändlerin Elisabeth Waldmann (1922-1996). In engem Kontakt mit Eltern, Lehrer:innen, Kindergärtner:innen und Bibliothekar:innen war ihr ein pädagogischer Zugang zum Kinderbuch besonders vertraut. Von den geschäftlich bestellten Titeln behielt sie oft ein Exemplar für sich beziehungsweise für ihre l d f ll ll hl h d b d hh dl d h h f h l Familie und füllte so allmählich die über der Buchhandlung an der Bahnhofstrasse Zürich gelegene 9 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Familienwohnung. Rund 9’000 Titel dieser umfangreichen Sammlung sowie zahlreiche Schriften und Korrespondenzen von Waldmann wurden 1998 dem SJI vermacht. Auch diese Sammlung ergänzt sich hervorragend mit den bisher vorgestellten. Der Schwerpunkt liegt hier nämlich auf der amerikanischen Bilderbuchproduktion des 20. Jahrhunderts. Rund 4’000 Titel sind englisch-, 2’500 deutsch- und 600 französischsprachig. Die Bilderbücher dominieren mit einem Anteil von etwa 80%. Auch das 19. Jahrhundert ist gut vertreten, und die deutschsprachigen Sachbücher reichen zurück bis ins 18. Jahrhundert. Abb. 4: Bild aus Froux le lièvre von Feodor Rojankowsky. Albums du Père Castor, Paris 1935. Abb. 4: Bild aus Froux le lièvre von Feodor Rojankowsky. Albums du Père Castor, Paris 1935. Das mit Bettina Hürlimann geteilte Interesse für das Bilderbuch führte zur gemeinsamen Publikation Die Welt im Bilderbuch (Hürlimann, 1965), diese wiederum zu einer lebenslangen Freundschaft. Später wirkte Waldmann bei dem von Verena Rutschmann herausgegebenen Lexikon Schweizer Bilderbuch-Illustratoren 1900-1980 (1983) mit. Daneben entstanden zahlreiche Ausstellungen mit zugehörigen Broschüren und Katalogen, welche die gewählten Perspektiven für die Nachwelt festhielten. So wurde etwa 1993 anlässlich des 25jährigen Jubiläums des SJI die amerikanische Bilderbuchproduktion der 1920er bis 1950er Jahre in den Fokus genommen. In der zugehörigen Publikation Passagen 1920-1960. Das Bilderbuch wird kosmopolitisch, vernehmen wir im Beitrag „Wege und Umwege einer Sammlerin“ (1993) Elisabeth Waldmanns aufschlussreiche Stimme zu ihrer eigenen Sammlertätigkeit. Wir erfahren hier, wie sie dabei zunächst thematisch vorging, vergleichend auch zwischen Ländern und Epochen, und dass dieses Sammeln stets mit dem Interesse für die zeitgeschichtlichen Bedingungen einherging, unter denen die Werke entstanden waren (1993, S. 53). So kam beispielsweise eine etwa 1’000 Bücher umfassende Rotkäppchen-Sammlung zustande, die allerdings an das Bilderbuch-Museum Burg Wissem von Troisdorf (D) gegeben wurde. 5. Pionierarbeit für die Kinder- und Jugendliteraturforschung Elisabeth Waldmann, Peter Keckeis und Bettina Hürlimann: Für alle drei gilt, dass sie mit ihren Publikationen und Kommentaren zu den eigenen Kollektionen Pionierarbeit leisteten für die akademisch damals noch junge Disziplin der Kinder- und Jugendliteraturforschung. Denn wie Sebastian Schmideler in einem Artikel zur Wiener Sammlerin Johanna Monschein darlegt, waren die Sammler:innen, zumindest im deutschen Sprachraum, neben den Antiquar:innen und Bibliothekar:innen, mehr als die Philolog:innen der Universitäten, die „eigentlichen Gründungsväter und -mütter der historischen Kinderbuchforschung“ (2018, S. 14). Bemerkenswert ist darüber hinaus, dass sie im Rückblick auch Fehleinschätzungen eingestehen, so etwa Waldmann und Hürlimann in Bezug auf die massenproduzierten „Golden Books“ oder amerikanischen Comics (vgl. Hürlimann, 1963, S. 119-132), denen beide mit Skepsis begegneten – was Lücken in den Sammlungen generierte, die sie später bedauerten (vgl. Hürlimann 1976, S. 121; Waldmann 1993, S. 48 f.) Hier wird nicht nur der individuelle Blickwinkel der Sammler:innen, sondern auch der Zeitgeist spürbar. Selbst wenn aus heutiger Sicht manches anders einzuordnen ist, so haben diese „Gründerväter- und mütter“ der Forschung doch viele Fäden in die Hand gegeben, die es aufzugreifen lohnt. 4. Amerikanische Bilderbücher von der Zürcher Bahnhofstrasse Deutlich wird überdies, ganz ähnlich wie bei Bettina Hürlimann, dass der Aufbau der Sammlung durch persönliche Begegnungen stark geprägt war. Neben ihrer Freundin Bettina Hürlimann nennt Waldmann drei weitere „Kinderbuchtanten“ (1993, S. 50 f.), allesamt aus dem englischsprachigen Raum, mit denen sie wegweisende 10 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Freundschaften schloss: Erstens Vera Peterson aus Portland, Oregon, der sie etwa die Entdeckung Taro Yashimas oder des Künstlerpaars Ingri und Edgar Parain d’Aulaire verdankt. Zweitens Susan Hirschman, tätig bei den Verlagen Macmillan und Greenwillow, deren Bücher so fast integral ihren Weg in die Sammlung Waldmann fanden. Schliesslich Janice Dohm aus der Buchhandlung Collett in London, von der weitere Impulse für englischsprachige Anschaffungen ausgingen. Ebenfalls bestimmend war die persönliche Begegnung mit Paul Faucher, Initiant und Verleger der legendären Sachbilderbücher „Albums du Père Castor“: Er war es, der sie auf Illustrator:innen wie Feodor Rojankovsky oder Nathalie Parain aufmerksam machte, beides Emigrant:innen aus Russland, von denen die Sammlung einige Erstausgaben enthält. 6. Johanna Spyri-Archiv und Nachlasse eils von Johanna Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutsc a Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutschmann, 2001, teils von Johanna Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutschmann, 2001, S. 5). Abb. 5: Spyri-Archiv, SIKJM Abb. 5: Spyri-Archiv, SIKJM Abb. 5: Spyri-Archiv, SIKJM Nicht nur Johanna Spyri selbst, auch weitere Mitglieder der Familien Schweizer und Heusser, denen sie entstammte, waren illustre Persönlichkeiten der Region Zürich und nehmen im Spyri-Nachlass einen entsprechenden Platz ein. Dieses reichhaltige Material ist durch die von Regine Schindler (einer Tochter Bettina Hürlimanns) im Auftrag der Spyri-Stiftung herausgegebene Reihe Pfarrherren, Dichterinnen, Forscher – Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Schindler, 2007-2015) teilweise erschlossen und beforscht. Angefangen beim Spyri-Grossvater, dem Pfarrer und Lavater-Freund Diethelm Schweizer- Gessner, über Spyris Mutter, die religiöse Dichterin Meta Heusser-Schweizer, bis hin zu Spyris Bruder, dem Naturwissenschaftler Christian Heusser, machen die fünf Bände die Quellen zugänglich und kontextualisieren das Schaffen der drei Generationen zeitgeschichtlich. Ebenso bieten diese Archivalien weitere Ansatzpunkte für Forschungsfragen zur Zürcher Literatur- und Gesellschaftsgeschichte. Nicht nur Johanna Spyri selbst, auch weitere Mitglieder der Familien Schweizer und Heusser, denen sie entstammte, waren illustre Persönlichkeiten der Region Zürich und nehmen im Spyri-Nachlass einen entsprechenden Platz ein. Dieses reichhaltige Material ist durch die von Regine Schindler (einer Tochter Bettina Hürlimanns) im Auftrag der Spyri-Stiftung herausgegebene Reihe Pfarrherren, Dichterinnen, Forscher – Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Schindler, 2007-2015) teilweise erschlossen und beforscht. Angefangen beim Spyri-Grossvater, dem Pfarrer und Lavater-Freund Diethelm Schweizer- Gessner, über Spyris Mutter, die religiöse Dichterin Meta Heusser-Schweizer, bis hin zu Spyris Bruder, dem Naturwissenschaftler Christian Heusser, machen die fünf Bände die Quellen zugänglich und kontextualisieren das Schaffen der drei Generationen zeitgeschichtlich. Ebenso bieten diese Archivalien weitere Ansatzpunkte für Forschungsfragen zur Zürcher Literatur- und Gesellschaftsgeschichte. Nicht nur Johanna Spyri selbst, auch weitere Mitglieder der Familien Schweizer und Heusser, denen sie entstammte, waren illustre Persönlichkeiten der Region Zürich und nehmen im Spyri-Nachlass einen entsprechenden Platz ein. Dieses reichhaltige Material ist durch die von Regine Schindler (einer Tochter Bettina Hürlimanns) im Auftrag der Spyri-Stiftung herausgegebene Reihe Pfarrherren, Dichterinnen, Forscher – Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Schindler, 2007-2015) teilweise erschlossen Nicht nur Johanna Spyri selbst, auch weitere Mitglieder der Familien Schweizer und Heusser, denen sie entstammte, waren illustre Persönlichkeiten der Region Zürich und nehmen im Spyri-Nachlass einen entsprechenden Platz ein. 6. Johanna Spyri-Archiv und Nachlasse Als Institut unter der Trägerschaft der Johanna Spyri-Stiftung hat das SIKJM den expliziten Auftrag, das Erbe Johanna Spyris (1827-1901) zu sammeln und zu bewahren. Neben dem literarischen Werk und zugehöriger Fachliteratur enthält das Spyri-Archiv handschriftliche Manuskripte und Briefe sowie Dokumente, Bilder und persönliche Erinnerungsgegenstände von Johanna Spyri und aus ihrem Umfeld, schliesslich zahlreiche Medien und Gegenstände aus dem Heidi-Medienverbund. Schon bei der Gründung 1968 erhielt das Johanna Spyri-Archiv etliche Schenkungen, und Gründer Franz Caspar sammelte systematisch die deutschsprachigen Spyri-Ausgaben wie auch Übersetzungen. So finden sich denn im SIKJM Heidi-Übersetzungen in mehr als 40 Sprachen, darunter auf Hindi, Mazedonisch oder Persisch. Die Sammlertätigkeit wurde in der Folge auch medienübergreifend fortgeführt. Aktuell befinden sich im Archiv 85 Laufmeter „Spyriana“. Rund 1’000 Dokumente aus dem Nachlass werden zusätzlich als Deposita in der Zentralbibliothek Zürich aufbewahrt. Allerdings wurden auch „grosse Teile des schriftlichen Nachlasses 11 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung teils von Johanna Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutschmann, 2001, S. 5). Nicht nur Johanna Spyri selbst, auch weitere Mitglieder der Familien Schweizer und Heusser, denen sie entstammte, waren illustre Persönlichkeiten der Region Zürich und nehmen im Spyri-Nachlass einen entsprechenden Platz ein. Dieses reichhaltige Material ist durch die von Regine Schindler (einer Tochter Bettina Hürlimanns) im Auftrag der Spyri-Stiftung herausgegebene Reihe Pfarrherren, Dichterinnen, Forscher – Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Schindler, 2007-2015) teilweise erschlossen und beforscht. Angefangen beim Spyri-Grossvater, dem Pfarrer und Lavater-Freund Diethelm Schweizer- Gessner, über Spyris Mutter, die religiöse Dichterin Meta Heusser-Schweizer, bis hin zu Spyris Bruder, dem Naturwissenschaftler Christian Heusser, machen die fünf Bände die Quellen zugänglich und kontextualisieren das Schaffen der drei Generationen zeitgeschichtlich. Ebenso bieten diese Archivalien weitere Ansatzpunkte für Forschungsfragen zur Zürcher Literatur- und Gesellschaftsgeschichte. Abb. 5: Spyri-Archiv, SIKJM teils von Johanna Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutschmann, 2001, S. 5). teils von Johanna Spyri selbst, teils durch die Kriegseinwirkung in Deutschland zerstört“ (Rutschmann, 2001, S. 5). 6. Johanna Spyri-Archiv und Nachlasse Dieses reichhaltige Material ist durch die von Regine Schindler (einer Tochter – Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Schindler, 2007-2015) teilweise erschlossen und beforscht. Angefangen beim Spyri-Grossvater, dem Pfarrer und Lavater-Freund Diethelm Schweizer- Gessner, über Spyris Mutter, die religiöse Dichterin Meta Heusser-Schweizer, bis hin zu Spyris Bruder, dem Naturwissenschaftler Christian Heusser, machen die fünf Bände die Quellen zugänglich und kontextualisieren das Schaffen der drei Generationen zeitgeschichtlich. Ebenso bieten diese Archivalien weitere Ansatzpunkte für Forschungsfragen zur Zürcher Literatur- und Gesellschaftsgeschichte. Neben literatur- und kunstgeschichtlich aufschlussreichen Materialien wie beispielsweise dem Briefwechsel zwischen Johanna Spyri und C.F. Meyer oder Originalillustrationen des Malers Rudolf Münger enthält das Archiv auch zahlreiche Kuriosa und Gegenstände aus dem Heidi-Medienverbund, die die Rezeptions- und Wirkungsgeschichte des Heidi-Stoffes dokumentieren: Von der Heidi-Puppe über japanische Essstäbchen mit Heidi-Aufdruck bis hin zu Briefmarken des karibischen Inselstaats Grenada, welche Standbilder aus dem Heidi- Film mit Shirley Temple zeigen. Am stärksten beforscht ist im Spyri-Feld, wenig erstaunlich, das Phänomen des Export-Schlagers Heidi. Als Figur, die den Siegeszug in die Welt antrat, wurde sie bekanntlich zur vielfältigen Projektionsfläche. Zum 100. Todestag Spyris wurden 2001 umfangreiche Projekte realisiert, unter anderem der Sammelband Heidi: Karrieren einer Figur (Halter, 2001), die Wanderausstellung Heidi 01 (unter Beteiligung des SJI) oder ein vom SJI ausgerichtetes internationales Kolloquium, aus dem die Publikation Johanna Spyri und ihr Werk – Lesarten (Schweizerisches Institut für Kinder- und Jugendmedien, 2004) hervorging. Sie alle setzten sich eingehend und Am stärksten beforscht ist im Spyri-Feld, wenig erstaunlich, das Phänomen des Export-Schlagers Heidi. Als Figur, die den Siegeszug in die Welt antrat, wurde sie bekanntlich zur vielfältigen Projektionsfläche. Zum 100. Todestag Spyris wurden 2001 umfangreiche Projekte realisiert, unter anderem der Sammelband Heidi: K i i Fi (H l 2001) di W d ll H idi 01 ( B ili d SJI) d i Karrieren einer Figur (Halter, 2001), die Wanderausstellung Heidi 01 (unter Beteiligung des SJI) oder ein vom SJI ausgerichtetes internationales Kolloquium, aus dem die Publikation Johanna Spyri und ihr Werk – Lesarten (Schweizerisches Institut für Kinder- und Jugendmedien, 2004) hervorging. 6. Johanna Spyri-Archiv und Nachlasse Lisa Tetzners neunbändige Reihe Die Kinder aus der Nummer Nr. 67 zählt zu den wichtigsten Werken der Exilliteratur. 2019 gründete das SIKJM in Kooperation mit der Humboldt-Universität zu Berlin und der Universität Göttingen die Tetzner/Kläber-Gesellschaft, die sich für die Erforschung und Sichtbarmachung des literarischen Werks der beiden Autor:innen einsetzt. Damit nahm die Tetzner/Kläber-Forschung große Fahrt auf: 2020 konnten Drittmittel akquiriert werden, um die im SIKJM lagernden Nachlassbestände zu ordnen. Nach einer ersten Tagung im Herbst 2019 auf Burg Ludwigstein bei Witzenhausen (D), Jugend bewegt Literatur: Lisa Tetzner, Kurt Kläber und die Literatur der Jugendbewegung (Becker, Benner, & Wassiltschenko, 2022) wird die Gesellschaft vom 6.-8. Mai 2022 an der Universität Zürich eine weitere Tagung zum Thema Exil in der Schweiz. Lisa Tetzner, Kurt Kläber und die Literatur im Exil ausrichten. Ihre kinderliterarischen Werke brachten es zu Welterfolgen, so etwa Kläbers unter dem Pseudonym Kurt Held veröffentlichte Rote Zora (1941) oder die in Co-Autorschaft verfassten Schwarzen Brüder (1940/41). Lisa Tetzners neunbändige Reihe Die Kinder aus der Nummer Nr. 67 zählt zu den wichtigsten Werken der Exilliteratur. 2019 gründete das SIKJM in Kooperation mit der Humboldt-Universität zu Berlin und der Universität Göttingen die Tetzner/Kläber-Gesellschaft, die sich für die Erforschung und Sichtbarmachung des literarischen Werks der beiden Autor:innen einsetzt. Damit nahm die Tetzner/Kläber-Forschung große Fahrt auf: 2020 konnten Drittmittel akquiriert werden, um die im SIKJM lagernden Nachlassbestände zu ordnen. Nach einer ersten Tagung im Herbst 2019 auf Burg Ludwigstein bei Witzenhausen (D), Jugend bewegt Literatur: Lisa Tetzner, Kurt Kläber und die Literatur der Jugendbewegung (Becker, Benner, & Wassiltschenko, 2022) wird die Gesellschaft vom 6.-8. Mai 2022 an der Universität Zürich eine weitere Tagung zum Thema Exil in der Schweiz. Lisa Tetzner, Kurt Kläber und die Literatur im Exil ausrichten. 7. Fazit und Ausblick Das SIKJM blickt auf eine lange Tradition von Sammel- und Forschungstätigkeiten zurück, die sich stets nahe an der Praxis von Vermittlung, literaler Förderung und kindlicher Rezeption bewegten. Als assoziiertes Institut der Universität Zürich verfügt es über eine kleine Forschungsabteilung, die diese Tradition aufrechterhält und sich gleichzeitig in der internationalen Forschungscommunity, auch im Rahmen von Kooperationsprojekten mit Universitäten und Pädagogischen Hochschulen, positioniert. Die intensiven Bemühungen um die Sammlungen und Nachlasse, die ein wichtiges „kulturelles Erbe der schweizerischen, deutschen und internationalen Kinder- und Jugendliteratur“ (Meyer & Wegmann, 2013) darstellen, werden fortgesetzt und haben weiterhin grosses Potenzial. Gleichzeitig nehmen aktuelle Fragestellungen nicht nur die einzelnen Sammlungen, sondern auch ihre Gesamtheit sowie darüber hinaus reichende internationale Bestände in den Blick. Neue Möglichkeiten dafür eröffnen die laufenden Initiativen für umfassende Online-Portale mit Kinder- und Jugendbüchern. Diese wiederum werden künftig die Chance bieten, auch von ferne einen Blick in die Wunderkammern des SIKJM zu werfen. 6. Johanna Spyri-Archiv und Nachlasse Sie alle setzten sich eingehend und 12 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung auch kritisch mit der weltweiten Verbreitung, Medialisierung und Trivialisierung der Heidi-Figur auseinander. Insbesondere die japanische Anime-Serie aus dem Jahre 1974, von der ausgehend eine neue Heidi-Welle den Globus überspülte, erregt immer wieder Interesse und zieht Forschende aus aller Welt, gerade auch aus Japan, ans SIKJM. auch kritisch mit der weltweiten Verbreitung, Medialisierung und Trivialisierung der Heidi-Figur auseinander. Insbesondere die japanische Anime-Serie aus dem Jahre 1974, von der ausgehend eine neue Heidi-Welle den Globus überspülte, erregt immer wieder Interesse und zieht Forschende aus aller Welt, gerade auch aus Japan, ans SIKJM. Abb. 6: Vietnamesische Ausgabe von Johanna Spyris Heidi, 1997. Abb. 6: Vietnamesische Ausgabe von Johanna Spyris Heidi, 1997. Abb. 6: Vietnamesische Ausgabe von Johanna Spyris Heidi, 1997. Abb. 6: Vietnamesische Ausgabe von Johanna Spyris Heidi, 1997. Für Forschungen können sämtliche Dokumente des Spyri-Archivs in der Bibliothek des SIKJM Anmeldung) beziehungsweise in der Handschriftenabteilung der Zentralbibliothek Zürich eingesehen werden. Zudem finden sich einige historische Werkausgaben auf e-rara (vgl. obige Ausführungen zur Sammlung Keckeis), darunter die Heidi-Erstausgaben von 1880 und 1881. Noch brach liegendes Archivmaterial wird zurzeit durch das SIKJM gesichtet und erschlossen und so der Weg für weiterführende Forschungen geebnet. Das Interesse der – auch internationalen – Forschung an den Spyri-Beständen ist ungebrochen, steuern wir doch auf wichtige Jubiläen zu: Im Jahr 2027 wird der 200. Geburtstag von Johanna Spyri gefeiert, 2030 wird der erste Heidi-Band 150 Jahre alt. Weitere bedeutende Nachlassdokumente im Besitz des SIKJM stammen von Lisa Tetzner und Kurt Kläber. Das Autorenpaar emigrierte 1933 ins Schweizer Exil, wo sie sich in Carona (TI) eine neue Existenz aufbauten. 13 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Ihre kinderliterarischen Werke brachten es zu Welterfolgen, so etwa Kläbers unter dem Pseudonym Kurt Held veröffentlichte Rote Zora (1941) oder die in Co-Autorschaft verfassten Schwarzen Brüder (1940/41). References Becker, M., Benner, J., & Wassiltschenko, J. (2022). Jugend bewegt Literatur: Lisa Tetzner, Kurt Kläber und die Literatur der Jugendbewegung. Stuttgart: Metzler (im Druck). ↩ Fassbind-Eigenheer, R. (1992). Die Kinderbuchsammlung Bettina Hürlimann – Gesamtkatalog. Zürich: Schweizerisches Jugendbuch-Institut. ↩ Frey, H. (2007). Peter Keckeis. Thurgauer Jahrbuch, 82, 219–220. ↩ Halter, E. (2001). Heidi-Karrieren einer Figur. Zürich: Offizin. ↩ Hürlimann, B. (1963). Europäische Kinderbücher in drei Jahrhunderten (2nd ed.). Zür Hürlimann, B. (1963). Europäische Kinderbücher in drei Jahrhunderten (2nd ed.). Zürich: Atlantis. ↩ Hürlimann, B. (1965). Die Welt im Bilderbuch. Moderne Kinderbilderbücher aus 24 Ländern. Zürich: Atlantis. ↩ Hürlimann, B. (1963). Europäische Kinderbücher in drei Jahrhunderten (2nd ed.). Zürich: Atlantis. ↩ Hürlimann, B. (1965). Die Welt im Bilderbuch. Moderne Kinderbilderbücher aus 24 Ländern. Zürich: Atlantis. ↩ Hürlimann, B. (1965). Die Welt im Bilderbuch. Moderne Kinderbilderbücher aus 24 Ländern. Zürich: Atlantis. ↩ 14 14 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung 027.7 Zeitschrift für Bibliothekskultur / Journal for Library Culture • Kinder und Jugendliche in Bibliotheken und Medienforschung Die Kinderbuchsammlungen des Schweizerischen Instituts für Kinder- und Jugendmedien SIKJM und ihre Impulse für die Forschung Hürlimann, B. (1966). Robinsonaden. Mit 5 farbigen und 13 schwarzweisen Illustrationen. Du, 26, 389–401. ↩ Hürlimann, B. (1976). Sieben Häuser. Aufzeichnungen einer Bücherfrau. 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In Arbeitsgemeinschaft Kinder- und Jugendliteraturforschung (Ed.), Kinder- und Jugendliteraturforschung 2000/2001 (pp. 3–5). Stuttgart: Metzler. ↩ Rutschmann, V. (2007). Weltoffenheit und Eigenart: internationale und schweizerische Sammlungen im SIKJM. Libri Liberorum (Sonderheft Johanna Monschein), 8, 152–169. ↩ Rutschmann, V. (Ed.). (1983). Lexikon Schweizer Bilderbuch-Illustratoren 1900-1980. References Zürich: Schweizerisches Jugendbuch-Institut. ↩ Schindler, R. (Ed.). (2007). Pfarrherren, Dichterinnen, Forscher: Lebenszeugnisse einer Zürcher Familie des 19. Jahrhunderts (Vols. 1–5). Zürich: Verlag Neue Zürcher Zeitung. ↩ Schmideler, S. (2018). In memoriam Johanna Monschein. Libri Liberorum, 50, 13–18. ↩ Schnyder, M., & Hahn, D. (2021). Staunen im Kinderbuch. Eine historische Ausstellung. Retrieved from https://staunenimkinderbuch.ch/about/ausstellung/ ↩ Schweizerisches Institut für Kinder- und Jugendmedien (Ed.). (2004). Johanna Spyri und ihr Werk – Lesarten. Zürich: Chronos. ↩ von Glasenapp, G. (2020). Die Insel als Metapher und Moratorium. Daniel Defoes Robinson Crusoe (1719/20) und Joachim Heinrich Campes Robinson der Jüngere (1779). Der Deutschunterricht. Beiträge Zu Seiner Praxis Und Wissenschaftlichen Grundlegung, (1), 2–12. ↩ Waldmann, E. (1993). Wege und Umwege einer Sammlerin. In J. H. Fraser... (Ed.), Passagen 1920-1960. Das Bilderbuch wird kosmopolitisch (pp. 44–55). Zürich: Schweizerisches Jugendbuch-Institut. ↩ Weilenmann, C. (1993). Annotierte Bibliographie der Schweizer Kinder- und Jugendliteratur von 1750 bis 1900 – Bibliographie annotée de livres suisses pour l’enfance et la jeunesse de 1750 à 1900. Stuttgart: Metzler. ↩ 15 15
https://openalex.org/W2547715637	https://amb-express.springeropen.com/track/pdf/10.1186/s13568-016-0276-y	English	null	Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass	AMB express	2,016	cc-by	9,017	© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and com- posites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitative protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. The participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natu- ral biomass fermentation experiments. Keywords: Myceliophthora thermophila, Biomass, Cellulose degradation, Secretome composition, Cellulose hydrolysis, Cellulose oxidation (Amorim et al. 2011; Lal 2005). However, currently the cost of cellulase enzyme cocktails are the bottleneck to the economical production of these second generation biofuels (Phillips et al. 2011) mainly because enzymatic conversion of lignocellulose into sugars is a slow and recalcitrant process and cellulose is an insoluble crys- talline substance (Himmel and Bayer 2009) clustered within phenolic lignin (benzene ether linkages) hinder- ing its ability to be enzymatically processed (Lacayo et al. 2013). dos Santos et al. AMB Expr (2016) 6:103 DOI 10.1186/s13568-016-0276-y ORIGINAL ARTICLE Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass Hévila Brognaro dos Santos1,3,5, Thaís Milena Souza Bezerra4,5, José G. C. Pradella3, Priscila Delabona3, Deise Lima3, Eleni Gomes2, Steve D. Hartson6, Janet Rogers6, Brian Couger5 and Rolf Prade5* Open Access Open Access Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass Hévila Brognaro dos Santos1,3,5, Thaís Milena Souza Bezerra4,5, José G. C. Pradella3, Priscila Delabona3, Deise Lima3, Eleni Gomes2, Steve D. Hartson6, Janet Rogers6, Brian Couger5 and Rolf Prade5* Introduction Lignocellulosic biomass polymers are a massive and renewable source for production of biofuels and bio- chemicals, because they trap about 60% of all sug- ars produced by plants on earth. Just as it happens in nature, man-made lignocellulosic biomass such as corn stover and sugar cane bagasse that pile up alongside bio refineries and could be broken down enzymatically *Correspondence: prade@okstate.edu 5 Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA Full list of author information is available at the end of the article For cellulase aided breakdown of cellulose to take place, a single chain must be separated from the crystalline fiber Strains, media, solutions and biomass sources The strain used in this work M. thermophila M77 was isolated from a sugar cane bagasse pile of the northwest region of São Paulo State, Brazil and was deposited at the Fungal Genetics Stock Center FGSC# 26436 (Moretti et al. 2012). A similar M. thermophila strain ATCC 42464 was recently sequenced by the DOE Joint Genome Insti- tute Fungal Genomics Program (Berka et al. 2011; Kol- busz et al. 2014) and was used for DNA sequence based interpretation of LC–MS/MS data. This recalcitrance towards the degradation of cellu- lose is abundantly illustrated in the repertoire of cellu- lose degrading enzymes produced by microorganisms that try to use this polymer as a carbon source (Segato et al. 2014). Most microorganisms produce at least three types of glycosidic bond breaking enzymes; cellobiohy- drolases (also defined as exo glucanases and/or proces- sive glucanases), endo glucanases and β-glucosidases. For comprehensive reviews of hydrolytic biomass breakdown refer to (Benz et al. 2013; Coutinho et al. 2009; Glass et al. 2013; Martens-Uzunova and Schaap 2009; Segato et al. 2014). Myceliophthora thermophila M77 was grown on 1.8% agar petri dishes in Mandels and Sternberg salts (Man- dels and Sternberg 1976) amended with 1.0% glucose and 0.1% peptone incubated at 45 °C for 7 days, or as otherwise stated. Spores were scraped off the plates with a platinum loop, suspended in 0.1% Tween 80 (Sigma- Aldrich, St Louis, MO, USA) and used to pre-inoculate (about 1 × 107 spores/mL) shaker flasks incubated at 45 °C, 250 rpm for 72 h prior to direct transfer to a biore- actor vessel or a large-scale shaker flask experiment. Recently oxidoreductase enzymes such as polysaccha- ride monooxygenases (PMO’s) that directly oxidize gly- coside bonds generating aldones and lactones have been discovered highlighting the role of oxidation reactions in the breakdown of biomass components (Beeson et al. 2012; Horn et al. 2012; Langston et al. 2011; Quinlan et al. 2011; Vaaje-Kolstad et al. 2010). gl p In experiments using biomass substrates and deriva- tives, the glucose was replaced with 1.0% (w/v) of com- mercial microcrystalline cellulose (EC) (Celuflok 200™, Celuflok Ind. Com. São Paulo, Brazil), “in natura” milled (200-μm particle size) sugar cane bagasse (SCBIN), lignin removed (sodium hydroxide extracted) and steam exploded sugar cane bagasse (SCBDL), steam exploded sugar cane bagasse only (SCBSE), wheat bran (WB), milled soybeans (SM), and fructooligosaccharides (FOS). Page 2 of 12 dos Santos et al. AMB Expr (2016) 6:103 Shaken flask experiments Here we report on the efficiency of biomass biocon- version by Myceliophthora thermophila M77, whereas in a traditional bioreactor, the fungus completely con- sumes biomass sources (sugar cane bagasse) but shows little cellulase filter paper activity, leading the research to determine global secretome composition of M. ther- mophila growing on biomass and purified biomass com- ponents (cellulose and hemicellulose). When purified cellulose was available, the fungus produced a secretome that included hydrolytic and oxidative enzymes, almost exclusively dedicated to the breakdown of cellulose and cellulose related molecules. When natural biomass was available, the fungus produced a comprehensive collec- tion of enzymes in addition to cellobiose dehydrogenase involved in oxidation and hydrolysis of cellulose, hemi- cellulose-pectin and lignin. Twenty millilitre of pre-inoculum was added to 1 L Erlen- meyer flasks containing 200 mL of Mandels and Stern- berg salts, 0.1% peptone amended with 1% (w/v) SCBSE, SCBDL, WB, EC, SM, glycerol (GLY), lactose (LAC), sucrose (SUC) and FOS alone or in combinations and proportions as indicated. Incubations were made in an orbital shaker (Innova 44R Stackable Incubator Shaker, New Brunswick, NJ, USA) for up to 120 h at 45 °C and 250 rpm and samples withdrawn daily for enzyme activ- ity and protein quantifications. Strains, media, solutions and biomass sources Sugar cane bagasse sources were prepared and chemi- cally defined as described in (Rocha et al. 2011) and milled powders washed with water and autoclaved prior to use. A direct role of cellobiose dehydrogenase on cellulose depolymerization via the oxidation of glycoside bonds aided by Fenton chemistry has been suggested (Canevas- cini et al. 1991; Divne et al. 1994; Henriksson et al. 2000a; Mansfield et al. 1997; Mason et al. 2003; Stahlberg et al. 1996; Westermark and Eriksson 1975; Zamocky et al. 2006). Moreover, the participation of cellobiose dehydro- genase in oxidation of other biomass components such as lignin has also been considered (Henriksson et al. 2000b; Hilden et al. 2000).fi Materials and methods and fitted into an enzyme binding site where catalytic Asp or Glu residues hydrolyze through a general acid/ base mechanism the glycoside bond (Divne et al. 1994). The disconnection of the glucan chain from crystalline cellulose fibers has been proposed to be the bottleneck in enzymatic hydrolysis of cellulose (Himmel and Bayer 2009). Enzymatic activity assays Myceliophthora thermophila M77 was grown in Erlen- meyer flasks on Mandels & Sternberg salts, 0.1% peptone containing SCBIN (natural sugar cane bagasse, milled at 200 μm particle size) as well as modified sugar cane bagasse versions such as SCBDL (delignified with sodium hydroxide), SCBSE (steam exploded), purified celluloses containing 0.5% of avicel and 0.5% carboxymethylcellu- lose (Sigma Aldrich, St Louis MO), purified hemicellu- loses containing 0.2% of each; birchwood-, beechwood-, oat spelt-xylan, arabinan and arabinoxylan (Megazyme International, Wicklow, Ireland) and glucose (control). Cellulase activity was determined by the method of Ghose (1987) that measures the release of detectable reducing sugars removed from filter paper (FPase). Xylanase activ- ity was determined by the method described by Bailey and Poutanen (1989). Both FPase and xylanase activities were performed measuring reducing sugars by the dini- trosalicylic acid (DNS) method (Miller 1959), using glu- cose and xylose standards as appropriate. β-glucosidase and cellobiohydrolase was measured using p-nitrophenol- β-d-glucopyranoside (pNPG) and p-nitrophenol-β-d- cellobioside (pNPC) (Sigma-Aldrich, USA) as substrate, respectively (Zhang et al. 2009). Total protein content was measured in micro plates using the Bio-Rad assay reagent (Bio-Rad Laboratories, Hercules, USA), using a procedure based on the Bradford method (Bradford 1976) with bovine serum albumin as standard. One enzyme unit (IU) corre- sponded to the amount of product (μmol) produced per minute and cellulase activity was expressed as filter paper units (FPU) calculated according to (Ghose 1987). Cellobi- ose dehydrogenase activity was assayed through 2,6-dichlo- rophenol-indophenol (DCPIP) reduction. The decrease in absorbance was measured continuously at 520 nm (ε = 6.8 × 103 M−1 cm−1) in sodium acetate buffer (50 mM; pH 5) containing DCPIP 0.3 mM, sodium lactate 30 mM and NaF 4 mM. One enzyme unit (IU) corresponded to the amount of enzyme reducing 1 μmol of DCPIP per minute (Baminger et al. 1999). Laccase activity was measured con- tinuously by the oxidation rate of ABTS2+ to ABTS●+ at 420 nm (ε = 3.6 × 104 M−1 cm−1) in acetate buffer (50 mM; pH 3.5) containing ABTS (5 mM) in a final volume of 2 mL at 25 °C. One enzyme unit (IU) corresponded to the amount of enzyme that oxidized 1 µmol of ABTS per min- ute (Bourbonnais et al. 1995). Secretome peptide mapping by liquid Secretome peptide mapping by liquid chromatography‑tandem mass spectrometry (LC–MS/MS) For secretome peptide mapping experiments two inde- pendent cultures and two protein separations through SDS-PAGE were carried out. For secretome LC–MS/MS analysis 20–30 μg of total secretome proteins were loaded onto an SDS-PAGE gel and while in Fig. 3 we show a fully resolved SDS-PAGE gel for proteomics experiments, for proteomics the SDS-PAGE was run for only about one inch into the 12% separation gel, stained with Comas- sie blue and the entire protein banding profile excised, processed for LC–MS/MS according to (Shevchenko et al. 1996) with modifications. Isolated gel bands were reduced with Tris (2-carboxyethyl) phosphine, alkylated by 2-Iodoacetamide, digested for 6–16 h with 8 μg/mL trypsin using ammonium bicarbonate buffer and ana- lyzed by LC–MS/MS using LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Scientific, Waltham, MA, Bioreactor experiments Bioreactor assays were performed in a lab-scale Bio- flo®115 (New Brunswick, NJ, USA) with a working vol- ume of 1.5 L, using SCBSE, WB and sucrose as carbon sources. The pre-inoculum was 10% of the final volume. Page 3 of 12 dos Santos et al. AMB Expr (2016) 6:103 dos Santos et al. AMB Expr (2016) 6:103 Cultivations were conducted in batch or pulse-fed batch mode (as indicated in Figures and Tables), the dis- solved O2 concentration was >30 % of air saturation and mechanical stirring was performed with two Rushton- type impellers, in the range of 200–400 rpm. Prior to use all equipment was sterilized for 30 min at 121 °C. Automatic pH control was done using a 0.4 M HCl and NH4OH aqueous solution 3:1 (v/v) and foaming was con- trolled as required by manual addition of sterile antifoam polypropylene glycol 2000 (Dow Chemical, São Paulo, Brazil). Samples were withdrawn under sterile conditions daily, centrifuged at 12,000 rpm for 25 min at 4 °C and supernatants collected for cellulase (FPase), xylanase, β-glucosidase activity and total protein quantification. 50 mM pH 5, 5% (w/v) of substrate (EC, SCBIN, SCBDL or SCBSE) and a protein load of 0.05 mg/g of glucan, incubated at 50 °C at 200 rpm. All experiments were per- formed in duplicates. Samples were withdrawn at 0, 6, 12, 24 and 48 h and the glucose, gluconic acid, cellobi- ose, cellobionic acid, xylose, arabinose, acetic, formic and levulinic acid concentrations were quantified by HPLC Dionex Ultimate 300 system equipped with a refractive index detector (HPLC-RI) using an Aminex ®HPX-87H column and eluted with 5 mM H2SO4 at 0.6 mL/min. Sugars and acids in control samples containing only the respective substrate and citrate buffer 50 mM pH 5.0 were also measured. All samples were filtered using a Millex TM 0.22 μm filter prior to further analysis. Enzymatic activity assays Secreted proteins were collected after a 36 h cultiva- tion period at 45 °C, 200 rpm supernatants cleared by centrifugation (5000×g), concentrated by ultra-filtration (10,000 MWCO, PES membrane, Vivaspin, Littleton USA), rinsed twice with 5 mL of sodium acetate buffer 50 mM pH 5 and the proteins were separated by SDS- PAGE (Weber and Osborn 1969). Enzymatic biomass hydrolysis Bioconversion assays were conducted in 50 mL (125 mL Erlenmeyer flasks) final volumes, buffered with citrate dos Santos et al. AMB Expr (2016) 6:103 Page 4 of 12 or sucrose (SUC) resulted in a slight increase in xylanase activity (Table 1). USA). For this analysis, an Eksigent LC pump was used to separate peptide populations on analytical C18 nano- columns, with the column effluent being sprayed directly into a New Objective Picoview ion source. Using a “Top Three” MS/MS method, the Orbitrap analyzer collected accurate (5 ppm) scans of intact peptides for one sec- ond, at the same time as the LTQ ion trap simultaneously performed MS/MS fragmentation analysis of each of the three most abundant peptides eluting in that 1 s chroma- tographic fraction (0.8 Da mass accuracy).hi USA). For this analysis, an Eksigent LC pump was used to separate peptide populations on analytical C18 nano- columns, with the column effluent being sprayed directly into a New Objective Picoview ion source. Using a “Top Three” MS/MS method, the Orbitrap analyzer collected accurate (5 ppm) scans of intact peptides for one sec- ond, at the same time as the LTQ ion trap simultaneously performed MS/MS fragmentation analysis of each of the three most abundant peptides eluting in that 1 s chroma- tographic fraction (0.8 Da mass accuracy). Table 2 shows a series of six bioreactor runs in which we varied pH and temperature, feeding schedule as well as the combination of biomass sources, designed to over- come process side effects such as the possible interference of proteases and the onset of carbon catabolite repression. With the exception of the presence of sucrose (Table 2, run #5) that doubled the amount of cellulase, none of the other variations seemed to enhance filter paper activity. i Figure 2 describes enzymatic biomass hydrolysis into sugars and corresponding aldonic acids of various forms of sugar cane bagasse, “in natura” (SCBIN), delignified (SCBDL), steam-exploded (SCBSE) and purified cellulose (EC) by a crude enzymatic cocktail from M. thermoph- ila M77 produced in a bioreactor with SCBIN as the carbon source. After 24 h incubation period, 6.31 g/L of glucose and gluconic acid was produced from cellulose (EC) and 4.31, 3.16 and 1.83 g/L from SCBIN, SCBDL and SCBSE, respectively (Fig. 2a). Enzymatic biomass hydrolysis When the conversion potential of each carbon source was considered a 19.53% conversion was determined for SCBIN and 15.89, 8.02 and 7.63% con- version for EC, SCBDL and SCBSE, respectively (Fig. 2b). The LC–MS/MS raw files were used for database Mascot (version 2.2.04, Matrix Science, London UK) searches run on a NCBI M. thermophila ATCC_42464 specific subset. The DNA and amino acid sequence of M. thermophila M77 are 98.95 and 99.45% identical to M. thermophila ATCC_42464, respectively. Searches were validated using Scaffold (version 4.0.7, Proteome Soft- ware Inc. Portland, OR) with a protein threshold of 5% FDR and a peptide threshold of 99%. Further manage- ment of spectral data were performed on downloaded Excel files, total spectral counts (TSC) were normalized (against the total spectral count of each sample) and finally duplicates averaged (Additional file 1: Table S1). Thus, the quantitative value NTSC (normalized total spectrum counts) for a given protein component of a secretome reflects the amount of protein secreted as a direct response to the applied carbon source. Secretome protein compositionh ( The secretome (all extracellular non-anchored proteins) produced by M. thermophila M77 grown in various car- bon sources; SCBIN, SCBDL and SCBSE as well as puri- fied cellulose (avicel and carboxymethylcellulose) and hemicelluloses (xylans, arabinan and arabinoxylan) were determined through LC–MS/MS (Additional file 1: Table S1; Figs. 4, 5). Total extracellular proteins (secretomes) were collected, concentrated by ultra-filtration (10 kDa cutoff), separated by SDS-PAGE, digested with trypsin, subjected to LC–MS/MS and peptides assigned through Mascot and Scaffold to M. thermophila ATCC_42464 predicted proteins. In total, 172 proteins were unambigu- ously identified with positive matching of 21,766 unique peptides (4019 SCBIN, 3661 SCBDL, 4269 SCBSE 3466 celluloses and 4716 hemicelluloses). The spectral counts from two independent experiments were normalized and duplicates averaged in order to enable quantitative com- parisons between samples (see Additional file 1: Table S1).i Bioreactors and shakers producing biomass‑degrading enzymes Figure 1 reports a typical bioreactor experiment in which the carbon source was SCBSE (steam-exploded sugar cane bagasse). Extracellular protein and expected enzyme activities, cellulase (measured as activity on filter paper (FPU), xylanase and β-glucosidase accumulated in the medium over time reaching a peak at or around 96 h.l Table 1 shows a series of shaking flask experiments modifying the forms of biomass and combinations with simple carbon sources such as glycerol, lactose, sucrose and glucose designed to improve enzyme production. The highest cellulase activity, as judged by the DNS assay, was observed with steam-exploded biomass (0.23 FPU/ mL) while lignin extracted biomass showed lower cel- lulase activity (0.10 FPU/mL) and other sources such as wheat bran and purified cellulose as well as combina- tions thereof did not improve cellulase activity (Table 1). Thus, none of the biomass variants produced significant improvement over cellulase activity (FPU). For xylanase activity a similar picture occurs, none of the biomass derivatives improve drastically xylanase activity, how- ever the addition of a non-repressive carbon source such as lactose (LAC), phospho-fructo-oligosacharides (FOS) i Figure 3 shows SDS-PAGE protein profiles of enzymes secreted to the medium as a response to sugar cane bagasse, purified cellulose and hemicellulose and Fig. 4 displays secretome protein abundance profiles of M. ther- mophila M77 grown with purified cellulose (left panel) and a mixture of purified hemicelluloses (right panel). All major proteins in hemicellulose were associated with hemicellulose and pectin breakdown while in cellulose all major proteins were related to cellulose hydrolysis or oxidation. dos Santos et al. AMB Expr (2016) 6:103 Page 5 of 12 Fig. 1 Myceliophthora thermophila M77 fed-batch bioreactor with steam exploded sugar cane bagasse (SCBSE) as the carbon source. Bioreactors containing Mandels and Sternberg salts amended with 0.1% peptone and 1% steam exploded sugar cane bagasse (SCBSE) were conducted for 120 h at 45 °C, constant pH 5.0 and feed-pulsed with SCBSE at the indicated time points (arrows). Extracellular protein accumulation (shaded sym- bols), cellulase (a), xylanase (b) and β-glucosidase (c) activities were followed in a single feed-pulse (open symbols) and a double feed-pulse (closed symbols) regimen Fig. 1 Myceliophthora thermophila M77 fed-batch bioreactor with steam exploded sugar cane bagasse (SCBSE) as the carbon source. Bioreactors and shakers producing biomass‑degrading enzymes Bioreactors containing Mandels and Sternberg salts amended with 0.1% peptone and 1% steam exploded sugar cane bagasse (SCBSE) were conducted for 120 h at 45 °C, constant pH 5.0 and feed-pulsed with SCBSE at the indicated time points (arrows). Extracellular protein accumulation (shaded sym- bols), cellulase (a), xylanase (b) and β-glucosidase (c) activities were followed in a single feed-pulse (open symbols) and a double feed-pulse (closed symbols) regimen In conditions where cellulose was the sole carbon source (Fig. 4, left panel) the most abundant proteins in the secretome were: GH7 cellobiohydrolase A (~10% of secretome), AA3 cellobiose dehydrogenase A (~7% of secretome), GH7 cellobiohydrolase B and C (~6 and ~4% of secretome, respectively), GH6 cellobiohydrolase A and AA3 cellobiose dehydrogenase B (~3% of each). A, catalase, hypothetical protein (MYCTH_2307339) and GH18 chitinase A contributed with less than 3% of secretome, each. Polysaccharide monooxygenases (seven in total) contributed with less than 1% each (Fig. 5). AA3 cellobiose dehydrogenase A (CdhA) on the other hand was the second most abundant protein (Fig. 4), accounting for about 6% of the secretome pro- tein content.i When M. thermophila M77 was grown in SCBIN, GH7 cellobiohydrolase A and AA3 cellobiose dehydro- genase A were the most abundant proteins both con- tributing with about 6% of the secretome each (Fig. 5), while other proteins such as GH55 β-1,3-glucanase, GH7 cellobiohydrolase B and C, GH81 endo-1,3-β- glucanase, GH74 xyloglucanase, GH43_62_32_68 ara- binoxylanase, GH31 α-xylosidase, GH3 β-glucosidase We thus decided to concisely define the core cellulose secretome (Fig. 5) and further corroborate gene/pro- tein complements. Typically, besides the classical cel- lobiohydrolase β-glucosidase set of proteins, cellobiose dehydrogenase, a glyoxal oxidase and an unknown GMC oxidoreductase make up the core cellulose secretome (Fig. 5). dos Santos et al. AMB Expr (2016) 6:103 Page 6 of 12 Table 1 Myceliophthora thermophila enzyme accumulation in shaking flask bioreactors SCBSE steam-exploded sugar cane bagasse; SCBDL delignified steam-exploded sugar cane bagasse; EC celuflok 200™; WB wheat bran; SM soybean mill; GLY glycerol; LAC lactose; SUC sucrose; FOS commercial phosphofructooligosacharide. Bioreactors and shakers producing biomass‑degrading enzymes Bioreactor time (h) of peak activity (p) Cellulase Xylanase β-glucosidase Protein FPU/mL p (h) IU/mL p (h) IU/mL p (h) mg/mL p (h) SCBSE 0.23 ± 0.03 48 2.90 ± 0.06 48 0.43 ± 0.10 72 0.12 ± 0.01 120 SCBDL 0.10 ± 0.01 48 2.60 ± 0.20 72 0.33 ± 0.06 96 0.07 ± 0.01 96 WB 0.12 ± 0.02 48 2.00 ± 0.04 24 1.00 ± 0.04 120 0.15 ± 0.02 120 EC 0.10 ± 0.01 48 2.60 ± 0.14 72 0.33 ± 0.08 96 0.04 ± 0.01 48 SCBSE + WB 0.10 ± 0.02 72 2.80 ± 0.05 72 0.55 ± 0.05 72 0.13 ± 0.02 96 SCBSE + SM 0.13 ± 0.02 72 2.50 ± 0.10 48 0.53 ± 0.07 120 0.24 ± 0.03 96 SCBSE + SM 0.13 ± 0.03 72 2.80 ± 0.08 72 0.42 ± 0.05 120 0.10 ± 0.01 120 3XSCBSE + 1XFOS – 3.50 ± 0.05 48 – 0.11 ± 0.01 24 1XSCBSE + 1XLAC – 3.50 ± 0.10 72 – 0.08 ± 0.01 24 3XSCBSE + 1XLAC 0.19 ± 0.02 72 3.10 ± 0.08 72 0.57 ± 0.05 120 0.12 ± 0.01 96 3XSCBSE + 1XGLY 0.18 ± 0.02 96 2.80 ± 0.05 72 0.46 ± 0.09 120 0.10 ± 0.02 48 3XSCBSE + 1XSUC 0.18 ± 0.04 96 3.30 ± 0.12 72 0.43 ± 0.08 120 0.10 ± 0.01 120 Table 1 Myceliophthora thermophila enzyme accumulation in shaking flask bioreactors SCBSE steam-exploded sugar cane bagasse; SCBDL delignified steam-exploded sugar cane bagasse; EC celuflok 200™; WB wheat bran; SM soybean mill; GLY glycerol; LAC lactose; SUC sucrose; FOS commercial phosphofructooligosacharide. Bioreactor time (h) of peak activity (p) SCBSE steam-exploded sugar cane bagasse; SCBDL delignified steam-exploded sugar cane bagasse; EC celuflok 20 LAC lactose; SUC sucrose; FOS commercial phosphofructooligosacharide. Bioreactors and shakers producing biomass‑degrading enzymes thermophila M77 secretomes developed on a variety of carbon sources; sugar cane bagasse (SCB) in natura (SCBIN), delignified (SCBDL) and steam-exploded (SCBSE) as well as purified cellulose (CEL) and hemicellulose (HCEL) were determined through LC–MS/MS from SDS-PAGE separated proteins. 172 proteins were identified with positive matching of 21,766 unique peptides (4019 SCBIN, 3661 SCBDL, 4269 SCBSE 3466 celluloses and 4716 hemicelluloses). CEL, purified cellulose mixture containing 0.5% of avicel and 0.5% carboxymethylcellulose; HCEL, purified hemicellulose mixture containing 0.2% of each; birchwood-, beechwood-, oat spelt-xylan, arabinan and arabinoxylan; SCBIN, milled “in natura” sugar cane bagasse; SCBDL, steam exploded and delignified with sodium hydroxide milled sugar cane bagasse and SCBSE, steam exploded milled sugar cane bagasse. +LIG, lignin present; -LIG, lignin absent; M.W., molecular weight markers shown in kDa Fig. 3 Myceliophthora thermophila M77 secretome composition. M. thermophila M77 secretomes developed on a variety of carbon sources; sugar cane bagasse (SCB) in natura (SCBIN), delignified (SCBDL) and steam-exploded (SCBSE) as well as purified cellulose (CEL) and hemicellulose (HCEL) were determined through LC–MS/MS from SDS-PAGE separated proteins. 172 proteins were identified with positive matching of 21,766 unique peptides (4019 SCBIN, 3661 SCBDL, 4269 SCBSE 3466 celluloses and 4716 hemicelluloses). CEL, purified cellulose mixture containing 0.5% of avicel and 0.5% carboxymethylcellulose; HCEL, purified hemicellulose mixture containing 0.2% of each; birchwood-, beechwood-, oat spelt-xylan, arabinan and arabinoxylan; SCBIN, milled “in natura” sugar cane bagasse; SCBDL, steam exploded and delignified with sodium hydroxide milled sugar cane bagasse and SCBSE, steam exploded milled sugar cane bagasse. +LIG, lignin present; -LIG, lignin absent; M.W., molecular weight markers shown in kDa Fig. 3 Myceliophthora thermophila M77 secretome composition. M. thermophila M77 secretomes developed on a variety of carbon sources; sugar cane bagasse (SCB) in natura (SCBIN), delignified (SCBDL) and steam-exploded (SCBSE) as well as purified cellulose (CEL) and hemicellulose (HCEL) were determined through LC–MS/MS from SDS-PAGE separated proteins. 172 proteins were identified with positive matching of 21,766 unique peptides (4019 SCBIN, 3661 SCBDL, 4269 SCBSE 3466 celluloses and 4716 hemicelluloses). CEL, purified cellulose mixture containing 0.5% of avicel and 0.5% carboxymethylcellulose; HCEL, purified hemicellulose mixture containing 0.2% of each; birchwood-, beechwood-, oat spelt-xylan, arabinan and arabinoxylan; SCBIN, milled “in natura” sugar cane bagasse; SCBDL, steam exploded and delignified with sodium hydroxide milled sugar cane bagasse and SCBSE, steam exploded milled sugar cane bagasse. Bioreactors and shakers producing biomass‑degrading enzymes Bioreactor time (h) of peak activity (p) Table 2 Substrate influence on enzyme accumulation in bioreactors SCBSE steam-exploded sugar cane bagasse; WB wheat bran; SUC sucrose; T temperature; (p), peak activity Run Bioreactor conditions Cellulase Xylanase β-glucosidase Protein Substrate T(°C) pH Pulse FPase/mL p (h) IU/mL p (h) IU/mL p (h) mg/mL p(h) #1 SCBSE 45 5 96 0.10 ± 0.01 72 3.88 ± 0.12 72 0.37 ± 0.04 120 0.06 ± 0.02 120 #2 SCBSE 45 5 48/96 0.13 ± 0.03 120 1.93 ± 0.13 96 0.30 ± 0.06 96 0.08 ± 0.02 96 #3 SCBSE 45 6 48/96 0.10 ± 0.02 96 1.80 ± 0.03 48 0.48 ± 0.03 120 0.10 ± 0.04 96 #4 SCBSE 38–29 6 96 0.18 ± 0.03 120 2.00 ± 0.01 120 0.44 ± 0.02 96 0.17 ± 0.01 120 #5 SCBSE + SUC 45 6 None 0.21 ± 0.02 24 2.70 ± 0.07 48 0.72 ± 0.01 120 0.16 ± 0.05 24 #6 WB 45 6 None 0.06 ± 0.01 24 2.88 ± 0.01 24 1.64 ± 0.10 96 0.13 ± 0.01 24 Table 2 Substrate influence on enzyme accumulation in bioreactors SCBSE steam-exploded sugar cane bagasse; WB wheat bran; SUC sucrose; T temperature; (p), peak activity Fig. 2 Conversion of various forms of sugar cane bagasse and crystalline cellulose into glucose, gluconic acid, cellobiose and cellobionic acid by M. thermophila M77 enzymes. M. thermophila M77 secretome produced on “in natura” sugar cane bagasse was concentrated and used to hydrolyze SCBIN (closed symbols), EC (cellufloc, dark shaded symbols), SCBDL (light shaded symbols) and SCBSE (±) at 50 °C. In a the release of glucose and gluconic acid is shown and in b the conversion percentage from total available sugars was estimated Fig. 2 Conversion of various forms of sugar cane bagasse and crystalline cellulose into glucose, gluconic acid, cellobiose and cellobionic acid by M. thermophila M77 enzymes. M. thermophila M77 secretome produced on “in natura” sugar cane bagasse was concentrated and used to hydrolyze SCBIN (closed symbols), EC (cellufloc, dark shaded symbols), SCBDL (light shaded symbols) and SCBSE (±) at 50 °C. In a the release of glucose and gluconic acid is shown and in b the conversion percentage from total available sugars was estimated dos Santos et al. AMB Expr (2016) 6:103 Page 7 of 12 Fig. 3 Myceliophthora thermophila M77 secretome composition. M. Bioreactors and shakers producing biomass‑degrading enzymes +LIG, lignin present; -LIG, lignin absent; M.W., molecular weight markers shown in kDa Cellulose hydrolases such as cellobiohydrolase A, B, C and D, GH74 xyloglucanase, GH3 β-glucosidase and GH81 endo-β-1,3 glucanase did not adjust in abun- dance between the three types of biomass. In addition, hemicellulose hydrolases such as GH55 β-1,3-glucanase, GH43_62_32_68 arabinoxylanase, GH7 α-mannosidase and GH2 β-galactosidase also did not vary significantly in abundance among those three biomass substrates. at differentiated levels in various forms of biomass and then sharply decreased and disappeared from the bio- mass cultures. β-glucosidase activity followed cellobio- hydrolase with no difference in various biomass sources and steady accumulation over time. Laccase however poorly accumulated at the early stages of growth and then disappeared. Discussion Figure 6 shows robust cellobiohydrolase, β-glucosidase and cellobiose dehydrogenase activity presence in the fluid of cultures grown on various forms of sugar cane bagasse (SCB). While cellobiohydrolase activity was pre- sent at similar levels in all biomass sources accumulat- ing over a 4-day period and remained steady for up to 15 days cellobiose dehydrogenase accumulated for 9 days Enzymatic cocktails are typically evaluated by their bio- mass conversion ability, which is specified by the types of enzymes involved in cellulose breakdown producing sug- ars (glucose and cellobiose) and their respective aldonic acids. Experiments aimed at the production of cellulases using sugar cane bagasse as the carbon source yielded dos Santos et al. AMB Expr (2016) 6:103 Page 8 of 12 Fig. 4 Biomass secretomes. M. thermophila M77 secretomes produced on cellulose (left panel), a mixture of 0,5% avicel and 0.5% carboxymethyl- cellulose, or hemicellulose (right panel), a mixture of 0.2% of each of three types of xylan, arabinan and arabinoxylan (see “Materials and methods” section) were analyzed by LC–MS/MS and protein abundance reported as normalized spectral counts. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Fig. 4 Biomass secretomes. M. thermophila M77 secretomes produced on cellulose (left panel), a mixture of 0,5% avicel and 0.5% carboxymethyl- cellulose, or hemicellulose (right panel), a mixture of 0.2% of each of three types of xylan, arabinan and arabinoxylan (see “Materials and methods” section) were analyzed by LC–MS/MS and protein abundance reported as normalized spectral counts. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Fig. 4 Biomass secretomes. M. thermophila M77 secretomes produced on cellulose (left panel), a mixture of 0,5% avicel and 0.5% carboxymethyl- cellulose, or hemicellulose (right panel), a mixture of 0.2% of each of three types of xylan, arabinan and arabinoxylan (see “Materials and methods” section) were analyzed by LC–MS/MS and protein abundance reported as normalized spectral counts. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Fig. 4 Biomass secretomes. M. Discussion thermophila M77 secretomes produced on cellulose (left panel), a mixture of 0,5% avicel and 0.5% carboxymethyl- cellulose, or hemicellulose (right panel), a mixture of 0.2% of each of three types of xylan, arabinan and arabinoxylan (see “Materials and methods” section) were analyzed by LC–MS/MS and protein abundance reported as normalized spectral counts. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 SCBIN and 15.89, 8.02 and 7.63% conversion for EC, SCBDL and SCBSE was observed, respectively (Fig. 2b). However, considering our experiment, the highest cel- lulase activity detected (0.23 FPU/mL) was applied on SCBIN 5% (w/v) representing an enzyme loading of only ~5 FPU/g of glucan in the hydrolysis experiment, making the observed 20% conversion rate unjustifiable, since other authors have loaded higher filter paper units (FPU) to get similar conversion rates (Adsul et al. 2005; da Silva et al. 2010; Ishihama et al. 2005; Pietrobon et al. 2011; Visser et al. 2015). enzyme cocktails with little FPase activity even though the fungus aggressively digested the food source (Figs. 1, 2; Tables 1, 2). We designed a series of bioreactor experiments (Table 2) to overcome process limitations. With the exception of the presence of sucrose (Table 2, run #5) that doubled the amount of cellulase, none of the other variations seemed to enhance filter paper activity. Other fungal systems such as Aspergillus nidulans and Phanero- chaete chrysosporium produce similar low levels of cellu- lases and xylanase when growing on solid sorghum stover (Ray et al. 2012; Saykhedkar et al. 2012). Measuring cellulose degradation by methods that only detect hydrolysis mechanisms (for instance FPase activity) may not be sufficient to evaluate the cellulose breakdown power of these enzyme mixtures because the fungus may secrete enzymes that instead of hydrolyzing cellulose, oxidize glycosidic linkages instead. We than designed a biomass hydrolysis experiment using the M. thermophila M77 enzymatic cocktail and determined the release of glucose and corresponding gluconic acid. Nevertheless, when the conversion poten- tial of each carbon source was considered, 19.53% for dos Santos et al. AMB Expr (2016) 6:103 Page 9 of 12 Fig. 5 Core cellulose secretome. M. thermophila M77 secretomes produced on purified cellulose (CEL, light shaded bars) and sugar cane bagasse (SCBIN, dark shaded bars). Total extracellular protein abundance was determined by LC–MS/MS and reported as normalized spectral counts. Discussion Each bar indicates the normalized spectral counts of one enzyme and abundance ranking was done for SCBIN. Seven AA9 lytic polysaccharide monooxy- genases did not rank at the top of any of the studied biomass sources however were added to the present table. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Fig. 5 Core cellulose secretome. M. thermophila M77 secretomes produced on purified cellulose (CEL, light shaded bars) and sugar cane bagasse (SCBIN, dark shaded bars). Total extracellular protein abundance was determined by LC–MS/MS and reported as normalized spectral counts. Each bar indicates the normalized spectral counts of one enzyme and abundance ranking was done for SCBIN. Seven AA9 lytic polysaccharide monooxy- genases did not rank at the top of any of the studied biomass sources however were added to the present table. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Fig. 5 Core cellulose secretome. M. thermophila M77 secretomes produced on purified cellulose (CEL, light shaded bars) and sugar cane bagasse (SCBIN, dark shaded bars). Total extracellular protein abundance was determined by LC–MS/MS and reported as normalized spectral counts. Each bar indicates the normalized spectral counts of one enzyme and abundance ranking was done for SCBIN. Seven AA9 lytic polysaccharide monooxy- genases did not rank at the top of any of the studied biomass sources however were added to the present table. A detailed list of annotated protein names along with spectral and quantitative measurement data can be found in Additional file 1: Table S1 Secretome protein abundance profiles of M. thermoph- ila M77 grown with purified cellulose (Fig. 4, left panel) and a mixture of purified hemicelluloses (Fig. 4, right panel) were constructed. Secretome protein profiles were substrate specific reflecting the nature of the substrate. All major proteins in hemicellulose were associated with hemicellulose and pectin breakdown while in cellulose all major proteins were related to cellulose hydrolysis or oxidation. When grown on SCBIN, GH7 cellobiohydrolase (CbhA) and AA3 cellobiose dehydrogenase (CdhA) were the most abundant proteins both contributing with about 6% of the secretome each (Fig. 5), while other proteins contributed with less than 3% of secretome, each. The M. Discussion thermophila M77 CdhA is a complete cellobi- ose dehydrogenase, a flavin-dependent dehydrogenase connected through a flexible linker to a heme-binding cytochrome and a true cellulose-binding domain (CBM1) (Tan et al. 2015). CdhA generates electrons by oxidation of cellobiose (perhaps generated by CbhA) and longer cellodextrins to 1-5-δ-lactones (Westermark and Eriks- son 1975). Lactones hydrolyze spontaneously in solu- tion, or enzymatically by lactonases (also present in the secretome), to generate aldonic acids (Beeson et al. 2011). Electrons generated by the flavin-dependent dehy- drogenase are shuttled via heme-binding cytochrome to the recently discovered copper dependent polysaccharide monooxygenases (PMO’s) that in turn oxidize glycoside GH7 cellobiohydrolases (CbhA) act at the reducing end of a single cellulose chain and CbhA is the only major GH7 cellobiohydrolase that contains a cellulose-binding domain (CBM1). The presence of cellobiohydrolases devoid of cellulose binding domains, CbhB and CbhC in M. thermophila M77 secretomes followed similar observations made in other fungi suggesting that these CBM-devoid enzymes collaborate with CBM-bearing exo enzymes on cellulosic chains that have already been pulled apart from the crystalline fiber (Segato et al. 2012). dos Santos et al. AMB Expr (2016) 6:103 Page 10 of 12 Fig. 6 Major enzyme activities on solid biomass degradation. Cellobiohydrolase (a) and β-glucosidase (c) are the major hydrolases and cellobiose dehydrogenase (b) the major oxidase secreted by M. thermophila during growth on SCBIN (closed circles) “in natura” as well as SCBDL (shaded circles) delignified and SCBSE (open circles) “steam exploded” biomass. Other oxidative enzymes laccase (d), lignin peroxidase (not shown) and Mn-peroxi- dase (not shown) were detected at very low levels Fig. 6 Major enzyme activities on solid biomass degradation. Cellobiohydrolase (a) and β-glucosidase (c) are the major hydrolases and cellobiose dehydrogenase (b) the major oxidase secreted by M. thermophila during growth on SCBIN (closed circles) “in natura” as well as SCBDL (shaded circles) delignified and SCBSE (open circles) “steam exploded” biomass. Other oxidative enzymes laccase (d), lignin peroxidase (not shown) and Mn-peroxi- dase (not shown) were detected at very low levels bonds in crystalline cellulose, hemicellulose and pectin (Beeson et al. 2011; Canevascini et al. 1991). clearly annotated by bioinformatics remain unclear and undefined.hi Remarkably, Fig. 5 shows that when the fungus grew in biomass (SCBIN) or purified cellulose (CEL) AA3 cel- lobiose dehydrogenase A was present in about the same concentration (about 50%) while GH7 and GH6 cellobio- hydrolases were present at higher levels in CEL. References Adsul MG, Ghule JE, Shaikh H, Singh R, Bastawde KB, Gokhale DV, Varma AJ (2005) Enzymatic hydrolysis of delignified bagasse polysaccharides. Carbohydr Polym 62(1):6–10. doi:10.1016/j.carbpol.2005.07.010 Amorim HV, Lopes ML, de Castro Oliveira JV, Buckeridge MS, Goldman GH (2011) Scientific challenges of bioethanol production in Brazil. Appl Microbiol Biotechnol 91(5):1267–1275. doi:10.1007/s00253-011-3437-6 Bailey M, Poutanen K (1989) Production of xylanolytic enzymes by strains of Aspergillus. Appl Microbiol Biotechnol 30(1):5–10. doi:10.1007/bf00255989 Baminger U, Nidetzky B, Kulbe KD, Haltrich D (1999) A simple assay for meas- uring cellobiose dehydrogenase activity in the presence of laccase. J Microbiol Methods 35(3):253–259 Abbreviations l Baminger U, Nidetzky B, Kulbe KD, Haltrich D (1999) A simple assay for meas- uring cellobiose dehydrogenase activity in the presence of laccase. J Microbiol Methods 35(3):253–259 AA3: auxiliary activities 3; ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt; CBM: carbohydrate binding domain; DCPIP: 2,6-dichlorophenol-indophenol; DNS: dinitrosalicylic acid; EC: commercial microcrystalline cellulose; FDR: false discovery rate; FOS: fructoligosaccharides; FPase: filter paper cellulase activity; FPU: filter paper unit; HPLC: high perfor- mance liquid chromatography; IU: enzyme unit; GH3 7, 18, 74, 81: glycoside hydrolase family 3, 7,18, 74 and 81; GLY: glycerol; LAC: lactose; LC–MS/MS: liquid chromatography–tandem mass spectrometry; NCBI: National Center for Biotechnology Information (NIH, US); NTSC: normalized total spectral counts; PMO: polysaccharide monoxygenase; pNPC: p-nitrophenol-β-d-cellobioside; pNPG: p-nitrophenol-β-d-glucopyranoside; SCB: sugar cane bagasse; SCBDL: lignin removed (sodium hydroxide extracted) and steam exploded sugar cane bagasse; SCBIN: in natura” milled (200-μm particle size) sugar cane bagasse; SCBSE: steam exploded sugar cane bagasse; SDS-PAGE: SDS polyacrylamide gel electrophoresis; SM: milled soybeans; SUC: sucrose; TSC: total spectral counts; WB: wheat bran. 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Bioresour Technol 101(19):7402–7409. doi:10.1016/j. biortech.2010.05.008 Author details 1 1 Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil. 2 Laboratório de Microbiologia e Bioquímica Aplicada, Departamento de Biologia, IBILCE/UNESP, Rua Cristovão Colombo, 2265 Bairro Jd. Nazareth, São José do Rio Preto, SP CEP 15054‑000, Brazil. 3 Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Rua Giuseppe Máximo Scolfaro, 10.000, Bairro Guará, Campinas, SP CEP 13083‑970, Brazil. 4 Laboratório de Enzimologia, Instituto de Química, UNESP, Araraquara, São Paulo, Brazil. 5 Department of Microbiol- ogy and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA. 6 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA. Bradford MM (1976) A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the princi- ple of protein-dye binding. Anal Biochem 72(1–2):248–254. doi:10.1016/0003-2697(76)90527-3 Bradford MM (1976) A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the princi- ple of protein-dye binding. Anal Biochem 72(1–2):248–254. doi:10.1016/0003-2697(76)90527-3 Canevascini G, Borer P, Dreyer JL (1991) Cellobiose dehydrogenases of Sporotri- chum (Chrysosporium) thermophile. Eur J Biochem 198(1):43–52 Coutinho PM, Andersen MR, Kolenova K, vanKuyk PA, Benoit I, Gruben BS, Trejo- Aguilar B, Visser H, van Solingen P, Pakula T, Seiboth B, Battaglia E, Aguilar- Osorio G, de Jong JF, Ohm RA, Aguilar M, Henrissat B, Nielsen J, Stalbrand H, de Vries RP (2009) Post-genomic insights into the plant poly- saccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae. Fungal Genet Biol 46(Suppl 1):S161–S169 Abbreviations l doi:10.1038/nbt.1976 Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass- degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29(10):922–927. doi:10.1038/nbt.1976 Additional file Additional file 1. Comprehensive LC–MS/MS secretome analysis. Additional file 1. Comprehensive LC–MS/MS secretome analysis. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Authors’ contributions JGCP, EG and RAP conceived and designed the study. HBS, TMSB SDH JR conducted protein analysis experiments; JGCP, PD, DL and EG conducted bioreactor experiments, BC acquired data, performed the bioinformatics analysis and RAP drafted the manuscript. All authors read and approved the final manuscript. Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M (1995) Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2′- azinobis (3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolym- erization. Appl Environ Microbiol 61(5):1876–1880 Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M (1995) Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2′- azinobis (3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolym- erization. Appl Environ Microbiol 61(5):1876–1880 Availability of data and materials which may directly oxidize glycosidic and phenolic bonds by a currently unknown mechanism. Strains used in this study are available from the Fungal Genetics Stock Center (Kansas City, MO). Ancillary data are submitted as Additional file 1: Table S1. Myceliophthora thermophila M77 produces specific secretomes that mirror the cell wall composition, for- mulate a mixed set of enzymes that in addition to hydro- lyze glycoside bonds also promote coupled oxidation of cellulose and other biomass components enhancing the overall biomass degradation process. The secretome protein signature of M. thermophila M77 revealed cel- lobiose dehydrogenase (21% of the total secretome) as the major player in cellulose oxidation partnering per- haps with oxidation proteins such as glyoxal oxidase (4% of the secretome content) or glucose oxidase (not very abundant). The exact function of these enzymes remains uncertain. Discussion Thus, the CdhA mediated electrons could be transferred to a wide range of oxygenases (Hemsworth et al. 2013; Westermark and Eriksson 1975; Zamocky et al. 2006). Other enzymes such as GH31 α-xylosidase, GH18 chitinase A (but not chitinase B), GMC oxidoreductase (an unknown oxidore- ductase), GH43 xylosidase arabinosidase and AA5 gly- oxal oxidase, followed a similar pattern, low abundance in delignified biomass and abundant in whole forms of biomass. The function of GMC oxidoreductase (GloA), glyoxal oxidase (GoxA) and GH18 chitinase even though i Thus, based on the protein profile of secreted proteins and enzymatic activity detected it appears that the fun- gus accesses all available polymers, cellulose hemicellu- lose-pectin and lignin but does not produce hydrolysis products exclusively.h The participation of oxidation reactions coupled to lignin decomposition in the breakdown of cellulose chains (Beeson et al. 2012; Phillips et al. 2011), may explain the discrepancy observed between absolute FPase activity values in bioreactor experiments and the real (total) power of cellulose breakdown observed in bio- mass hydrolysis experiments. Furthermore, it is possible that CdhA, GloA and GoxA fail to interact and transfer electrons to cellulose, lignin and hemicellulose acceptor proteins, they generate an excess of hydrogen peroxide, dos Santos et al. AMB Expr (2016) 6:103 Page 11 of 12 Availability of data and materials Funding g Department of Energy, awards 06103-OKL, ZDJ-7-77608-01 and CNPq (Brazil) Science Without Borders Program awards 403090/2012-1 to RAP and 201319/2012-8 to HBS. Received: 18 October 2016 Accepted: 27 October 2016 Received: 18 October 2016 Accepted: 27 October 2016 annurev-micro-092611-150044 Hemsworth GR, Davies GJ, Walton PH (2013) Recent insights into copper- containing lytic polysaccharide mono-oxygenases. Curr Opin Struct Biol 23(5):660–668. doi:10.1016/j.sbi.2013.05.006 Henriksson G, Johansson G, Pettersson G (2000a) A critical review of cellobiose dehydrogenases. J Biotechnol 78(2):93–113 Henriksson G, Zhang L, Li J, Ljungquist P, Reitberger T, Pettersson G, Johans- son G (2000b) Is cellobiose dehydrogenase from Phanerochaete chrysosporium a lignin degrading enzyme? 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https://openalex.org/W1965238961	https://pubs.rsc.org/en/content/articlepdf/2013/an/c3an00872j	English	null	Optimization of Fe<sub>3</sub>O<sub>4</sub>@Ag nanoshells in magnetic field-enriched surface-enhanced resonance Raman scattering for malaria diagnosis	Analyst (London. 1877. Online)/Analyst	2,013	cc-by	6,979	Division of Bioengineering, School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457. E-mail: quanliu@ntu.edu.sg; Fax: +65 6791 1761; Tel: +65 6316 8748 Analyst View Article Online View Journal \| View Issue Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Analyst, 2013, 138, 6494 Clement Yuen and Quan Liu* The great potential of magnetic ﬁeld enriched surface enhanced resonance Raman spectroscopy (SERRS) for early malaria diagnosis has been demonstrated previously. This technique is able to detect b-hematin, which is equivalent to a malaria biomarker (hemozoin) in Raman features, at a concentration of 5 nM. In this study, we present the optimization of nanoparticles used in the magnetic ﬁeld enriched SERRS by tuning the core size and shell thickness of nanoparticles with an iron oxide core and a silver shell (Fe3O4@Ag). The discrete dipole approximation (DDA) model was introduced to investigate the localized electromagnetic ﬁeld distributions and extinction eﬃciencies of the aggregate of Fe3O4@Ag and b-hematin, in correlation with their magnetic ﬁeld enriched SERRS performance. We ﬁnd that the optimal core–shell size of Fe3O4@Ag leading to the eﬀective aggregation of Fe3O4@Ag and b-hematin under an external magnetic ﬁeld with superior extinction eﬃciencies is the key to realize highly augmented Raman signals in this strategy. Furthermore, it is noted that the optimized result diﬀers from the case without the external magnetic ﬁeld to that with the external magnetic ﬁeld. Therefore, this work demonstrates experimentally and theoretically the potential of tuning the core–shell Fe3O4@Ag for achieving the eﬃcient magnetic ﬁeld-enriched SERRS detection of b-hematin for early malaria diagnosis. Optimization of Fe3O4@Ag nanoshells in magnetic ﬁeld-enriched surface-enhanced resonance Raman scattering for malaria diagnosis Cl Y d Q Li * Cite this: Analyst, 2013, 138, 649 Received 29th April 2013 Accepted 20th August 2013 DOI: 10.1039/c3an00872j www.rsc.org/analyst cle. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. s article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Raman measurements of analytes Raman measurements of analytes that without the magnetic eld enrichment strategy. The discrete-dipole approximation (DDA) method,12 which is exible in handling irregular geometries,13 is exploited to evaluate the performance of Fe3O4@Ag with diﬀerent core–shell sizes in the magnetic eld-enriched SERRS technique. The aggregation of nanoshells due to the external magnetic eld for a range of core and shell sizes during SERRS measurement is also discussed. The preparation of analytes [Rhodamine 6G (R6G) and b-hematin], the setup for magnetic eld-enriched SERRS, and Raman instrumentation were the same as reported in the previous publication.8 Briey, for the SERS measurements of R6G (Sigma-Aldrich, USA), aqueous R6G at concentrations ranging from 106 to 109 M was adsorbed onto Fe3O4@Ag nanoshells. In the sample preparation for the SERRS evaluation of b-hematin with and without magnetic eld enrichment, b-hematin crystals were fabricated by using the same acid-catalyzed method as dis- cussed,8 at concentrations ranging from 103 to 107 M were mixed with an equal volume of Fe3O4@Ag nanoshells suspension of 0.33 ml and the number of nanoshells was about 5 1011. These analytes were transferred to a small vial made of aluminum foil for SERS measurements with the bottom of the vial being exposed to a magnetic eld of 0.198 T and a magnetic eld gradient of 26.6 T m1. These samples were excited by a 633 nm laser (Renishaw, UK) with an excitation power of 0.1 mW that was focused through a microscope objective (20, NA ¼ 0.4, Leica) to a spot about 3 mm in diameter. The emitted Raman signals were fed into a Czerny–Turner type spectrograph (f ¼ 250 mm) and a holographic grating (1800 g mm1) dispersed the incoming signal into a RemCam CCD detector (inVia, Renishaw, UK) with a spectral resolution of 2 cm1. All spectra were acquired with an integration time of 15 s and averaged from more than ve diﬀerent samples, in which the typical standard deviation of the signal intensity is smaller than 5% for R6G and smaller than 10% for b-hematin. These raw data were corrected for the baseline level, then smoothed by averaging over a window of ve points, and processed for removing the uorescence background to yield the nal spectra. Fabrication of core–shell Fe3O4@Ag nanoparticles The Fe3O4 nanoparticles were fabricated by a coprecipitation technique.14 0.168 M of FeCl2$4H2O (Alfa Aesar, USA) and 0.333 M of FeCl3$6H2O (Alfa Aesar, USA) were prepared and dissolved in deionized water, prior to the introduction of 4 M NaOH (Sigma-Aldrich, USA) drop-wise to the mixture at 60 C under vigorous mixing (15k rpm, SilentCrusherM, Heidolph, Ger- many). To synthesize Fe3O4 with three diﬀerent radii, the addition of NaOH was stopped at pH values of 11, 13, and 14, respectively, and the mixture was heated subsequently at 70 C for another 1 hour and cooled to room temperature under stirring, followed by separation using a magnet and then washing with deionized water. 16.2 mM of the fabricated Fe3O4 nanoparticles in 20 ml of ethanol was surface graed with 0.15 g of polyacrylic acid (Sigma-Aldrich, USA) in 80 ml of ethanol, prior to sonication (Elma E30H, Elma, Switzerland) for 15 min. Fe3O4 was separated with a magnet, followed by washing with ethanol. About 2.1 mM of Fe3O4 were re-dispersed in ethanol and deionized water (80.6 : 19.4% v/v) for Ag shell synthesis. The Ag shells were coated by using the seed-growth reduc- tion method.8,15 The number of Fe3O4 nanoparticles used, which was estimated by considering the weight and the size of these Fe3O4 nanoparticles, was kept identical in each of the following sets. For each size of Fe3O4, we coated the Ag shell with three diﬀerent thicknesses by introducing the following mixtures drop-wise to the aforesaid Fe3O4 suspension in an ultrasonic bath. Discrete-dipole approximation (DDA) model The discrete-dipole approximation (DDA) model12,16 was used to calculate the extinction eﬃciency (Qext) of Fe3O4@Ag nano- particles with diﬀerent core and shell dimensions under the excitation wavelength of 633 nm. Qext was calculated for comparison with the experimental SERS performance of these nanoparticles, since Qext and SERS signals are closely related to the induced electromagnetic eld (Eind).12,13 Qext is related to the induced electromagnetic eld by the imaginary (Im) part in the equation,12 Set 1. AgNO3 (2.8 mM, Merck, USA) was added and mixed for 30 min, prior to introduction of hydroxylamine hydrochloride (4.1 mM, MP biomedical, USA), NaOH (8.1 mM), in Triton X-100 (Bio-Rad laboratories, USA), ethanol, and deionized water (0.9 : 70.8 : 28.3% v/v/v); Set 2. The chemicals and their concentrations added were the same as the previous set prior to the introduction of AgNO3 (9.7 mM) in Triton X-100, ethanol, and deionized water (2 : 65.3 : 32.7% v/v/v). Qext¼ 4pk EO 2 X N j¼1 Im E* ind;jPj . aeff; (1) (1) Set 3. The same procedure as stated in set 2, except that 19.4 mM instead of 9.7 mM of AgNO3 was used during the second-time addition of AgNO3. where k is the wavevector, EO is the amplitude of the incident electromagnetic eld, Einc,j is the induced electromagnetic eld incident on the j-th dipole, with a polarization of Pj (j ¼ 1, 2, ., N) for N discretized cube-shaped dipoles with sides of length 5 nm and aeﬀis the eﬀective area. For SERS signals, the inten- sities can be approximated to be proportional to \|Eind\|4.17 Moreover, the electromagnetic eld distributions of these Fe3O4@Ag nanoparticles in close contact with the irregular- shaped b-hematin crystal were investigated. The ADDA code16 was used in the DDA calculations with the assumption that Set 4. Additional AgNO3 (9.7 mM) was mixed into set 3, prior to the introduction of Triton X-100, ethanol, and deionized water (2 : 65.3 : 32.7% v/v/v). Set 4. Additional AgNO3 (9.7 mM) was mixed into set 3, prior to the introduction of Triton X-100, ethanol, and deionized water (2 : 65.3 : 32.7% v/v/v). Finally, mixtures in these sets were washed and a magnet was used to separate the Fe3O4@Ag nanoparticles. The nal nanoparticles were obtained by ltering (0.2 mm supor syringe lters, Pall, USA) a suspension of the Fe3O4@Ag nanoparticles in 15 ml of methanol. Introduction Our result shows that the detection limit of this technique in terms of the parasitemia level is comparable to that in early malaria infection at the ring stage, which suggests the great potential of this technique for early malaria diag- nosis.8 The unique feature in this technique is that resonance In this paper, we present the trends in the magnetic eld- enriched SERRS performance of b-hematin as a function of the core and shell size of Fe3O4@Ag nanoshells in comparison to This journal is ª The Royal Society of Chemistry 2013 6494 \| Analyst, 2013, 138, 6494–6500 Analyst View Article Online Analyst View Article Online Paper This journal is ª The Royal Society of Chemistry 2013 Introduction Raman,9 surface enhanced Raman,6 and the magnetic eld enrichment10 eﬀects are integrated to give further augmentation of the Raman signal, which allows more sensitive b-hematin detection (at a concentration of 5 nM) than each of these aforesaid techniques alone.8 Hemozoin is a unique biomarker in human malaria disease with isostructural and paramagnetic properties similar to those of the chemically fabricated b-hematin.1–3 The detection of these crystals (hemozoin or b-hematin) has been demonstrated by Raman spectroscopy and its variations (e.g. resonance Raman, surface enhanced Raman, and tip-enhanced Raman spectroscopy).4–7 These techniques have the advantages of fast data acquisition, minimal need for labor, and minimal requirement of skilled workers, in comparison to the micro- scopic examination of a blood smear that is the “gold standard” of malaria diagnosis.8 Although the optimizations of core and shell size-correlated plasmonic properties (e.g. Raman performance, extinction, and electromagnetic eld distributions) for similar plasmonic structures have been studied in other experiments without an external magnetic eld,11 these core and shell-related proper- ties could be modied due to nanoparticle aggregation induced by the external magnetic eld. Moreover, b-hematin crystals, which are the target analyte generating Raman signals for enhancement, possess an elongated shape, a large size and paramagnetic properties compared to those commonly studied molecules such as Rhodamine 6G (R6G). These factors deter- mine that the optimization of nanoshells for the enhancement of b-hematin Raman signals would be diﬀerent from other common Raman molecules. In this study, we explore the variation in the core diameter and shell thickness of Fe3O4@Ag in relation to the magnetic eld-enriched SERRS of para- magnetic b-hematin crystals to maximize Raman signal enhancement. Recently, our group has demonstrated magnetic eld- enriched surface-enhanced resonance Raman spectroscopy (SERRS) for the sensitive detection of b-hematin by using nanoshells with an iron oxide core and a silver shell, i.e. Fe3O4@Ag. Discrete-dipole approximation (DDA) model This journal is ª The Royal Society of Chemistry 2013 Analyst, 2013, 138, 6494–6500 \| 6495 6495 View Article Online View Article Online Analyst Paper Fig. 2 (a) Experimental SERS spectra of R6G solution at concentrations of 106 M adsorbed on Fe3O4@Ag nanoparticles with core–shell parameters of C40S60, C40S50, C40S40, C40S10, C25S60, C25S45, C25S25, C25S10, C10S50, C10S40, C10S30, and C10S10. C10S50 means Fe3O4@Ag with a core radius of 10 nm and an outer shell thickness of 50 nm. (b) Raman peak intensities at 1508 cm1 (aromatic C–C stretching) for R6G solution at concentrations of 106, 107, 108, 109 and 1010 M by using these Fe3O4@Ag nanoparticles. these nanoparticles were comprised of Fe3O4, Ag, and b- hematin only, using electric constants18–20 reported in the literature. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2(a) shows the SERS spectra of aqueous R6G at a concentration of 1 mM adsorbed on these Fe3O4@Ag nano- particles whose SEM images are shown in Fig. 1 and Fig. 2(b) plots the corresponding R6G Raman peak intensities at 1508 cm1 (aromatic C–C stretching). R6G adsorbed on these nano- particles provides diﬀerent Raman enhancements [Fig. 2(a)], which are revealed by the Raman intensities of prominent peak locations at about 615 cm1 (C–C–C ring in-plane bending), 775 cm1 (CH out-of-plane bending), 1310 cm1 and 1365 cm1 (C– C/C–N stretching), and 1508 cm1 (aromatic C–C stretching).8 For the shell dimensions C40S60, C40S50, C40S40, C40S10, C25S60, C25S45, C25S25, C25S10, C10S50, C10S40, and C10S30, in which the rst number indicates the Fe3O4 core radius and the second number gives the shell thickness, the signal enhancement is roughly 36.0, 26.4, 6.2, 1.6, 23.9, 7.8, 3.9, 1.3, 4.7, 4.3, and 2.2 times higher than that observed for C10S10. Fig. 2 (a) Experimental SERS spectra of R6G solution at concentrations of 106 M adsorbed on Fe3O4@Ag nanoparticles with core–shell parameters of C40S60, C40S50, C40S40, C40S10, C25S60, C25S45, C25S25, C25S10, C10S50, C10S40, C10S30, and C10S10. C10S50 means Fe3O4@Ag with a core radius of 10 nm and an outer shell thickness of 50 nm. (b) Raman peak intensities at 1508 cm1 (aromatic C–C stretching) for R6G solution at concentrations of 106, 107, 108, 109 and 1010 M by using these Fe3O4@Ag nanoparticles. Fig. 1 FESEM images of Fe3O4@Ag nanoparticles with diﬀerent Fe3O4 core radii (C) and Ag shell thicknesses (S). (a) Raw 10 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 30, 40 and 50 nm. (b) Raw 25 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 25, 45 and 60 nm. (c) Raw 40 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 40, 50 and 60 nm. The scale bar indicates a length of 200 nm and is shared by all ﬁgures. These SERS intensities are linearly proportional to the R6G concentrations adsorbed on the Fe3O4@Ag with diﬀerent core and shell sizes [Fig. 2(b)], which are typical13 in SERS measurement. The general trend is that a larger Fe3O4 core and a thicker Ag shell yield higher Raman signal enhancement, with the highest Raman signal enhancement noted for C40S60. Results Fig. 1 shows the eld emission scanning electronic microscopy (FESEM, JEOL JSM-6700F, JEOL, Japan) images of the fabricated Fe3O4@Ag nanoparticles. These Fe3O4 nanoparticles show radii of (a) 10, (b) 25, and (c) 40 nm serving as the cores of nanoshells. The cores with a radius of 10 nm were coated with 10, 30, 40, and 50 nm thick Ag [Fig. 1(a)]. The cores with a radius of 25 nm were coated with 10, 25, 45 and 60 nm thick Ag [Fig. 1(b)]. The cores with a radius of 40 nm were coated with 10, 40, 50 and 60 nm thick Ag [Fig. 1(c)]. The total size typically had a range of less than 30 nm according to zetasizer measurements. The compositions of nanoshells were conrmed by taking energy- dispersive X-ray graphs, which show Fe, O, and Ag peaks origi- nated from the Fe3O4 core and Ag shell (result not shown). Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The trend in the enhancement factor with the shell and core dimensions of R6G conforms to the typical variation of the core and shell-related SERS performance. These SERS intensities are linearly proportional to the R6G concentrations adsorbed on the Fe3O4@Ag with diﬀerent core and shell sizes [Fig. 2(b)], which are typical13 in SERS measurement. The general trend is that a larger Fe3O4 core and a thicker Ag shell yield higher Raman signal enhancement, with the highest Raman signal enhancement noted for C40S60. The trend in the enhancement factor with the shell and core dimensions of R6G conforms to the typical variation of the core and shell-related SERS performance. Fig. 3 compares the diﬀerent enhancement trends in the representative SERRS spectra of b-hematin (a) without and (b) with the inuence of an external magnetic eld by using diﬀerent Fe3O4@Ag nanoparticles. Interestingly, the trend in magnetic eld-enriched SERRS performance is no longer a monotonic function of the core size or shell thickness of Fe3O4@Ag nanoparticles, based on intensities corresponding to the most prominent vibrational features of b-hematin, such as n15 (754 cm1, D4h notation system for resonance Raman peak studies on myoglobin),21 n22 (1120 cm1), n2 (1570 cm1), or n10 Fig. 1 FESEM images of Fe3O4@Ag nanoparticles with diﬀerent Fe3O4 core radii (C) and Ag shell thicknesses (S). (a) Raw 10 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 30, 40 and 50 nm. (b) Raw 25 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 25, 45 and 60 nm. (c) Raw 40 nm Fe3O4 nanoparticles and Fe3O4@Ag with Ag thicknesses of 10, 40, 50 and 60 nm. The scale bar indicates a length of 200 nm and is shared by all ﬁgures. This journal is ª The Royal Society of Chemistry 2013 6496 \| Analyst, 2013, 138, 6494–6500 View Article Online Paper Analyst Analyst Paper Fig. 4 SERRS intensities at Raman peak n10 (1628 cm1) for b-hematin at 5 103, 5 104, 5 105, 5 106, 5 107, and 5 108 M by using Fe3O4@Ag nanoparticles of diﬀerent core–shell parameters (a) with and (b) without magnetic enrichment. y Fig. 3 Experimental SERRS spectra of b-hematin at (a) concentration of 0.5 mM without magnetic-ﬁeld enrichment and (b) concentration of 0.5 mM with magnetic-ﬁeld enrichment by using Fe3O4@Ag nanoparticles of diﬀerent core– shell parameters. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In contrast, R6G molecules are allowed to chemisorb with the Triton X-100 molecules24 in close proximity to the Ag shell for giving rise to stronger adsorption. Thus, attachment of Fe3O4@Ag to b-hematin can be enabled by magnetic forces under the inuence of a magnetic eld instead of weak adsorption. A similar observation, i.e. the weak adsorption of molecules on Ag yields SERRS spectra similar to ordinary Raman spectra, has been reported previously.25 The shell dimension of C25S45 yields the largest SERRS signal among (1628 cm1). Note that these SERS spectra of b-hematin are similar to RRS spectra reported5 and the SERS spectra in tip- enhanced Raman measurements of hemozin7 without any inuence of the magnetic eld. Such a similarity can originate from the fact that the free-carboxylate COOH group in b-hematin, which is supposed to be responsible for the forma- tion of hydrogen bonds in a physisorption process,22 is diﬃcult to attach to the OH-terminated group of Triton X-100 capped Ag surfaces because the hydrogen bond formed between the OH group of Triton X-molecules and water molecules is much stronger23 than that established with a COOH group. In contrast, R6G molecules are allowed to chemisorb with the Triton X-100 molecules24 in close proximity to the Ag shell for giving rise to stronger adsorption. Thus, attachment of Fe3O4@Ag to b-hematin can be enabled by magnetic forces under the inuence of a magnetic eld instead of weak adsorption. A similar observation, i.e. the weak adsorption of molecules on Ag yields SERRS spectra similar to ordinary Raman spectra, has been reported previously.25 The shell dimension of C25S45 yields the largest SERRS signal among Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Some spectra are scaled by the speciﬁed magnifying factors to facilitate comparison. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Downloaded on 10/24/2024 6:23:26 AM. Fig. 4 SERRS intensities at Raman peak n10 (1628 cm1) for b-hematin at 5 103, 5 104, 5 105, 5 106, 5 107, and 5 108 M by using Fe3O4@Ag nanoparticles of diﬀerent core–shell parameters (a) with and (b) without magnetic enrichment. these nanoparticles, which is about 10.3 times higher in Raman intensity than that for C10S10. Therefore, the core and shell size-correlated enhancement trends of Fe3O4@Ag are modied in the magnetic eld-enriched SERRS measurement of b-hematin. Fig. 4 shows the corresponding b-hematin SERRS intensities at Raman peak n10 (1628 cm1) in the case with magnetic-eld enrichment in comparison to those without magnetic-eld enrichment. The magnetic-eld enriched b-hematin SERRS performance is more sensitive and displays the highest SERRS intensities for C25S45 among these nanoparticles [Fig. 4(a)], which is diﬀerent from the trends observed without magnetic enrichment [Fig. 4(b) and 2] that show a higher SERRS signal for a larger core. Thus, selecting the optimal core–shell dimensions of Fe3O4@Ag can improve the magnetic eld-enriched SERRS detection of b-hematin. Fig. 3 Experimental SERRS spectra of b-hematin at (a) concentration of 0.5 mM without magnetic-ﬁeld enrichment and (b) concentration of 0.5 mM with magnetic-ﬁeld enrichment by using Fe3O4@Ag nanoparticles of diﬀerent core– shell parameters. Some spectra are scaled by the speciﬁed magnifying factors to facilitate comparison. (1628 cm1). Note that these SERS spectra of b-hematin are similar to RRS spectra reported5 and the SERS spectra in tip- enhanced Raman measurements of hemozin7 without any inuence of the magnetic eld. Such a similarity can originate from the fact that the free-carboxylate COOH group in b-hematin, which is supposed to be responsible for the forma- tion of hydrogen bonds in a physisorption process,22 is diﬃcult to attach to the OH-terminated group of Triton X-100 capped Ag surfaces because the hydrogen bond formed between the OH group of Triton X-molecules and water molecules is much stronger23 than that established with a COOH group. This journal is ª The Royal Society of Chemistry 2013 Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Schematic diagram used in DDA modeling of b-hematin and (a) 1, (b) 4, and (c) 5 Fe3O4@Ag nanoparticles (C25S25) at 633 nm wavelength excitation polarized along the axis as indicated by the arrows. The scale bars represent 100 nm. (d) Ratio of \|E\|2 distributions obtained by a point by point ratio of \|E\|2 distributions between (b) to (a); (e) ratio of \|E\|2 for (c) to (a), and (f) ratio of \|E\|2 for (c) to (b). The intensity in (d) and (f) is rescaled to saturate at higher values for clear illustration of the enhancement at the interface between Fe3O4@Ag and b- hematin. The white line outlines the nanoparticles and the b-hematin. improvement inuenced by neighboring Fe3O4@Ag based on the DDA method, since Raman intensities are closely related to \|E\|2 and Qext as discussed. These coexisting phenomena are illustrated separately for the ease of discussion. For the calculation of \|E\|2 distributions of a b-hematin crystal in contact with Fe3O4@Ag (Fig. 5 and 6), the b-hematin topography is obtained from FESEM [Fig. 5(f)]. A horizontally (Fig. 5 and 6) and vertically (Fig. 6) polarized 633 nm illumination is excited onto each of the investigated volume of 875 nm 435 nm 120 nm. The Fe3O4@Ag is modeled as a Fe3O4 spherical core of diameter 50 nm with a constant Ag shell thickness of 25 nm (unless otherwise stated) to show clearly the dependence of \|E\|2 distribution and Qext on the number of Fe3O4@Ag attached to a b-hematin. We analyze the electromagnetic eld (\|E\|2) distribution or the extinction eﬃciency (Qext) enhancement of these nano- particles without (Fig. 5) and (Fig. 6 and 7) with the Fig. 5 Calculated electromagnetic (\|E\|2) distributions of (a) 9, (b) 5, (c) 1, Fe3O4@Ag C25S25 and (d) 13 Fe3O4@Ag C20S15, in contact with the b-hematin crystal, in comparison with that (e) without Fe3O4@Ag nanoparticles at 633 nm wavelength excitation polarized along the horizontal axis. (a) is obtained by introducing four nanoparticles to (b) with the positions of the original ﬁve nanoparticles unchanged, and (b) is obtained from (c) likewise. Colors in the color bar have been tuned in (e) to avoid misconception that high \|E\|2 distributions can be achieved without nanoparticles. The white line demarcates the position of a b- hematin crystal. The topography of the b-hematin crystal is obtained from (f) the FESEM image. Analyst Paper Fig. 6 Schematic diagram used in DDA modeling of b-hematin and (a) 1, (b) 4, and (c) 5 Fe3O4@Ag nanoparticles (C25S25) at 633 nm wavelength excitation polarized along the axis as indicated by the arrows. The scale bars represent 100 nm. (d) Ratio of \|E\|2 distributions obtained by a point by point ratio of \|E\|2 distributions between (b) to (a); (e) ratio of \|E\|2 for (c) to (a), and (f) ratio of \|E\|2 for (c) to (b). The intensity in (d) and (f) is rescaled to saturate at higher values for clear illustration of the enhancement at the interface between Fe3O4@Ag and b- hematin. The white line outlines the nanoparticles and the b-hematin. One factor that may play an important role in the formation of aggregates between Fe3O4@Ag nanoshells and b-hematin as well as among nanoshells could be inter-particle forces. Among these forces, the magnetic dipole–dipole attractive and repul- sive force is likely most signicant in the presence of a magnetic eld due to its much longer eﬀective range than other forces such as steric interaction and electrostatic interaction.27 The magnetic force can bring two or more nanoparticles or b-hematin crystals that are originally out of the critical distance for SERS close enough (<40 nm)8 for eﬀective SERS activities. Fe3O4@Ag nanoshells with a larger Fe3O4 core have a larger magnetic dipole moment than that of a smaller core since the dipole moment is proportional to the cube of the core diameter. As a result, these nanoparticles will form aggregates easily along the direction parallel to the magnetic eld resulting in increased Raman activities. In addition, the magnetic force can be increased with a decreasing Ag shell thickness because a large Ag shell could shield the magnetic force. From the point of view of increasing the formation of aggregates, nanoshells with a large Fe3O4 core and a thin Ag shell are desirable, since the magnetic shielding eﬀect should be minimized with a thinner outer Ag shell (similar to other magnetic nanoparticles28 reported in the literature). In our experiment, the shielding eﬀect is nominal for the same diameter iron core with a thicker shell (e.g. C40S10, C40S40, and C40S50 in Fig. 3), which can be attributed to the greater weight that allows faster aggregation settlement at the laser focused spot. Discussion The trend in Raman enhancement with diﬀerent core–shell dimensions shown above can be explained in terms of the extinction eﬃciencies and magnetic enrichment eﬀectiveness contributed by these Fe3O4@Ag nanoparticles with diﬀerent core diameters and shell thicknesses. Higher SERS enhancement resulted in (1) a thicker Ag shell with the same Fe3O4 core size (e.g. C40S10, C40S40, C40S60, and C40S60) or (2) a larger core size with the same shell thickness (e.g. C10S10, C25S10, and C40S10) for the R6G SERS (Fig. 2) and b-hematin SERRS in the case without magnetic enrichment [Fig. 4(b)]. This observation is consistent with the enhancement trends derived from the extinction eﬃciencies in other types of core–shell nanoparticles reported in the literature.13,26 In contrast, b-hematin SERRS in the case of magnetic-eld enrichment [Fig. 3(b) and 4(a)] gives a diﬀerent trend and an improved sensitivity, in comparison to that without the magnetic eld. These ndings will be explained as follows. This journal is ª The Royal Society of Chemistry 2013 Analyst, 2013, 138, 6494–6500 \| 6497 View Article Online Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The FESEM image of Fe3O4@Ag (g) C25S25 and (h) C20S15 with b- hematin in magnetic ﬁeld-enrichment. Fig. 5 shows the \|E\|2 distributions of a b-hematin in contact with Fe3O4@Ag spaced at least a radius from each other, to exclude neighboring Fe3O4@Ag enhancement as shown in our results later. High \|E\|2 intensities are observed at contacts between b-hematin and Fe3O4@Ag [Fig. 5(a)–(d)], known as “hot spots”, in comparison to the situation without Fe3O4@Ag nanoparticles [Fig. 5(e)]. The number of hot spots increases with the number of nanoparticles attached to b-hematin with Qext of 4.4, 3.4, and 1.81 for these arrangements as shown in Fig. 5(a)–(c), respectively, which is in agreement with our previous published work8 that magnetic enrichment leads to nanoparticle aggregation with better Raman signals. Moreover, we nd that smaller Fe3O4@Ag [C20S15 in Fig. 5(d) with Qext of 3.4] allows more of the Fe3O4@Ag to be attached around a b-hematin crystal [Fig. 5(g) and (h)] to create more hot spots. The comparison between Fig. 5(c) and (d) demonstrates that the increase in the number of hot spots in the arrangement C20S15, as shown in Fig. 5(d), could enlarge Qext so that its maximum Fig. 5 Calculated electromagnetic (\|E\|2) distributions of (a) 9, (b) 5, (c) 1, Fe3O4@Ag C25S25 and (d) 13 Fe3O4@Ag C20S15, in contact with the b-hematin crystal, in comparison with that (e) without Fe3O4@Ag nanoparticles at 633 nm wavelength excitation polarized along the horizontal axis. (a) is obtained by introducing four nanoparticles to (b) with the positions of the original ﬁve nanoparticles unchanged, and (b) is obtained from (c) likewise. Colors in the color bar have been tuned in (e) to avoid misconception that high \|E\|2 distributions can be achieved without nanoparticles. The white line demarcates the position of a b- hematin crystal. The topography of the b-hematin crystal is obtained from (f) the FESEM image. The FESEM image of Fe3O4@Ag (g) C25S25 and (h) C20S15 with b- hematin in magnetic ﬁeld-enrichment. This journal is ª The Royal Society of Chemistry 2013 6498 \| Analyst, 2013, 138, 6494–6500 Analyst View Article Online View Article Online View Article Online Paper Fig. 7 FESEM of Fe3O4@Ag aggregates at (a) the tip of a b-hematin crystal and (b) two sides of a b-hematin crystal, and the corresponding simpliﬁed schematic diagrams (c) and (d), respectively. (e) Unpolarized extinction eﬃciencies of Fe3O4@Ag (C25S25) for diﬀerent dimers, 3-nanoparticle chain, trimers, and single nanoparticle conﬁgurations. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (e) Unpolarized extinction eﬃciencies of Fe3O4@Ag (C25S25) for diﬀerent dimers, 3-nanoparticle chain, trimers, and single nanoparticle conﬁgurations. (a) From the point of view of increasing the formation of aggregates, nanoshells with a large Fe3O4 core and a thin Ag shell are desirable according to the discussion earlier about the magnetic force. (a) From the point of view of increasing the formation of aggregates, nanoshells with a large Fe3O4 core and a thin Ag shell are desirable according to the discussion earlier about the magnetic force. \|E\|2 value is comparable to the arrangement of C25S25 in Fig. 5(c), although monomer C20S15 shows a lower Qext than C25S25 theoretically,26 due to a smaller core and a thinner shell. In contrast, Fig. 6 illustrates the ratio of \|E\|2 distributions for diﬀerent congurations of Fe3O4@Ag nanoparticles in contact with b-hematin inuenced by the neighboring Fe3O4@Ag on the original nanoparticle. Real nanoparticle arrangements are simplied to spatial arrangement [e.g. Fig. 6(a)–(c)] to study the enhancement of an electromagnetic eld on the original Fe3O4@Ag (marked with “” and “o” at the same position next to the identical b-hematin) due to additional Fe3O4@Ag attached to the b-hematin crystal. We found that additional Fe3O4@Ag nanoparticles [e.g. Fig. 6(a)–(c)] that are in close contact with each other and attached to a b-hematin crystal improve the \|E\|2 intensity of the original nanoparticle [Fig. 6(d)– (f)]. The \|E\|2 intensity vicinity between the nanoparticle and b-hematin improves Raman intensity since SERS is approxi- mated to be proportional to \|Eind\|4, although reduced \|E\|2 intensity is observed at other locations. These observations imply the same aforesaid point that the magnetic enrichment (b) However, nanoshells with a smaller size are more likely to create more hot spots around the b-hematin crystal (Fig. 5) and have much improved extinction (Fig. 6), which would result in higher Raman enhancement. Thus, the evaluation of the above factors and calculated results (e.g. \|E\|2 distributions and Qext) for the diﬀerent core and shell dimensions allows tuning of the magnetic eld-enriched SERRS, causing C25S45 to give optimal SERRS performance instead of C40S50 in the case of magnetic enrichment. This study provides more insight into SERS performance under an external eld for the paramagnetic test molecules and ferro- magnetic SERS-active metal nanoshells, which would be otherwise diﬃcult to predict based on the commonly used DDA model alone, since the forecast of the geographical distribu- tions for these molecules and nanoparticles is unknown. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. strategy leads to improved Raman performance for arrange- ments with more aggregations and smaller Fe3O4@Ag size. gg g g We also account for the enhancement attributed from other Fe3O4@Ag located at the ends and opposite sides along the length [Fig. 7(a) and (b)] of the b-hematin crystal as illustrated in the simplied schematic diagrams in Fig. 7(c) and (d), respec- tively. Fig. 7(e) presents the extinction eﬃciency (Qext) for diﬀerent spatial congurations of Fe3O4@Ag C25S25. At our excitation wavelength of 633 nm, a higher Qext is observed [Fig. 7(e)] for Fe3O4@Ag nanoparticle aggregations [Fig. 7(a) and (c)] that form under an external magnetic eld in comparison to single-nanoparticle Qext which is analogous to dispersed nano- particles without magnetic enrichment [Fig. 7(e), resonance peak at about 420 nm typically observed29 in individual Ag nanoparticles]. Moreover, Fe3O4@Ag positioned on opposite sides of the b-hematin crystal at about 5 to 40 nm apart creates high SERS enhancement, which is analogous to other types of SERS nanoparticles reported25 in the literature. Thus, further Raman enhancement and dissimilar core and shell size-corre- lated enhancement trends are noted [Fig. 3(b) and 4(a)] for aggregated nanoparticles. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To briey summarize our investigation to explain the nding that C25S45 yields the largest Raman intensity for b-hematin SERRS, the following factors would play diﬀerent roles in the determination of Raman intensity from b-hematin SERRS measurements. 1. In the absence of a magnetic eld, a larger Fe3O4 core and a larger Ag shell in nanoshells would yield more signicant Raman enhancement, which is evident from Fig. 2, 3(a) and 4(b). 2. When an external magnetic eld is present, the magnetic attractive force would signicantly increase the chance of forming the aggregates of b-hematin and nanoshells according to the SEM image shown in Fig. 5 and 6 and our previous publication.8 This observation could result in two opposing trends. Fig. 7 FESEM of Fe3O4@Ag aggregates at (a) the tip of a b-hematin crystal and (b) two sides of a b-hematin crystal, and the corresponding simpliﬁed schematic diagrams (c) and (d), respectively. This journal is ª The Royal Society of Chemistry 2013 Acknowledgements 17 C. Yuen, W. Zheng and Z. Huang, J. Innov. Opt. Health Sci., 2008, 1, 267. This research was funded by the Singapore Lee Kuan Yew (LKY) start-up grant. The authors are also grateful to the LKY research fellowship (Clement Yuen) for sponsoring him to carry out his postdoctoral research and the New Investigator Grant (Project no. NMRC/NIG/1044/2011) funded by the National Medical Research Council (NMRC) in Singapore (Quan Liu). 18 P. B. Johnson and R. W. Christy, Phys. Rev. B: Solid State, 1972, 6, 4370. 19 A. Schegel, S. F. Alvarado and P. Wachter, J. Phys. C: Solid State Phys., 1979, 12, 1157. 20 Y. M. Serebrennikova, J. Patel and L. H. Garcia-Rubio, Appl. Opt., 2010, 49, 180. 21 S. Hu, K. M. Smith and T. G. Spiro, J. Am. Chem. Soc., 1996, 118, 12638. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 10 D. M. Newman, J. Heptinstall, R. J. Matelon, L. Savage, 10 D. M. Newman, J. Heptinstall, R. J. Matelon, L. Savage, M. L. Wears, J. Beddow, M. Cox, H. D. Schallig and P. F. Mens, Biophys. J., 2008, 95, 994. 11 S. Lal, S. Link and N. J. Halas, Nat. Photonics, 2007, 1, 641. 12 B. T. Draine and P. J. Flatau, J. Opt. Soc. Am. A, 1994, 11, 1491. 13 C. Yuen, W. Zheng and Z. Huang, J. Raman Spectrosc., 2010, 41, 374. 14 K. T. Wu, P. C. Kuo, Y. D. Yao and E. H. Tsai, IEEE Trans. Magn., 2001, 37, 2651. 15 Y. Zhai, J. Zhai, Y. Wang, S. Guo, W. Ren and S. Dong, J. Phys. Chem. C, 2009, 113, 7009. 16 M. A. Yurkin and A. G. Hoekstra, J. Quant. Spectrosc. Radiat. Transfer, 2011, 112, 2234. Open Access Article. Published on 21 August 2013. Downloaded on 10/24/2024 6:23:26 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The theoretical predictions presented in this work are in agreement with the experimental results (Fig. 3 and 4), which enable the This journal is ª The Royal Society of Chemistry 2013 Analyst, 2013, 138, 6494–6500 \| 6499 View Article Online Analyst Paper 6 S. Cinta-Pinzaru, N. Peica, B. Kustner, S. Schlucker, M. Schmitt, T. Frosch, J. H. Faber, G. Bringmann and J. Popp, J. Raman Spectrosc., 2006, 37, 326. 7 B. R. Wood, E. Bailo, M. A. Khiavi, L. Tilley, S. Deed, T. D. Gaudig, D. McNaughton and V. Deckert, Nano Lett., 2011, 11, 1868. 6 S. Cinta-Pinzaru, N. Peica, B. Kustner, S. Schlucker, M. Schmitt, T. Frosch, J. H. Faber, G. Bringmann and nanoshell C25S45 to achieve a b-hematin detection limit of 5 nM (equivalent to detection of 30 parasites per ml in the earlier malaria stage), as reported in our previous work.8 6 S. Cinta-Pinzaru, N. Peica, B. Kustner, S. Schlucker, M. Schmitt, T. Frosch, J. H. Faber, G. Bringmann and J. Popp, J. Raman Spectrosc., 2006, 37, 326. J. Popp, J. Raman Spectrosc., 2006, 37, 326. 7 B. R. Wood, E. Bailo, M. A. Khiavi, L. Tilley, S. Deed, 7 B. R. Wood, E. Bailo, M. A. Khiavi, L. Tilley, S. Deed, T. D. Gaudig, D. McNaughton and V. Deckert, Nano Lett., 2011, 11, 1868. Conclusion In conclusion, we report the dependence of magnetic eld- enriched b-hematin SERRS on the variations of the Fe3O4@Ag core size and shell thickness. We explain that the magnetic eld-enriched SERRS performance of b-hematin can be further improved by tuning the extinction eﬃciencies, localized elec- tromagnetic eld and magnetic properties of the Fe3O4@Ag to an optimal core size and shell thickness. Hence, the optimized magnetic eld-enriched SERRS performance shows the poten- tial for sensitive b-hematin detection for early malaria diag- nosis. The methodologies developed in this study will be applicable to nanoparticles of other shapes such as nanostars for SERS measurements from b-hematin, which could yield higher enhancement than nanoshells. This strategy can be also generalized to optimize SERS enhancement for nanocrystals of interest with irregular shapes in other applications. 8 C. Yuen and Q. Liu, J. Biomed. Opt., 2012, 17, 017005. 9 T. Frosch, S. Koncarevic, K. Becker and J. Popp, Analyst, 2009, 134, 1126. Notes and references 1 T. J. Egan, J. M. Combrinck, J. Egan, G. R. Hearne, H. M. Marques, S. Ntenteni, B. T. Sewell, P. J. Smith, D. Tayor, D. A. V. Schalkwyk and J. C. Walden, Biochem. J., 2002, 365, 343. 1 T. J. Egan, J. M. Combrinck, J. Egan, G. R. Hearne, H. M. Marques, S. Ntenteni, B. T. Sewell, P. J. Smith, D. Tayor, D. A. V. Schalkwyk and J. C. Walden, Biochem. J., 2002, 365, 343. 22 M. T. Shio, F. A. Kassa, M. J. Bellemare and M. Olivier, Microbes Infect., 2010, 12, 889. 23 T. Kar and S. Scheiner, J. Phys. Chem. A, 2004, 108, 9161. 24 D. W. Phillion, D. J. Kuizenga and A. E. Siegman, J. Chem. Phys., 1974, 61, 3828. 2 D. J. Sullivan, I. Y. Gluzman and D. E. Goldberg, Science, 1996, 271, 219. 2 D. J. 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Bambery, G. T. Webster, M. A. Khiavi, B. M. Cooke, S. Deed, D. Naumann and D. McNaughton, Analyst, 2009, 134, 1119. 5 B. R. Wood, A. Hermelink, K. R. Bambery, G. T. Webster, 29 L. Wang, C. Clavero, Z. Huba, K. J. Carroll, E. E. Carpenter, D. Gu and R. A. Lukaszew, Nano Lett., 2011, 11, 1237. M. A. Notes and references Khiavi, B. M. Cooke, S. Deed, D. Naumann and D. McNaughton, Analyst, 2009, 134, 1119. 6500 \| Analyst, 2013, 138, 6494–6500 This journal is ª The Royal Society of Chemistry 2013
https://openalex.org/W2675166493	https://europepmc.org/articles/pmc5484771?pdf=render	English	null	A Review of Quantitative Tools Used to Assess the Epidemiology of Porcine Reproductive and Respiratory Syndrome in U.S. Swine Farms Using Dr. Morrison’s Swine Health Monitoring Program Data	Frontiers in veterinary science	2,017	cc-by	5,778	Review published: 27 June 2017 doi: 10.3389/fvets.2017.00094 published: 27 June 2017 doi: 10.3389/fvets.2017.00094 Keywords: porcine reproductive and respiratory syndrome, Swine Health Monitoring Project, data sharing, epidemiology, spatiotemporal analysis Carles Vilalta1, Andreia G. Arruda1,2, Steven J. P. Tousignant 3,4, Pablo Valdes-Donoso1,5, Petra Muellner 6, Ulrich Muellner 6, Moh A. Alkhamis 1,7, Robert B. Morrison 1 and Andres M. Perez 1 Carles Vilalta1, Andreia G. Arruda1,2, Steven J. P. Tousignant 3,4, Pablo Valdes-Donoso1,5, Petra Muellner 6, Ulrich Muellner 6, Moh A. Alkhamis 1,7, Robert B. Morrison 1 and Andres M. Perez 1 Edited by: Francisco Ruiz-Fons, Spanish Research Council, Spain 1 Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, United States, 2 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, United States, 3 Swine Vet Center PA, St. Peter, MN, United States, 4 Boehringer Ingelheim Animal Health, St. Joseph, MO, United States, 5 Department of Agriculture and Resource Economics, University of California, Davis, Davis, CA, United States, 6 Epi-interactive, Wellington, New Zealand, 7 Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Kuwait City, Kuwait Spanish Research Council, Spain Reviewed by: Dinko Novosel, University of Zagreb, Croatia Alberto Allepuz, Universitat Autònoma de Barcelona, Spain Correspondence: Carles Vilalta cvilalta@umn.edu Reviewed by: Dinko Novosel, University of Zagreb, Croatia Alberto Allepuz, Universitat Autònoma de Barcelona, Spain Porcine reproductive and respiratory syndrome (PRRS) causes far-reaching financial losses to infected countries and regions, including the U.S. The Dr. Morrison’s Swine Health Monitoring Program (MSHMP) is a voluntary initiative in which producers and veterinarians share sow farm PRRS status weekly to contribute to the understanding, in quantitative terms, of PRRS epidemiological dynamics and, ultimately, to support its control in the U.S. Here, we offer a review of a variety of analytic tools that were applied to MSHMP data to assess disease dynamics in quantitative terms to support the decision-making process for veterinarians and producers. Use of those methods has helped the U.S. swine industry to quantify the cyclical patterns of PRRS, to describe the impact that emerging pathogens has had on that pattern, to identify the nature and extent at which environmental factors (e.g., precipitation or land cover) influence PRRS risk, to identify PRRS virus emerging strains, and to assess the influence that voluntary reporting has on disease control. Results from the numerous studies reviewed here pro- vide important insights into PRRS epidemiology that help to create the foundations for a near real-time prediction of disease risk, and, ultimately, will contribute to support the prevention and control of, arguably, one of the most devastating diseases affecting the North American swine industry. The review also demonstrates how different approaches to analyze and visualize the data may help to add value to the routine collection of surveillance data and support infectious animal disease control. Correspondence: Carles Vilalta cvilalta@umn.edu Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Edited by: Francisco Ruiz-Fons, Spanish Research Council, Spain Edited by: Francisco Ruiz-Fons, Spanish Research Council, Spain A Review of Quantitative Tools Used to Assess the epidemiology of Porcine Reproductive and Respiratory Syndrome in U.S. Swine Farms Using Dr. Morrison’s Swine Health Monitoring Program Data Carles Vilalta1*, Andreia G. Arruda1,2, Steven J. P. Tousignant 3,4, Pablo Valdes-Donoso1,5, Petra Muellner 6, Ulrich Muellner 6, Moh A. Alkhamis 1,7, Robert B. Morrison 1 and Andres M. Perez 1 INTRODUCTION receives weekly data from sow farms corresponding to approxi- mately 45% (n = 2,854,000) of the sows in the country according to the U.S. Census (3), and for that reason, it is, arguably, one of the largest datasets of voluntarily shared information by producers on swine health in the world. The MSHMP, there- fore, demonstrates how epidemiological knowledge on one of the most pressing diseases for a large industry may be gained through the analysis of routinely collected data, as part of a private–academic partnership and in the absence of a regulatory framework.h The U.S. is home to one of the largest swine industries world- wide; although the country is one of the largest pork exporters globally, the domestic market is still the most important for the industry, with 112 million hogs processed in national slaughter- houses in 2013 and with an average breeding stock of 6 million sows in the last 10 years (1–3). Approximately 50% of the hog production is concentrated in three states, namely, Iowa, North Carolina, and Minnesota (4). The concentration of such a large population in a relatively small area resulted in high density, and consequently, relatively high vulnerability of the industry to the introduction and spread of infectious diseases. The industry has tried to mitigate this vulnerability by increasing biosecurity and other prevention strategies, including air filtration and quarantine of incoming pigs (5, 6). The objective of the paper here was to summarize the methods and results of a variety of analytic tools applied to MSHMP data to elucidate the epidemiological dynamics of PRRS in the U.S. This review provides a summary of knowledge gained over the last years on PRRS dynamics through the use of different types of analytical methods for the analysis of routinely collected surveil- lance data. In addition, the review will ultimately contribute to demonstrate the application of epidemiological analytic tools in supporting the prevention and control of, arguably, one of the most economically challenging diseases for the swine industry worldwide. Porcine reproductive and respiratory syndrome (PRRS) is, arguably, one of the most economically devastating diseases affecting the U.S. swine industry, causing annual losses estimated to be between $550 and $664 million (7, 8). The disease, caused by the infection of an Arterivirus, referred to as PRRS virus (PRRSv), emerged in the U.S. in the late 1980s and, since then, has remained prevalent in the country (9, 10). Space and Time Clusteringf Two different approaches and tools were used to quantify the spatial dependence of PRRS incidence since 2011. Specifically, a. Spatial scan statistical models were used to determine the repeatability of annual patterns of disease incidence in space and to compare the spatial distribution of the disease before and after the country was affected by PED (23, 24). An exponentially weighted moving average and an upper confidence limit were used to signal the onset of the epidemic season. b. Space and time point process assessed by G- and K-functions were used to quantify the clustering of PRRS outbreaks, as well as data sharing to evaluate its relationship with PRRS incidence on a subset of participants (25). The University of Minnesota started a voluntary project in 2011 referred to as the Dr. Morrison’s Swine Health Monitoring Program (MSHMP) with two main objectives, namely, (1) to, in the short term, translate shared data into knowledge that informs veterinarian and producer decisions and (2) to, in the long term, build capacity to respond, in an effective and timely manner, to emerging and reemerging pathogens that may threaten the swine industry. Initially, participants shared weekly PRRS status data for sow farms, following and adapting AASV guidelines (21). As the program progressed, the MSHMP expanded, and some participants are also sharing data for porcine epidemic diarrhea (PED) and porcine delta corona virus. The MSHMP is quite unique because, despite its voluntary nature, it currently Citation: Vilalta C, Arruda AG, Tousignant SJP, Valdes-Donoso P, Muellner P, Muellner U, Alkhamis MA, Morrison RB and Perez AM (2017) A Review of Quantitative Tools Used to Assess the Epidemiology of Porcine Reproductive and Respiratory Syndrome in U.S. Swine Farms Using Dr. Morrison’s Swine Health Monitoring Program Data. Front. Vet. Sci. 4:94. doi: 10.3389/fvets.2017.00094 June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org 1 Quantitative Tools Applied in MSHMP Vilalta et al. INTRODUCTION PRRSv is an RNA virus, which has led to the appearance of a wide array of emerg- ing or reemerging strains with a large variety of phenotypic and genotypic features (11). Commercial vaccines do not prevent infection although they are reported to reduce virus shedding and decrease clinical impact. Protection is reported to be more profound after infection with homologous than heterologous strains (12, 13). Modified live vaccines are more effective than those formulated with inactivated virus (14). Many alternative mechanisms of transmission have been described for PRRSv, including direct transmission through the movement of pigs and indirect through fomites, insects, semen, or airborne spread (6, 15–20). The American Association of Swine Veterinarians (AASV) developed a classification system with four categories based on infection status in sow herds. A herd was defined as “unstable” if field virus could be detected within the herd. These categories are referred to as positive unstable (Category I), positive stable (Category II), provisional negative (Category III), and negative (Category IV) (21). In the absence of a regulatory framework, data on farm-level infection incidence can only be collected on a voluntary basis in the U.S.h MATERIALS AND METHODS A systematic pathway was used to direct the application of epide- miological tools to assess the epidemiological dynamics of PRRS and its related components, as described elsewhere (22). Frontiers in Veterinary Science \| www.frontiersin.org 1 Alkhamis M, Arruda AG, Vilalta C, Morrison R, Perez A. Surveillance of porcine reproductive and respiratory syndrome virus in the United States using risk map- ping and species distribution modeling. Prev Vet Med. Factors That Influence PRRS Riskhi l a. The significance of quarterly and annual PRRS incidence from 2009 to 2013 was assessed using a chi-square test. Furthermore, logistic regression was used to estimate the likelihood of the presence of PRRSv infection in a given year increasing the odds of that herd being infected the following year (23). Chi-square test and logistic regression models were also used to compare PRRS incidence between 2009–2012 and 2013, the year that PED was first detected in the U.S., and used to explain how its emergence might have affected the temporal dynamics, and overall incidence of PRRS (24). a. The significance of quarterly and annual PRRS incidence from 2009 to 2013 was assessed using a chi-square test. Furthermore, logistic regression was used to estimate the likelihood of the presence of PRRSv infection in a given year increasing the odds of that herd being infected the following year (23). Chi-square test and logistic regression models were also used to compare PRRS incidence between 2009–2012 and 2013, the year that PED was first detected in the U.S., and used to explain how its emergence might have affected the temporal dynamics, and overall incidence of PRRS (24). June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org 2 Vilalta et al. Quantitative Tools Applied in MSHMP b. A general linear mixed-effects logistic regression model was used to investigate the association between factors hypoth- esized to influence PRRS risk (i.e., month, farm type, county density, active participation in a voluntary control program, and proportion of farms stable) and PRRS incidence (25).f Factors That Influence PRRS Risk l Farms that were PRRS infected in a given year were more likely to be infected in consecutive years compared to those that were not affected by PRRS. However, during the year in which PED emerged in the U.S., that pattern changed and the described association could no longer be observed. Furthermore, PRRS incidence was significantly lower, and the beginning of the PRRS season was delayed, compared to previous years (23, 24). Modeling Time-dependent reproduction (TD-R) numbers for PRRS across different regions and production systems within the U.S. were estimated. The uses of TD-R include monitoring of epidemic trends over time, identification of “super-spreader events,” measurement of progress of interventions over time, and extrac- tion of parameters for mathematical models (e.g., models to test interventions) (28). Evolutionary Analysis A large dataset (n = 6,774) of open reading frame 5 (ORF5) com- plete sequences provided by five production systems was used to study the evolution and spread of PRRSv across included systems. Bayesian phylogenetic analysis with divergence time, growth rate, population size, and estimation of viral dispersal history between systems was applied to this dataset to understand the order of events and spatiotemporal progression of this specific cluster (27). Analysis of data collected on a regional project in Minnesota, referred to as RCP-N212, suggested an increased interest of producers in participating in regional control programs during the study period, from July 2012 to June 2014. That analysis also showed, however, that participation on a regional project is not necessarily highly correlated with probability of data sharing. Time within the analyzed period (24 months) and the probability of sharing PRRS status to the voluntary regional control program were negatively associated with disease outbreaks, while density (as number of farms per square mile) was positively associated with PRRS outbreaks. Findings in this study showed that despite a statistical decreasing trend of the disease, spatiotemporal cluster- ing of PRRS was observed along the whole study period. In turn, farm type (i.e., farms with and without breeding herds) did not show a specific association with PRRS, although farms with sows were more prone to report PRRS status in the RCP-N212 (25). Space and Time Clustering Porcine reproductive and respiratory syndrome showed a repetitive pattern within a cohort of sow farms in time and space. It was determined that the onset of the PRRS season began every year during the fall (between the months of October and November). Furthermore, spatial clusters of PRRS positive farms were consistently identified in similar locations throughout the study period (23, 24). Spatial trends in PRRS case distribution after PED emergence were similar to those found in previous studies at the national level (23, 24), although PRRS incidence decreased during that year. These data suggest that changes in management and biosecurity to avoid spread of PED may have affected PRRS incidence nationally without affecting the spatial dependence of PRRS incidence. c. A mixed-effect Poisson regression model was built to evaluate the effects of land coverage (e.g., cultivated fields and shrubs or trees) and geographical features (including terrain slope and altitude) on the incidence of PRRS outbreaks considering the years of 2009–2016. The model included a random effect at the production system level and also included explanatory variables that were known to affect the risk of PRRS outbreaks such as pig density, farm size, and geographical region (26). d. Species distribution models, such as presence-only maximum entropy machine learning algorithm, were used to build risk maps through predicting the spatial distribution of PRRSv out- breaks using a set of environmental variables.1 That algorithm can extract associations to characterize the demographic and climatic requirements for PRRSv, and subsequently deploying those associations to predict suitable geographical locations in non-sampled areas, where most likely PRRSv outbreaks will be detected (see text footnote 1). Modeling Although most PRRS outbreaks were reported in areas with the highest hog density, between farm PRRS transmission patterns differed substantially among regions. Differences in transmissi- bility were reported between different regions and systems, with evident peaks of disease occurring in different regions at different times. Results suggested that factors other than season and swine density might have a major impact on PRRS spread at the regional level, but such factors need to be elucidated (28). Evolutionary Analysis Spatial distribution of farms and production systems has an important role in the maintenance of endemic PRRSv strains and the occurrence of epidemic PRRS episodes. Recent emergence of novel PRRSv occurred in certain production systems, escalating to epidemic proportions when other, highly connected systems were reached. Sow farms seem to have a potential role in maintaining and spreading the disease within those production systems (27). Prototype of a Real-time Data Land-related variables and land coverage appeared to have an impact on the incidence of PRRS outbreaks. Sow farms located in areas of cultivated/managed areas had higher incidence of PRRS outbreaks when compared to sow farms located in areas characterized by having herbaceous areas or trees. Altitude, as expressed in meters above sea level, did not appear to be associ- ated with the occurrence of PRRS outbreaks; however, farms in slopes larger than 9%, when compared with the neighbor, had lower incidence of PRRS outbreaks compared to those located in areas characterized by slopes less than 2% (26). Finally, an application was developed in RStudio Shiny (29) to allow for real-time visualization of MSHMP data including analytical outputs of different types of analysis [Pig/Statistical Analysis and Visual Insights (SAVI)]. Pig/SAVI allows for data visualization and analysis using web-based and standalone versions. JavaScript and CSS were used to enable some of the functionality and visual elements. Several functional elements were utilized, including reactivity, isolation, and download of outputs into CSV format. Results of the maximum entropy model suggested that hog density was the most important predictor for PRRS incidence, whereas a number of environmental factors accounted for much of the remaining risk. More specifically, when considering differ- ent regions, swine density still has a role in impacting the number of cases being a driven factor in swine denser areas. However, June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org 3 Quantitative Tools Applied in MSHMP Vilalta et al. some other variables such as precipitation or temperature can have an impact on the spread of the disease (see text footnote 1). data sharing, visualization, and analysis to support disease pre- vention and control. Its aim is also to improve the accessibility of the data and to add value to the program for different system stakeholders. The current array of tools developed as part of the epidemiological platform includes descriptive visualizations of prevalence and incidence over time, real-time risk maps, network visualizations, and molecular epidemiology tools, including the ability of the user to build different types of phylogenetic trees (Figure 1). DISCUSSION The MSHMP efforts resulted in a unique dataset generated through a voluntary system, which demonstrates U.S. producers’ commitment to sharing data toward the common goal of mitigat- ing PRRS impact in the country. However, collection of data by itself has no value unless it is analyzed to bring knowledge and wisdom to improve speed and quality of decisions (30). Here, the value of sharing, aggregating data, and creating knowledge has been demonstrated with the use of multidisciplinary analytic tools that helped to maintain producer’s interest in a voluntary data sharing project. In the absence of a regulatory framework, data sharing is a prerequisite for increasing disease awareness and, ultimately, supporting its control. Lessons learnt from the systematic analysis of the MSHMP relate to the repetitive time and time–space patterns associated with PRRS incidence in the U.S. and the regional variation, and the influence that Visualization Dashboardh Visualization Dashboardh The RStudio Shiny application platform integrates portions of the data described above into a system that allows near real-time Figure 1 \| Dashboard of the RStudio Shiny-based application [Pig/Statistical Analysis and Visual Insights (SAVI)] with its four core analytical components: frequency, genetics, space time analysis, and movement. For example, the proportional importance of transmis- sion routes (air, pig movement, or fomites) in the spread of the disease and whether that pattern is mostly influenced by system or region remain unknown. The application of different analyti- cal tools to the rich dataset assessed here has helped producers and practitioners to better understand the epidemiological dynamics of PRRS. For example, we learned that the onset of the disease had a repetitive pattern and a similar starting point from the first 4 years of data of the project, from 2009 to 2012 (23). However, data from 2013 and 2014, when PED emerged in the country, failed to signal the epidemics at the same date as predicted by previous years, showing a delay of 3 weeks (24). Also during that same season (2013 and 2014), the same cohort of farms showed a 50% decrease in the number of PRRS outbreaks between October and March when compared with the average observed in the same season in the previous four previous years. Many factors may have impacted the change in disease pattern observed that year, including an increase of bios- ecurity practices associated with PED emergence, an increase of PRRS vaccination, secular cycles, or difficulty performing the routine PRRSv diagnostics following high PED mortality rates. Furthermore, no changes in the centroid of the epidemic clusters were observed between 2009 and 2014, suggesting that although incidence decreased that year, the spatial dependence of the disease prevailed. Preliminary results of disaggregating national data and aggregating cases from different states (not published) seem to point to a different temporal incidence trend between geographical regions and/or systems, and those could be caused by different factors as management, density or different climate conditions as pointed by the ecological niche modeling (see text footnote 1). yii g Because sharing information was perceived by at least some companies as a competitive advantage as they can learn new insights on the disease epidemiology, some MSHMP participants started sharing PRRSv ORF5 sequences. ORF 5 sequencing has recently become a relatively common practice within the swine industry. The amount of available data from Veterinary Diagnostic Laboratories, and the use of new molecular epidemiology tools may help elucidate the relative importance of different transmis- sion routes during outbreak investigations. Figure 1 \| Dashboard of the RStudio Shiny-based application [Pig/Statistical Analysis and Visual Insights (SAVI)] with its four core analytical components: frequency genetics space time analysis and movement Figure 1 \| Dashboard of the RStudio Shiny-based application [Pig/Statistical Analysis and Visual Insights (SAVI)] with its four core analytical components: frequency, genetics, space time analysis, and movement. June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org Quantitative Tools Applied in MSHMP Vilalta et al. changes on epidemiological conditions, may have in those pat- terns. In addition, quantitative evidence was collected on how phylogenetic analysis may support investigations on the most likely mechanisms of spread of the virus, and how collaboration and data sharing may contribute to the control of the disease at the regional level in the absence of a regulatory framework. signal PRRS epidemics on those areas where outbreaks are rela- tively rare. For areas in which PRRSv is endemic, TD-R did not show any fluctuation and revolved around 1, which is expected for an endemic disease such as PRRS.hi The finding that having crops or trees around the farm could influence the occurrence of new cases was intriguing and may have multiple interpretations and applications (26). It would be, indeed, interesting to assess whether the use of natural tree buffers could make a significant impact on the protection of the herd. To the best of our knowledge, there is no information on how vegetative buffers could prevent the spread of the diseases or protect the herd from PRRS infection. Similar conclusions were extracted using an ecological niche modeling approach in which land cover accounted for much of the variation in PRRS risk, after hog density and precipitation (see text footnote 1). However, further analysis is needed to confirm these findings. A negative association between sharing PRRS status and incidence was found. It is unknown if this relationship is because negative farms are more inclined to share the data or if the increase in awareness has an effect on the incidence of the disease. However, in that specific study, the participation of farms, as well as PRRS status sharing, increased in the time frame studied suggesting that farmers and veterinarians may perceive collaboration as an advantage (25).f Noteworthy, even though a substantial amount of effort has been devoted to describing PRRSv transmission routes (15–18), many aspects of the disease epidemiology are yet-to-be-eluci- dated. For example, using ORF5 sequences from five different companies and Bayesian phylodynamic methods that could integrate time, space, and phylogenetic analysis allowed for a better estimate on the history of the virus, helping us to understand the underlying connections in the disease spread process (27). As part of the same project, a platform that could provide access to data and analytical outputs in real time was also explored and developed. 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Measuring progress on porcine reproductive and respiratory syndrome (PRRS) control at a regional level: the Minnesota N212 regional control project (RCP) as a working example. PLoS One (2016) 11(2):e0149498. doi:10.1371/journal.pone. ACKNOWLEDGMENTS The authors of this study would like to acknowledge the support of the swine producers, veterinarians, and the trust of sharing their data with us. The authors would also like to acknowledge the contribution made by Christina Ahlstrom (Epi-interactive) to the development of the Pig/SAVI dashboard. CONCLUSION Higher disease incidence was observed and linked to new PRRSv introduction that quickly disseminated in an area or system (27). Those observations were also supported by the description of PRRS transmissibility across regions and systems using the TD-R (28). Interestingly, the two densest swine regions showed different patterns of disease transmission, which may be somehow explained by a difference in management and PRRS immunization. The TD-R showed to be a valuable method to The review here summarizes the knowledge gained over the last 6 years on the epidemiological dynamics of the most devastating disease affecting the U.S. swine industry through the voluntary MSHMP. When evaluating PRRS incidence data at a national scale, there is a predictable incidence pattern, with winter offer- ing environmental conditions most favorable for virus spread at long (transport and fomites) and short (airborne) distances, and June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org Vilalta et al. Quantitative Tools Applied in MSHMP data analysis process. All the authors reviewed the manuscript and participated in the review process. hog density and system being the most relevant factors associated with the spatial distribution of disease. However, when incidence data are explored at low levels of aggregation (state or county), factors such as presence of trees, being located in a hilly area, or fine-scale weather conditions are most influential on the risk for PRRS spread. Finally, this review demonstrates the level of contribution that the use of novel multidisciplinary analysis tools applied to routine collection of large surveillance data may provide to prevention and control of livestock diseases in the U.S. and at a global scale. FUNDING CV—primary author. AA, PM, UM, MA, PV-D, ST, RM, and AP—contributing authors. RM designed and supervised the data collection and storage process. AP designed and supervised the Funding in support of the different studies of this paper, as well as SHMP, is from the Swine Health Information Center (SHIC), the University of Minnesota MnDrive, and National Pork Board. Funding in support of the different studies of this paper, as well as SHMP, is from the Swine Health Information Center (SHIC), the University of Minnesota MnDrive, and National Pork Board. REFERENCES 0149498 p 13. Murtaugh MP, Xiao Z, Zuckermann F. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol (2002) 15:533–47. doi:10.1089/088282402320914485 26. Arruda AG, Vilalta C, Perez A, Morrison R. Land altitude, slope, and coverage as risk factors for porcine reproductive and respiratory syndrome (PRRS) outbreaks in the United States. PLoS One (2017) 12(4):e0172638. doi:10.1371/ journal.pone.0172638 14. Lyoo YS. Porcine reproductive and respiratory syndrome virus vaccine does not fit in classical vaccinology. Clin Exp Vaccine Res (2015) 4:159–65. doi:10.7774/cevr.2015.4.2.159 27. Alkhamis MA, Perez AM, Murtaugh MP, Wang X, Morrison RB. Applications of Bayesian phylodynamic methods in a recent U.S. porcine reproductive and 15. Dee S, Deen J, Rossow K, Wiese C, Otake S, Joo HS, et al. Mechanical trans- mission of porcine reproductive and respiratory syndrome virus throughout June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org 6 Vilalta et al. Quantitative Tools Applied in MSHMP surveillance data in Switzerland. Front Vet Sci (2015) 2:47. doi:10.3389/ fvets.2015.00047 surveillance data in Switzerland. Front Vet Sci (2015) 2:47. doi:10.3389/ fvets.2015.00047 respiratory syndrome virus outbreak. Front Microbiol (2016) 7:67. doi:10.3389/ fmicb.2016.00067 surveillance data in Switzerland. Front Vet Sci (2015) 2:47. doi:10.3389/ fvets.2015.00047 respiratory syndrome virus outbreak. Front Microbiol (2016) 7:67. doi:10.3389/ fmicb.2016.00067 28. Arruda AG, Alkhamis MA, VanderWaal K, Morrison RB, Perez AM. Estimation of time-dependent reproduction numbers for porcine repro- ductive and respiratory syndrome across different regions and production systems of the US. Front Vet Sci (2017) 4:46. doi:10.3389/fvets.2017. 00046 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Vilalta, Arruda, Tousignant, Valdes-Donoso, Muellner, Muellner, Alkhamis, Morrison and Perez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 29. Chang W, Cheng J, Allaire J, Xie Y, McPherson J. Shiny: Web Application Framework for R. R Package Version 011. (2015). Available from: ftp:// cran.r-project.org/pub/R/web/packages/SpatialEpiApp/vignettes/manual.pdfh 30. Rowley J. The wisdom hierarchy: representations of the DIKW hierarchy. J Inf Sci (2007) 33:163–80. Frontiers in Veterinary Science \| www.frontiersin.org June 2017 \| Volume 4 \| Article 94 REFERENCES doi:10.1177/0165551506070706 31. Muellner UJ, Vial F, Wohlfeder F, Hadorn D, Reist M, Muellner P. Timely reporting and interactive visualization of animal health and slaughterhouse June 2017 \| Volume 4 \| Article 94 Frontiers in Veterinary Science \| www.frontiersin.org
https://openalex.org/W3012380988	https://www.e3s-conferences.org/10.1051/e3sconf/202015605005/pdf	English	null	Evaluation of seismic capacity on the building law Faculty Tadulako University due to Palu earthquake 2018	E3S web of conferences	2,020	cc-by	3,760	1 Corresponding author: hamdenimedriosa@itp.ac.id Evaluation of seismic capacity on the building law Faculty Tadulako University due to Palu earthquake 2018 Hamdeni Medriosa1,2* 1Doctoral Student of Civil Engineering Department, Andalas University, Padang, 25163, Indonesia 2Civil Engineering Department, Padang Institute of Technology, Padang, 25143, Indonesia Abstract. Indonesia is located in a high level of the earthquake risk area. According to USGS data, from December 2004 to October 2009, more than ten large earthquakes occurred and exceeded 5.0 Scale Richter in magnitude. Recently, the major earthquake occurred on September 28, 2018, in Palu City, Central Sulawesi, with 7.4 on the Scale Richter in magnitude and triggered a lot of building damaged. In this study, the seismic capacity of a 3-story reinforced concrete building, which collapsed due to a large earthquake in the city of Palu, was evaluated. The Standard for Seismic Evaluation of Existing Reinforced Concrete Buildings, 2001 from Japan, was used to evaluate the seismic capacity of reinforced concrete. In this analysis only reviews the column structure elements. However, the brick wall elements are considered to determine net column height. The analysis has been done only for the first floor, where there is the maximum shear force on the structure. Seismic capacity is determined by the relationship between the lateral strength and the ductility index. The obtained results of the seismic capacity analysis showed that the total strength index value of the building was 0.307 for the north to south and 0.455. The seismic capacity of this building is compared with the seismic capacity of a reinforced concrete building that survived due to a large earthquake with 7.6 on the Scale Richter in West Sumatra in September 2009. The obtained results show that the strength index value of this building is smaller than that of a building that could survive the 2009 West Sumatra earthquake. E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 https://doi.org/10.1051/e3sconf/202015605005 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2.1.1.5 Column Strength Index (C) The strength index (C) in the second level calculation method can be calculated by the following equation: (The Japan Building Disaster Prevention Association, 2001)[7]. Ultimate Flexural Strength of Structure (Mu) is calculated based on equations (2.1), (2.2) or (2.3) (The Japan Building Disaster Prevention Association, 2001). a. For Nmax ≥ N > 0.4 x b x D x Fc Mu = {0,8. ܽ୲ . ߪy. ܦ + 0,12ܾܦ2 Fc } ே௠௔௫ିே ே௠௔௫ି଴,ସ௕.஽.ி௖ (2.1) a. For Nmax ≥ N > 0.4 x b x D x Fc Mu = {0,8. ܽ୲ . ߪy. ܦ + 0,12ܾܦ2 Fc } ே௠௔௫ିே ே௠௔௫ି଴,ସ௕.஽.ி௖ (2.1) C = ொೠ Ʃௐ (2.12) (2.12) ( ) b. For 0.4 b x D x Fc ≥ N > 0 Mu = {0,8.ߙt .ߪy. D + 0,5.N.D }(1 − ே ௕஽ி௖) (2.2) 2.1.1.4 Column failure type 2.1.1.4 Column failure type The collapse of a structure is determined by the ultimate shear strength (Qsu) and the ultimate flexural strength (Qmu). If the Qsu / Qmu <1, it is mean the collapsed form of the building is a shear collapse. The shape of the collapse of a structure is an important point to calculate the ductility of a structure. According to The Japan Building Disaster Prevention Association (2001)[7], the collapsed form of a building is used to see the ratio of building strength index. 2.2.1 Plastic deviation of Column The ultimate shear strength of the structure (Qsu) is calculated based on equation (2.7) (The Japan Building Disaster Prevention Association, 2001). Plastic deviation of column can be calculated with considering some part according to equation 2.18 (The Japan Building Disaster Prevention Association, 2001)[7] su = { ଴.଴ହଷ.௉೟బ.మయ(ଵ଼ା୊ୡ) ಾ ೂ.೏ା଴.ଵଶ + 0,85ඥܲ௪. ݏ. ߪ௪+ 0.1ߪ଴}b.j (2.7) Pt = ఈ೟ ௕.஽. 100% (2.8) ெ ொ.ௗ = ௛బଶ ൗ ஽ (2.9) Pw = ஺ೡ ௕.௦. 100% (2.10) ߪ0 = ே ௕.஽ (2.11) Qsu = { ଴.଴ହଷ.௉೟బ.మయ(ଵ଼ା୊ୡ) ಾ ೂ.೏ା଴.ଵଶ + 0,85ඥܲ௪. ݏ. ߪ௪+ 0.1ߪ଴}b.j (2.7) Pt = ఈ೟ ௕.஽. 100% (2.8) ெ ொ.ௗ = ௛బଶ ൗ ஽ (2.9) Pw = ஺ೡ ௕.௦. 100% (2.10) ߪ0 = ே ௕.஽ (2.11) Qsu = { ଴.଴ହଷ.௉೟బ.మయ(ଵ଼ା୊ୡ) ಾ ೂ.೏ା଴.ଵଶ + 0,85ඥܲ௪. ݏ. ߪ௪+ 0.1ߪ଴}b.j cRmp = 10 ( ௖ொೞೠ ௖ொ೘ೠ− ݍ) . cRmy ≥ 0 (2.18) q = 1.0 for S ≤ 100 mm (2.19) q = 1,1 for S > 100 mm Pt = ఈ೟ ௕.஽. 100% ெ ொ.ௗ = ௛బଶ ൗ ஽ Pw = ஺ೡ ௕.௦. 100% ߪ0 = ே ௕.஽ (2.9) (2.10) 2.1.1 Column strength According to the level two methods, the building strength is reviewed vertically by the structural strength of the column. In general, the structural strength is divided into the shear strength and flexural strength. 2.1.1.2 Shear strength of column when ultimate bending (Qmu) : h0 / H0 ≤ 1 cRmu = cRmy + cRmp (2.14) Rmy = h0/H0. cRmy ≥ cR250 (2.15) cRmy = R150 for h0/H0 ≥ 3 (2.16) cRmy = R250 for h0/H0 ≤ 2 (2.17) h0 / H0 ≤ 1 cRmu = cRmy + cRmp The column shear force on ultimate bending is calculated by equation (2.6) (The Japan Building Disaster Prevention Association, 2001). Qmu = ଶெ௨ ௛బ (2.6) (2.6) 2.1.1.3 The Ultimate Shear Strength Column (Qsu) 1 Introduction Based on the damage due to the earthquake, it is interesting to evaluate the performance of the seismic capacity of the reinforced concrete building (existing building) of the Faculty of Law, Tadulako University. The Standard for Seismic Evaluation of Existing Reinforced Concrete Buildings, 2001 from Japan, was used to evaluate the seismic capacity of reinforced concrete. Indonesia is located in a high level of the earthquake risk area. According to USGS data, from December 2004 to October 2009, more than ten large earthquakes occurred exceed 5.0 Scale Richter and triggered a lot of damage to buildings. The resilience of buildings during earthquake loads is influenced by several things, which are the earthquake-resistant house planning standards, construction implementation, and construction planning (Liza, N. M. et al., 2014). Several major earthquakes have occurred in West Sumatra, which resulted in many reinforced concrete buildings that were damaged and collapsed (Maidiawati and Sanada 2008, EERI, 2009).[6] Fig. 1: Law Faculty Tadulako University Recently, the earthquake occurred in Palu City, Central Sulawesi, on September 28, 2018, with a magnitude of 7.4 SR triggered many damages on reinforced concrete buildings. The damage of the building can be classified on from minor to heavy damage and collapsed. One of them is the Tadulako University Faculty of Law Building, Palu City, Central Sulawesi. This building is a 3-story building with reinforced concrete structures that collapsed when an earthquake of 7.4 on the Richter Scale struck Palu City. The building collapsed only on the first floor, while the second and third floors did not experience such significant damage. Fig. 1: Law Faculty Tadulako University E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 https://doi.org/10.1051/e3sconf/202015605005 2.2 Deformation Ultimate of Column ≥ Mu = {0,8.ߙt .ߪy. D + 0,5.N.D }(1 − ே ௕.஽.ி௖) (2.2) c. For 0 >N ≥ Nmin Mu =0,8.at.ߪy. D + 0,4.N.D (2.3) Nmax = b.D.Fc + at .ߪy (2.4) Nmin = - ag .ߪy (2.5) The concept of deformation is a change in shape or size due to the loads. In general, compressive stress is applied to the column, due to shortening. Equation 2.13 was using to calculate that. (The Japan Building Disaster Prevention Association, 2001). Rmu = ( ௛బ ுబ ) . cRmu ≥ R250 (2.13) (2.13) (2.13) When : 2.1.1.2 Shear strength of column when ultimate bending (Qmu) a. Shear column The ductility index of the shear column is calculated using equation (2.26) based on the angle when the deformation in the building occurs. It can be translated to the ultimate deformation in the failure of the shear column (The Japan Building Disaster Prevention Association, 2001). , ) F = 1 + 0,27 ோೞೠି ோమఱబ ோ೤ି ோమఱబ (2.26) Rsu = ௖ொೞೠ௖ொ೘ೠ ൗ ି ଵ ଴,଻ . Rmy ≥ R250 for cߙ, Qmu < Qsu (2.27) Rsu = R250 for cߙ, Qmu ≥ Qsu (2.28) cߙ = 0,3 + 0,7 (R250/ Rmy) (2.29) b. Bending column (2.26) 2.2.3 Ductility Index (F) Definition of ductility is the ability of a building structure to experience large post-elastic deviations repeatedly and cyclic due to the earthquake loads that cause of the first yield stage, while maintaining sufficient strength and rigidity so that the structure of the building remains to stand, even though it is already in critical condition of collapse. αs = Q(F1)/ Qsu = αm Qmu/ Qsu ≤ 1.0 (2.32) αm = Q(F1)/ Qmu = 0.3 + 0.7 x R1/ Rmy (2.33) αs = Q(F1)/ Qsu = αm Qmu/ Qsu ≤ 1.0 (2.32) αm = Q(F1)/ Qmu = 0.3 + 0.7 x R1/ Rmy (2.33) Ductility index, F is a certain deformability which is calculated according to structural specifications based on stiffness, strength, dimensions, etc. (JBDPA, 2005). Which is determined by equations (2.26), (2.30), (2.31) (The Japan Building Disaster Prevention Association, 2001) 3 Methodology The observation was done by examining the breakdown of reinforced concrete structures in buildings. The existing compressive strength of concrete is obtained by conducting non-destructive testing using the hammer test. The reinforcement used in the damaged reinforced concrete structure is also measured as the diameter of the reinforcement used, the type of reinforcement, and the length of the dispensing. The reinforcement of the stirrups is a measurement of the distance between the stirrups and the large angle and the length of the curve in the stirrups. The analysis was carried out based on The Standard for Seismic Evaluation of Existing Reinforced Concrete Buildings published by The Japan Building Disaster Prevention Association[7]. Furthermore, the next procedure is using the Microsoft Excel program. a. Shear column 2.2.2 Limit of Plastic Deviations in the Column The limit of plastic deviation from the column can be determined with (2.21), (2.22), (2.23), (2.24), (2.25) 2 2 E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 https://doi.org/10.1051/e3sconf/202015605005 equations. (The Japan Building Disaster Prevention Association, 2001). cRmax : min { cRmax(n), cRmax(s), cRmax(t), cRmax(b), cRmax(h) } (2.20) cRmax(n) = cR250 untuk ߟ > ߟH (2.21) cRmax(n) = cR30(cR250/ cR30) ߟ’ ≤ cR30 Where : ߟ = (ߟ- ߟL) (ߟH – ߟL) ߟ = Ns / (b.D Fc) ߟL = 0,25 dan ߟH = 0,5 for s ≤ 100 mm ߟL = 0,2 dan ߟH = 0,4 for s > 100 mm cRmax(s) = cR250 for c߬u /Fc > 0,2 (2.22) cRmax(s) = cR30 cRmax(t) = cR250 for Pt > 1 % (2.23) cRmax(t) = cR30 cRmax(b) = cR50 for s/db > 8 (2.24) cRmax(b) = cR30 cRmax(h) = cR50 for h0/D ≤ 2 (2.25) cRmax(h) = cR30 2 2 3 Ductility Index (F) umn (The Japan Building Disaster Prevention ociation, 2001). (i) For case Rmu< Ry F = 1 + 0,27 ோ೘ೠି ோమఱబ ோ೤ି ோమఱబ (2.30) (ii) For case Rmu ≥ Ry F = ට ଶ ோ೘ೠ/ோ೤ି ଵ ଴.଻ହ(ଵା଴.଴ହ ோ೘ೠ/ ோ௬) ≤3.2 (2.31) column (The Japan Building Disaster Prevention Association, 2001). 2001). For case Rmu< Ry F = 1 + 0,27 ோ೘ೠି ோమఱబ ோ೤ି ோమఱబ (2.30) (ii) For case Rmu ≥ Ry F = ට ଶ ோ೘ೠ/ோ೤ି ଵ ଴.଻ହ(ଵା଴.଴ହ ோ೘ೠ/ ோ௬) ≤3.2 (2.31) (2.31) 2.3 Effective Strength Factor (α) The value of the effective strength factor (α) can be seen in table 1. αs is the effective strength factor of the shear column which is calculated using equation (2.32). αm is the effective factor strength factor of the bending column which is determined using equation (2.33). Rmy is the angle of deformation that occurs when bending is calculated using equation (2.15). Rsu is the angle of deformation when the shear strength is calculated using equations (2.16) and (2.17). Q (F1) is the shear force when the deformation capacity R1 of a column in the second or higher group. Qsu is the shear strength of a column in the second group or higher. Qmu is the load when flexural yielding of a column in the second group or higher (The Japan Building Disaster Prevention Association, 2001). 2.2.3 Ductility Index (F) 4.1 Building description The ductility index of the bending column can be calculated using equation (2.30) or (2.31) based on the angle formed on each floor of the building during the ultimate deformation in the bending failure of the The structural details for calculations such as column cross-section size, column details, reinforcement quality are obtained from the DED (Detail 3 3 https://doi.org/10.1051/e3sconf/202015605005 E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 Engineering Design) of the building. The first-floor plan of the building is illustrated in Fig. 2 Engineering Design) of the building. The first-floor plan of the building is illustrated in Fig. 2 direction). In the analysis of the brick wall is ignored in the calculation by considering the wall as a non- structure. The seismic capacity of a building is expressed in the relationship between the strength index and the ductility index as shown in Fig. 3.a for north- south direction and Fig. 3.b for east-west direction. Fig. 3.a shows the collapsing stages of the north-south column. The building has a total strength index of 0.307. At the ductility index of 0.8, the building collapses before the plastic limit by reducing the strength index to 0.044. 4.2 Seismic Capacity of Reinforced Concrete Buildings The seismic capacity of the Tadulako University Faculty of Law building is evaluated only for the first floor, where the floor has a maximum load. The analysis is calculated in two (2) directions, from north-south direction (X direction) and east-west direction (Y Table 1. Effective strength factors (α) The value of F1 for the first group = 0.8 (R1 = R500 = 1/500) F1 F1 = 0,8 R1 R1 = R500 First Group Shear (Rsu = R250) ߙs Shear (Rsu < R250) ߙs Bending (Rmy = R250) 0,65 Bending (R250 < Rmy < R150) ߙm Bending (Rmy = R150) 0,51 Shear wall dan bending 0,65 If in the first group, the F1 value > 1.0 (R1 ≥ R250 = 1/250) F1 F1 = 1.0 1.0 < F1 < 1.27 1.27 ≤ F1 R1 R250 R250 < Rmy < R150 R250 = R1 Second Group Shear (Rsu = R250) 1.0 0.0 0 Shear (Rsu < R250) ߙs ߙs 0 Bending (Rmy = R250) 1.0 1.0 1.0 Bending (R250 < Rmy < R150) ߙm ߙm 1.0 Bending (Rmy = R150) 0.72 ߙm 1.0 g. 2: The floor load area for one column K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K1 K1 K1 K1 800 240 1040 500 900 900 900 300 236 305 4040 U T Table 1. Effective strength factors (α) The value of F1 for the first group = 0.8 (R1 = R500 = 1/500) F1 F1 = 0,8 R1 R1 = R500 First Group Shear (Rsu = R250) ߙs Shear (Rsu < R250) ߙs Bending (Rmy = R250) 0,65 Bending (R250 < Rmy < R150) ߙm Bending (Rmy = R150) 0,51 Shear wall dan bending 0,65 If in the first group, the F1 value > 1.0 (R1 ≥ R250 = 1/250) F1 F1 = 1.0 1.0 < F1 < 1.27 1.27 ≤ F1 R1 R250 R250 < Rmy < R150 R250 = R1 Second Group Shear (Rsu = R250) 1.0 0.0 0 Shear (Rsu < R250) ߙs ߙs 0 Bending (Rmy = R250) 1.0 1.0 1.0 Bending (R250 < Rmy < R150) ߙm ߙm 1.0 Bending (Rmy = R150) 0.72 ߙm 1.0 Fig. 4.2 Seismic Capacity of Reinforced Concrete Buildings 2: The floor load area for one column K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K1 K1 K1 K1 800 240 1040 500 900 900 900 300 236 305 4040 U T Fig. 2: The floor load area for one column K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K2 K1 K1 K1 K1 K1 K1 800 240 1040 500 900 900 900 300 236 305 4040 U T Fig. 2: The floor load area for one column west column. The building has a total strength index of 0.364. At the ductility index of 0.8, some columns collapse before reaching the plastic limit by reducing the strength index to 0.090. The collapsed column at the 0.8 ductility index is the shear column. This column experiences sliding collapse because there is a brick wall that locks so that it is as a short column with a net height of 1.8 meters. These columns include x1-y3 and x2-y3 columns. The collapse of this column caused a drastic reduction in the building strength index so that the building strength index became 0.223. The collapsed column at the 0.8 ductility index is the shear column. This column experiences sliding collapse because there is a brick wall that locks so that it is as a short column with a net height of 1.8 meters. These columns include the x1-y2 column. The collapse of this column caused a drastic reduction in the building strength index so that the building strength index became 0.138. Furthermore, at the 2.48, ductility index the building collapsed again by reducing the strength index to 0.238. The building continues to collapse the column until all columns collapse at the ductility limit of 3.2. Fig. 3.b shows the collapsing stages of the east- E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 https://doi.org/10.1051/e3sconf/202015605005 a. Relationship between Index C and F in the North-South Direction b. 4.2 Seismic Capacity of Reinforced Concrete Buildings Relationship between Index C and F Index in East-West Direction 0,000 0,050 0,100 0,150 0,200 0,250 0,300 0 0,5 1 1,5 2 2,5 3 3,5 Strength Indexes C Ductility Indexes F 0,000 0,050 0,100 0,150 0,200 0,250 0,300 0,350 0,400 0 0,5 1 1,5 2 2,5 3 3,5 Strength Indexes C Ductility Indexes F 0,000 0,050 0,100 0,150 0,200 0,250 0,300 0 0,5 1 1,5 2 2,5 3 3,5 Strength Indexes C Ductility Indexes F a. Relationship between Index C and F in the North-South Direction a. Relationship between Index C and F in the North-South Direction a. Relationship between Index C and F in the North-South Direction a. Relationship between Index C and F in the North South Direction b. Relationship between Index C and F Index in East-West Direction 0,000 0,050 0,100 0,150 0,200 0,250 0,300 0,350 0,400 0 0,5 1 1,5 2 2,5 3 3,5 Strength Indexes C Ductility Indexes F b. Relationship between Index C and F Index in East-West Direction Fig. 3. Graph of Relationship between C and F Index be too large for the building to become more rigid so it is expected to withstand the earthquake load. Decreasing the strength index can be a possible reason why the building collapsed when the major quake struck. Furthermore, at the ductility index of 1.03, the building collapsed again by reducing the strength index to 0.332. The building continues to collapse the column until all columns collapse at the ductility limit of 3.2. 5 Conclusion Based on the results of the analysis and evaluation of the Tadulako University Faculty of Law building according to The Standard for Seismic Evaluation Of Existing Reinforced Concrete Building, 2001 (The Japan Building Disaster Prevention Association, 2005) [7] the second level calculation method, it can be concluded : 1. The lateral strength index of the Tadulako University Faculty of Law building north-south is 0.264. In the north-south direction, some of the columns experienced shear collapse because there were brick walls which can be translated to the short columns with a net height of 1.27 m. Most of the columns in the north-south direction are quite ductile because they have a ductility index 3.2. 2. The lateral strength index of the Tadulako University Faculty of Law building east-west direction is 0.364. Few columns in the east-west direction have a ductility index 3.2. Based on the results of the analysis and evaluation of the Tadulako University Faculty of Law building according to The Standard for Seismic Evaluation Of Existing Reinforced Concrete Building, 2001 (The Japan Building Disaster Prevention Association, 2005) [7] the second level calculation method, it can be concluded : 4.3 Mitigation efforts In the earthquake-prone area for reinforced concrete buildings, the quality of concrete K-300 (fc' 25 MPa). The floor is recommended to be used in the diameter of the minimum stroke is reinforcement screw diameter 10 mm (D10) and Rainwater drain pipes should not be entered into the column because it will reduce the column cross-section so that the column strength will also decrease. Based on SNI 2847:2013 [9], the length of the main reinforcement of the beam to the column is 12d with a curvature of 90°. Long overlapping on a major reinforcement connection of 40d. The length of the stirrups latch is 6d with a curve of 135° [8]. The distance between the columns in the building should not 1. The lateral strength index of the Tadulako University Faculty of Law building north-south is 0.264. In the north-south direction, some of the columns experienced shear collapse because there were brick walls which can be translated to the short columns with a net height of 1.27 m. Most of the columns in the north-south direction are quite ductile because they have a ductility index 3.2. 2. The lateral strength index of the Tadulako University Faculty of Law building east-west direction is 0.364. Few columns in the east-west direction have a ductility index 3.2. 5 E3S Web of Conferences 156, 05005 (2020) 4th ICEEDM 2019 https://doi.org/10.1051/e3sconf/202015605005 4.Liza, N M., Maidiawati., dan Tanjung, J., Proc. 1st Andalas Civil Engineering National Conference; Padang (2014) References 1.Agus. dan Maidiawati, Perbandingan Kapasitas Seismik Gedung Beton Bertulang dengan dan tanpa pengaruh Dinding Bata. Padang : Institut Teknologi Padang, In Indonesia language (2017) 5.Maidiawati, Agus, Jurnal Teknik Sipil ITB 23 (2016) 5.Maidiawati, Agus, Jurnal Teknik Sipil ITB 23 (2016) 6.Maidiawati, Yasushi Sanada DOI : 10.1002/eqe.2787 (2016) 6.Maidiawati, Yasushi Sanada DOI : 10.1002/eqe.2787 (2016) 2.Akbar, F. A. Tugas Akhir Analisis Kontribusi Dinding Batu Bata terhadap Kekuatan Lateral Gedung Beton Bertulang (Studi Kasus Gedung Perkuliahan Institut Teknologi Padang). Padang : Institut Teknologi Padang. In Indonesia language (2017) 7.The Japan Building Disaster Prevention Association (JBDPA), English Version, 1st, Standard for seismic evaluation of existing reinforced concrete buildings, 2001 (2005) 8. Badan Standardisasi Nasional Persyaratan Beton Struktural untuk Bangunan Gedung SNI2847:2013. Jakarta (2013) 3.Earthquake Engineering Research Institute, Learning from Earthquakes, The Mw 7.6 Western Sumatra earthquake of September 30, 2009, EERI Special Earthquake Report (2009) 6
https://openalex.org/W1986806664	https://europepmc.org/articles/pmc4613723?pdf=render	English	null	Autonomous and non-autonomous roles of DNase II during cell death in <i>C. elegans</i> embryos	Bioscience reports	2,015	cc-by	10,479	Biosci. Rep. (2015) / 35 / art:e00203 / doi 10.1042/BSR20150055 Biosci. Rep. (2015) / 35 / art:e00203 / doi 10.1042/BSR20150055 Biosci. Rep. (2015) / 35 / art:e00203 / doi 10.1042/BSR20150055 Biosci. Rep. (2015) / 35 / art:e00203 / doi 10.1042/BSR20150055 ............................................................................................................................................................................................................................................................................................................ Abbreviations: CAD, caspase-associated DNase; ER, endoreticulum; EtBr, ethidium bromide; HDEL, tetrapeptide of His–Asp–Glu–Leu; MADA, metachromatic agar-diffusion assay; NGM, nematode growth medium; PCD, programmed cell death; RT, room temperature; ToLFP, topoisomerase ligation ﬂuorescence probes; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. 1 To whom correspondence should be addressed (email losj@mail.cgu.edu.tw). Synopsis y p Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulﬁng cells (non-autonomous). The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method is used as an in situ assay of apoptosis by labelling DNA fragments generated by caspase-associated DNase (CAD), but not those by the downstream DNase II. In the present study, we report a method of ToLFP (topoisomerase ligation ﬂuorescence probes) for directly visualizing DNA fragments generated by DNase II in Caenorhabditis elegans embryos. ToLFP analysis provided the ﬁrst demonstration of a cell autonomous mode of DNase II activity in dying cells in ced-1 embryos, which are defective in engulﬁng apoptotic bodies. Compared with the number of ToLFP signals between ced-1 and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was ∼3–7. Among three DNase II mutant embryos (nuc-1, crn-6 and crn-7), nuc-1 embryos exhibited the least number of ToLFP. The ToLFP results conﬁrmed the previous ﬁndings that NUC-1 is the major DNase II for degrading apoptotic DNA. To further elucidate NUC-1′s mode of action, nuc-1- rescuing transgenic worms that ectopically express free or membrane-bound forms of NUC-1 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed NUC-1 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA. Cite this article as: Bioscience Reports (2015) 35, e00203, doi:10.1042/BSR20150055 Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos Hsiang Yu, Huey-Jen Lai†, Tai-Wei Lin* and Szecheng J. Lo1 Hsiang Yu, Huey-Jen Lai†, Tai-Wei Lin and Szecheng J. Lo1 Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan †Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80302, U.S.A. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. Generation of cguIs3 transgenic worm g g Plasmid of pPnuc-1nuc-1::lmp-1153–237::gfp was constructed by in- sertion of a PCR fragment of lmp-1153–237 into the MscI and AgeI restriction enzyme sites of pPnuc-1nuc-1::gfp and was conﬁrmed by sequencing. The primers for amplifying the lmp-1153–237 frag- ment were as follows: forward primer: CCAAtgctccaccgcca; re- verse primer: TTCTACCGGTTTgacgctggcatatccttg. The integ- ration line of cguIs3 was obtained by microinjection with the target plasmid (100 μg/ml) and a plasmid with the rol-6 selection marker (40 μg/ml) and following to use the UV/TMP method as described previously for selection of transgene integration [27]. The integration line was back-crossed by mating to nuc-1(e1392) males for four times. INTRODUCTION However, questions with regard to these modes of DNase II action in worms, particularly related to spatial mani- festation and function representation, remain unanswered. In the present study, we employed a method, ToLFP (topoisomerase ligation of ﬂuorescence probes), for directly labelling the DNA breaks generated by DNase II with ﬂuorescence probes [22–25]. By applying ToLFP to examine worms of various genetic back- grounds, our current results show that the relative representation of the autonomous and non-autonomous actions of DNase II is ∼70%–30% and further demonstrate that the ToLFP method can complement with the method of TUNEL in studying apoptotic DNA degradation. [10,11,20]. However, questions with regard to these modes of DNase II action in worms, particularly related to spatial mani- festation and function representation, remain unanswered. In the present study, we employed a method, ToLFP (topoisomerase ligation of ﬂuorescence probes), for directly labelling the DNA breaks generated by DNase II with ﬂuorescence probes [22–25]. By applying ToLFP to examine worms of various genetic back- grounds, our current results show that the relative representation of the autonomous and non-autonomous actions of DNase II is ∼70%–30% and further demonstrate that the ToLFP method can complement with the method of TUNEL in studying apoptotic DNA degradation. MATERIALS AND METHODS Figure 1 Illustration of in situ TUNEL assay in consecutive steps of DNA degradation during apoptosis During cell apoptosis, inside of dying cells CADs cleavage the intact DNA into fragments exposing 3′-hydroxy ends which can be extended with poly-dUTP by terminal deoxynucleotidy transferase (TdT) and shown as TUNEL positive. Subsequently, the DNA fragments are then digested by DNase II, either in dying cells or in engulﬁng cells, to yield products with 5′-hydroxy ends and 3′-phsophate ends which are unable to be reacted with tdt and shown as TUNEL negative. In the present study, a ToLFP was employed to directly label the 5′-hydroxy ends and shown as ToLFP positive. INTRODUCTION by in situ extension of poly-dUTP from the 3′-hydroxy ends (Figure 1) [6,7]. The later steps of further degradation of fragmented DNA in cell apoptosis rely on DNase II (EC 3.1.22.1), an acidic deoxyribonuclease. Due to its lysosomal localization and optimal activity at pH 4.5–5.5, DNase II was initially thought to play a role in the digestion of exogenous DNA engulfed by phagocytosis in many animals [8,9]. Subsequent studies in Caenorhabditis el- egans found that NUC-1 (a key member of three C. elegans DNase II enzymes) is also involved in DNA fragmentation and degradation during cell apoptosis [10]. DNase II is known to act downstream of CAD to further digest large DNA fragments into small DNA fragments or mononucleotides by hydrolysing the phosphodiester linkages in both native and denatured DNA to yield products with 5′-hydroxy ends and 3′-phosphate ends (Figure 1). As these structures are not substrates for terminal Programmed cell death (PCD) or apoptosis plays a vital role in animal development and tissue homoeostasis and is tightly regu- lated by a multitude of hydrolytic enzymes [1,2]. Subsequent to the activation of caspases, cells trigger a cascade of enzymes in- volved in digesting DNA of apoptotic bodies [3]. The early events of internucleosomal chromatin DNA fragmentation are mediated by a family of endonucleases called CAD (caspase-associated DNase) [4]. CAD is a magnesium-dependent endonuclease spe- ciﬁc for cutting dsDNA to generate 3′-hydroxy ends [5], which is a hallmark of apoptosis. Accordingly, established assays for cell apoptosis entail detection of the appearance of DNA ladders (internucleosomal DNA) by agarose gels and signals of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) 1 H. Yu and others Figure 1 Illustration of in situ TUNEL assay in consecutive steps of DNA degradation during apoptosis During cell apoptosis, inside of dying cells CADs cleavage the intact DNA into fragments exposing 3′-hydroxy ends which can be extended with poly-dUTP by terminal deoxynucleotidy transferase (TdT) and shown as TUNEL positive. Subsequently, the DNA fragments are then digested by DNase II, either in dying cells or in engulﬁng cells, to yield products with 5′-hydroxy ends and 3′-phsophate ends which are unable to be reacted with tdt and shown as TUNEL negative. In the present study, a ToLFP was employed to directly label the 5′-hydroxy ends and shown as ToLFP positive. Fig 1 Ill t ti f i it TUNEL i ti t [10,11,20]. Strain maintenance All i i i All strains were maintained with standard procedures and raised at 20 ◦C [26]. Bristol N2 was used as a wild-type animal. Two apoptosis mutants [LGIV: ced-3(n717) and LGI: ced- 1(e1735)] and three DNase II mutants: LGIII: crn-6(tm890), crn- 7(ok866); LGX: nuc-1(e1392) were described previously [21]. Three DNase II double mutants [crn-7(ok886) crn-6(tm890), crn-7(ok866); nuc-1(e1392) and crn-6(tm890); nuc-1(e1392)] and one triple mutant (crn-7 crn-6; nuc-1) were obtained from a previous study [21]. A transgenic worm of smIs172 [nuc-1 (e1392); Pnuc-1nuc-1::gfp] was created previously [21] whereas another transgenic worm of cguIs3 [nuc-1 (e1392); Pnuc-1nuc- 1::lmp-1153–237::gfp] was generated in the present study. transferase [8,11,12], DNase II activity in situ is correlated with decreases in TUNEL signals, albeit indirectly. In mammalian apoptosis, the action of DNase II is mostly restricted to the phagocytes or engulﬁng cells that digest DNA fragments of apoptotic cells and belongs to a unique class that exhibits cell non-autonomous activity [9,13–15]. Whether DNase II functions as a cell-autonomous nuclease remains controversial. However, the view that DNase II can function autonomously has been supported by two lines of evidence: (1) DNase II have been detected in the nuclei of Chinese hamster ovary (CHO) and HL-60 cells and (2) intracellular acidiﬁcation occurs in many apoptotic cells [16,17]. It was suggested that DNase II could be released from lysosomes for cleaving nuclear DNA when the intracellular acidiﬁcation occurs during cell apoptosis [16,18]. Furthermore, a cell autonomous action of DNase II has been found in Drosophila, through which the DNA of dying nurse cells under starvation-induced necrosis is degraded [19]. In C. elegans, the autonomous action has been demonstrated by the ﬁndings that DNA is digested into small pieces in the ced-1 (encoding a transmembrane receptor for cell corpse recognition) mutant background [20]. In addition to the ced-1 mutant, the TUNEL signals were increased in other engulfment-defective mutants (ced-2, ced-5,ced-6 and ced-10) in the absence of NUC- 1 [10,21], thus suggesting that the DNA fragments are normally digested within dying cells in an autonomous manner. Topoisomerase ligation of ﬂuorescence probe to detect the DNase II acting sites ToLFP was applied to detect the DNA fragmentation that is spe- ciﬁcally generated by DNase II in embryos. As previously de- scribed by Minchew and Didenko [22–25], a ﬂuorescent probe was able to label the DNA fragments having 5′-hydroxy ends (DNase II-type breaks) by topoisomerase. ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit (APT1000) was purchased Previous reports have demonstrated that the functional roles of NUC-1 in PCD and engulfment-mediated DNA degradation in C. elegans comprises two phases: an autonomous action shown by a negative TUNEL staining in dying cells followed by a non-autonomous action of DNA elimination in phagocytic cells access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 2 DNase II action in embryos DNase II action in embryos DNase II action in embryos an internal control of loaded protein for the in vitro DNase II activity assay. from Merck Millipore. We followed the standard protocol that was provided by Merck Millipore with a slight modiﬁcation. Fixation of embryos was conducted as previously described [28]. Fixed samples were added into a solution that was premixed with topoisomerase and ﬂuorescent probes. After 12–16 h of in- cubation at 20 ◦C, embryos were washed three times with PBST (1× PBS with 0.1% Tween 20) and then mounted on to a slide which contains 2% agar pad. Z-stack images were detected and acquired under a ﬂuorescence microscope (Leica DM IRE2 with BD CARV II) with a FITC ﬁlter. Immunoﬂuorescence of staining ER-marker and GFP Embryos from smIs172 and cguIs3 were collected by hypochlor- ite/NaOH treatment. After freeze cracking, embryos were ﬁxed with 3.7% formaldehyde, 75% methanol, 0.5× PBS for 15 min at −20 ◦C followed by 15 min in 100% methanol and then in- cubated in a blocking solution (30% goat serum in PBS/0.1% Tween 20) for 1 h at room temperature (RT). The primary anti- bodies of anti-GFP (GeneTex) and anti-HDEL (tetrapeptide of His–Asp–Glu–Leu) antibody (Santa Cruz) with 1:200 dilution in blocking buffer were reacted with embryos for 12–16 h at 4 ◦C, the secondary antibodies anti-rabbit AlexFluor-488 (for staining GFP) and anti-mouse AlexFluor-594 (for staining ER, endoretic- ulum; Invitrogen, 1:500) were reacted for 2–4 h at RT, following by 5 min DAPI staining. Metachromatic agar diffusion assay DNase II activities of embryo lysates were also examined by metachromatic agar-diffusion assay (MADA), as previously de- scribed [29] with some modiﬁcations. The boiled 1% agarose gel contained DNase II assay buffer (100 mM sodium acetate, pH 4.5, 10 mM EDTA) and mixed with salmon sperm DNA (40 μg/ml; Sigma) and EtBr (400 μg/ml) at 65–70 ◦C, was then poured into a 9-cm plastic petridish. The wells were made on solidiﬁed gels with a Pasteur pipettes using 5 μg of samples, as determined by the measurement of an A595, where the amount of tubulin was veriﬁed by Western blotting as a loading con- trol. Crude extracts were prepared from embryos, which were collected by hypochlorite/NaOH treatment and homogenized in PBST (by douncing 20 times with pre-cooled Dura-Grind stain- less steel dounce tissue grinder (Wheaton). After 16-h incubation at 37 ◦C, a ring like DNA consumption pattern was observed un- der UV light, which represents the DNase II activity for each sample. The area of dark ring was measured by ImageJ software. A DNase II activity standard was included employing porcine DNase II (Sigma) in 0.5–50 KUs (Kunitz units). non-autonomous manner Since the TUNEL method is an indirect measurement of the DNase II activity in vivo, it is hardly to distinguish the autonom- ous and non-autonomous action of DNase II in C. elegans. We ad- apted a newly established method called ToLFP to directly label the 5′-hydroxy ends of DNA fragments, which are the products of DNase II digestion, with the FITC-labelled DNA fragments [22]. To distinguish the autonomous and non-autonomous action of DNase II, we ﬁrst examined ToLFP signals in the wild-type worm (N2) embryos and two mutant embryos of ced-3 (encod- ing a homologue of mammalian caspase) and ced-1 (encoding a transmembrane receptor for cell corpse recognition). As com- pared with positive signals of ToLFP in N2 embryos, no ToLFP STYO 11 staining The undigested bacteria DNA in the gut lumen was stained by vital DNA-binding dye SYTO11 (Molecular Probe, Invitrogen) as previously described [21]. The L3 and L4 stage worms were collected and washed twice with M9 buffer then transferred into a 1.5 ml of tube containing SYTO11 dye (10 μM in M9) at RT for 2 h in the dark. The stained worms were washed twice with M9 and recovered on Escherichia coli OP50 seeded NGM (nematode growth medium) plates at RT for 1 h in the dark. After recovery, the worms were washed twice with M9 and anaesthetized by lavamisole (200 μM), then mounted on to 2% agar pads and observed under a ﬂuorescence microscope (Leica DM2500) with a FITC ﬁlter. In vitro assay of DNase II activity DNase II activities of embryo lysates were analysed by gel electrophoresis. Embryos were harvested by hypochlorite/NaOH treatment and lysed by a sonication method in PBST buffer con- taining protease inhibitors (Roche). Protein quantiﬁcation was performed by measurement of A595 using a Dye Reagent Con- centrate (Bio-Rad). One microgram of each embryo extract was incubated with 25 μg of salmon sperm DNA (Sigma) at 37 ◦C in 100 μl of DNase II assay buffer (100 mM sodium acetate, pH 4.5, 10 mM EDTA) and collected at desired time point. Samples were separated by 1% agarose gel and stained with EtBr (ethidium bromide) for determination of DNase II activity. Topoisomerase ligation of ﬂuorescence probe to detect the DNase II acting sites Samples were washed three times with PBST after each step of reaction. Images were taken using Zeiss Axio Imager.Z2 with apotome 2. Western blotting Total proteins from various genetic backgrounds (N2, ced-3, ced- 1, smIs172 and cguIs3) embryos were separated by SDS/gel elec- trophoresis and processed for Western blot analysis. Five micro- grams of each embryo extract was added into 1× sample buffer in a 20 μl of total volume and boiled at 100 ◦C for 15 min. The lysate was cooled on ice for 5 min and then centrifuged to remove debris and stored at −80 ◦C. Anti-actin (1:100000) was used as c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. H. Yu and others H. Yu and others H. Yu and others ced-3 N2 (phase) N2 (FITC) A ced-1 d c b a A P A P B time N2 N2 ced-3 ced-1 5 . 4 : H p 5 . 7 : H p M N2 (pH:7.5) ced-3 ced-1 N2 (pH:4.5) actin C 2 1 6 5 3 2 1 7 1 1 4 10 9 8 ced-1 3 1 4 2 3 1 4 2 5 6 5 6 7 5 6 7 7 7 8 10 8 9 7 8 9 8 10 11 10 11 4 4 3 3 2 1 N2 8 7 6 8 7 6 8 7 6 1 2 3 4 5 1 2 3 4 5 8 7 8 7 9 10 7 9 11 10 12 9 11 13 10 12 11 13 11 14 15 14 15 16 17 16 17 18 D E a b i g c j k d l e f h a b i g c j k d l e f h ure 2 ToLFP staining in embryos from wild-type animals (N2) and two apoptosis related mutants, ced-3 (caspase defective mutant) and ced-1 (a defective mutant of engulﬁng apoptotic bodies) (A) A representative image of ToLFP labelling patterns from N2, ced-3 and ced-1 embryos. The same embryo of N2 is shown in a phase image (a) and ﬂuorescence image (b), a ced-3 embryo in a ﬂuorescence image (c) and a ced-1 embryo in a ﬂuorescence image (d). The imaginary line in (a) and (b) indicates anterior (A) and posterior (P) half of embryo. Scale bar: 20 μm. (B) An in vitro assay of DNase II activity. Western blotting The samples of each strain were collected and analysed at three time points: 0, 15 and 30 min. The lysates from various genetic background worms are indicated above the gel. M lane shows DNA ladder markers. (C) The Western blot analysis of actin is used as loading amount of lysates. (D) Quantitative analysis of ToLFP number of whole embryos. A series section of images (a–l) from a single N2 embryo shows the total ToLFP signals (labelled with numbers). (E) A series section of images (a–l) from a single ced-1 embryo shows the total ToLFP signals (labelled with numbers). Scale bar: 20 μm. Figure 2 ToLFP staining in embryos from wild-type animals (N2) and two apoptosis related mutants, ced-3 (caspase defective mutant) and ced-1 (a defective mutant of engulﬁng apoptotic bodies) Figure 2 ToLFP staining in embryos from wild-type animals (N2) and two apoptosis related mutants, ced-3 (caspase defective mutant) and ced-1 (a defective mutant of engulﬁng apoptotic bodies) (A) A representative image of ToLFP labelling patterns from N2, ced-3 and ced-1 embryos. The same embryo of N2 is shown in a phase image (a) and ﬂuorescence image (b), a ced-3 embryo in a ﬂuorescence image (c) and a ced-1 embryo in a ﬂuorescence image (d). The imaginary line in (a) and (b) indicates anterior (A) and posterior (P) half of embryo. Scale bar: 20 μm. (B) An in vitro assay of DNase II activity. Embryo extracts from N2, ced-3 and ced-1 were analysed the DNase II activity under the condition of pH 7.5 (lane 1–3) or pH 4.5 (lane 4–12). The samples of each strain were collected and analysed at three time points: 0, 15 and 30 min. The lysates from various genetic background worms are indicated above the gel. M lane shows DNA ladder markers. (C) The Western blot analysis of actin is used as loading amount of lysates. (D) Quantitative analysis of ToLFP number of whole embryos. A series section of images (a–l) from a single N2 embryo shows the total ToLFP signals (labelled with numbers). (E) A series section of images (a–l) from a single ced-1 embryo shows the total ToLFP signals (labelled with numbers). Scale bar: 20 μm. of pH 4.5 (Figure 2B). Western blotting Embryo extracts from N2, ced-3 and ced-1 were analysed the DNase II activity under the condition of pH 7.5 (lane 1–3) or pH 4.5 (lane 4–12). The samples of each strain were collected and analysed at three time points: 0, 15 and 30 min. The lysates from various genetic background worms are indicated above the gel. M lane shows DNA ladder markers. (C) The Western blot analysis of actin is used as loading amount of lysates. (D) Quantitative analysis of ToLFP number of whole embryos. A series section of images (a–l) from a single N2 embryo ced-3 N2 (phase) N2 (FITC) A ced-1 d c b a A P A P B time N2 N2 ced-3 ced-1 5 . 4 : H p 5 . 7 : H p M 2 1 6 5 3 2 1 7 1 1 4 10 9 8 B N2 (pH:7.5) ced-3 ced-1 N2 (pH:4.5) actin C C C N2 8 7 6 8 7 6 8 7 6 1 2 3 4 5 1 2 3 4 5 8 7 8 7 9 10 7 9 11 10 12 9 11 13 10 12 11 13 11 14 15 14 15 16 17 16 17 18 D a b i g c j k d l e f h ced-1 3 1 4 2 3 1 4 2 5 6 5 6 7 5 6 7 7 7 8 10 8 9 7 8 9 8 10 11 10 11 4 4 3 3 2 1 E a b i g c j k d l e f h E Figure 2 ToLFP staining in embryos from wild-type animals (N2) and two apoptosis related mutants, ced-3 (caspase defective mutant) and ced-1 (a defective mutant of engulﬁng apoptotic bodies) (A) A representative image of ToLFP labelling patterns from N2, ced-3 and ced-1 embryos. The same embryo of N2 is shown in a phase image (a) and ﬂuorescence image (b), a ced-3 embryo in a ﬂuorescence image (c) and a ced-1 embryo in a ﬂuorescence image (d). The imaginary line in (a) and (b) indicates anterior (A) and posterior (P) half of embryo. Scale bar: 20 μm. (B) An in vitro assay of DNase II activity. Embryo extracts from N2, ced-3 and ced-1 were analysed the DNase II activity under the condition of pH 7.5 (lane 1–3) or pH 4.5 (lane 4–12). Western blotting The quantitative data of total ToLFP signals from at least 15 embryos are summarized in Figure 3(C), which showed that both N2 and crn-7 had on average 19–20 labelled signals in contrast with 7 and 14 exhibited by nuc-1 and crn-6 respectively. These ﬁndings were consistent with the in vitro MADA analyses that NUC-1 is the primary DNase II for the clearance of apoptotic DNA in dying cells during embryogenesis (Figure 4). The semi-quantitative data of MADA were summarized from three independent experiments in which the N2 activity was regarded as 100% (Figure 4C). The cell extracts from embryos of double mutants, crn-7(ok886) crn- 6(tm890), crn-7(ok866); nuc-1(e1392) and crn-6(tm890); nuc- 1(e1392) respectively, showed 90.6%, 1.4% and 3.6% of the total DNase II activity of N2 (Figure 4C), indicating that NUC-1 alone had 30–50 folds higher activity than either CRN-6 or CRN- 7. This conclusion was further supported by the signiﬁcantly reduced DNase II activity (7.5% of total) in the nuc-1 mutant, which presumably was contributed by CRN-6 and CRN-7. but the presence of functional DNase II enzyme. Furthermore, the quantitative difference of ToLFP numbers between N2 and ced-1embryos (Figure 2A frame b compared with frame d) arose from defective apoptotic body engulfment in ced-1 embryos. The ToLFP signals from ced-1 embryos were presumably due to the autonomous action of DNase II only. To quantify the total ToLFP number in each embryo from N2 and ced-1 mutants, we photographed each individual em- bryo under a ﬂuorescence microscope with Z-axis sections. Rep- resentative results from N2 and ced-1 embryos are shown in Figures 2(D) and 2(E) respectively. Approximately 14 of ToLFP signals were observed in the ced-1 embryos (Table 1) clearly illustrating DNase II activity in the dying cells (autonomous ac- tion) but lack thereof in the engulﬁng cells (non-autonomous action). Since only part of embryonic cells expresses NUC-1 and given that not all the cells destined to die autonomously ex- press NUC-1, the six additional ToLFP signals in N2 embryos in comparison with the ced-1 embryos were suggested to be from non-autonomous action (Table 1). As a whole embryo, the percentage of non-autonomous to autonomous degradation of apoptotic DNA was ∼30%–70%; however, a higher percentage (∼45%) of non-autonomous degradation of DNA was found in the posterior half of embryos (Table 1). Western blotting The differential detection of DNase II activity of ced-3 embryos between in vitro assay and in vivo assay (Figure 2B, lanes 7–9 compared with Figure 2A, frame c) was probably attributed to the lack of CED-3 (caspase) in worms, which leads to no apoptosis and DNA fragmentation, signal was observed in ced-3 embryos, whereas signiﬁcant num- bers of ToLFP signals were observed in ced-1 embryos at the comma stage (Figure 2A). By contrast, similar levels of DNase II activities were observed in N2, ced-3 and ced-1 embryo lys- ates when the lysates were assayed in vitro under the condition c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. DNase II action in embryos Table 1 The summarized data of autonomous and non-autonomous action of DNase II in C. elegans through ToLFP staining Embryos were scored at the 1.5-fold stage. The data shown are mean +−S.E.M. N-number: N2 = 13 and ced-1(e1735) = 12. Table 1 The summarized data of autonomous and non-autonomous action of DNase II in C. elegans through ToLFP staining Embryos were scored at the 1.5-fold stage. The data shown are mean +−S.E.M. N-number: N2 = 13 and ced-1(e1735) = 12. Embryos were scored at the 1.5-fold stage. The data shown are mean +−S.E.M. N-number: N2 = 13 and ced-1(e1735) = 12. Percentage of apoptotic DNA degradation by autonomous and non-autonomous manners Genotype Number of ToLFP Autonomous Non-autonomous Whole embryo N2 19.85 +−0.7 70 % (13.92/19.85) 30 % (5.93/19.85) ced-1 13.92 + 0.6 100 % (13.92/13.92) 0 % (0/13.92) Head N2 16.54 + 0.7 74 % (12.17/16.54) 26 % (4.37/16.54) ced-1 12.17 + 0.6 100 % (12.17/12.17) 0 % (0/12.17) Tail N2 3.3 + 0.3 55 % (1.8/3.3) 45 % (1.5/3.3) ced-1 1.8 + 0.2 100 % (3.3/3.3) 0 % (0/3.3) exhibited the least number of ToLFP signals among the four strains. However, the average size of the ToLFP signal was the largest in nuc-1 embryos (Figure 3B), suggesting a distinct mode of action of NUC-1 from that of CRN-6 or CRN-7. To further quantify the total ToLFP-foci numbers in all examined embryos, we photographed each individual embryo under a ﬂuorescence microscope with Z-axis sections as described above and compiled each image into a movie (Supplementary movies S1–S12). Western blotting It was probably a result of higher level of NUC-1 expression in the anterior half of embryos and subsequent secretion to and uptake by the posterior cells of embryos. Phenotypic characterization of two transgenic lines expressing different NUC-1 fusion proteins ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants Since nuc-1 mutants showed the least ToLFP signals, an increas- ing number of ToLFP signals in transgenic embryos which ex- press various forms of NUC-1 fusion proteins can be easily de- tectable. We then employed two transgenic lines, smIs172[nuc-1 (e1392); Pnuc-1nuc-1::gfp] and cguIs3[nuc-1 (e1392); Pnuc-1nuc- 1::lmp-1153–237::gfp] to examine their capability of rescuing of nuc-1 mutant phenotypes and the distribution of ToLFP signals in embryos. Transgenic worms of smIs172 express NUC-1::GFP To support the above results and to conﬁrm our previous TUNEL study showing that NUC-1 plays the major role of DNase II and CRN-6 is auxiliary to NUC-1 in the apoptosis during the early embryonic development [21], we then compared ToLFP signals in three strains of DNase II mutant embryos (nuc-1, crn-6 and crn-7) with the wild-type (N2) embryos at the comma, 1.5- fold and 2-fold stages. As shown in Figure 3(A), nuc-1 embryos c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licen H. Yu and others H. Yu and others H. Yu and others N2 nuc-1 crn-6 crn-7 A N2 nuc-1 crn-6 crn-7 0 100 200 300 400 spot size (pixel) B n=65 n=64 n=57 n=75 N2 crn-6 crn-7 nuc-1 N2 crn-6 crn-7 nuc-1 N2 crn-6 crn-7 nuc-1 0 10 20 30 No. of ToLFP signals / embryo dlo f 2 dlo f 5 . 1 a m m o c n=22 n=24 0 2 = n 8 1 = n 7 2 = n 6 1 = n 5 1 = n 5 2 = n 8 1 = n 6 2 = n 0 2 = n 5 2 = n C Figure 3 ToLFP assay of embryos from N2 and three DNase II mutants (A) ToLFP labelling patterns of N2, nuc-1, crn-6 and crn-7 embryos at the 2-fold stage. Green spots are ToLFP positive signals, which are indicated with white arrows. The anterior of the embryo is shown towards the left. Scale bar: 20 μm. (B) The spot size of ToLFP signals in DNase II mutants. The spot sizes were determined by ImageJ software. More than 10 embryos and over 50 signals were scored for each strain at the comma stage. ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants (C) Quantitative analysis of ToLFP signals in three DNase II mutants at the comma, 1.5-fold and 2-fold stages. At least 16 embryos were scored for each stage and strain. Error bars indicate S.E.M., *P > 0.001. ns, non-signiﬁcant. N2 nuc-1 crn-6 crn-7 A N2 nuc-1 crn-6 crn-7 0 100 200 300 400 spot size (pixel) B n=65 n=64 n=57 n=75 N2 nuc-1 crn-6 crn-7 0 100 200 300 400 spot size (pixel) B n=65 n=64 n=57 n=75 n=24 0 2 = n 8 6 1 = n 5 1 = n 8 1 = n 0 2 = n N2 nuc-1 crn-6 crn-7 A B A N2 spot size (pixel) C n=24 0 2 = n 6 1 = n 8 1 = n N2 crn-6 crn-7 nuc-1 N2 crn-6 crn-7 nuc-1 N2 crn-6 crn-7 nu 0 10 20 30 No. of ToLFP signals / embryo dlo f 2 dlo f 5 . 1 a m m o c n=22 n=24 0 2 = n 8 1 = n 7 2 = n 6 1 = n 5 1 = n 5 2 = n 6 2 = n 0 2 = n 5 2 = n C dlo f 5 . 1 Figure 3 ToLFP assay of embryos from N2 and three DNase II mutants P assay of embryos from N2 and three DNase II mutant Figure 3 ToLFP assay of embryos from N2 and three DNase II mutants (A) ToLFP labelling patterns of N2, nuc-1, crn-6 and crn-7 embryos at the 2-fold stage. Green spots are ToLFP positive signals, which are indicated with white arrows. The anterior of the embryo is shown towards the left. Scale bar: 20 μm. (B) The spot size of ToLFP signals in DNase II mutants. The spot sizes were determined by ImageJ software. More than 10 embryos and over 50 signals were scored for each strain at the comma stage. (C) Quantitative analysis of ToLFP signals in three DNase II mutants at the comma, 1.5-fold and 2-fold stages. At least 16 embryos were scored for each stage and strain. Error bars indicate S.E.M., P > 0.001. ns, non-signiﬁcant. fusion protein whereas cguIs3 worms express a different version of fusion protein (NUC-1::LMP-1153–237::GFP) with the insertion of the membrane domain of lysosomal protein LMP-1 between NUC-1 and GFP. ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants This additional transmembrane sequence is known to promote recruitment into lysosomal membrane with the C-terminal portion facing the cytoplasm [30]. The results of MADA showed that lysates from smIs172 and cguIs3 had lower DNase II activity in comparison with N2 (respectively 28.3% and 18.7%), but higher activity than that of nuc-1 mutant (7.5%; Figure 4C), suggesting that the fusion proteins are functional but slightly compromised by the addition of GFP. A lower DNase II activity of cguIs3 than that of smIs172, as well as the observa- tion that both transgenic worms could not exert the full activity of DNase II as N2 worms are probably due to additional GFP and lysosomal segments that disrupt the NUC-1′s proper folding. Nevertheless, double staining of anti-GFP and anti-HDEL (ER marker) in smIs172 and cguIs3 transgenic worms revealed that both NUC-1 fusion proteins appeared at the anterior blastomeres of embryos and were co-localized with ER with a slightly differ- ent pattern (Figure 5). c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. DNase II action in embryos N2 nuc-1 crn-6 crn-7 crn-6;nuc-1 crn-7 crn-6 crn-7;nuc-1 crn-7 crn-6;nuc-1 smIs172 cguIs3 N2 nuc-1 crn-6 crn-7 crn-6;nuc-1 crn-7;crn-6 crn-7;nuc-1 crn-7 crn-6;nuc-1 smIs172 cguIs3 A C B 7.5% 3.6% 80% 95.3% 90.6% 1.4% 0% N2 nuc-1 crn-6 crn-7 crn-6;nuc-1 crn-7 crn-6 crn-7;nuc-1 crn-7 crn-6;nuc-1 smIs172 cguIs3 0% 25% 50% 75% 100% * * * relative enzyme activity ** * 28.3% 18.7% tubulin Figure 4 Analysis of DNase II enzymatic activity of embryo lysate in various genetic backgrounds as indicated by MADA (A) Western blotting with anti-tubulin antibody (in 1:5000 dilution) to show a similar amount of each lysate was loaded. (B) A representative MADA result showing DNase II enzymatic activity. (C) Quantitative analysis was determined by the area of the clear zone on agarose from three independent experiments. The enzymatic activity of DNase II in N2 is served as 100 % and the relative percentage of DNase II activity from each strain of worms is indicated. N=3, Error bars indicate S.E.M., P > 0.05, P > 0.01, P > 0.001. ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants A C 7.5% 3.6% 80% 95.3% 90.6% 1.4% 0% N2 nuc-1 crn-6 crn-7 crn-6;nuc-1 crn-7 crn-6 crn-7;nuc-1 7 crn-6;nuc-1 smIs172 cguIs3 0% 25% 50% 75% 100% * * * * relative enzyme activity * 28.3% 18.7% in C C B N2 nuc-1 crn-6 crn-7 crn-6;nuc-1 crn-7;crn-6 crn-7;nuc-1 crn-7 crn-6;nuc-1 smIs172 cguIs3 Figure 4 Analysis of DNase II enzymatic activity of embryo lysate in various genetic backgrounds as indicated by MADA (A) Western blotting with anti-tubulin antibody (in 1:5000 dilution) to show a similar amount of each lysate was loaded. (B) A representative MADA result showing DNase II enzymatic activity. (C) Quantitative analysis was determined by the area of the clear zone on agarose from three independent experiments. The enzymatic activity of DNase II in N2 is served as 100 % and the relative percentage of DNase II activity from each strain of worms is indicated. N=3, Error bars indicate S.E.M., P > 0.05, P > 0.01, *P > 0.001. ated to 13 and 10 respectively, in smIs172 and cguIs3 embryos (Figure 7A), indicating a higher extent of DNA digestion in the presence of NUC-1::GFP and NUC-1:: LMP-1153–237::GFP. How- ever, the increased number of ToLFP in smIs172 was also signi- ﬁcantly higher than that in cguIs3, suggesting that the two fusion proteins function in different modes or with different levels of activity. Since NUC-1 is expressed in the anterior portion (head) of embryos, we next sought to evaluate any non-autonomous action of the two NUC-1fusion proteins in the posterior (tail) of embryos. The counts of ToLFP signals in the embryos were grouped according to spatial distribution (head or tail) for the nuc-1, smIs172 and cguIs3 strains. Such analysis showed that whereas ToLFP signals remained signiﬁcantly elevated in the head region of smIs172 and cguIs3 embryos as compared with nuc-1 (Figure 7B), they dropped considerably in the tail of cguIs3 embryos in all three stages examined (Figure 7C). Conversely for the smIs172 embryos, signiﬁcantly increased ToLFP signals were observed in the tail at the comma and 1.5-fold stages, but not at the 2-fold stage. Together, these data revealed that NUC-1::GFP, but not NUC-1:: LMP-1153–237::GFP, could execute degradation of apoptotic DNA non-autonomously in the tail cells of em- bryos. We reasoned that the difference might be contributed by the presence of membrane domain of LMP-1 in the protein of NUC-1::LMP-1153–237::GFP, which blocks the secretion of this protein. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants Two NUC-1 fusion proteins are biologically functional in digesting bacterial DNA but varied in non-autonomous degradation of apoptotic DNA After having obtained MADA results showing a partial re- covery of NUC-1 activity by NUC-1::GFP and NUC-1::LMP- 1153–237::GFP fusion proteins (Figure 4C), we then examined if the fusion proteins in two transgenic worms can rescue the nuc-1 phenotypes. Four strains of worms (N2, nuc-1, smIs172 and cguIs3) were stained with DNA dye and then examined for the presence of bacterial DNA in the intestinal lumen under a ﬂuorescence microscope. In contrast with 90% positive DNA staining in the intestine lumen of nuc-1 worms, 10% and 12% of DNA staining were found in the smIs172 and cguIs3 worms respectively, with 7% positive staining in wild-type worms (N2; Figure 6). Alleviation of the defective phenotype by the ectopic NUC-1::GFP and NUC-1::LMP-1153–237::GFP indicated that they are functional counterpart of the wild-type protein. Next, we applied ToLFP to examine whether these two NUC-1 fusion proteins could remove apoptotic DNA in transgenic em- bryos during embryogenesis. To quantify the number of ToLFP signal from embryos of nuc-1, smIs172 and cguIs3 at the comma, 1.5-fold and 2-fold stages, we performed Z-axis sectioning pho- tography as described in Figure 3. Data from at least 18 embryos showed that nuc-1 embryos exhibited approximately six signals at each stage, whereas numbers of ToLFP signals were elev- en access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. H. Yu and others H. Yu and others H. Yu and others cguIs3 Anti-GFP Anti-HDEL (ER marker) DAPI Merge smIs172 Figure 5 Characterization of the two NUC-1 fusion proteins in transgenic animals of smIs172 and cguIs3 The subcellular localization of NUC-1 fusion proteins in smIs172 and cguIs3 embryos. Immunostaining with antibodies against GFP (in 1:500 dilution) and against HDEL (ER marker, in 1:200 dilution) were performed on embryos of smIs172 and cguIs3. Images are indicated with conditions of microscopy for photography. Nucleus was labelled with DAPI. The anterior of embryo is shown toward left and those cells located in the anterior region are enlarged to show co-localization of NUC-1 fusion proteins with ER proteins portion in detail. Imperfect merged of GFP and ER markers are indicated by arrows. Scale bar: 20 μm. ToLFP analysis is consistent with TUNEL analysis for DNA degradation in three DNase II mutants cguIs3 smIs172 Figure 5 Characterization of the two NUC-1 fusion proteins in transgenic animals of smIs172 and cguIs3 The subcellular localization of NUC-1 fusion proteins in smIs172 and cguIs3 embryos. Immunostaining with antibodies against GFP (in 1:500 dilution) and against HDEL (ER marker, in 1:200 dilution) were performed on embryos of smIs172 and cguIs3. Images are indicated with conditions of microscopy for photography. Nucleus was labelled with DAPI. The anterior of embryo is shown toward left and those cells located in the anterior region are enlarged to show co-localization of NUC-1 fusion proteins with ER proteins portion in detail. Imperfect merged of GFP and ER markers are indicated by arrows. Scale bar: 20 μm. Figure 5 DISCUSSION DNase II action in embryos smIs172 12% of strong gut staining in 94 animals cguIs3 10% of strong gut staining in 103 animals N2 7% of strong gut staining in 84 animals nuc-1 90% of strong gut staining in 82 animals Figure 6 Functional assays for the two NUC-1 fusion proteins in digesting bacterial DNA in the gut Staining images of undigested bacteria DNA in the gut lumen by SYTO11. The positive signals are indicated with white arrowheads. The number of scored animals and the percentage of positive DNA staining are shown beneath the strain name in each frame. Scale bar: 100 μm. N2 Figure 6 Functional assays for the two NUC-1 fusion proteins in digesting bacterial DNA in the gut Staining images of undigested bacteria DNA in the gut lumen by SYTO11. The positive signals are indicated with white arrowheads. The number of scored animals and the percentage of positive DNA staining are shown beneath the strain name in each frame. Scale bar: 100 μm. expresses NUC-1 only, also supports this supposition (Supple- mentary Figure S1). The two transgenic worms also provided a new line of evidence that fusion proteins of NUC-1 are enzymat- ically functional (Figures 4B and 4C). However, both transgenic worms showed a similar capability in digesting bacteria DNA in the intestine lumen (Figure 6) but cguIs3 worms appeared less efﬁciently in removing apoptotic DNA in embryos (Figure 7). Accordingly, a hypothetical model shown in Figure 8 depicts the maturation pathways of two fusion proteins and their possible distinct actions on the clearance of bacterial DNA and apop- totic DNA. In this model, both NUC-1 fusion proteins complete the biosynthesis in ER then either enter a secretory pathway or become localized to lysosomes, in response to bacterial DNA or apoptotic signalling. In this context, the NUC-1::GFP fusion protein is probably released into the lumen of intestine for de- grading bacterial DNA or the extracellular space followed by engulfment into neighbouring cells for cleaving apoptotic DNA non-autonomously (Figures 8A and 8B). Whereas NUC-1::LMP- 1153–237::GFP may follow a similar fate (Figures 8C and 8D), upon trafﬁcking it probably remains on the plasma membrane with the DNase II domain facing the intestine lumen, thus retaining activ- ity towards bacterial DNA but not its non-autonomous action in the neighbouring cells. DISCUSSION ToLFP signals in nuc-1 mutant embryos represent the functional sites of CRN-6 and/or CRN-7, their distribution in the precursor of intestine cells evidence the probably non-autonomous action of CRN-6. The larger size of ToLFP signals present in the nuc-1 mutant, indicative of greater accumulation of DNA fragments detected by ﬂuorescent probes (Figures 3A and 3B), also im- plies that NUC-1 acts differently from CRN-6 and CRN-7, the less active enzymes. Whether the different sizes of ToLFP sig- nal represent various stages of apoptotic body, similarly to those reported in germ cell apoptosis stained by the DNA dye [34], remains unknown. Elucidating how NUC-1 acts more actively than CRN-6 and CRN-7 awaits the solution of the 3D structure of each DNase II. In the past, most reports on detecting DNase II activity of vari- ous animal samples were based on in vitro assays [29,31–33] and only in a few papers from Didenko’s laboratory were as- says done in vivo [22–25]. In the present study, we employed a newly established in vivo method of ToLFP to directly illus- trate DNA breaks generated by DNase II in the embryonic cells, for which TUNEL was previously used for indirect observa- tion. In addition, in combination of various genetic backgrounds of worms, the ToLFP method can differentiate the autonomous or non-autonomous action of DNase II in the embryonic cells and conﬁrms that NUC-1 is principally responsible for degrada- tion of apoptotic DNA during embryogenesis (Figures 3 and 4). Importantly, ToLFP showed for the ﬁrst time that the autonom- ous degradation of apoptotic DNA, presumably contributed by NUC-1 and CRN-6, occurs in ced-1 embryos at the comma stage (Figures 2D compared with 2E; Table 1). Furthermore, since the Further ToLFP studies of smIs172, but not cguIs3, showed a signiﬁcantly higher number of ToLFP signals in the tail re- gion of embryos, conﬁrming that NUC-1 in vivo can act non- autonomously to remove apoptotic DNA in the tail region em- bryos at speciﬁc stages (Figure 7). The appearance of ToLFP in the tail of embryos of the double mutant of crn-6 and crn-7, which pen access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. DISCUSSION In addition, this notion further hints that NUC-1::LMP- 1153–237::GFP can function as a membrane bound form and in a single-polypeptide form. Many reports support the possibil- ity that DNase II can function without any further cleavage into smaller polypeptides [35–37]. However, a recent paper indicated that cathepsin L is responsible for cleaving mammalian DNase II into smaller fragments inside of lysosomes [32]. It remains un- known whether the processed DNase II has up-regulated enzyme activity. We speculate that activation of DNase II may require further cleavage by cathespin L in C. elegans, since the mutation of clp-1 (the homologue of cathespin L) prolongs the timing of re- moving the germ cell corpses [38], similarly to what we observed in crn-7 mutants (Hsiang Yu and Szecheng J. Lo, unpublished en access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. H. Yu and others H. Yu and others C comma 1.5 fold 2 fold tail nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 1 2 3 No. of ToLFP signals/ embryo B No. of ToLFP signals/ embryo head nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 5 10 15 comma 1.5 fold 2 fold A n=22 8 1 = n 1 2 = n n=25 n=20 n=24n=26 n=18n=22 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 5 10 15 No. of ToLFP signals/ embryo comma 1.5 fold 2 fold Figure 7 Quantitative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 and cguIs3 animals (A) The ToLFP signals of nuc-1 mutants and transgenic worms in whole embryos at the comma, 1.5-fold and 2-fold stages. (B) The ToLFP signals of nuc-1 mutants and transgenic worms in the head region of embryo at the comma, 1.5-fold and 2-fold stages. (C) The ToLFP signals of nuc-1 mutants and transgenic worms in the tail region of embryo at the comma, 1.5-fold and 2-fold stages. The n number of executed embryos in each strain and stage is indicated above the bar. At least 18 embryos were scored for each stage and strain. Error bars indicate S.E.M., P > 0.05, P > 0.01, P > 0.001. ns, non-signiﬁcant. DISCUSSION A n=22 8 1 = n 1 2 = n n=25 n=20 n=24n=26 n=18n=22 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 5 10 15 No. of ToLFP signals/ embryo comma 1.5 fold 2 fold A B No. of ToLFP signals/ embryo head nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 5 10 15 comma 1.5 fold 2 fold C comma 1.5 fold 2 fold tail nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 nuc-1 smIs172 cguIs3 0 1 2 3 No. of ToLFP signals/ embryo C C B 2 fold ative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 uIs3 animals Figure 7 Quantitative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 and cguIs3 animals (A) The ToLFP signals of nuc-1 mutants and transgenic worms in whole embryos at the comma 1 5-fold and 2-fold stages Figure 7 Quantitative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 and cguIs3 animals (A) The ToLFP signals of nuc-1 mutants and transgenic worms in whole embryos at the comma, 1.5-fold and 2-fold stages. (B) The ToLFP signals of nuc-1 mutants and transgenic worms in the head region of embryo at the comma, 1.5-fold and 2-fold stages. (C) The ToLFP signals of nuc-1 mutants and transgenic worms in the tail region of embryo at the comma, 1.5-fold and 2-fold stages. The n number of executed embryos in each strain and stage is indicated above the bar. At least 18 embryos were scored for each stage and strain Error bars indicate S E M P > 0 05 P > 0 01 P > 0 001 ns Quantitative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 and cguIs3 animals Figure 7 Quantitative analysis of ToLFP signals shows the rescue of nuc-1 phenotypes by two transgenes in smIs172 and cguIs3 animals (A) The ToLFP signals of nuc-1 mutants and transgenic worms in whole embryos at the comma, 1.5-fold and 2-fold stages. DISCUSSION (B) The ToLFP signals of nuc-1 mutants and transgenic worms in the head region of embryo at the comma, 1.5-fold and 2 f ld t (C) Th T LFP i l f 1 t t d t i i th t il i f b t th and cguIs3 animals (A) The ToLFP signals of nuc-1 mutants and transgenic worms in whole embryos at the comma, 1.5-fold and 2-fold stages. (B) The ToLFP signals of nuc-1 mutants and transgenic worms in the head region of embryo at the comma, 1.5-fold and 2-fold stages. (C) The ToLFP signals of nuc-1 mutants and transgenic worms in the tail region of embryo at the comma, 1.5-fold and 2-fold stages. The n number of executed embryos in each strain and stage is indicated above the bar. At least 18 embryos were scored for each stage and strain. Error bars indicate S.E.M., P > 0.05, P > 0.01, *P > 0.001. ns, non-signiﬁcant. data). To test whether a higher DNase II activity is achieved by CLP-1 cleavage in the lysosome, RNAi knockdown experiments of clp-1 can be done to verify the hypothesis. degradation. Since there are three waves of apoptosis occurring at the embryonic and the ﬁrst larval stages and the germ cells of gonad in adults of C. elegans [39], we anticipate that this method can be further used to extend our knowledge of the roles and mechanisms of DNase II activity in the third wave of apop- tosis in C. elegans germ cells. This useful method can be applied to other parasitic nematodes and invertebrates [19,35], in which both autonomous and non-autonomous actions of DNase II have been demonstrated. In summary, we employed the newly established method of ToLFP to illustrate the sites of the autonomous or non- autonomous action of DNase II in the embryonic cells and which DNase II is principally responsible for degradation of apoptotic DNA during embryogenesis. We suggest that the ToLFP method can complement the TUNEL assay in studying apoptotic DNA c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 10 DNase II action in embryos Figure 8 A hypothetical model illustrates the maturation pathway and action mode of two NUC-1 fusion proteins The N-terminal leader sequence present in the NUC-1 of fusion proteins leads the polypeptide to ER for completion of biosynthesis. DISCUSSION The NUC-1::GFP protein is then released into the cisternae of ER as a free form of protein in smIs172 animals (left panel). The additional LMP-1 sequence present in the NUC-1::LMP-1153–237::GFP protein renders the fusion protein appearing as a transmembrane protein, in which the GFP is located outside of ER and the DNase II domain is facing to ER lumen (right panel). Most of fusion proteins are resided in ER, upon signalling of bacterial DNA or apoptosis, both move to Golgi apparatus for further glycosylation and being secreted through vesicles transportation to the plasma membrane. After the vesicles are fused with plasma membrane, the NUC-1::GFP fusion protein is released into the lumen of intestine for degradation of bacterial DNA (B) or into the extracellular space and then being retaken up by neighbouring cells (A) for degradation of apoptotic DNA (pathway I). The secreted fusion protein can also go through endocytosis mechanism back to lysosomes of cells (pathway II). Whereas the NUC-1::LMP-1153–237::GFP protein remains on the plasma membrane of intestine cells or embryonic blastomeres (D) (pathway I), it is able to digest bacterial DNA but unable to be retaken up by posterior cells for degradation of apoptotic DNA non-autonomously (C). However, it can be recycled through endocytosis pathway to target to lysosomes (pathway II). Both fusion proteins have a regular pathway targeting to lysosomes (pathway III). Symbols of fusion proteins are indicated by green circle as GFP and a pacman as DNase II. Figure 8 A hypothetical model illustrates the maturation pathway and action mode of two NUC-1 fusion proteins The N-terminal leader sequence present in the NUC-1 of fusion proteins leads the polypeptide to ER for completion of biosynthesis. The NUC-1::GFP protein is then released into the cisternae of ER as a free form of protein in smIs172 animals (left panel). The additional LMP-1 sequence present in the NUC-1::LMP-1153–237::GFP protein renders the fusion protein appearing as a transmembrane protein, in which the GFP is located outside of ER and the DNase II domain is facing to ER lumen (right panel). Most of fusion proteins are resided in ER, upon signalling of bacterial DNA or apoptosis, both move to Golgi apparatus for further glycosylation and being secreted through vesicles transportation to the plasma membrane. FUNDING This work was supported by the Chang Gung Memorial Hospital grants (CMRPD180451, CMRPD180452 and CMRPD180453) to S.J.L. Hsiang Yu and Szecheng Lo conceived and designed the exper- iments. Hsiang Yu, Huey-Jen Lai and Tai-Wei Lin performed the experiments. Hsiang Yu, Huey-Jen Lai and Szecheng Lo analysed the data. Hsiang Yu and Szecheng Lo wrote the paper. DISCUSSION After the vesicles are fused with plasma membrane, the NUC-1::GFP fusion protein is released into the lumen of intestine for degradation of bacterial DNA (B) or into the extracellular space and then being retaken up by neighbouring cells (A) for degradation of apoptotic DNA (pathway I). The secreted fusion protein can also go through endocytosis mechanism back to lysosomes of cells (pathway II). Whereas the NUC-1::LMP-1153–237::GFP protein remains on the plasma membrane of intestine cells or embryonic blastomeres (D) (pathway I), it is able to digest bacterial DNA but unable to be retaken up by posterior cells for degradation of apoptotic DNA non-autonomously (C). However, it can be recycled through endocytosis pathway to target to lysosomes (pathway II). Both fusion proteins have a regular pathway targeting to lysosomes (pathway III). Symbols of fusion proteins are indicated by green circle as GFP and a pacman as DNase II. REFERENCES and Torriglia, A. (2006) Acid DNases and their interest among apoptotic endonucleases. Biochimie 88, 1851–1858 CrossRef PubMed 28 Lee, M.H. and Schedl, T. (2006) RNA in situ hybridization of dissected gonads. WormBook: The Online Review of C. elegans Biology, NCBI Bookshelf, Pasadena 1–7 9 Kawane, K., Fukuyama, H., Yoshida, H., Nagase, H., Ohsawa, Y., Uchiyama, Y., Okada, K., Iida, T. and Nagata, S. 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Received 2 March 2015/9 March 2015; accepted 23 April 2015 REFERENCES (2006) Developmental apoptosis in C. elegans: a complex CEDnario. Nat. Rev. Mol. Cell Biol. 7, 97–108 CrossRef PubMed Received 2 March 2015/9 March 2015; accepted 23 April 2015 Published as Immediate Publication 27 April 2015, doi 10.1042/BSR20150055 c⃝2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0. 12
https://openalex.org/W4366829997	http://eprints.uanl.mx/25412/2/25412.pdf	English	null	Electrochemical Corrosion Behavior of Passivated Precipitation Hardening Stainless Steels for Aerospace Applications	Metals	2,023	cc-by	13,007	Citation: Villegas-Tovar, J.; Gaona-Tiburcio, C.; Lara-Banda, M.; Maldonado-Bandala, E.; Baltazar- Zamora, M.A.; Cabral-Miramontes, J.; Nieves-Mendoza, D.; Olguin-Coca, J.; Estupiñan-Lopez, F.; Almeraya- Calderón, F. Electrochemical Corrosion Behavior of Passivated Precipitation Hardening Stainless Steels for Aerospace Applications. Metals 2023, 13, 835. https://doi.org/ 10.3390/met13050835 Academic Editor: Eric Hug Received: 10 April 2023 Revised: 17 April 2023 Accepted: 20 April 2023 Published: 24 April 2023 Citation: Villegas-Tovar, J.; Gaona-Tiburcio, C.; Lara-Banda, M.; Maldonado-Bandala, E.; Baltazar- Zamora, M.A.; Cabral-Miramontes, J.; Nieves-Mendoza, D.; Olguin-Coca, J.; Estupiñan-Lopez, F.; Almeraya- Calderón, F. Electrochemical Corrosion Behavior of Passivated Precipitation Hardening Stainless Steels for Aerospace Applications. Metals 2023, 13, 835. https://doi.org/ 10.3390/met13050835 Keywords: corrosion; potentiodynamic polarization; precipitation hardening; stainless steels Article Electrochemical Corrosion Behavior of Passivated Precipitation Hardening Stainless Steels for Aerospace Applications José Villegas-Tovar 1, Citlalli Gaona-Tiburcio 1 , María Lara-Banda 1, Erick Maldonado-Bandala 2,, Miguel Angel Baltazar-Zamora 2 , Jose Cabral-Miramontes 1 , Demetrio Nieves-Mendoza 2, Javier Olguin-Coca 3, Francisco Estupiñan-Lopez 1 and Facundo Almeraya-Calderón 1, 1 Centro de Investigación e Innovación en Ingeniería Aeronáutica (CIIIA), Universidad Autónoma de Nuevo León, FIME, San Nicolás de los Garza 66455, Mexico; miguel.villegastvr@uanl.edu.mx (J.V.-T.); citlalli.gaonatbr@uanl.edu.mx (C.G.-T.); maria.laraba@uanl.edu.mx (M.L.-B.); jose.cabralmr@uanl.edu.mx (J.C.-M.); francisco.estupinanlp@uanl.edu.mx (F.E.-L.) 1 Centro de Investigación e Innovación en Ingeniería Aeronáutica (CIIIA), Universidad Autónoma de Nuevo León, FIME, San Nicolás de los Garza 66455, Mexico; miguel.villegastvr@uanl.edu.mx (J.V.-T.); citlalli.gaonatbr@uanl.edu.mx (C.G.-T.); maria.laraba@uanl.edu.mx (M.L.-B.); jose.cabralmr@uanl.edu.mx (J.C.-M.); francisco.estupinanlp@uanl.edu.mx (F.E.-L.) 2 Facultad de Ingeniería Civil, Universidad Veracruzana, Xalapa 91000, Mexico; dnieves@uv.mx (D.N.-M.) 3 Área Académica de Ingeniería y Arquitectura, Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo Km. 4.5. Hidalgo, Pachuca 42082, Mexico; olguinc@uaeh.edu.mx * Correspondence: erimaldonado@uv.mx (E.M.-B.); facundo.almerayacld@uanl.edu.mx (F.A.-C.) g y q , g , Pachuca-Tulancingo Km. 4.5. Hidalgo, Pachuca 42082, Mexico; olguinc@uaeh.edu.mx * Correspondence: erimaldonado@uvmx (E M B ); facundo almerayacld@uanl edu mx (F A C ) Abstract: Precipitation-hardening (PH) stainless steels (SS) are widely used in various aerospace applications. These steels exhibit good mechanical and corrosion resistance. The electrochemical behavior of 15-5PH, 17-4PH, Custom450 and AM 350 stainless steels passivated with citric and nitric acid baths for 60 and 90 min at 25 and 49 ◦C were evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. The electrochemical behavior was studied with potentiody- namic polarization curves (PPC) according to the ASTM G5-13 standard. The results indicated that there are two characteristic mechanisms that are present in the potentiodynamic polarization curves. When the PHSS is immersed in an H2SO4 solution, there is a secondary passivation, and in the NaCl solution, there is a pseudo-passivation (not stable passivation ﬁlm). The current densities in the NaCl solution were between 10−4 and 10−5 mA/cm2, while those of H2SO4 were recorded around 10−2 and 10−3 mA/cm2. Citric acid does work as a passivating solution, and in some cases, the corrosion resistance of the stainless steel was comparable to that of nitric acid. Article Article Electrochemical Corrosion Behavior of Passivated Precipitation Hardening Stainless Steels for Aerospace Applications metals metals 1. Introduction Environmental regulations in the aeronautical industry suggest using ecological and environmentally sustainable corrosion protection treatments. The chemical treatment known as passivation is a typical coating on stainless steel that helps improve its resistance to corrosion [1–3]. Iron-based alloys containing at least 11% chromium are known as stainless steels. With chromium content and other elements, stainless steel can provide an extraordinary range of corrosion resistance. The SS are classiﬁed into ﬁve distinct families according to their crystalline structure and precipitates [1,4]. metals metals metals The use of precipitation-hardening stainless steels (PHSS) in the aerospace industry is low, but they are essential for some components. It is a family that can be used for its excellent properties (low weight and high mechanical and corrosion resistance) [3,5]. PHSS can be semi-austenitic and martensitic. The semi-austenitic type is essentially austenitic stainless steel with annealing heat treatment, which is then heat treated where the austenitic phase transforms into the martensitic phase for subsequent precipitation hardening. Marten- sitic types are already stainless steel in the solution annealed condition and only require precipitation hardening after manufacturing [6–10]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/metals Metals 2023, 13, 835. https://doi.org/10.3390/met13050835 Metals 2023, 13, 835 2 of 18 The most widely used PHSS in the aerospace industry are 15-5PH, 17-4PH, Custom450 (martensitic) and AM350 (semi-austenitic) [8]. The most important applications of these PHSS are in the manufacture of turbine blades for Custom450, while AM350 is used for shafts, rotors, and turbine blades. Finally, 15-5PH and 17-4PH are used in structural components such as ﬂaps [11–13]. Stainless steels have good corrosion resistance because they naturally form a thin and invisible surface oxide ﬁlm. This ﬁlm is an oxide that protects the steel from chemical attack in an aggressive environment, thus preventing corrosion. The passivation process is a surface treatment that allows the steel to improve its resistance to corrosion, having a more resistant chromium oxide ﬁlm that forms. Naturally, this ﬁlm is continuous, adherent, and compact [8,14]. To obtain a chromium oxide ﬁlm, it is necessary that the steel contain at least 11% chromium: the passivity increases as the chromium content is higher in the steel. For this reason, many stainless steels contain 17–18% chromium as an alloying element. The passive ﬁlm is self-healing, unlike other coatings such as paints [13–15]. In the aeronautical industry, the corrosion costs are high, in addition to aircraft down- time, environmental risks, and possible human losses [8]. In the passivation process, nitric acid (HNO3) is a strong oxidant and promotes the formation of passive ﬁlms on stain- less steels [15]. In recent years, citric acid (C6H8O7) has become a sustainable alternative passivating agent for HNO3 since it is not toxic and is friendly to the environment. This acid can be extracted from fruits and vegetables and is also low-cost. C6H8O7 is generally applied in a solution of around 10%, while the HNO3 can reach solutions up to 40%. Both solutions are used for the same time and temperature, although the HNO3 is used in a lower temperature range [16,17]. Electrochemical techniques used in corrosion studies are an important tool for under- standing the behavior of metallic materials. Potentiodynamic polarization is a technique where the potential of an electrode (stainless steel) is polarized to determine the corrosion rate. One of the most widely used methods to calculate the corrosion rate is the Tafel extrapolation method, where it can be appreciated that both the anodic branch (upper) and the cathodic branch (lower) present a linearity between 50 to 100 mV [18,19]. ( ) p y El-Taib Heakal et al. [20] studied the corrosion behavior of austenitic stainless steels in aerated and deaerated solutions. The electrochemical characterization was carried out by using potentiodynamic polarization and electrochemical impedance spectroscopy, and the results indicated that the pH decreases with the current density due to the alloying elements. In another study, El-Taib Heakal et al. [21] evaluated stainless steels containing molybdenum in order to investigate the behavior of the natural growth of the passive ﬁlm. Ameer et al. [22] reported icorr values that increased with increasing either Cl−or SO42−concentration, characterized by potentiodynamic polarization in stainless steel. This represents a decrease in the formation of the passive ﬁlm. The evaluation of passivated 304, 15-5PH, and 17-4PH stainless steel employing electrochemical noise and potentiodynamic polarization indicated that a similar passive layer was formed [23]. Bragaglia et al. [24] used PP to observe the behavior of passivated and unpassivated 304 austenitic stainless steel in acid electrolytes. The pitting potential in nitric acid increases when the steel is passivated. Marcelin et al. [25] studied the corrosion behavior of martensitic stainless steel. The results showed that the electrochemical process was controlled by passive ﬁlm properties. Recent investigations on precipitation-hardening stainless steels have focused on fatigue behavior, hydrogen diffusion, and microstructural characterization [13,26–28]. Gaona et al. [29] studied the corrosion behavior of AM350 passivated PHSS steels using electrochemical noise, potentiodynamic polarization and electrochemical impedance spectroscopy in acid baths. The martensitic precipitation hardening stainless steels showed the best results for corrosion behavior in acid solutions. 2. Materials and Methods 2.1. Materials The employed stainless steels (AMS Aerospace Material Speciﬁcations) were 15-5PH (AMS 5659), 17-4PH (AMS 5643), Custom 450 (AMS 5773), and AM 350 (AMS 5548) in cylindrical bar form. The nominal chemical composition of these PHSS is shown in Table 1. Table 1. The chemical composition of the used stainless steel (wt.%) [30–33]. Table 1. The chemical composition of the used stainless steel (wt.%) [30–33]. Table 1. The chemical composition of the used stainless steel (wt.%) [30–33]. PHSS Elements Cr Ni Mo Mn Cu Ti Nb N Si S C Fe 15-5PH 14.0–15.5 3.5–5.5 – 1.0 max. 2.5–4.5 – 0.15–0.45 – 1.0 max. 0.03 max. 0.07 max Balance 17-4PH 15.0–17.5 3.0–5.0 0.50 1.0 max. 3.0–5.0 – 0.15–0.45 – – 0.03 max. 0.07 max. Balance Custom 450 14.0–16.0 5.0–7.0 0.50–1.0 1.00 1.25–1.75 0.90–1.40 0.5–0.75 ≤0.1 1.00 0.030 ≤0.05 Balance AM350 16.0–17.0 4.0–5.0 2.50–3.25 0.50–1.25 – – – 0.07–0.13 ≤0.50 0.030 0.07–0.11 Balance From the cylindrical steel bar, coupons of approximately 0.5 cm thickness were ob- tained according to ASTM A380/A380M [34]. Each sample was roughed with silicon carbide abrasive paper up to #600 [35]. In this way, the working electrode (anode) was obtained, which was washed in an acetone solution in ultrasound to obtain a homogeneous surface without contaminants. Optical microscopy (OM, Olympus, Hamburg, Germany) was used to determine the microstructure of PHSS. Over the past few years, the corrosion behavior of austenitic stainless steels has been extensively studied. There are few studies on passivated PHSS, so it is important to know the behavior of electrochemical corrosion in environments that simulate aircraft working conditions, such as marine and industrial atmospheres. Metals 2023, 13, 835 3 of 18 3 of 18 The present work aims to study the electrochemical behavior of PHSS (15-5PH, 17- 4PH, Custom450, and AM350) from the Potentiodynamic Polarization (PP) using C6H8O7 and HNO3 as passivating agents, having as variables the passivation temperature and immersion time, exposed to NaCl and H2SO4 solutions. 2.2. Passivation Treatment The passivation treatment was performed according to ASTM G967 and SAE/ASM2700 standards [17,36]. The following were control variables: passivation solution, passivation temperature, and passivation time (see Figure 1). W 4 of 18 Figure 1. Diagram of passivation treatment of PHSS in citric and nitric acid baths. Figure 1. Diagram of passivation treatment of PHSS in citric and nitric acid baths. Figure 1. Diagram of passivation treatment of PHSS in citric and nitric acid baths. Figure 1. Diagram of passivation treatment of PHSS in citric and nitric acid baths. .3. Corrosion Tests The passivation treatment consisted of the following steps: 2.3. Corrosion Tests The passivation treatment consisted of the following steps: Corrosion tests were performed at room temperature using Gill AC equipment (po- tentiostat/galvanostat), evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. A typical corrosion cell with three electrodes was used: a working electrode WE (passivated stainless steel) a reference electrode (saturated calomel (SCE)) (a) Pretreatment: degreased and pickled stainless steel in a 50 wt.% HCl solution (ana- lytical grade reagents (J.T. Baker, Nuevo León, México) for 5 s at 25 ◦C, and rinsed in distilled water. Corrosion tests were performed at room temperature using Gill AC equipment (po- tentiostat/galvanostat), evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. A typical corrosion cell with three electrodes was used: a working electrode WE (passivated stainless steel) a reference electrode (saturated calomel (SCE)) (a) Pretreatment: degreased and pickled stainless steel in a 50 wt.% HCl solution (ana- lytical grade reagents (J.T. Baker, Nuevo León, México) for 5 s at 25 ◦C, and rinsed in distilled water. Corrosion tests were performed at room temperature using Gill AC equipment (po- tentiostat/galvanostat), evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. A typical corrosion cell with three electrodes was used: a working l t d WE ( i t d t i l t l) f l t d ( t t d l l (SCE)) (a) Pretreatment: degreased and pickled stainless steel in a 50 wt.% HCl solution (ana- lytical grade reagents (J.T. Baker, Nuevo León, México) for 5 s at 25 ◦C, and rinsed in distilled water. Metals 2023, 13, 835 4 of 18 (b) Passivation: two passivation bath solutions were used [citric acid (55%v) and nitric acid (20%v), the rest is distilled water (analytical grade reagents (J.T. 2.2. Passivation Treatment Baker))]. A constant temperature of 25 and 49 ◦C. Samples were immersed in the solutions for 60 and 90 min [28,29]. (c) Final Stage: the specimens were rinsed in distilled water (analytical grade reagents (J.T. Baker)). (c) Final Stage: the specimens were rinsed in distilled water (analytical grade reagents (J.T. Baker)). All these processes were completed for each passivating solution and for each material. According to the experimental matrix, a total of 32 experiments were performed. Figure 1. Diagram of passivation treatment of PHSS in citric and nitric acid baths. 3.1. OM Microstructural Analysis 3.1. OM Microstructural Analysis The microstructure of the steels under study was obtained by optical microscopy. In Figure 4, the martensitic steel (17-4PH, 15-5PH, and Custom 450) showed a martensitic (α′) phase, while the AM350 semi-austenitic stainless steel presented a microstructure of delta (δ) ferrite phase and austenite (γ) [41–43]. The microstructure of the steels under study was obtained by optical microscopy. In Figure 4, the martensitic steel (17-4PH, 15-5PH, and Custom 450) showed a martensitic (α‘) phase, while the AM350 semi-austenitic stainless steel presented a microstructure of delta (δ) ferrite phase and austenite (γ) [41–43]. Figure 4. OM microstructure of PHSS. (a) 15-5PH, (b) 17-4PH, (c) Custom 450, and (d) AM 350. Figure 4. OM microstructure of PHSS. (a) 15-5PH, (b) 17-4PH, (c) Custom 450, and (d) AM 350. Figure 4. OM microstructure of PHSS. (a) 15-5PH, (b) 17-4PH, (c) Custom 450, and (d) AM 350. Figure 4. OM microstructure of PHSS. (a) 15-5PH, (b) 17-4PH, (c) Custom 450, and (d) AM 350. In stainless steel, the carbon content decreases corrosion resistance. Still, it increases toughness and causes greater susceptibility to the formation of chromium carbides that can embrittle the material due to precipitation at grain boundaries. In stainless steel, the carbon content decreases corrosion resistance. Still, it increases toughness and causes greater susceptibility to the formation of chromium carbides that can embrittle the material due to precipitation at grain boundaries. Resistance to pitting corrosion can be measured using the pitting resistance equivalent number (PREN). This parameter is based on the chemical composition of stainless steels, and the PREN result indicated greater resistance to pitting corrosion at high values [44–46]: see Table 2. It is calculated (Equation (1)) based on the chromium (Cr), molybdenum (Mo), tungsten (W), and nitrogen (N) content of an alloy [47]. In this sense, of the four materials evaluated in this work, the AM350 and 15-5PH materials were the ones that presented the highest PREN values of 29.80 and 23.5, respectively, while the Custom 450 and 17-4PH materials presented lower values. Therefore, based on these results, PHSS AM350 and 15-5PH should present greater resistance to pitting corrosion (See Table 2). PREN = Cr + 3.3Mo + 16N (1) (1) PREN = Cr + 3.3Mo + 16N 3. Results and Discussion 3. Results and Discussion 3.1. OM Microstructural Analysis 3.1. OM Microstructural Analysis 2.3. Corrosion Tests 2.3. Corrosion Tests Corrosion tests Corrosion tests were performed at room temperature using Gill AC equipment (po- tentiostat/galvanostat), evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. A typical corrosion cell with three electrodes was used: a working electrode, WE (passivated stainless steel), a reference electrode (saturated calomel (SCE)), and a counter electrode (CE), a platinum mesh (Figure 2). The following parameters were used for the electrochemical technique of potentiodynamic polarization (PP): a potential range was used between −1.0 and 1.2 V of OCP, and a sweep rate of 0.06 V/min was applied, according to ASTM G5-11 [37–40]. Corrosion tests were performed at room temperature using Gill AC equipment (po- tentiostat/galvanostat), evaluated in 5 wt.% sodium chloride (NaCl) and 1 wt.% sulfuric acid (H2SO4) solutions. A typical corrosion cell with three electrodes was used: a working electrode, WE (passivated stainless steel), a reference electrode (saturated calomel (SCE)), and a counter electrode (CE), a platinum mesh (Figure 2). The following parameters were used for the electrochemical technique of potentiodynamic polarization (PP): a potential range was used between −1.0 and 1.2 V of OCP, and a sweep rate of 0.06 V/min was ap- plied, according to ASTM G5-11 [37–40]. Figure 2. Conventional three-electrode corrosion cell. Figure 2. Conventional three-electrode corrosion cell. Figure 2. Conventional three-electrode corrosion cell. Figure 2. Conventional three-electrode corrosion cell. 5 of 18 Figure 2. Conventional three-electrode corrosion cell. Figure 2. Conventional three-electrode corrosion cell. Figure 3 shows the ﬂow diagram of the experimentation of the Electrochemical Cor- rosion Behavior of Passivated Precipitation Hardening Stainless Steels. Figure 3 shows the ﬂow diagram of the experimentation of the Electrochemical Corro- sion Behavior of Passivated Precipitation Hardening Stainless Steels. igure 3. Experimentation ﬂow diagram. Figure 3. Experimentation ﬂow diagram. gure 3. Experimentation ﬂow diagram. Figure 3. Experimentation ﬂow diagram. Metals 2023, 13, 835 5 of 18 5 of 18 3.2. Potentiodynamic Polarization Figure 6. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 ◦C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. Table 3. Electrochemical parameters obtained by PPC for passivated PHSS in citric acid at 60 min exposed to H2SO4 and NaCl solutions. mp. (°C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) In general, the PPC represents the corrosion potential vs. the logarithm of the current, indicating a mixed control by activation which, in turn, reveals the behavior of the corrosion kinetics. The parameters obtained from potentiodynamic polarization (PP) curves are summarized in Tables 3–6. (mA/cm2) (mV) (mm/y) 25 −326 862 3.96 × 10−3 7.73 × 10−3 632 8.13 × 10−5 49 −305 837 2 17 × 10−3 3 51 × 10−3 616 1 55 × 10−5 Table 3. Electrochemical parameters obtained by PPC for passivated PHSS in citric acid at 60 min exposed to H2SO4 and NaCl solutions. (mA/cm2) (mV) (mm/y) 25 −326 862 3.96 × 10−3 7.73 × 10−3 632 8.13 × 10−5 49 305 837 2 17 × 10−3 3 51 × 10−3 616 1 55 × 10−5 Table 3. Electrochemical parameters obtained by PPC for passivated PHSS in citric acid at 60 min exposed to H2SO4 and NaCl solutions. H2SO4 17-4PH 25 −332 789 1.44 × 10−3 3.18 × 10−3 394 3.93 × 10−6 49 −353 863 1.52 × 10−2 7.61 × 10−3 640 6.58 × 10−5 Custom 450 25 −312 768 9.52 × 10−3 5.64 × 10−3 722 8.68 × 10−3 49 −279 916 1.13 × 10−2 7.69 × 10−3 778 1.45 × 10−2 AM350 25 −247 586 4.64 × 10−4 1.53 × 10−2 737 1.06 × 10−2 49 −337 840 5.35 × 10−3 7.31 × 10−3 680 7.16 × 10−3 NaCl 15-5PH 25 −263 161 1.03 × 10−4 1.41 × 10−4 329 5.22 × 10−7 49 −322 45 1.29 × 10−4 1.76 × 10−4 267 5.54 × 10−7 17-4PH 25 −340 105 1.63 × 10−4 2.37 × 10−4 * 335 * 7.91 × 10−7 49 −334 95 1.51 × 10−4 2.52 × 10−4 * 338 * 9.64 × 10−7 Custom 450 25 −619 251 8.29 × 10−4 2.59 × 10−3 518 7.57 × 10−4 49 −369 203 3.68 × 10−4 7.42 × 10−4 304 3.91 × 10−4 Solution PHSS Temp. 3.2. Potentiodynamic Polarization The corrosion kinetic behavior using potentiodynamic polarization can be observed through cathodic and anodic reactions in polarization curves to obtain the electrochemical parameters (corrosion current density, icorr (µA·cm2), potential corrosion, Ecorr (mV), and corrosion rate). The Tafel extrapolation technique is used [48–50]. Figures 5 and 6 show the PP curves obtained for PHSS passivated in acid baths at 25 and 49 ◦C for 60 and 90 min and immersed in 5 wt.% NaCl and 1 wt.% H2SO4 solutions. Metals 2023, 13, 835 6 of 18 Table 2. Pitting resistance equivalent numbers of the martensitic and semi-austenitic precipitation hardening stainless steel. PHSS Cr Mo N PREN 15-5PH 14.0–15.5 – 0.50 23.5 17-4PH 15.0–17.5 – – 17.5 Custom 450 14.0–16.0 0.50–1.0 ≤0.1 20.9 AM350 16.0–17.0 2.50–3.25 0.07–0.13 29.80 Metals 2023, 13, x FOR PEER REVIEW 7 of 18 Figure 5. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 °C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Fi 6 d h th CPP bt i d f PHSS f b th it i id t 25 d 49 (a) (b) (c) (d) Figure 5. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 ◦C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Table 2. Pitting resistance equivalent numbers of the martensitic and semi-austenitic precipitation hardening stainless steel. Table 2. Pitting resistance equivalent numbers of the martensitic and semi-austenitic precipitation hardening stainless steel. (b) (a) (d) (c) Figure 5. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 °C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Figure 5. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 ◦C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Metals 2023, 13, 835 Metals 2023, 13, x FO 7 of 18 8 of 18 7 of 18 8 of 18 Figure 6. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 °C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. (a) (b) (c) (d) Figure 6. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 ◦C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. (b) (d) (c) (d) Figure 6. Potentiodynamic polarization curves for passivated PHSS in citric acid, at 25 and 49 °C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. 3.2. Potentiodynamic Polarization (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) H2SO4 15-5PH 25 −326 862 3.96 × 10−3 7.73 × 10−3 632 8.13 × 10−5 49 −305 837 2.17 × 10−3 3.51 × 10−3 616 1.55 × 10−5 17-4PH 25 −332 789 1.44 × 10−3 3.18 × 10−3 394 3.93 × 10−6 49 −353 863 1.52 × 10−2 7.61 × 10−3 640 6.58 × 10−5 Custom 450 25 −312 768 9.52 × 10−3 5.64 × 10−3 722 8.68 × 10−3 49 −279 916 1.13 × 10−2 7.69 × 10−3 778 1.45 × 10−2 AM350 25 −247 586 4.64 × 10−4 1.53 × 10−2 737 1.06 × 10−2 49 −337 840 5.35 × 10−3 7.31 × 10−3 680 7.16 × 10−3 Metals 2023, 13, 835 8 of 18 Table 3. Cont. Solution PHSS Temp. (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) NaCl 15-5PH 25 −263 161 1.03 × 10−4 1.41 × 10−4 329 5.22 × 10−7 49 −322 45 1.29 × 10−4 1.76 × 10−4 267 5.54 × 10−7 17-4PH 25 −340 105 1.63 × 10−4 2.37 × 10−4 * 335 * 7.91 × 10−7 49 −334 95 1.51 × 10−4 2.52 × 10−4 * 338 * 9.64 × 10−7 Custom 450 25 −619 251 8.29 × 10−4 2.59 × 10−3 518 7.57 × 10−4 49 −369 203 3.68 × 10−4 7.42 × 10−4 304 3.91 × 10−4 AM350 25 −249 586 2.96 × 10−4 6.83 × 10−4 * 711 * 8.52 × 10−5 49 −417 539 4.12 × 10−4 8.08 × 10−4 * 849 * 9.01 × 10−4 * pseudo-passivation. Table 4. Electrochemical parameters obtained by PPC for passivated PHSS in nitric acid at 60 min exposed to H2SO4 and NaCl solutions. Solution PHSS Temp. 3.2. Potentiodynamic Polarization (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) H2SO4 15-5PH 25 −329 831 1.30 × 10−3 3.17 × 10−3 731 3.67 × 10−6 49 46 601 3.87 × 10−5 1.34 × 10−4 657 1.01 × 10−6 17-4PH 25 −296 815 1.39 × 10−2 3.12 × 10−3 508 1.17 × 10−5 49 −352 819 2.85 × 10−3 3.09 × 10−4 530 4.47 × 10−7 Custom 450 25 −279 936 2.97 × 10−3 5.14 × 10−3 711 1.52 × 10−2 49 −241 937 5.36 × 10−3 1.47 × 10−3 878 6.41 × 10−3 AM350 25 −288 874 1.22 × 10−3 3.92 × 10−3 575 4.74 × 10−3 49 −360 882 4.78 × 10−3 1.43 × 10−2 994 5.11 × 10−2 NaCl 15-5PH 25 −291 116 4.53 × 10−5 6.42 × 10−5 212 3.63 × 10−7 49 −239 425 4.34 × 10−5 6.16 × 10−5 251 2.28 × 10−7 17-4PH 25 −200 275 8.75 × 10−5 1.24 × 10−4 * 394 * 5.66 × 10−7 49 −275 400 6.43 × 10−5 7.93 × 10−5 214 3.67 × 10−7 Custom 450 25 −285 315 3.02 × 10−4 5.82 × 10−4 485 3.70 × 10−4 49 −223 536 1.86 × 10−4 3.46 × 10−4 * 673 * 2.58 × 10−4 AM350 25 −258 906 3.13 × 10−4 5.41 × 10−4 * 1049 * 3.09 × 10−4 49 −231 921 2.61 × 10−4 5.52 × 10−4 * 1038 * 3.71 × 10−4 * pseudo-passivation. Table 3. Cont. Solution PHSS Temp. (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) NaCl 15-5PH 25 −263 161 1.03 × 10−4 1.41 × 10−4 329 5.22 × 10−7 49 −322 45 1.29 × 10−4 1.76 × 10−4 267 5.54 × 10−7 17-4PH 25 −340 105 1.63 × 10−4 2.37 × 10−4 * 335 * 7.91 × 10−7 49 −334 95 1.51 × 10−4 2.52 × 10−4 * 338 * 9.64 × 10−7 Custom 450 25 −619 251 8.29 × 10−4 2.59 × 10−3 518 7.57 × 10−4 49 −369 203 3.68 × 10−4 7.42 × 10−4 304 3.91 × 10−4 AM350 25 −249 586 2.96 × 10−4 6.83 × 10−4 * 711 * 8.52 × 10−5 49 −417 539 4.12 × 10−4 8.08 × 10−4 * 849 * 9.01 × 10−4 * pseudo-passivation. Table 4. * pseudo-passivation. 3.2. Potentiodynamic Polarization Electrochemical parameters obtained by PPC for passivated PHSS in nitric acid at 60 min exposed to H2SO4 and NaCl solutions. Table 4. Electrochemical parameters obtained by PPC for passivated PHSS in nitric acid at 60 min exposed to H2SO4 and NaCl solutions. Table 4. Electrochemical parameters obtained by PPC for passivated PHSS in nitric acid at 60 min exposed to H2SO4 and NaCl solutions. Solution PHSS Temp. (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) H2SO4 15-5PH 25 −329 831 1.30 × 10−3 3.17 × 10−3 731 3.67 × 10−6 49 46 601 3.87 × 10−5 1.34 × 10−4 657 1.01 × 10−6 17-4PH 25 −296 815 1.39 × 10−2 3.12 × 10−3 508 1.17 × 10−5 49 −352 819 2.85 × 10−3 3.09 × 10−4 530 4.47 × 10−7 Custom 450 25 −279 936 2.97 × 10−3 5.14 × 10−3 711 1.52 × 10−2 49 −241 937 5.36 × 10−3 1.47 × 10−3 878 6.41 × 10−3 AM350 25 −288 874 1.22 × 10−3 3.92 × 10−3 575 4.74 × 10−3 49 −360 882 4.78 × 10−3 1.43 × 10−2 994 5.11 × 10−2 NaCl 15-5PH 25 −291 116 4.53 × 10−5 6.42 × 10−5 212 3.63 × 10−7 49 −239 425 4.34 × 10−5 6.16 × 10−5 251 2.28 × 10−7 17-4PH 25 −200 275 8.75 × 10−5 1.24 × 10−4 * 394 * 5.66 × 10−7 49 −275 400 6.43 × 10−5 7.93 × 10−5 214 3.67 × 10−7 Custom 450 25 −285 315 3.02 × 10−4 5.82 × 10−4 485 3.70 × 10−4 49 −223 536 1.86 × 10−4 3.46 × 10−4 * 673 * 2.58 × 10−4 AM350 25 −258 906 3.13 × 10−4 5.41 × 10−4 * 1049 * 3.09 × 10−4 49 −231 921 2.61 × 10−4 5.52 × 10−4 * 1038 * 3.71 × 10−4 * pseudo passivation Metals 2023, 13, 835 9 of 18 Table 5. Electrochemical parameters obtained from PPC by PHSS passivated in citric acid at 90 min exposure to H2SO4 and NaCl solutions. Table 5. Electrochemical parameters obtained from PPC by PHSS passivated in citric acid at 90 min exposure to H2SO4 and NaCl solutions. Table 5. Electrochemical parameters obtained from PPC by PHSS passivated in citric acid at 90 min exposure to H2SO4 and NaCl solutions. Solution PHSS Temp. 3.2. Potentiodynamic Polarization (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) H2SO4 15-5PH 25 −329 831 1.30 × 10−3 6.43 × 10−3 621 6.41 × 10−5 49 46 601 3.87 × 10−5 1.87 × 10−3 718 6.91 × 10−6 17-4PH 25 −296 815 1.39 × 10−2 7.27 × 10−3 761 1.12 × 10−4 49 −352 819 2.85 × 10−3 6.24 × 10−3 692 9.00 × 10−5 Custom 450 25 −279 936 2.97 × 10−3 7.42 × 10−3 722 1.31 × 10−2 49 −241 937 5.36 × 10−3 5.14 × 10−3 758 2.58 × 10−2 AM350 25 −288 874 1.22 × 10−3 7.34 × 10−3 698 2.46 × 10−2 49 −360 882 4.78 × 10−3 5.41 × 10−3 596 2.11 × 10−3 NaCl 15-5PH 25 −291 116 4.53 × 10−5 1.04 × 10−4 319 4.46 × 10−7 49 −239 425 4.34 × 10−5 1.23 × 10−4 312 4.75 × 10−7 17-4PH 25 −200 275 8.75 × 10−5 2.52 × 10−4 * 433 * 9.12 × 10−7 49 −275 400 6.43 × 10−5 2.01 × 10−4 * 355 * 5.63 × 10−7 Custom 450 25 −285 315 3.02 × 10−4 9.81 × 10−4 * 422 * 6.26 × 10−4 49 −223 536 1.86 × 10−4 1.08 × 10−3 445 5.74 × 10−4 AM350 25 −258 906 3.13 × 10−4 8.09 × 10−4 * 351 * 6.95 × 10−4 49 −231 921 2.61 × 10−4 8.78 × 10−4 * 1048 * 8.95 × 10−4 * pseudo-passivation. Figure 5a–d shows the PPC for passivated PHSS samples in citric acid at 25 and 49 ◦C for 60 min and exposure to sulfuric acid and sodium chloride solutions. The anodic and cathodic branches presented activation in a range of 500 mV. The corrosion potential (Ecorr) in most cases was around −400 mV. However, the corrosion potential in sodium chloride at 25 ◦C is −600 mV. All the passivated samples resulted in a stable passivation range in both solutions, being larger for the samples exposed to the sulfuric acid solution at 25 and 49 ◦C. Under these conditions, the samples continued with the second passivation. AM 350 steel presented a pseudo-passivation followed by transpassivation in the sodium chloride solution. The corrosion current densities (icorr) for the PHSS samples in the sulfuric acid solution were found at 10−3 mA/cm2, and for the sodium chloride solution at 10−4 mA/cm2, respectively. 3.2. Potentiodynamic Polarization Such behaviors occurred in PHSS passivated with citric acid, i.e., a strong acid obtained from citrus fruits, in order to have an “eco-friendly” passivation treatment [28,50]. Figure 6a–d shows the CPPs obtained for PHSS from bath nitric acid at 25 and 49 ◦C for 60 min, exposing the passivated PHSS to sulfuric acid or sodium chloride solutions. Nitric is a strong acid that, with an increasing temperature of around 83 ◦C, can generate toxic nitrate vapors harmful to health. The anodic and cathodic branches present activation in a range of 500 mV. However, the cathode branches of the PHSS samples exposed to a sodium chloride solution present a concentration polarization effect. The Ecorr of the anodic branch varies from 300 to 200 mV, with 15-5PH being at nobler potentials, also at a temperature of 49 ◦C either in sulfuric acid or sodium chloride solutions. The formation of the passive layer is variable. In sulfuric acid, there is even secondary passivation. In contrast, in a sodium chloride solution, there is the activation of the system followed by a small passive layer of 200 mV. The icorr for the PHSS samples in sulfuric acid solution was found at 10−3 mA/cm2 and for the sodium chloride solution at 10−4 and 10−5 mA/cm2, respectively. Metals 2023, 13, 835 10 of 18 10 of 18 Table 6. Electrochemical parameters obtained by PPC for PHSS passivated in nitric acid at 90 min exposure to H2SO4 and NaCl solutions. Solution PHSS Temp. 3.2. Potentiodynamic Polarization (◦C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) H2SO4 15-5PH 25 −320 844 1.66 × 10−3 7.37 × 10−3 813 1.65 × 10−4 49 72 901 5.64 × 10−5 4.65 × 10−3 * 752 * 7.07 × 10−7 17-4PH 25 −418 860 2.11 × 10−2 1.92 × 10−3 715 5.19 × 10−6 49 −377 800 1.54 × 10−3 3.13 × 10−4 562 5.31 × 10−7 Custom 450 25 −305 934 3.91 × 10−3 5.48 × 10−3 883 1.65 × 10−2 49 −314 899 1.86 × 10−3 6.38 × 10−3 911 1.23 × 10−2 AM350 25 −307 895 1.29 × 10−3 4.93 × 10−3 735 1.24 × 102 49 −327 737 1.33 × 10−3 7.88 × 10−3 847 1.22 × 10−2 NaCl 15-5PH 25 −238 426 4.14 × 10−5 6.48 × 10−5 288 2.60 × 10−7 49 −227 739 3.47 × 10−5 5.53 × 10−5 217 2.39 × 10−7 17-4PH 25 −206 285 8.33 × 10−5 1.14 × 10−4 259 5.10 × 10−7 49 −282 269 6.23 × 10−5 1.17 × 10−4 * 452 * 4.61 × 10−7 Custom 450 25 −247 260 2.27 × 10−4 4.43 × 10−4 * 405 * 4.01 × 10−4 49 −248 777 1.95 × 10−4 3.31 × 10−4 212 4.45 × 10−4 AM350 25 −257 417 3.38 × 10−4 7.22 × 10−4 * 558 * 3.89 × 10−4 49 −250 522 2.56 × 10−4 5.39 × 10−4 * 671 * 5.62 × 10−4 * pseudo-passivation. Table 6. Electrochemical parameters obtained by PPC for PHSS passivated in nitric acid at 90 min exposure to H2SO4 and NaCl solutions. Table 6. Electrochemical parameters obtained by PPC for PHSS passivated in nitric acid at 90 min exposure to H2SO4 and NaCl solutions. The electrochemical parameters obtained from the potentiodynamic polarization of the materials passivated in citric acid can be seen in Tables 3 and 4, where the steel is exposed to a sulfuric acid solution forming more stable layers followed by transpassivation and secondary passivation. The values of the passivation range showed only a tendency in the PHSS steels (marked with the symbol ) since it was not completely deﬁned. Pitting potential (Epit) is the potential value at which the current increases and the pitting attack occurs. 3.2. Potentiodynamic Polarization The passivated PHSS in citric and nitric acid at 60 min had pitting potential values from 45 mV (15-5 PH) to 921 mV (AM350) in NaCl solution and values from 586 mV (AM350) to 937 mV (Custom 450) in H2SO4 solution, respectively. Figure 7a–d shows the potentiodynamic polarization curves of the passivated PHSS in a nitric acid bath at 25 and 49 ◦C for 90 min and exposing the passivated PHSS to a sulfuric acid or sodium chloride solution. The anodic and cathodic branches presented activation in a range of 600 mV. The corrosion potential of these steels is around −300 mV. 15-5 PH steel has a corrosion potential (Ecorr) value of −20 mV in sulfuric acid solution at 49 ◦C. The PHSS samples exposed to sulfuric acid solution have a stable passivation range that goes from −200 to 800 mV, followed by a transpassivation, and the samples in sodium chloride solution have an unstable passivation range because they present a pseudo-passivation at both temperatures. The passivated PHSS have current densities (icorr) of 10−3 mA/cm2 in sulfuric acid solution and 10−4 and 10−5 mA/cm2 in sodium chloride solution. Metals 2023, 13, 835 Metals 2023, 13, x FO 11 of 18 10 of 18 Figure 7. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 °C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. (a) (b) (c) (d) Figure 7. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 ◦C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. (b) (a) (d) (c) (d) Figure 7. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 °C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Figure 7. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 ◦C for 60 min: (a,c) H2SO4 and (b,d) NaCl solutions. Figure 8a–d shows the potentiodynamic polarization curves of the passivated PHSS in a bath of nitric acid at 25 and 49 °C for 90 min and exposing the passivated PHSS to sulfuric acid or sodium chloride solutions. The anodic and cathodic branches present ac- tivation in a range of 500 mV. A concentration polarization eﬀect is observed in the cath- ode branches of the samples exposed to sodium chloride solution. The Ecorr range of −200 to −400 mV for all samples. 3.2. Potentiodynamic Polarization The PHSS samples passivated in citric and nitric acid present the same behavior when exposed to sulfuric acid and sodium chloride solutions. Pas- sivation ranges are stable, followed by transpassivation and secondary passivation when samples are in sulfuric acid. They have an unstable passivation range when exposed to sodium chloride because they present a pseudo-passivation at both temperatures. The icorr for the PHSS samples in sulfuric acid solution was found at 10−3 mA/cm2, and for the so- dium chloride solution at 10−4 and 10−5 mA/cm2 respectively Figure 8a–d shows the potentiodynamic polarization curves of the passivated PHSS in a bath of nitric acid at 25 and 49 ◦C for 90 min and exposing the passivated PHSS to sulfuric acid or sodium chloride solutions. The anodic and cathodic branches present activation in a range of 500 mV. A concentration polarization effect is observed in the cathode branches of the samples exposed to sodium chloride solution. The Ecorr range of −200 to −400 mV for all samples. The PHSS samples passivated in citric and nitric acid present the same behavior when exposed to sulfuric acid and sodium chloride solutions. Passivation ranges are stable, followed by transpassivation and secondary passivation when samples are in sulfuric acid. They have an unstable passivation range when exposed to sodium chloride because they present a pseudo-passivation at both temperatures. The icorr for the PHSS samples in sulfuric acid solution was found at 10−3 mA/cm2, and for the sodium chloride solution at 10−4 and 10−5 mA/cm2, respectively. dium chloride solution at 10 and 10 mA/cm , respectively. The electrochemical parameters obtained from the potentiodynamic polarization of PHSS passivated in nitric acid are presented in Tables 5 and 6. The values of the pas- sivation range showed only a tendency in the PHSS steels (marked with the symbol ) since it was not completely deﬁned. The corrosion potential ranges from −400 to 70 mV, where the noblest values occur at higher passivation temperatures and in sulfuric acid. The pitting potential was above 700 mV in the sulfuric acid solution, while sodium chlo- ride was above 300 mV. Current densities (icorr) are of the order of 10−3 mA/cm2 in sulfuric acid medium and 10−3 mA/cm2 in sodium chloride The electrochemical parameters obtained from the potentiodynamic polarization of PHSS passivated in nitric acid are presented in Tables 5 and 6. 3.2. Potentiodynamic Polarization The values of the passivation range showed only a tendency in the PHSS steels (marked with the symbol ) since it was not completely deﬁned. The corrosion potential ranges from −400 to 70 mV, where the noblest values occur at higher passivation temperatures and in sulfuric acid. The pitting potential was above 700 mV in the sulfuric acid solution, while sodium chloride was above 300 mV. Current densities (icorr) are of the order of 10−3 mA/cm2 in sulfuric acid medium and 10−3 mA/cm2 in sodium chloride. Metals 2023, 13, 835 Metals 2023, 13, x FO 12 of 18 11 of 18 12 of 18 11 of 18 Figure 8. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 °C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. (a) (b) (c) (d) Figure 8. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 ◦C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. (b) (a) (d) (c) (d) Figure 8. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 °C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. Figure 8. Potentiodynamic polarization curves for passivated PHSS in nitric acid, at 25 and 49 ◦C for 90 min: (a,c) H2SO4 and (b,d) NaCl solutions. Table 5. Electrochemical parameters obtained from PPC by PHSS passivated in citric acid at 90 min exposure to H2SO4 and NaCl solutions. . (°C) Ecorr (mV) Epit (mV) icorr (mA/cm2) ipass (mA/cm2) Range Passive (mV) Corrosion Rate (mm/y) 5 −329 831 1.30 × 10−3 6.43 × 10−3 621 6.41 × 10−5 9 46 601 3.87 × 10−5 1.87 × 10−3 718 6.91 × 10−6 The current results of the potentiodynamic polarization curves of the passivated PHSS in nitric acid bath acid at 25 and 49 ◦C for 60 and 90 min and exposure to solutions of sulfuric acid and sodium chloride allowed us to ﬁnd out the behavior of the corrosion kinetics of precipitation hardening stainless steels. Hence, mixed activation and passivation (formation of a passive ﬁlm) was shown, followed by a transpassivation or secondary passivation trend. In the PPC, more stable passivation was observed in sulfuric acid solution, and pseudo-passivation when the steels were exposed to sodium chloride solution. 3.2. Potentiodynamic Polarization [29] indicate that the corrosion kinetics of stainless steels are associated with an increase in current density as a result of an unstable passivation ﬁlm when the samples are immersed in NaCl solution. However, austenitic stainless steels immersed in H2SO4 solution showed transients associated with rupture of the passivation ﬁlm (transpas- sivation) and regeneration of the passive layer (secondary passivation). Research by Lara et al., Samaniego et al., Noh et al., and Gaydos et al. [13,16,23,55,56] have indicated that stainless steels passivated in nitric acid showed a higher trend of pitting corrosion, concluding that nitric acid increases the chromium presence of the passive layer, removing MnS inclusions from the surface. The probability of individual pitting also increases. In this research, the samples passivated in nitric acid presented more corrosion than those passivated within citric acid due to the presence of MnS. To reduce the presence of MnS, changes in the acid concentration or the use of other solutions similar to citric acid, where the pitting process was more controlled, should be used. See Equations (2) and (3). From the above reactions, MnS can be removed, and the passivation stability could be related to the acid concentration. MnS + 2H+ →Mn2+ + H2S (2) 2MnS + 3H2O →2Mn2+ + S2O2− 3 + 6H+ + 8e− (3) (2) (3) According to some authors [57–59], the results indicated that during passivation in the PHSS, there is a relatively stable range of passive potential. The passive current density is the same, which indicates that it forms a relatively stable passive ﬁlm. However, if the electrochemical process is still active in the anodic reaction, the passive current density is not the same, and the passive ﬁlm is unstable. Some authors say stainless steels present passive ﬁlms before and after transpassiva- tion [60,61]. Transpassivation is a dissolution mechanism where the steel is activated and begins to dissolve, having noble electrode potentials [62,63]. A characteristic of passivated stainless steel is that passivation occurs in the anodic branch. This is where most of the studies have been focused on, forgetting that some acid solutions can cause a second ﬁlm for passivation, as observed in this study. The passive zone involves the formation of chromium and iron oxide ﬁlms that is commonly present in PHSS [28,64–66]. 3.2. Potentiodynamic Polarization 5 −296 815 1.39 × 10−2 7.27 × 10−3 761 1.12 × 10−4 9 −352 819 2.85 × 10−3 6.24 × 10−3 692 9.00 × 10−5 5 −279 936 2.97 × 10−3 7.42 × 10−3 722 1.31 × 10−2 9 −241 937 5.36 × 10−3 5.14 × 10−3 758 2.58 × 10−2 5 −288 874 1.22 × 10−3 7.34 × 10−3 698 2.46 × 10−2 9 −360 882 4.78 × 10−3 5.41 × 10−3 596 2.11 × 10−3 5 −291 116 4.53 × 10−5 1.04 × 10−4 319 4.46 × 10−7 9 −239 425 4.34 × 10−5 1.23 × 10−4 312 4.75 × 10−7 5 −200 275 8.75 × 10−5 2.52 × 10−4 433 * 9.12 × 10−7 9 −275 400 6.43 × 10−5 2.01 × 10−4 * 355 * 5.63 × 10−7 5 −285 315 3.02 × 10−4 9.81 × 10−4 * 422 * 6.26 × 10−4 p p p The shapes of the potentiodynamic polarization curves (Figures 5–8) of the PHSS steels are different, which indicates since the electrochemical processes were not similar in NaCl and H2SO4 test solutions, there is passivation in the anodic reaction, but their pitting potential is different. However, two characteristic mechanisms are present in the potentiodynamic polarization curves when the PHSS is immersed in the H2SO4 solution. This passivation protection mechanism occurs when the passivation ﬁlm formed on the surface of the Cr-Fe alloy determines its corrosion resistance. Chromium oxides play an important role in passive ﬁlms, and the behavior is attributed to the anodic reactions of the OH−. The increase in the current density in the PHSS samples gives rise to the transpassivation and formation of secondary passivation. In the case of samples immersed in NaCl solution, there is a pseudo-passivation not representing stable passivation ﬁlm. This protection mechanism occurs due to the formation of a passive layer formed by oxides and oxy/hydroxides rich in Cr that prevents the propagation of oxygen to the internal Metals 2023, 13, 835 13 of 18 13 of 18 layer and protects the base material from the penetration of corrosive ions such as the Cl- to which the samples were exposed [50–54]. layer and protects the base material from the penetration of corrosive ions such as the Cl- to which the samples were exposed [50–54]. Gaona et al. 3.2. Potentiodynamic Polarization Hence, selective dissolution on the surface of stainless steels generates the presence of Cr3+, leading to the formation of the chromium trihydroxide compound Cr(OH)3 (see Equation (4)). When Cr(OH)3 is on the surface, and the hydroxides continue to react, the dissolution leads to the formation of a continuous passive ﬁlm of chromium oxide Cr2O3 (see Equation (5)) [28,67,68]. Cr3+ + 3OH−→Cr(OH)3 + 3e− (4) Cr(OH)3 + Cr + 3OH−→Cr2O3 + 3H2O + 3e− (5) (4) (5) As mentioned before, the anodic reactions during the passivation ﬁlm growth period come mainly from the oxidation of iron and chromium. The oxidation reactions of iron can be seen in Equations (6)–(8) [69–72]: 3Fe + 8OH−→Fe3O4 + 4H2O + 8e− (6) 2Fe3O4 + 2OH−+ 2H2O →6FeOOH + 2e− (7) 2Fe3O4 + 2OH−→3Fe2O3 + H2O + 2e− (8) (6) (7) (7) (8) (8) Pseudo-passivation occurred in the passivated samples of PHSS immersed in 3.5 wt.% NaCl. The current density continues to increase with increases in the anodic potential instead of reaching the stable state within the passive region, thus suggesting that the Metals 2023, 13, 835 14 of 18 14 of 18 passive ﬁlms formed on the PHSS are in an incomplete steady state. Chloride ions (Cl−) cause this instability since they have a great ability to adhere to the steel surface and then diffuse into the steel through defects in the passive surface ﬁlm, thus impairing the effectiveness of the passive ﬁlm of the PHSS [23,28,73,74]. The pseudo-passivation phenomenon observed in the different passivated samples in the polarization curve may be associated with the formation of the Cr(OH)3 ﬁlm, and the rupture of the pseudo- passivation is accompanied by the detachment of the Cr(OH)3 ﬁlm. Therefore, the Cr(OH)3 ﬁlm functions as a pseudo-passive ﬁlm [75,76]. As some papers have reported, Cr(OH)3 could block the path of the dissolution of the iron, isolate the corrosion media and reduce the number of active sites of the iron dissolution [77,78]. 14 of 18 d in the diﬀerent passivated samples in the polarization curve may be associ- formation of the Cr(OH)3 ﬁlm, and the rupture of the pseudo-passivation is by the detachment of the Cr(OH)3 ﬁlm. Therefore, the Cr(OH)3 ﬁlm func- udo-passive ﬁlm [75,76]. As some papers have reported, Cr(OH)3 could block e dissolution of the iron, isolate the corrosion media and reduce the number of the iron dissolution [77,78]. 3.2. Potentiodynamic Polarization ematic diagram shows the corrosion mechanism for 15-5PH, 17-4PH, CUS- The schematic diagram shows the corrosion mechanism for 15-5PH, 17-4PH, CUSTOM 450, and AM 350 stainless steels after the passivation process in C6H8O7 and HNO3 baths (see Figure 9). The passive protective ﬁlm formed on stainless steel surfaces is highly attributed to the corrosion resistance in these steels. The double-layer structure of SS passive ﬁlm has a double-layer structure that is rich in Fe and Cr, respectively. Chromium oxides play a signiﬁcant role in the corrosion resistance of PHSS. Cr3+ has higher anticorrosion stability compared to FeO and Fe2O3 oxides. Therefore, the Cr2O3 content in the stainless steel passive ﬁlm is a primary factor for the stability and anticorrosive property of the steel. Defects will form in the passive ﬁlm, leading to the nucleation of localized corrosion (generating pitting). On the other hand, the defect density of the iron-rich outer layer is higher than that of the chromium-rich inner layer, which could lead to the absorption of a large amount of Cl−in the passive ﬁlm in PHSS [13,29,79,80]. In Figure 9a, the protection mechanism occurs due to the formation of a passive layer formed by oxides and oxy/hydroxides rich in Cr that prevents the propagation of oxygen to the internal layer and protects the base material from the penetration of corrosive ions such as the Cl−to which the samples were exposed. In the case of Figure 9b, the protection mechanism is different since passivation occurs. The passivation ﬁlm formed on the surface of the Cr–Fe alloy determined its corrosion resistance. Chromium oxides play an important role in passive ﬁlms [81]. d AM 350 stainless steels after the passivation process in C6H8O7 and HNO3 ure 9). The passive protective ﬁlm formed on stainless steel surfaces is highly the corrosion resistance in these steels. The double-layer structure of SS pas- a double-layer structure that is rich in Fe and Cr, respectively. Chromium signiﬁcant role in the corrosion resistance of PHSS. Cr3+ has higher anticor- ty compared to FeO and Fe2O3 oxides. Therefore, the Cr2O3 content in the l passive ﬁlm is a primary factor for the stability and anticorrosive property Defects will form in the passive ﬁlm, leading to the nucleation of localized nerating pitting). 3.2. Potentiodynamic Polarization On the other hand, the defect density of the iron-rich outer r than that of the chromium-rich inner layer, which could lead to the absorp- e amount of Cl in the passive ﬁlm in PHSS [13,29,79,80]. In Figure 9a, the echanism occurs due to the formation of a passive layer formed by oxides roxides rich in Cr that prevents the propagation of oxygen to the internal tects the base material from the penetration of corrosive ions such as the Cl samples were exposed. In the case of Figure 9b, the protection mechanism is e passivation occurs. The passivation ﬁlm formed on the surface of the Cr– rmined its corrosion resistance. Chromium oxides play an important role in [81]. Figure 9. Schematic diagram of passivation treatment in citric and nitric acid baths for 15-5PH, 17- 4PH, CUSTOM 450, and AM 350 stainless steels exposed to (a) 5 wt.% NaCl solution; (b) 1 wt.% H2SO4 solution. Figure 9. Schematic diagram of passivation treatment in citric and nitric acid baths for 15-5PH, 17-4PH, CUSTOM 450, and AM 350 stainless steels exposed to (a) 5 wt.% NaCl solution; (b) 1 wt.% H2SO4 solution. matic diagram of passivation treatment in citric and nitric acid baths for 15-5PH, 17- M 450, and AM 350 stainless steels exposed to (a) 5 wt.% NaCl solution; (b) 1 wt.% . Figure 9. Schematic diagram of passivation treatment in citric and nitric acid baths for 15-5PH, 17-4PH, CUSTOM 450, and AM 350 stainless steels exposed to (a) 5 wt.% NaCl solution; (b) 1 wt.% H2SO4 solution. ng to the literature [16,28,29,82–87], citric acid can be an alternative to nitric ric acid passivation indicates better results than the nitric acid solution. Fur- mples passivated within citric acid presented a lower trend to localized cor- According to the literature [16,28,29,82–87], citric acid can be an alternative to nitric acid since citric acid passivation indicates better results than the nitric acid solution. Further- more, samples passivated within citric acid presented a lower trend to localized corrosion. 4. Conclusions ns earch shows the passive state of PHSS passivated in acid baths at 25 and 49 90 min and immersed in NaCl and H2SO4 solutions From these results the This research shows the passive state of PHSS passivated in acid baths at 25 and 49 ◦C for 60 and 90 min and immersed in NaCl and H2SO4 solutions. From these results, the following can be concluded: 90 min and immersed in NaCl and H2SO4 solutions. From these results, the n be concluded: racterization indicated that the martensitic PHSS presented a microstructure • OM characterization indicated that the martensitic PHSS presented a microstructure with a martensitic (α′) phase and a semi-austenitic PHSS containing a microstructure 90 min and immersed in NaCl and H2SO4 solutions. From these results, the n be concluded: racterization indicated that the martensitic PHSS presented a microstructure • OM characterization indicated that the martensitic PHSS presented a microstructure with a martensitic (α′) phase and a semi-austenitic PHSS containing a microstructure Metals 2023, 13, 835 15 of 18 15 of 18 of austenite (γ) and delta (δ) ferrite phases, respectively. Based on the values ob tained from PREN, the AM 350 (semi-austenitic) (29.80) presented a higher corrosion resistance than the martensitic 15-5 PH (23.5), 17-4 PH (17.5), and CUSTOM 450 (20.9) of austenite (γ) and delta (δ) ferrite phases, respectively. Based on the values ob- tained from PREN, the AM 350 (semi-austenitic) (29.80) presented a higher corrosion resistance than the martensitic 15-5 PH (23.5), 17-4 PH (17.5), and CUSTOM 450 (20.9). of austenite (γ) and delta (δ) ferrite phases, respectively. Based on the values ob- tained from PREN, the AM 350 (semi-austenitic) (29.80) presented a higher corrosion resistance than the martensitic 15-5 PH (23.5), 17-4 PH (17.5), and CUSTOM 450 (20.9). • Potentiodynamic polarization results allowed us to determine the corrosion kinetic behavior of PHSS passivated samples immersed in H2SO4 and NaCl solutions and passivity in the anodic branch. p y • The current density levels in NaCl solution were between 10−4 and 10−5 mA/cm2, while those of H2SO4 were recorded around 10−2 and 10−3 mA/cm2. • Using the citric acid bath as a substitute for nitric acid in the passivation process gen- erates a system in which the electrochemical behavior is similar, mixed by activation, where the anodic branch presents a series of events such as pseudo-passivation and/or passivation–transpassivation–secondary passivation. 4. Conclusions • PHSS passivated in nitric acid and immersed in sodium chloride have higher pitting potentials than samples passivated in citric acid. • The citric acid passivation treatment on PHSS could be a green alternative to the currently employed nitric acid passivation treatment because it is not toxic and is friendly to the environment. y • Based on the results obtained from the corrosion behavior of passivated PHSS, it is considered that future work may use electrochemical impedance spectroscopy to analyze and complement the corrosion mechanism and characterize the oxides through the XPS technique. Author Contributions: Conceptualization, J.V.-T., C.G.-T. and F.A.-C. methodology, J.V.-T., E.M.-B., M.L.-B., F.E.-L. and C.G.-T.; data curation, F.A.-C., J.C.-M., D.N.-M., F.A.-C., J.O.-C. and F.E.-L.; formal analysis, F.A.-C., M.A.B.-Z. and C.G.-T.; writing—review and editing, F.A.-C., E.M.-B. and C.G.-T. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, J.V.-T., C.G.-T. and F.A.-C. methodology, J.V.-T., E.M.-B., M.L.-B., F.E.-L. and C.G.-T.; data curation, F.A.-C., J.C.-M., D.N.-M., F.A.-C., J.O.-C. and F.E.-L.; formal analysis, F.A.-C., M.A.B.-Z. and C.G.-T.; writing—review and editing, F.A.-C., E.M.-B. and C.G.-T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: The authors would like to thank the UANL-CA-316 working group and Univer- sidad Autónoma de Nuevo León (UANL) for the facilities given to developing this investigation. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Mouritz, P.A. 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https://openalex.org/W2277766036	https://hull-repository.worktribe.com/file/446423/1/Article.pdf	English	null	The role of biophysical cohesion on subaqueous bed form size	Geophysical research letters	2,016	cc-by	6,731	1. Introduction Estuaries, the coastal zone, and the continental shelf make up 8% of the world’s oceans [Meadows et al., 2012], and in these environments subaqueous dune bed forms are the primary sedimentary structures, acting as principal contributors to bed roughness and mediating sediment ﬂuxes [e.g., Lefebvre et al., 2011; Naqshband et al., 2014]. These coastal and nearshore locations are among the most sensitive regions in terms of sea level rise, a pro- blem exacerbated by the predicted increased frequency of extreme weather events, which will act to alter a range of sediment transport processes [e.g., FitzGerald et al., 2008]. Consequently, a robust understanding of how dune dimensions relate to controlling hydrodynamics is crucial for informing estuarine and coastal management, including prediction of the impacts of sea level rise, the maintenance of navigable channels [van der Mark et al., 2008], scour around engineering infrastructure, and roughness parameterizations in numerical models [Ganju and Sherwood, 2010]. Understanding the transport of organic material is important for determining ecological interactions and overall organic carbon ﬂuxes [Battin et al., 2008] in the coastal zone, and habitat modeling also requires appropriate dynamic models of bed form development in order to better predict spatial distributions of biological activity [e.g., Habersack et al., 2014]. Finally, preserved bed forms in the geological record are ﬁrst- order predictors for minimum water depth and hence environmental reconstruction [e.g., Leclair and Bridge, 2001]. Prediction of sediment transport rates presently relies on bed form phase diagrams and empirical bed form pre- diction formulae that are based exclusively on cohesionless silt, sand, and gravel [e.g., van den Berg and van Gelder, 1993; van Rijn, 1984]. However, substrates composed of mixtures of sand and mud are common to many coasts, deltas, estuaries, and lowland rivers [Healy et al., 2002]. The organic portion of these habitats is often ignored in sedimentological studies, but the importance of living organisms, their products, constructions, and remains can strongly mediate the physical behaviors and functionality of depositional systems [Black et al., 2002]. Substrata composed of sand-mud mixtures are important habitats for benthic biota. Moreover, where light pene- trates to the bed, microbial communities driven by oxygenic photosynthesis [Staats et al., 2000] also have the capacity to alter the surrounding physical and chemical nature of the substratum [e.g., Meadows et al., 2012]. Many subaqueous environments consist of mixtures of cohesionless sand, physically cohesive mud, and benthic organisms. 10.1002/2016GL067667 Daniel R. Parsons1, Robert J. Schindler1,2, Julie A. Hope3, Jonathan Malarkey4, Jaco H. Baas4, Jeffrey Peakall5, Andrew J. Manning1,2,6, Leiping Ye1, Steve Simmons1, David M. Paterson3, Rebecca J. Aspden3, Sarah J. Bass2, Alan G. Davies7, Ian D. Lichtman4,8, and Peter D. Thorne8 Key Points: • Subaqueous bed form dimensions are controlled more by biological than physical cohesion 1Department of Geography, Environment and Earth Sciences, University of Hull, Hull, UK, 2School of Marine Science and Engineering, Plymouth University, Plymouth, UK, 3School of Biology, University of St Andrews, Saint Andrews, UK, 4School of Ocean Sciences, Bangor University, Anglesey, UK, 5School of Earth and Environment, University of Leeds, Leeds, UK, 6HR Wallingford, Wallingford, UK, 7Centre for Applied Marine Sciences, Bangor University, Anglesey, UK, 8National Oceanography Centre, Liverpool, UK • Existing predictors require reformulation for combined biophysical cohesive effects • Small amounts of substrate biological cohesion act to reduce bed roughness 2 orders of magnitude Abstract Biologically active, ﬁne-grained sediment forms abundant sedimentary deposits on Earth’s surface, and mixed mud-sand dominates many coasts, deltas, and estuaries. Our predictions of sediment transport and bed roughness in these environments presently rely on empirically based bed form predictors that are based exclusively on biologically inactive cohesionless silt, sand, and gravel. This approach underpins many paleoenvironmental reconstructions of sedimentary successions, which rely on analysis of cross-stratiﬁcation and bounding surfaces produced by migrating bed forms. Here we present controlled laboratory experiments that identify and quantify the inﬂuence of physical and biological cohesion on equilibrium bed form morphology. The results show the profound inﬂuence of biological cohesion on bed form size and identify how cohesive bonding mechanisms in different sediment mixtures govern the relationships. The ﬁndings highlight that existing bed form predictors require reformulation for combined biophysical cohesive effects in order to improve morphodynamic model predictions and to enhance the interpretations of these environments in the geological record. Received 6 JAN 2016 Accepted 22 JAN 2016 Accepted article online 28 JAN 2016 ©2016. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1. Introduction The latter can increase sedimentary stabilization via burrow formation, cast constructions, and more pervasively, the secretion of cohesive extracellular polymeric substances (EPS) [Tolhurst et al., 2002]. As a result, the erosion thresholds of both cohesive and noncohesive sedimentary fractions are known to signiﬁcantly COHESION IN SUBAQUEOUS BED FORMS 1 PARSONS ET AL. Geophysical Research Letters 10.1002/2016GL067667 Table 1. Experimental Parameters for Series A–Ca Run m (%) e (%) H (mm) L (mm) H/L () ks (mm) A1 1.9 0.0 75 1549 0.0482 90.77 A2 4.7 0.0 65 1135 0.0571 94.07 A3 8.9 0.0 25 1011 0.0252 17.87 A4 9.8 0.0 24 894 0.0266 16.18 A5 11.9 0.0 22 741 0.0292 16.41 A6 12.7 0.0 11 625 0.0170 4.91 A7 14.1 0.0 18 537 0.0326 14.37 B1 2.8 0.027 37 990 0.0372 39.85 B2 6.8 0.038 13 772 0.0170 5.58 B3 15.4 0.030 4 979 0.0042 0.44 C1 9.1 0.075 4 121 0.0364 3.99 C2 9.9 0.071 4 116 0.0332 3.19 C3 12 0.073 3 115 0.0275 2.16 C4 17.7 0.100 - - - - am is initial bed mud fraction, e is initial bed EPS fraction, H is mean bed form height, L is mean bed form length, H/L is bed form steepness, and ks = 25H2/L is bed roughness. increase in the presence of EPS [e.g., Tolhurst et al., 2002]. Most studies have focused on the mechanical protection derived from high concentrations of EPS in the form of surface bioﬁlms, where surface scour is more likely than bed form development [Hagadorn and McDowell, 2012]. However, EPS are also distributed at lower con- centrations (0.01–0.1%) throughout the sediment substratum [Lanuru et al., 2007], where their inﬂuence on bed form development has been observed experimentally [Malarkey et al., 2015]. A handful of studies has examined sediment transport and bed forms in cohesive sediment under controlled conditions. However, these studies have treated biological and physical cohesion separately [Baas et al., 2013; Malarkey et al., 2015; Schindler et al., 2015]. While these studies have elucidated the differences between biological and physical cohesion, their applicability to the natural aquatic environment is limited, because physical and biological cohesion almost always occur together [Friend et al., 2008]. Furthermore, the interaction between physical and biological cohesion for bed form dynamics is largely unknown. Here we provide the ﬁrst results under controlled conditions of the inﬂuence of physical and biological cohesion on equilibrium dune morphology by means of laboratory experiments and examine the nature of the cohesive bonding mechanisms in three-way mixtures of mud, sand, and EPS. 2. Methods Experiments were undertaken in a recirculating ﬂume channel, 10 m long and 2 m wide, in the Total Environment Simulator at the University of Hull. Uniform ﬂow conditions were maintained over the test section (Figure S1 in the supporting information), and the ﬂow velocity was monitored at an acquisition rate of 25 Hz throughout each experimental run, using four vertically stacked 10 MHz acoustic Doppler velocimeters (ADVs) located close to the ﬂume centerline (Figure S1). Flow depth (d) was 0.38 m for all runs, and depth-mean ﬂow velocity (U) over an initially ﬂat bed was 0.80 m s1, yielding subcritical and fully turbulent ﬂow. Salinity was set, using sodium chloride, to approximate estuarine conditions, at 16 practical salinity units (psu), which is equivalent to a density of 1010 kg m3. These experimental settings, together with the mean grain size of the substrata used, are known to generate three-dimensional equilibrium dunes [van den Berg and van Gelder, 1993] (Figure S2). Three types of substrata were prepared, corresponding to series A–C (Table 1). In series A, only physical cohesion was considered [Schindler et al., 2015], with sand-mud mixtures made using two sediment frac- tions: upper ﬁne sand with a median diameter, D50, of 239 μm and kaolinite clay with a D50 of 3.4 μm. Seven substrata, labeled runs A1–A7, were prepared by incrementally increasing initial substratum mud content (1.9% < m < 14.1% by dry weight). In series B and C, various ratios of sand, mud, and EPS were combined to form a homogenous mixture. Xanthan gum was used as a proxy for EPS found in natural sediment [e.g., Tolhurst et al., 2002]. The range of EPS content used in the experiments is comparable with background ranges measured at intertidal sites in the Eden and Dee Estuaries, U.K. (0–0.1% EPS per dry weight of sediment), collected as part of parallel ﬁeld investigations [Malarkey et al., 2015], which tended to be relatively constant with depth in the upper centimeter of the bed. The initial and ﬁnal depth-mean EPS contents were determined by applying the phenol-sulphuric acid assay [Dubois et al., 1956] to millimetric slices taken from 100 mm long, 10 mm diameter syringe core samples. Standard grain size analysis techniques were used to quantify bed mud contents after each experiment. COHESION IN SUBAQUEOUS BED FORMS 2 PARSONS ET AL. 2 PARSONS ET AL. Geophysical Research Letters 10.1002/2016GL067667 Figure 1. 2. Methods Planform contour maps of the ﬁnal bed morphology of the experimental runs taken over a central swath of t domain (x is the distance downstream). First row shows selected runs for series A (no EPS), where a reduction in bed dimensions occurs as mud content is increased, resulting in a transition from fully three-dimensional dune-scale bed fo ripples superimposed on dunes to surfaces that approach a ﬂat bed. The second row shows bed forms from series B (low These bed forms are small compared with series A, and a transition from irregular, low-steepness 3-D dunes (run B1) almost featureless surface (B3) is evident. The third row show bed forms from series C (high EPS). These bed forms are to 2-D ripples and approach a featureless surface at the highest mud content (run C4). m = initial bed mud content; e = bed EPS content. Note the dramatic changes in bed form type and size for mere trace amounts of EPS. In series B, EPS was added at a low concentration (mean EPS content e=0.032± 0.006%) in order to represent environments with low primary production rates [Lanuru et al., 2007]. Three substrata with increasing mud content were made (2.8< m< 15.4%). In series C, EPS content was set to approximately 3 times the concentrations in ser Figure 1. Planform contour maps of the ﬁnal bed morphology of the experimental runs taken over a central swath of the tes domain (x is the distance downstream). First row shows selected runs for series A (no EPS), where a reduction in bed form dimensions occurs as mud content is increased, resulting in a transition from fully three-dimensional dune-scale bed forms via ripples superimposed on dunes to surfaces that approach a ﬂat bed. The second row shows bed forms from series B (low EPS) These bed forms are small compared with series A, and a transition from irregular, low-steepness 3-D dunes (run B1) to an almost featureless surface (B3) is evident. The third row show bed forms from series C (high EPS). These bed forms are limited to 2-D ripples and approach a featureless surface at the highest mud content (run C4). m = initial bed mud content; e = initia bed EPS content. Note the dramatic changes in bed form type and size for mere trace amounts of EPS. Figure 1. 2. Methods Planform contour maps of the ﬁnal bed morphology of the experimental runs taken over a central swath of the test domain (x is the distance downstream). First row shows selected runs for series A (no EPS), where a reduction in bed form dimensions occurs as mud content is increased, resulting in a transition from fully three-dimensional dune-scale bed forms via ripples superimposed on dunes to surfaces that approach a ﬂat bed. The second row shows bed forms from series B (low EPS). These bed forms are small compared with series A, and a transition from irregular, low-steepness 3-D dunes (run B1) to an almost featureless surface (B3) is evident. The third row show bed forms from series C (high EPS). These bed forms are limited to 2-D ripples and approach a featureless surface at the highest mud content (run C4). m = initial bed mud content; e = initial bed EPS content. Note the dramatic changes in bed form type and size for mere trace amounts of EPS. In series B, EPS was added at a low concentration (mean EPS content e=0.032± 0.006%) in order to represent environments with low primary production rates [Lanuru et al., 2007]. Three substrata with increasing mud content were made (2.8< m< 15.4%). In series C, EPS content was set to approximately 3 times the concentrations in ser- ies B (mean e=0.086±0.015%) to represent environments with high primary production rates [Lanuru et al., 2007]. Four substrata with increasing mud content were made (9.1< m< 17.7%). Each substratum was manually ﬂat- tened across the whole ﬂume to a thickness of 0.20m and subjected to the 0.80ms1 ﬂow for a period of 10.5 h. Geophysical Research Letters 10.1002/2016GL067667 Figure 2. (a) Relationship between bed form height, H, and initial mud content, m, for series A (blue, no EPS), B (red, low EPS), and C (green, high EPS). (b) Relationship between bed form wavelength, L, and initial mud content for series A–C. (c) Relationship between bed roughness, ks = 25H2/L, and initial mud content for series A–C. Error bars represent the variability from the mean across three longitudinal transects. All graphs also show predictions, based on noncohesive sand experiments, as dotted lines (VR1984 and N2014), after van Rijn [1984] and Naqshband et al. [2014], respectively. The linear ﬁts to H and L in series A can be used to infer clean sand values of H = 83 mm and L = 1627 mm. Bed topography was measured across a swathe of the channel bed at the end of each experiment, using a 2 MHz ultrasonic ranging sensor system mounted on an automated traverse oriented along the center of the ﬂume, spanning a test section distance of 4.7m (Figure S1). The dune dimensions were quantiﬁed from three transects in the swathe. Bed form length (L) was deﬁned as the distance between consecutive crests, and height (H) was deﬁned as the vertical distance from the crest to the upstream trough. Some dunes exhibited superimposed ripples. Dunes were distinguished from ripples by their order of magnitude longer lengths and laterally continuous crest lines that stretched across the width of the chan- nel [cf. Reesink and Bridge, 2007]. The height and length of each bed form along each transect were averaged together to produce a representative height, H, and length, L, for each experiment. In order to examine the nature of the cohe- sive bonding mechanisms, bed samples were also taken prior to the experiments in all three series of runs and compared using low-temperature scanning electron microscopy (LTSEM) [Paterson, 1995]. Figure 2. (a) Relationship between bed form height, H, and initial mud content, m, for series A (blue, no EPS), B (red, low EPS), and C (green, Figure 2. (a) Relationship between bed form height, H, and initial mud content, m, for series A (blue, no EPS), B (red, low EPS), and C (green, high EPS). (b) Relationship between bed form wavelength, L, and initial mud content for series A–C. (c) Relationship between bed roughness, ks = 25H2/L, and initial mud content for series A–C. Geophysical Research Letters Error bars represent the variability from the mean across three longitudinal transects. All graphs also show predictions, based on noncohesive sand experiments, as dotted lines (VR1984 and N2014), after van Rijn [1984] and Naqshband et al. [2014], respectively. The linear ﬁts to H and L in series A can be used to infer clean sand values of H = 83 mm and L = 1627 mm. COHESION IN SUBAQUEOUS BED FORMS 3 PARSONS ET AL. PARSONS ET AL. 3. Results The experimental results reveal a substan- tial inﬂuence of initial bed mud and EPS content on bed form height (H), length (L), steepness (H/L), and bed roughness (ks=25H2/L) [e.g., van Rijn, 1984] (Figures 1 and 2 (Figures S3 and S4) and Table 1). When EPS was absent (series A), the ﬁnal bed topographies show a clear transition the variability from the mean across three longitudinal transects. All graphs also show predictions, based on noncohesive sand experiments, as dotted lines (VR1984 and N2014), after van Rijn [1984] and Naqshband et al. [2014], respectively. The linear ﬁts to H and L in series A can be used to infer clean sand values of H = 83 mm and L = 1627 mm. from steep, more three-dimensional, dunes at low mud contents (m = 1.9%, A1) to low-steepness dunes (4.7% < m < 11.9%, A2 to A5), and very low steepness dunes (12.7%< m < 14.1%, A6 and A7), which are more two-dimensional, at higher mud contents (Figures 1 and 2). The dunes in runs A4 to A7 contained superim- posed current ripples. Bed form height, length, steepness, and roughness all decreased signiﬁcantly as initial bed mud fraction was increased (Figure 2 (Figure S4) and Table 1). A linear ﬁt to H and L for series A allows the dimensions of clean sand equivalent bed forms (m = 0%) to be estimated at H = 83 mm and L = 1627 mm. The addition of EPS to sand-mud mixtures in series B and C prevented the formation of steep, fully 3-D, dunes and substantially reduced bed form heights across the same range of initial mud contents as series A. The ﬁnal bed morphologies for the low EPS cases (approximately 0.03%; Table 1 and Figure 2) showed irregular, low- steepness, 3-D dunes. As in series A, bed form height, steepness, and roughness decreased with increasing mud fraction (Figure 2), whereas bed form length remained approximately constant. At low mud fractions, the bed form height, length, steepness, and roughness were smaller than at equivalent mud contents in the EPS-free experiments (Table 1). COHESION IN SUBAQUEOUS BED FORMS COHESION IN SUBAQUEOUS BED FORMS 4 PARSONS ET AL. PARSONS ET AL. Geophysical Research Letters 10.1002/2016GL067667 Figure 3. LTSEM images, comparing initial substratum microstructure for selected runs in series A, B, and C. Top and bottom rows show low- and high-resolution images, respectively. Scale bar units are in micrometers. (a) Run A2 (m = 4.7%), with plated kaolin particle aggregates found predominantly between sand grains rather than on the exposed sand grain surfaces. (b) Run B2 (m = 6.8%; e = 0.038%), showing kaolin-EPS aggregates dominated by EPS sheathes and partial coatings of sand grain surfaces. (c) Run C1 (m = 9.1%; e = 0.075%), showing EPS lining sand grain socket (top) and EPS strands and webs linking individual sand grains (bottom). Images obtained using procedures outlined in Tolhurst et al. [2002]. Figure 3. LTSEM images, comparing initial substratum microstructure for selected runs in series A, B, and C. Top and bottom rows show low- and high-resolution images, respectively. Scale bar units are in micrometers. (a) Run A2 (m = 4.7%), with plated kaolin particle aggregates found predominantly between sand grains rather than on the exposed sand grain surfaces. (b) Run B2 (m = 6.8%; e = 0.038%), showing kaolin-EPS aggregates dominated by EPS sheathes and partial coatings of sand grain surfaces. (c) Run C1 (m = 9.1%; e = 0.075%), showing EPS lining sand grain socket (top) and EPS strands and webs linking individual sand grains (bottom). Images obtained using procedures outlined in Tolhurst et al. [2002]. At the higher initial bed EPS fractions examined in series C (0.07–0.1%), the size of the bed forms was reduced substantially (Table 1 and Figures 1 and 2 (Figures S3 and S4)), with bed form types being limited to two- dimensional ripples for 9.1% < m < 12.0% (C1–C3) and a ﬂat bed at m = 17.7% (C4). Notably, the dominant bed form height of 4 mm was an order of magnitude smaller than the heights for equivalent mud contents in the absence of EPS (series A). Moreover, in runs C1 to C3, the bed form height, length, steepness, and roughness were independent of m. According to the widely used bed form predictor of van Rijn [1984], the dunes in this study should have reached an equilibrium height of 80 mm and an equilibrium length of 2774 mm (Figure 2). COHESION IN SUBAQUEOUS BED FORMS With the exception of the bed forms in run A1, which had the lowest mud substratum fraction (m = 1.9%), observed dune heights were all lower than the predicted height by up to an order of magnitude. The predicted dune length was also well in excess of all measured lengths, with the difference rapidly increasing as bed mud fraction and bed EPS fraction were increased. The initial compositions for selected experimental substrata are compared using LTSEM (Figure 3). In the abiotic samples (run A2; m = 4.7%), sand grain surfaces were largely free of clay particles (Figure 3a), and clay platelet aggregates formed distinctive layers between sand grains. At low fractions of EPS (run B1; m = 6.8%; e = 0.038%), there are EPS-bound sheaths resembling an “open card structure” (Figure 3b). Aggregates of mud and EPS span voids between grains, and there is a greater adhesion of cohesive material to sand grain surfaces compared with the abiotic (series A) case. At high EPS fractions (run C1; m = 9.1%; e = 0.075%) the matrix is visibly denser than in series A and B (Figure 3c, top). This density variability is apparent in the void created by the removal of a sand grain that exposed an EPS lining, indicating that the sand grains are enveloped by EPS (in direct contrast to clay-derived edge-to-plate bonds). At higher resolution, strands and webs of EPS link individual sand grains within a matrix (Figure 3c, bottom). COHESION IN SUBAQUEOUS BED FORMS 4.1. Comparison of Physical and Biological Cohesion 4.1. Comparison of Physical and Biological Cohesion The different types of bonding that mud and EPS exert within the substratum can explain the different sensitivities of bed form development to mud-induced physical cohesion and EPS-induced biological cohesion. The presence of clay platelet aggregates between the sand grains leads to physical cohesion imparted by mass attractive London-van der Waals forces and interparticle electrostatic bonding of cohesive particles [Mehta, 2014] (Figure 3a). Such electrostatic bonding would increase with salinity, in turn affecting the zeta potential (a measure of the net electrical charge around particles) of clay particles [Mietta et al., 2009]. In contrast, biological cohesion occurs when the polymer creates surface bonding through long chain molecular polymeric strands and gel surface coatings that physically link or envelope the sediment grains (Figures 3b and 3c) [cf. Underwood and Paterson, 2003]. In addition, if clay particles and EPS are both present, EPS can enhance the physicochemical cohesive properties of the mud fraction by increasing the molecular attractive forces between clay particles to form physical interparticle bonds that increase the tensile strength of the mud fraction [e.g., Chenu and Guerif, 1991]. Although the physicochemical cohesive properties of the mud may be enhanced by EPS, the order of magnitude changes in bed form properties suggests that the polymeric strands and gel surface coatings are the dominant mechanism for enhanced cohesion. This enhanced cohesion restricts the heights and lengths of mixed mud-EPS bed forms, and high levels of EPS modify the bed form type from dunes to ripples and ultimately ﬂat beds. Flow separation in the lee of bed forms is important for their development, in particular at the point where the shear layer between the main ﬂow and the vortex reattaches to the bed, con- trolling substratum erosion. It is postulated that the cohesive strength of the bed limits the ability of the ﬂow to erode sediment, thus limiting the height to which the bed form can ultimately grow. Geophysical Research Letters Geophysical Research Letters 10.1002/2016GL067667 Both the dunes and the ripples migrated during the experiments, resulting in an active layer in the bed, down to a level corresponding to the bed form troughs, and an inactive substratum underneath. Analysis of the postexperiment mud and EPS content in the bed revealed that both components had mostly been removed from the active layer by winnowing, but in the underlying substratum both components remained largely unchanged. This is in agreement with previous experimental work on ripples in mud-sand and EPS-sand mixtures [Baas et al., 2013; Malarkey et al., 2015], which indicated that the removal of EPS and mud from the troughs limits bed form growth. 4. Discussion The results presented herein show the dramatic effect that substratum fractions of physically cohesive mud and biologically cohesive EPS have on bed form height, length, and steepness compared with noncohesive sediment substrates that are exposed to similar shear stresses. The addition of mud and EPS signiﬁcantly reduced both bed form height and length across all the experiments. While substratum mud in isolation has a signiﬁcant effect, the addition of even small amounts of EPS dominates the combined effect of cohesion and has a dramatic inﬂuence on bed form dimensions and bed form type (Figure 2). COHESION IN SUBAQUEOUS BED FORMS PARSONS ET AL. PARSONS ET AL. 5 4.3. Implications for Ancient Environments The effects of cohesion on bed form size and shape have signiﬁcant implications for paleohydraulic and paleoenvironmental interpretations. Ancient strata record the modiﬁcation of sedimentary environments, and the formation of characteristic sedimentary structures, by microfauna and meiofauna since the ﬁrst appearance of microbial life in the Precambrian [Schopf, 1992], and the expansion and diversiﬁcation of microbial life to speciﬁc environments, such as tidal ﬂats, in the Phanerozoic [Ericksson et al., 2004]. This geological evidence is typically based on sedimentary facies where microfauna are present in large quan- tities, such as in microbial mats and stromatolites. However, smaller communities of microbial life may have inﬂuenced sedimentary processes in a wider range of settings during the Precambrian and possibly also earlier in the Precambrian than is detectable by direct fossil evidence. The results of the present experiments suggest that dune dimensions and thus their cross-set thicknesses are reduced even at low mud and extremely low EPS fractions. This observation may provide a tool for framing the search for early life on Earth to indirect evidence from the shape and average size of subaqueous dunes in the early Precambrian. This approach would rely on an improved quantitative understanding of how bed form cross-set thickness in mixed sand-mud is related to the height of bed forms, as such under- standing in clean sand underpins prediction of bed form height from the thickness distribution of ancient cross sets [e.g., Paola and Borgman, 1991; Leclair and Bridge, 2001]. The experimental data pre- sented herein suggest that the scales of preserved bed form sets and cross stratiﬁcation within deposits formed in natural substrates containing sand, mud, and microbiota are likely to diverge signiﬁcantly from those established for substrates devoid of such cohesion. Moreover, if not accounted for, the reduction in bed form size and steepness in response to physical and biological cohesion may lead to ﬂaws in the reconstruction of paleohydraulic variables, such as ﬂow discharge and channel depth and width [e.g., Rubin and Carter, 2006], which has already been suggested elsewhere for bioﬁlms formed by cyanobac- teria [Hagadorn and McDowell, 2012]. For instance, ancient ripple cosets could be misinterpreted as being produced by low ﬂow velocities, based on cohesionsionless predictions, when high levels of substrate cohesion would suggest much higher ﬂow velocities. Geophysical Research Letters Geophysical Research Letters 10.1002/2016GL067667 hydrodynamics and morphodynamics has recently been highlighted [Hauer et al., 2009]. The results pre- sented herein indicate that biological hydrodynamic coupling requires an understanding of how bed form dimensions inﬂuence habitat availability and the distribution of sediments and organisms. Furthermore, the results also indicate that microbial communities could play a key role in reducing bed form dimensions through the secretion of EPS, a system feedback that is not presently incorporated in morphodynamic and biological predictions. 4.2. Implications for Modern Environments 4.2. Implications for Modern Environments The changes in dominant bed form geometries and dimensions described herein have a number of impor- tant implications for morphodynamics, sediment transport, and biophysical habitat modeling. Our results demonstrate that form roughness (ks = 25H2/L) in numerical models may be overestimated by as much as 2 orders of magnitude in areas of signiﬁcant biophysical cohesion. Models that overpredict bed roughness would underestimate ﬂow velocities and overestimate background turbulence levels, which in turn has impli- cations for ﬂocculation processes, sediment ﬂuxes, and ultimately morphodynamic change in regional mod- els [e.g., Sutherland et al., 2004]. Our experimental data also suggest that bed form scour depths may be overestimated, by up to an order of magnitude, in engineering design of bridge piers, pylons, buried pipelines, and cables, because bed forms with natural biophysical cohesion do not reach the heights and trough scour depths as those produced in cohesionless sand. Similarly, maintenance of navigable channels by dredging requires information on the highest crest elevations for operational under keel clearance [van der Mark et al., 2008], which may be overpredicted by up to an order of magnitude. The experimental data presented herein also suggest that sediment transport rates may vary signiﬁcantly in sediment beds containing EPS and at scales that could be relevant for the dynamics of biological activity [e.g., Maddock, 1999]. The need to couple biological habitat models with simulations of time-varying COHESION IN SUBAQUEOUS BED FORMS PARSONS ET AL. PARSONS ET AL. 6 5. Conclusions Our experiments examined the importance of combined physical and biological cohesion on current- generated bed form morphology. The experimental data reveal that both have signiﬁcant inﬂuence and that biologically produced extracellular polymeric substances (EPS) are by far the most effective of the two components in reducing bed form dimensions and steepness, due to their stronger interpar- ticle bonding. The combined effect of biological and physical cohesion has been shown to alter bed form dimensions by up to an order of magnitude and bed roughness by up to 2 orders of magnitude. These large changes result from the suppression of dunes in favor of ripples as the dominant bed form. Changes induced by physical and biological cohesion render existing and widely adopted bed form predictors, based on cohesionless grains, inadequate for many naturally occurring sedimentary environ- ments, particularly coastal and estuarine systems that tend to comprise signiﬁcant levels of biologically active ﬁne-grained sediment. The present results have signiﬁcant implications for modern coastal management and engineering, the interpretation of ancient sedimentary environments, and the role of biological mediation in such sedimentary systems. Physical and biological cohesion require incorporation into new generations of bed form predictors, morphodynamic models, and biological habitat models. The results also provide a basis for reassessing the impact of early life on bed form dynamics and sedimentary systems more generally. COHESION IN SUBAQUEOUS BED FORMS COHESION IN SUBAQUEOUS BED FORMS 7 PARSONS ET AL. PARSONS ET AL. 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https://openalex.org/W4386010876	https://jerkin.org/index.php/jerkin/article/download/144/93	Indonesian	null	Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo	Jurnal Pengabdian Masyarakat dan Riset Pendidikan	2,023	cc-by-sa	3,642	ARTICLE INFO Penelitian ini bertujuan untuk mengetahui implementasi pembelajaran Al- Qur’an menggunakan metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo, dan untuk mengetahui faktor pendukung dan penghambat implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo. Jenis penelitian yang digunakan adalah kualitatif deskriptif. Proses pengumpulan data melalui observasi, wawancara, dan dokumentasi. Hasil penelitian menunjukkan bahwa: 1) implementasi metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo memperoleh hasil yang baik yakni berdasarkan hasil observasi yang dilakukan oleh peneliti hanya 5% santri atau 5 santri dari 54 santri yang belum dapat menguasai secara maksimal kemampuan membaca Al-Qur’an dengan menggunakan metode Bil Qolam.2) faktor pendukung dalam penerapan metode Bil Qolam adalah kemempuan membaca Al-Qur’an santri masuk dalam kategori baik, antusiasme santri, motivasi dan dukungan pengajar, metode Bil Qolam menarik, dan dapat menambah wawasan dan pengetahuan santri. Sedangkan faktor penghambatnya adalah suasana yang kurang kondusif, santri yang saling menganggu, dan alokasi waktu pembelajaran yang kurang. This study aims to determine the implementation of Al-Qur’an learning using the Bil Qolam method in improving the ability to read the Qur’an at Nurul Ulum MAN Purworejo Dormitory, and to determine the supporting and inhibiting factors for implementing Al-Qur’an learning using the Bil Qolam method. In improving the ability to read the Qur’an in Nurul Ulum MAN Purworejo dormitory. The type of research used is dercriptive qualitative. The process of collecting data trough observation, interviewa, and documentation. The research results sgow that: 1) the implementation of the Bil Qolam method in improving the ability to read the Al-Qur’an at the Nurul Ulum MAN Purworejo Dormitory obtained good result, namely based on observations made by researches only 5% of student or 5 students from 54 students who have not been able to fully master the ability to read the Qur’an using the Bil Qolam method. 2) supporting factors in the application of the Bil Qolam method are the ability to read the Qur’an for students in the good category, the enthusiasm of the students, the support and motivation of the teacher, the Bil Qolam method is interesting, and can add to the insight and knowledge of the studentas. While the inhibiting factors are the atmosphere that is not conducive, students who disturb each other, and the allocation of learning time that less. ARTICLE INFO Penelitian ini bertujuan untuk mengetahui implementasi pembelajaran Al- Qur’an menggunakan metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo, dan untuk mengetahui faktor pendukung dan penghambat implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo. Jenis penelitian yang digunakan adalah kualitatif deskriptif. Proses pengumpulan data melalui observasi, wawancara, dan dokumentasi. Hasil penelitian menunjukkan bahwa: 1) implementasi metode Bil Qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo memperoleh hasil yang baik yakni berdasarkan hasil observasi yang dilakukan oleh peneliti hanya 5% santri atau 5 santri dari 54 santri yang belum dapat menguasai secara maksimal kemampuan membaca Al-Qur’an dengan menggunakan metode Bil Qolam.2) faktor pendukung dalam penerapan metode Bil Qolam adalah kemempuan membaca Al-Qur’an santri masuk dalam kategori baik, antusiasme santri, motivasi dan dukungan pengajar, metode Bil Qolam menarik, dan dapat menambah wawasan dan pengetahuan santri. Sedangkan faktor penghambatnya adalah suasana yang kurang kondusif, santri yang saling menganggu, dan alokasi waktu pembelajaran yang kurang. Article history Received: 15 August 2023 Revised: 15 August 2023 Accepted: 15 August 2023 Article history Received: 15 August 2023 Revised: 15 August 2023 Accepted: 15 August 2023 Kata Kunci: Implementasi, Metode Bil Qolam, Membaca Al-Qur’an. Keywords: Implementation, Bil Qolam Method, Read Al- Qur’an. This study aims to determine the implementation of Al-Qur’an learning using the Bil Qolam method in improving the ability to read the Qur’an at Nurul Ulum MAN Purworejo Dormitory, and to determine the supporting and inhibiting factors for implementing Al-Qur’an learning using the Bil Qolam method. In improving the ability to read the Qur’an in Nurul Ulum MAN Purworejo dormitory. The type of research used is dercriptive qualitative. The process of collecting data trough observation, interviewa, and documentation. The research results sgow that: 1) the implementation of the Bil Qolam method in improving the ability to read the Al-Qur’an at the Nurul Ulum MAN Purworejo Dormitory obtained good result, namely based on observations made by researches only 5% of student or 5 students from 54 students who have not been able to fully master the ability to read the Qur’an using the Bil Qolam method. p-ISSN: 2963-7856 \| e-ISSN: 2961-9890 Available online at jerkin.org/index.php/jerkin Jurnal Pengabdian Masyarakat dan Riset Pendidikan Volume 2 No 1, Juli-September 2023, pp 208-212 Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo Rina Aminatul Hasna1*, Muchotob Hamzah2, Vava Imam Agus Faisal3 1,2,3Pendidikan Agama Islam, Universitas Sains Al-Qur’an, Jl. Kyai Hasyim Asy’ari No. Km, Rw.03, Kalibeber, Kec. Mojotengah, Kabupaten Wonosobo, Jawa Tengah 56351. E-mail: rinanana2018@gmail.com https://doi.org/10.31004/jerkin.v2i1.144 PENDAHULUAN Al-Qur’an merupakan kalamullah yang menjelaskan tentang suatu kebenaran, dan dengan hadirnya Al-Qur’an manusia dapat berhubungan langsung kepada Tuhan-Nya dan sesama manusia. Tujuan diturunkannya Al-Qur’an adalah sebagai pedoman dan petunjuk hidup manusia untuk mencapai kesejahteraan di dunia dan akhirat.(Mohammad Daud Ali, 2013) Dalam membaca Al-Qur’an sebaiknya harus berhati-hati dan tidak boleh asal-asalan dalam pengucapan makhrajul hurufnya, karena apabila dalam melafalkan ayat Al-Qur’an salah maka nantinya akan salah juga dalam mengartikannya. Sebagai umat muslim Allah SWT menganjurkan untuk membaca Al-Qur’ansecara baik dan benar dengan bacaan tartil. Karena asal-Qur’an merupakan pedoman hidup bagi ummat muslim dan wajib mempelajarinya.(Kohn, 2011) Seperti yang difirmankan oleh Allah SWT dalam Q.S Al-Muzammil ayat 4 yang artinya: “Dan bacalah Al-Qur’an dengan perlahan-lahan (tartil)” Q.S Al-Muzammil:4).(Al-Qur’an dan Terjemahnya) Ayat diatas menjelaskan bahwa dalam membaca Al-Qur’an sebaiknya harus perlahan agar makhrajul hurufnya dan tajwidnya sesuai kaidah yang berlaku. Membaca Al-Qur’an secara perlahan, tidak tergesa-gesa, dengan bacaan yang baik dan benar sesuai makhraj dan ciri-cirinya sebagaimana dijelaskan dalam ilmu tajwid. Hal ini mempengaruhi pada kemampuan membaca Al-Qur’an. Salah satu faktor yang mempengaruhi bagi kemampuan membaca dan pemahaman Al-Qur’an siswa bisa disebabkan oleh pengajar. Apabila pengajar memiliki kemampuan yang mendukung maka akan berpengaruh ke kemampuan siswa yang dapat meningkat, begitu pula sebaliknya apabila pengajar memiliki kemampuan yang rendah maka akan berpengaruh ke kemampuan siswa yang dulit berkembang. g Salah satu metode pembelajaran Al-Qur’an ini menggunakan metode Bil Qolam. Metode Bil Qolam dianggap metode yang cocok digunakan untuk membaca Al-Qur’an dan dapat diterapkan oleh semua kalangan. Dengan menggunakan metode ini siswa mudah memahami materi yang diberikan oleg gurunya, karena seorang guru mempraktikkan secara langsung kepada siswanya. Menurut karya dari K.H Basori Alqi Pesantren Ilmu Al-Qur’an (PIQ) Singosari mengenai metode Bil Qolam. Metode ini menggunakan teknik taqlid (menirukan) dan bersifat teacher-centris yaitu seorang guru membaca satu ayat, lalu siswa menirukan apa yang dibaca oleh gurunya. Seorang guru membaca lagi satu sampai dua kali, lalu siswa menirukan lagi. Kemudian membaca ayat berikutnya, lalu siswa menirukan lagi begitupun seterusnya.(Tim Pusat Metode Bil Qolam, 2004). ARTICLE INFO 2) supporting factors in the application of the Bil Qolam method are the ability to read the Qur’an for students in the good category, the enthusiasm of the students, the support and motivation of the teacher, the Bil Qolam method is interesting, and can add to the insight and knowledge of the studentas. While the inhibiting factors are the atmosphere that is not conducive, students who disturb each other, and the allocation of learning time that less. This is an open access article under the CC–BY-SA license. 208 Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo, Rina Aminatul Hasna, Muchotob Hamzah, Vava Imam Agus Faisal 209 How to Cite: Rina Aminatul Hasna, Muchotob Hamzah, Vava Imam Agus Faisal (2023). Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al- Qur’an di Asrama Nurul Ulum MAN Purworejo, 2(1) 208-212. https://doi.org/10.31004/jerkin.v2i1.144 How to Cite: Rina Aminatul Hasna, Muchotob Hamzah, Vava Imam Agus Faisal (2023). Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al- Qur’an di Asrama Nurul Ulum MAN Purworejo, 2(1) 208-212. https://doi.org/10.31004/jerkin.v2i1.144 Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Implementasi pembelajaran Al-Qur’an di Asrama Nurul Ulum MAN Purworejo dengan menerapkan metode bil qolam dalam meningkatkan kemampuan membaca al-qur’an sudah masuk kategori baik, hal ini dibuktikan dengan hasil observasi yang didapatkan yakni dari 54 santri hanya 9% atau 5 santri yang belum dapat membaca al-qur’an sesuai dengan indikator yang diterapkan di dalam metode bil qolam. Dari kelima santri tersebut dikatakan belum lancar dalam menguasi metode bil qolam untuk membaca al-qur’an karena belum bisa maksimal dalam pelafalan makhraj, tajwid, masih ada yang belum sesuai, shifatul huruf belum sesuai dan belum terlalu lancar dalam membaca Al-Qur’an. Pada proses pembelajaran menggunakan metode bil qolam di Asrama Nurul Ulum dibagi menjadi 3 kegiatan yakni kegiatan pembuka, kegiatan inti, dan kegiatan penutup. Pada kegiatan pembuka yang dilakukan oleh pengajar bertujuan untuk mengkoordinasikan para santri agar kondusif serta agar lebih mudah dalam menyampaikan materi yang akan diajarkan oleh pengajar dan tuntas menguasi indikator dari keberhasilan dari metode bil qolam yang meliputi makhraj, tajwid, shifatul huruf, dan kelancaran dalam membaca Al-Qur’an. Kemudian pada kegiatan inti pengajar menerapkan metode bil qolam dengan cara talqin, ittiba’, urdhoh, dan urdhoh bi nafsi. Dengan pengajar memberikan contoh membaca 1 ayat kemudian para santri mengikutinya, selanjutnya pengajar mengetes beberapa santri secara bergilir guna untuk mengetahui seberapa jauh kemampuan santri dalam menguasai materi akan tetapi untuk kegiatan santri di tes untuk membaca kembali materi yang diajarkan belum bisa maksimal karena keterbatasan waktu. Kemudian pengajar mencontohkan kembali bacaan ayat secraa berulang-ulang sebelum berganti pada materi di ayat berikutnya cara ini dilakukan guna untuk memperdalam materi dan memperkuat materi yang diajarkan pengajar kepada para santri. Pada kegiatan penutup pengajar memberikan motivasi kepada santri dengan adanya motivasi yang diberikan oleh pengajar diharapkan dapat meningkatkan semangat para santrinya dalam menuntut ilmu dan berdampak baik bagi kemampuan membaca Al-Qur’an para santri, kemudian dilanjutkan dengan santri membaca doa penutup. Proses implementasi metode Bil Qolam di Asrama Nurul Ulum MAN Purworejo terdapat empat indikator yang diajarkan dan diperhatikan oleh pengajar, yang bertujuan untuk melihat seberapa jauh pemahaman santri terhadap materi yang diajarkan, sehingga santri memiliki kemampuan membaca Al-Qur’an dengan lancar dan fasih. Keempat indikator tersebut diantaranya yaitu: makhraj, tajwid, sifatul huruf, dan kelancaran, dimana santri harus menguasai keempat indikator tersebut sesuai dengan ketentuan yang diajarkan oleh pengajar. METODE Jenis penelitian yang digunakan adalah penelitian kualitatif lapangan. Penelitian kualitatif lapangan adalah penelitian yang dilakukan secara sistematis dengan mengangkat data yang ada dilapangan.(Arikunto, 1995)Penelitian ini dilaksanakan di Asrama Nurul Ulum MAN Purworejo kurang lebih dilaksanakan selama satu bulan, penelitian dimulai pada dari bulan Juni sampai dengan bulan Juli. Adapun subjek dalam penelitian ini adalah para santri di Asrama Nurul Ulum MAN Purworejo. Teknik pengumpulan data dalam penelitian ini menggunakan teknik wawancara, Observasi dan studi dokumentasi. Adapun responden yang diwawancarai adalah kepala asrama dan beberapa pengajar serta santri dari Asrama Nurul Ulum MAN Purworejo. Obervasi yang dilakukan di dalam penelitian ini adalah observasi terhadap bagaimana kemampuan membaca santri dari Asrama Nurul ULUM MAN Purworejo. Instrument di dalam penelitian ini adalah peneliti, di sini peneliti memiliki peran sebagai alat peneliti yang utama yang berarti peneliti harus ikut perpartisipasi didalam proses pembelajaran Al-Qur’an menggunkan metode bil-qolam guna untuk melihat seberapa jauh kemampuan membaca santri dalam membaca Al-Qur’an.(Sugiyono, 2013) Untuk menguji keabsahan data di dalam penelitian ini menggunakan dua jenis triangulasi untuk mengecek keabsahan data yaitu triangulasi sumber dan triangulasi teknik. Kemudian analisis data yang digunakan di dalam penelitian ini meliputi pengumpulan data, reduksi data, penyajian data, dan Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 1, Juli-September 2023, hal. 208-212 210 penarikan kesimpulan. (Sugiyono, 2019). penarikan kesimpulan. (Sugiyono, 2019). HASIL DAN DISKUSI Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Apabila santri mampu menguasai keempat indikator tersebut maka dapat dikatakan santri tersebut memiliki kemampuan yang baik dalam membaca Al-Qur’an, namun sebaliknya apabila santri belum menguasai keempat indikator tersebut maka kemampuan membaca Al-Qur’an masih harus diasah secara terus menerus. Pada indikator pelafalan makhraj diperoleh hasil observasi sebesar 74,07% dengan jumlah santri yang tuntas sebanyak 40 santri dari 54 santri. Hal tersebut menunjukkan bahwa pada indikator tersebut kemampuan membaca Al-Qur’an dalam kategori baik karena hampir seluruh santri menguasai dalam pelafalan Makhrajul huruf. Pada indikator tajwid diperoleh hasil observasi sebesar 83,33% dengan jumlah santri yang tuntas sebanyak 43 dari 54 santri. Hal tersebut menunjukkan bahwa pada indikator tersebut kemampuan membaca Al-Qur’an santri masuk kedalam kategori sangat baik karena hamper seluruh santri menguasai dalam pelafalan tajwid dalam membaca Al-Qur’an. Pada indikator sifatul huruf diperoleh hasil observasi sebesar 85,18% dengan jumlah santri yang tuntas sebanyak 46 dari 54 santri. Hal tersebut menunjukkan bahwa pada indikator tersebut kemampuan membaca Al-Qur’an santri masuk dalam kategori sangat baik, karena hampir seluruh santri menguasai dalam pelafalan sifatul huruf dalam membaca Al-Qur’an. g p Kemudian pada indikator kelancaran diperoleh hasil observasi sebesar 88,88% dengan jumlah santri yang tuntas sebanyak 48 dari 54 santri. Hal tersebut menunjukkan bahwa pada indikator tersebut Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) Implementasi Pembelajaran Al-Qur’an Menggunakan Metode Bil-Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo, Rina Aminatul Hasna, Muchotob Hamzah, Vava Imam Agus Faisal 211 kemampuan membaca Al-Qur’an santri masuk dalam kategori sangat baik, karena hampir seluruh santri menguasai dan memiliki kelancaran yang baik dalam membaca Al-Qur’an. Keempat indikator tersebut memberikan hasil dan dampak positif yakni memudahkan santri dalam proses pembelajaran Al-Qur’an menggunakan metode Bil Qolam. Penerapan metode Bil Qolam dapat meningkatkan kemampuan membaca Al-Qur’an santri, karena penerapan dari metode itu sendiri diberikan secara bertahap sehingga santri lebih mudah dalam mengikuti arahan dan bimbingan dari pengajar. Proses pelaksanaan pembelajaran Al-Qur’an menggunakan metode Bil Qolam diikuti oleh seluruh santri secara bersama-sama, dimana santri mengikuti kegiatan tersebut sesuai dengan arahan dari pengajar dan sesuai dengan penjelasan yang disampaikan oleh pengajar, sehingga proses tersebut dalam berjalan dengan lancar dan memberikan hasil yang maksimal. Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Hal tersebut juga tak lepas dari peran pengajar, dimana dukungan dan motivasi dari pengajar sangat dibutuhkan oleh santri, apabila pengajar bisa memberikan dukungan dan motivasi penuh maka akan diperoleh hasil yang maksimal dan mampu melahirkan generasi muda yang Qur’ani dan dapat mejadi santri yang baik, memiliki kemampuan membaca Al-Qur’an yang baik sesuai dengan ajaran dan ketentuan-ketentuan yang sesuai. Faktor Pendukung dan Penghambat Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Faktor Pendukung dan Penghambat Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Q Q Proses implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam tentu ada faktor pendukung dan penghambat berjalannya kegiatan tersebut. Adapun faktor pendukung dari implementasi pembelajaran Al-Qur’an yang dilaksanaka rama Nurul Ulum MAN Purworejo adalah sebagai berikut: Adapun faktor pendukung dari implementasi pembelajaran Al-Qur’an yang dilaksanakan di asrama Nurul Ulum MAN Purworejo adalah sebagai berikut: 1. Pertama, kemampuan membaca Al-Qur’an santri masuk kategori baik, kemampuan membaca Al-Qur’an di asrama Nurul Ulum MAN Purworejo sudah masuk kedalam kategori baik, hal ini dibuktikan dengan kelancaran santri dalam membaca Al-Qur’an dan kemampuan santri dalam menghafal Al-Qur’an serta banyaknya prestasi-prestasi yang diraih oleh para santri, yaitu beberapa santri sudah mampu menghafal Al-Qur’an bahkan lebih dari 5 sampai 10 Juz, dan beberapa sanatri yang mengikuti ajang perlombaan di tingkat nasional. 2. Kedua, antusiasme santri dalam proses pembelajaran Al-Qur’an, antusiasme santri dalam mengikuti proses pembelajaran Al-Qur’an di asrama Nurul Ulum merupakan salah satu faktor pendukung yang sangat penting, apabila santri dapat mengikuti kegiatan dengan baik dan semangat maka akan memudahkan proses implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam, dan memudahkan bagi para pengajar di asrama tersebut. 3. Ketiga, pengajar memotivasi dan memberikan dukungan penuh, dalam suatu proses pembelajaran pengajar harus memberikan motivasi dan dukungan penuh terhadap anak didik atau santrinya, hal ini bertujuan agar proses pembelajaran berjalan secara maksimal dan memberikan hasil yang memuaskan. 4. Keempat, metode pembelajaran Al-Qur’an yang diterapkan menarik dan mudah dipahami, metode pembelajaran Al;Qur’an yang menarik dan mudah dipahami dalam meningkatkan kemampuan membaca Al-Qur’an santri, salah satunya metode Bil Qolam dimana metode tersebut merupakan metode yang sangat praktis dan cukup mudah diterapkan dalam proses pembelajaran Al-Qur’an, selain itu metode Bil-Qolam juga memiliki nada yang khas, mudah, dan menyentuh. y 5. KESIMPULAN Adapun kesimpulan dari penelitian ini meliputi: p p p p 1. Implementasi metode bil qolam dalam meningkatkan kemampuan membaca Al-Qur’an di Asrama Nurul Ulum MAN Purworejo diterapkan dengan melakukan kegiatan pembuka, kegiatan inti yang meliputi talqin, ittiba’, urdhoh, dan urdhoh bi nafsi dan kegiatan penutup. Hasil dari implementasi metode bil qolam ini mendapakan hasil yang yang baik yakni berdasarkan hasil observasi yang dilakukan oleh peneliti hanya 5% santri atau 5 santri dari 54 santri yang belum dapat menguasi secara maksimal kemampuan membaca al-qur’an dengan menggunakan metode bil qolam. Keempat indikator yang diberikan dalam proses pembelajaran Al-Qur’an menggunakan metode Bil Qolam yang diantaranya yaitu makhraj, tajwid, sifatul huruf, dan kelancaran menunjukkan hasil yang baik, dimana pada indikator makhraj diperoleh hasil observasi sebesar 74, 07%, pada indikator tajwid diperoleh hasil observasi sebesar 83,33%, pada indikator sifatul huruf diperoleh hasil observasi sebesar 85,18%, dan pada indikator kelancaran diperoleh hasil observasi sebesar 88,88%. Keempat indikator tersebut menunjukkan hasil dan dampak yang positif bagi santri karena memudahkan santri dalam membaca Al-Qur’an sesuai dengan metode dan ketentuan yang diberikan oleh pengajar. p g j 2. Proses implementasi metode Bil Qolam terdapat faktor pendukung dan penghamba pelaksanaan proses pembelajaran tersebut. Diantara faktor pendukung tersebut adalah sebaga berikut: a. kemampuan membaca Al-Qur’an santri masuk kedalam kategori baik, b. antusiasme santri dalam proses pembelajaran Al-Qur’an, c. pengajar memotivasi dan memberikan dukungan penuh, d. metode pembelajaran Al-Qur’an yang diterapkan menarik dan mudah dipahami, dan e. menambah wawasan dan ilmu baru dalam membaca Al-Qur’an. Sedangkan faktor penghambatnya adalah sebagai berikut: a. suasana kurang kondusif, b. jumlah santri yang banyak membuat waktu kurang efisien, c. beberapa Bahasa yang digunakan oleh pengajar sulit dipahami, dan d. diganggu santri lain. 2. Proses implementasi metode Bil Qolam terdapat faktor pendukung dan penghambat pelaksanaan proses pembelajaran tersebut. Diantara faktor pendukung tersebut adalah sebagai berikut: c. pengajar memotivasi dan memberikan dukungan penuh, d. metode pembelajaran Al-Qur’an yang diterapkan menarik dan mudah dipahami, dan e. menambah wawasan dan ilmu baru dalam membaca Al-Qur’an. Sedangkan faktor penghambatnya adalah sebagai berikut: a. suasana kurang kondusif, b. jumlah santri yang banyak membuat waktu kurang efisien, c. beberapa Bahasa yang digunakan oleh pengajar sulit dipahami, dan Implementasi Pembelajaran Al-Qur’an menggunakan Metode Bil Qolam dalam Meningkatkan Kemampuan Membaca Al-Qur’an Kelima, enambah pengetahuan dan ilmu baru dalam membaca Al-Qur’an, metode Bil Qolam merupakan metode yang menarik dan mudah dipahami, sehingga dapat menambah wawasan dan pengetahuan santri dalam membaca Al-Qur’an dan dapat meningkatkan kemampuan santri dalam membaca Al-Qur’an. Q Selain faktor pendukung juga terdapat faktor penghambat dalam implementasi pembelajaran Al- ur’an di asrama Nurul Ulum MAN Purworejo, yaitu sebagai berikut: Selain faktor pendukung juga terdapat faktor penghambat dalam implementasi pembelajaran Al- Q r’an di asrama N r l Ul m MAN P r orejo ait sebagai berik t: 1. Pertama, Suasana yang kurang kondusif dapat menghambat kelancaran proses implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam di asrama Nurul Ulum MAN Purworejo, hal tersebut disebabkan oleh pelaksanaan pembelajaran Al-Qur’an yang hanya dilaksanakan 1 kali dalam satu minggu dan diikuti oleh seluruh santri. Mungkin akan lebih efektif lagi apabila disusun jadwal secara bergantian agar mendapatkan hasil yang maksimal. g p j g g p y g 2. Kedua, alokasi waktu dalam implementasi pembelajaran Al-Qur’an di asrama tersebut Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 1, Juli-September 2023, hal. 208-212 212 terkadang berjalan kurang efisien, hal tersebut disebabkan karena jumlah santri yang cukup banyak tetapi jadwal pembelajaran menggunakan metode Bil Qolam hanya dilaksanakan 1 kali salam satu minggu, mungkin akan lebih efektif lagi apabila diterapkan jadwal pembelajaran secara bergantian atau dibentuk pengelompokkan santri berdasarkan tingkat kemampuan membaca Al-Qur’an santri. p Q 3. Ketiga, beberapa Bahasa yang digunakan pengajar sulit dipahami, dalam proses implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam terkadang beberapa Bahasa yang digunakan oleh pengajar sulit dipahami oleh santri seperti Bahasa Arab dan Tajwid, karena beberapa santri masih terkendala dalam mengenali Bahasa yang diterapkan oleh pengajar. 3. Ketiga, beberapa Bahasa yang digunakan pengajar sulit dipahami, dalam proses implementasi pembelajaran Al-Qur’an menggunakan metode Bil Qolam terkadang beberapa Bahasa yang digunakan oleh pengajar sulit dipahami oleh santri seperti Bahasa Arab dan Tajwid, karena beberapa santri masih terkendala dalam mengenali Bahasa yang diterapkan oleh pengajar. 4. Keempat, beberapa santri yang menganggu sesame santri lainya merupakan salah satu faktor penghambat dalam proses pembelajaran Al-Qur’an menggunakan metode Bil Qolam, hal tersebut dapat menyebabkan kegaduhan dan kurang dapat memahami penjelasan yang diberikan oleh pengajar. Sehingga pengajar harus dengan penuh perhatian memberikan arahan yang baik agar tidak terjadi kegaduhan pada saat proses pembelajaran. Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) REFERENSI Al-Qur’an dan Terjemahnya, Dapartemen Agama RI. Al-Qur’an dan Terjemahnya, Dapartemen Agama RI. Al-Qur’an dan Terjemahnya, Dapartemen Agama RI. Arikunto (1995) Dasar-Dasar Research. Bandung: Tarsoto. Kohn, A.M. (2011) Praktikum Qira’at Keanehan Bacaan Al- Qur’an Qira’at Ashim dari Hafs. Mohammad Daud Ali (2013) Pendidikan Agama Islam. Jakarta: PT Raja Grafindo. Sugiyono (2013) Metode Penelitian Kuantitatif dan Kualitatif R&D. Bandung: ALFABETA. Tim Pusat Metode Bil Qolam (2004) Buku Panduan Metode Praktor Belajar Al- Qur’an Bil Qolam Malang: PIQ Singosari. Arikunto (1995) Dasar-Dasar Research. Bandung: Tarsoto. n, A.M. (2011) Praktikum Qira’at Keanehan Bacaan Al- Qur’an Qira’at Ashim dari Hafs. Mohammad Daud Ali (2013) Pendidikan Agama Islam. Jakarta: PT Raja Grafindo. Sugiyono (2013) Metode Penelitian Kuantitatif dan Kualitatif R&D. Bandung: ALFABETA. Tim Pusat Metode Bil Qolam (2004) Buku Panduan Metode Praktor Belajar Al- Qur’an Bil Qolam. Malang: PIQ Singosari. Tim Pusat Metode Bil Qolam (2004) Buku Panduan Metode Praktor Belajar Al- Qur’an Bil Qolam. Malang: PIQ Singosari. Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online)
https://openalex.org/W3119603099	https://repositori.udl.cat/bitstream/10459.1/70245/4/030841.pdf	English	null	Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens	Animals	2,021	cc-by	11,735	Abstract: The current study was conducted to examine the effects of deoxynivalenol (DON) at different levels (5 and 15 mg/kg feed) on the metabolism, immune response and welfare parameters of male broiler chickens (Ross 308) at 42 days old. Forty-ﬁve 1 day-old broiler chickens were randomly distributed into three different dietary treatments: (1) control, (2) DON-contaminated diet with 5 mg DON/kg of feed (guidance level), and (3) DON-contaminated diet with 15 mg DON/kg of feed. Five replicated cages with three birds each were used for each treatment in a randomized complete block design. The results showed that DON was detected in excreta of birds fed contaminated diets compared with controls. The metabolite DON-3 sulphate (DON-3S) was detected in plasma and excreta in both treated groups, as well as in the liver (but only at 15 mg/kg feed). The increase in the level of DON decreased the hemoglobin concentration (p < 0.001), whereas the erythrocyte counts were only decreased at 15 mg DON/kg feed. No effect of DON on the responses to common vaccines was observed. In plasma, interleukin 8 levels in both contaminated groups were signiﬁcantly higher than in the control group. The expression of interleukin 6, interleukin 1β and interferon-γ increased in jejunum tissues of broilers fed 5 mg/kg of DON compared with controls. The stress index (heterophil to lymphocyte ratio) was not affected by DON-contaminated diets compared with controls. The plasma corticosterone level was signiﬁcantly lower in both DON groups compared with controls. In conclusion, DON-3S could be used as a speciﬁc biomarker of DON in different biological matrices, while the immune response in broiler chickens is stimulated by the presence of DON at the guidance level, but no adverse effect was observed on physiological stress parameters. Citation: Riahi, I.; Marquis, V.; Pérez-Vendrell, A.M.; Brufau, J.; Esteve-Garcia, E.; Ramos, A.J. Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens. Animals 2021, 11, 147. https://doi.org/10.3390/ani11010147 Citation: Riahi, I.; Marquis, V.; Pérez-Vendrell, A.M.; Brufau, J.; Esteve-Garcia, E.; Ramos, A.J. Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens. Animals 2021, 11, 147. https://doi.org/10.3390/ani11010147 Received: 26 November 2020 Accepted: 8 January 2021 Published: 11 January 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. Article Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens. Animals 2021, 11, 147. https://doi.org/10.3390/ani11010147 Received: 26 November 2020 Accepted: 8 January 2021 Published: 11 January 2021 animals animals animals animals Article Article Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens Insaf Riahi 1,* , Virginie Marquis 2 , Anna Maria Pérez-Vendrell 1 , Joaquim Brufau 1, Enric Esteve-Garcia 1 and Antonio J. Ramos 3 irginie Marquis 2 , Anna Maria Pérez-Vendrell 1 , Joaquim Brufau 1, Enric Esteve-Garcia 1 mos 3 Insaf Riahi 1,* , Virginie and Antonio J. Ramos 3 1 Institute of Agrifood Research and Technology (IRTA Mas Bové), Animal Nutrition Department, 43120 Constanti, Spain; anna.perez@irta.cat (A.M.P.-V.); joaquim.brufau@irta.cat (J.B.); enric.esteve@irta.cat (E.E.-G.) 1 Institute of Agrifood Research and Technology (IRTA Mas Bové), Animal Nutrition Department, 43120 Constanti, Spain; anna.perez@irta.cat (A.M.P.-V.); joaquim.brufau@irta.cat (J.B.); enric.esteve@irta.cat (E.E.-G.) 1 Institute of Agrifood Research and Technology (IRTA Mas Bové), Animal Nutrition Department, 43120 Constanti, Spain; anna.perez@irta.cat (A.M.P.-V.); joaquim.brufau@irta.cat (J.B.); enric.esteve@irta.cat (E.E.-G.) 1 Institute of Agrifood Research and Technology (IRTA Mas Bové), Animal Nutrition Department, 43120 Constanti, Spain; anna.perez@irta.cat (A.M.P.-V.); joaquim.brufau@irta.cat (J.B.); enric.esteve@irta.cat (E.E.-G.) 2 Phileo by Lesaffre, 137 Rue Gabriel Péri, 59700 Marcq en Baroeul, France; v.marquis@phileo.lesaffre.com 3 Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio, Av.Rovira Roure 191, 25198 Lleida, Spain; antonio.ramos@udl.cat 2 Phileo by Lesaffre, 137 Rue Gabriel Péri, 59700 Marcq en Baroeul, France; v.marquis@phileo.lesaffre.com 3 Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio, Av.Rovira Roure 191, 25198 Lleida, Spain; antonio.ramos@udl.cat p * Correspondence: insaf.riahi@irta.cat Simple Summary: Mycotoxin contamination in feed is a signiﬁcant problem worldwide because these toxic metabolites can have serious adverse effects on animals, resulting great economic loss. Deoxynivalenol (DON) is the most commonly detected mycotoxin in cereals and therefore in poultry feed. This mycotoxin could adversely affect the health of birds. In this work, broiler chickens were exposed to two different levels of DON (5 and 15 mg/kg feed) for 42 days. The results revealed that broilers fed the high level (15 mg/kg) had DON-3-sulphate (a speciﬁc metabolite of DON) deposited in the liver and had reduced blood hematological parameters. The ingestion of the guidance level (5 mg DON/kg fee) affected the immune system parameters in broiler chickens. Both levels did not adversely affect the broiler’s welfare related-parameters. Citation: Riahi, I.; Marquis, V.; Pérez-Vendrell, A.M.; Brufau, J.; Esteve-Garcia, E.; Ramos, A.J. Effects of Deoxynivalenol-Contaminated Diets on Metabolic and Immunological Parameters in Broiler Chickens. Animals 2021, 11, 147. https://doi.org/10.3390/ani11010147 Received: 26 November 2020 Accepted: 8 January 2021 Published: 11 January 2021 Citation: Riahi, I.; Marquis, V.; Pérez-Vendrell, A.M.; Brufau, J.; Esteve-Garcia, E.; Ramos, A.J. 1. Introduction Deoxynivalenol (DON) is a secondary toxic metabolite mainly produced by Fusarium species that belongs to the trichothecenes family. DON frequently occurs in cereals, includ- ing wheat, maize, barley, rye and oats [1]. A 10-year survey from 2008 to 2017 of the global mycotoxin occurrence in feed revealed that DON was the most prevalent of mycotoxins and was detected in 64% of 74,821 samples collected from 100 countries [2]. Therefore, DON is considered to be the most frequently found mycotoxin in poultry feed, as chicken diets consist of high levels of cereals. The guidance level of DON in poultry feed is 5 mg/kg [3]. In term of productive parameters, it has been reported in some studies that poultry could tolerate up 15 mg/kg feed [4,5]. This tolerance could be related to the metabolism of DON in this specie [6]. The metabolism of DON or other mycotoxins is deﬁned as the conversion of the native toxin (DON) to various degradation metabolites in the organism and in the digestive tract by microbes [7]. The analysis of the metabolites in the plasma of broiler chickens after a single intravenous injection or oral bolus of the synthetic or labeled DON at the guidance level, revealed that the main metabolite is DON- 3-sulphate (DON-3S) of all metabolites [6,8]. However, the determination of DON-3S in plasma, liver and excreta of broiler chickens fed chronic DON at the guidance level has not been evaluated to date. Knowledge on the metabolites of mycotoxins is essential to test the efﬁcacy of detoxifying agents in vivo afterwards [9]. On the other hand, the immune system is a target of DON mycotoxicosis [10]. As with other trichothecenes, DON can induce either immunostimulation or immunosuppression, depending on the dose and the duration of exposure [11]. A Low to moderate concen- tration induces the up-regulation of cytokines, whereas a high concentration induces the apoptosis of immune cells [12]. However, results are not conclusive regarding the effect of DON on parameters related to the poultry immune system. Furthermore, DON-induced physiological stress has been observed in chickens, but few studies have evaluated the related stress indicators [13,14]. The purposes of this study were to investigate the metabolism of DON in broiler chickens after chronic feeding and to better understand its effects on chicken immune response at both 5 mg/kg and 15 mg/kg levels. 1. Introduction We also tested the hypothesis that DON can affect the physiological stress parameters of birds. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: deoxynivalenol; broiler chickens; deoxynivalenol-3-sulphate; blood hematology; im- mune response Animals 2021, 11, 147. https://doi.org/10.3390/ani11010147 https://www.mdpi.com/journal/animals Animals 2021, 11, 147 2 of 15 2. Materials and Methods 2.1. Ethical Approval All animal care procedures were approved by the Ethical Committee for Animal Experimentation of IRTA, in accordance with current regulations on the use and handling of experimental animals (Decree 214/97, Generalitat de Catalunya, Catalonia, Spain). 2.2. Birds, Diets, and Experimental Design 2.2. Birds, Diets, and Experimental Design Birds, management, the production of DON and contaminated diets, and experimental design for this current study have already been detailed in a recently published paper [15]. Forty-ﬁve 1-day-old male chicks (Ross 308) were randomly allotted to 15 battery cages (0.62 m × 0.62 m × 0.41 m). Birds were vaccinated against infectious bronchitis virus (IBV) at the hatchery and against Newcastle disease virus (NDV) at the farm at start of the trial. At the farm, the lighting program was 24 h of light per day for the ﬁrst two days, 18 h of light per day until 7 days, and 14 h of light per day afterwards. The birds were maintained at 34 ◦C for the ﬁrst two days and the temperature was gradually decreased by 3 ◦C per week until reaching 21 ◦C, and then was maintained. Chickens were fed starter diets from 1 to 21 days and grower diets from 22 to 42 days based on corn, soybean meal, soy oil, and a premix with vitamins, minerals, and amino acids; diets were formulated according the nutrient requirements for Ross 308 strain broilers (Table 1). Feed was provided in mash form ad libitum, in a metal feeder connected to each battery cage. Unlimited access to water was provided from individual nipple drinkers. Three dietary treatments with ﬁve cages per treatment and three birds per cage were used. The treatment 1 received a non- Animals 2021, 11, 147 3 of 15 contaminated diet, and treatments 2 and 3 received DON contaminated diets at 5 mg/kg feed or 15 mg/kg feed, respectively for 42 d. DON used in this trial was produced by inoculating wheat with Fusarium graminearum strain I159 as reported by Metayer et al. [16] (ENVT, Toulouse, France). Table 1. Formulation and proximate analysis of control diet. 2.2. Birds, Diets, and Experimental Design Ingredients (%) Starter: Control 1–21 Days Grower: Control 21–42 Days Maize 54.00 59.49 Soy-meal 48% 36.93 31.02 Soybean oil 4.91 5.73 Monocalcium phosphate 1.42 1.30 Calcium carbonate 1.23 1.13 Sodium chloride 0.19 0.21 Sodium bicarbonate 0.27 0.24 DL-methionine 0.30 0.26 L-Lysine HCl 0.23 0.18 Noxyfeed 0.02 0.02 Premix 1 0.49 0.44 Calculated content (%) Metabolizable energy (Kcal/kg) 3050 3150 Crude protein 22.0 19.5 Ether extract 7.01 7.92 Crude ﬁbre 2.36 2.25 Lysine 1.38 1.18 Methionine + cysteine 0.91 0.87 Threonine 0.81 0.70 Tryptophan 0.21 0.18 Calcium 0.90 0.82 Inorganic phosphorus 0.64 0.59 Sodium 0.16 0.16 1 Vitamin-mineral premix provided following nutrients per kg of diet: vitamin A, 13,500 IU; vitamin D3, 4, 800 IU: vitamin E, 67 IU; vitamin B1: 3 mg; vitamin B2, 9 mg; vitamin B6, 4.5 mg; vitamin B12, 16.5 µg; vitamin K3, 3 mg; calcium pantothenate, 16.5 mg; nicotinic acid, 51 mg; folic acid 1.8 mg, biotin: 30 µg; Fe, 54 mg; I, 1.2 mg; Co, 0.6 mg; Cu, 12 mg; Mn, 90 mg; Zn, 66 mg; Se, 0.18 mg; Mo, 1.2 mg. Table 1. Formulation and proximate analysis of control diet. 1 Vitamin-mineral premix provided following nutrients per kg of diet: vitamin A, 13,500 IU; vitamin D3, 4, 800 IU: vitamin E, 67 IU; vitamin B1: 3 mg; vitamin B2, 9 mg; vitamin B6, 4.5 mg; vitamin B12, 16.5 µg; vitamin K3, 3 mg; calcium pantothenate, 16.5 mg; nicotinic acid, 51 mg; folic acid 1.8 mg, biotin: 30 µg; Fe, 54 mg; I, 1.2 mg; Co, 0.6 mg; Cu, 12 mg; Mn, 90 mg; Zn, 66 mg; Se, 0.18 mg; Mo, 1.2 mg. 2.3. Analysis of Mycotoxins in Experimental Feeds 2.3. Analysis of Mycotoxins in Experimental Feeds The presence of DON and other mycotoxins in the feeds used in the assay was evalu- ated. Regarding DON analysis, 5 g of ground feed from each diet was mixed with 40 mL of distilled water and stirred for 10 min at 600 rpm. Thereafter, the mixture was ﬁltered through Whatman paper no4, and 2 mL as passed through a DONPREP® immunoafﬁnity column (r-Biopharm, Rhone LTD, Glasgow, UK) which was cleaned up with 5 mL of MiliQ water. To eluate DON, 3 mL of HPLC-grade methanol was passed through the column and evaporated to dryness under a gentle stream of nitrogen. Dry extract was reconstituted in 1 mL of HPLC-grade mobile phase and 100 µL was analyzed by HPLC using a Waters (Milford, MA, USA) Module Alliance 2695®, coupled to a UV/Visible dual λ absorbance Detector Waters 2487®. A Waters Spherisorb® 5 µm ODS2, 4.6 × 250 mm column was used. Absorption wavelength was set at 220 nm. The mobile phase was methanol:acetonitrile:water (4:4:92, v/v/v) and was set at a ﬂow rate of 1.2 mL min−1, and the column temperature was set at 40 ◦C. The limit of detection (50 µg/kg) was considered to be three times the signal of the blank. g With regard to Aﬂatoxin B1 (AFB1) analysis, 5 g of ground feed sample from each diet was mixed with 15 mL of 60% methanol and stirred for 10 min at 600 rpm. Thereafter, the mixture was ﬁltered through Whatman paper nº 4, and 2 mL of ﬁltrate were added to 14 mL of phosphate buffered saline (PBS) and mixed well. The whole solution was passed through an Easy-extract® Aﬂatoxin immunoafﬁnity column (r-Biopharm), which was cleaned up Animals 2021, 11, 147 4 of 15 with 20 mL of PBS. To eluate AFB1, 1.5 mL HPLC-grade methanol and 1.5 mL MiliQ water were sequentially passed through the column and 100 µL of the joint eluates was analyzed by HPLC coupled with a ﬂuorescence detector (FLD). The chromatographic equipment and column were the same as those used for DON analysis, but coupled to a Multi λ Fluorescence Detector Waters 2475®. The excitation wavelength was set at 365 nm and the emission wavelength was set at 465 nm. The derivatization of AFB1 was obtained using a post-column photochemical derivatization device (UVE™Derivatizer LC Tech). 2.3. Analysis of Mycotoxins in Experimental Feeds The mobile phase consisted of a solution of water:methanol:acetonitrile (70:17:17) and was set at a ﬂow rate of 1.2 mL min−1, and the column temperature was set at 40 ◦C. The limit of detection (0.3 µg/kg) was considered to be three times the signal of the blank. g g g The detection of zearalenone (ZEN), total fumonisins (FBs) and ochratoxin A (OTA) in feed samples was carried out using the Ridascreen® Zearalenon, Ridascreen® Fumonisin and Ridascreen® Ochratoxin A enzyme-linked-immunosorbent assay (ELISA) kits (R- Biopharm), following the manufacturer’s instructions, with detection limits of 1.75, 25, and 2.5 µg/kg for ZEN, FBs and OTA, respectively. All mycotoxin analyses were carried out by the Applied Mycology Unit of the Food Technology Department of the University of Lleida (Spain). 2.5.2. In Plasma Plasma extract preparation was carried out according to the method of Broekaert et al. [17]. Brieﬂy, 5 µL of 13C15-DON IS solution (at 1 µg/mL and 750 µL of ACN were added to 250 µL of chicken plasma. ACN was added to precipitate plasma proteins. The samples were vortexed approximately for 1 min. Afterwards, the samples were centrifuged at 8517× g for 10 min. The supernatant (1 mL) was transferred to a new tube, evaporated to dryness under nitrogen ﬂow over a heating block and reconstituted in 1 mL of ammonium formate 5 mM/MeOH (50:50, v/v) solution, and 10 µL was injected for HPLC-MS/MS analysis. 2.5.3. In Liver and Excreta Samples of lyophilized liver or excreta were weighed (1 g) in 50 mL centrifugation tubes. In total, 5 µL of 1 µg/mL of IS working solution (13C15-DON) and 10 mL of ACN: water: acetic acid (79:20:1, v/v/v) were added. Samples were vortex mixed for 2 min. Four grams of sodium sulphate and 1.5 g of sodium acetate were added and then each tube was vortex mixed for 5 min and then with an orbital shaker for 20 min (IKA™KS 260, Fisher Scientiﬁc, Madrid, Spain). After extraction, a centrifugation was made at 2716× g for 10 min, and supernatants were transferred to another tube and 5 mL of hexane was added and vortexed. After the separation of two phases, the hexane phase was removed. A 5 mL aliquot of the extract was evaporated to dryness under nitrogen ﬂow over a heating block and reconstituted in 0.5 mL of water/ammonium formate (5 mM):MeOH (50:50, v/v) and then ﬁltrated through nylon syringe ﬁlters (0.20 µm) from Agilent (Santa Clara, CA, USA) and injected directly in HPLC-MS/MS. 2.4. Sampling and Analysis Chemicals, Products and Reagents The DON analytical standard was supplied by Sigma-Aldrich Chemie GmbH (Stein- heim, Germany). The DON standard was dissolved in acetonitrile (ACN) as stock solution (1 mg/mL), and then diluted with HPLC-grade ACN to obtain an individual working standard solution of 1 µg/mL. The stable internal standard (IS) isotope (13C15-DON) was obtained from Romer labs (Bioser, Barcelona, Spain) as 1.2 mL of a solution of 25 µg/mL in ACN. An individual working standard solution of 5 µg/mL was prepared by dilut- ing the above stock solution with HPLC-grade ACN and was stored at −15 ◦C. Acetic acid (LC-MS gradient grade) was purchased from Montplet & Esteban SA (Barcelona, Spain). Sodium sulfate, sodium acetate and hexane were from PanReac Quimica SLU (Barcelona, Spain). Ammonium formate was from Sigma-Aldrich Chemie GmbH (Stein- heim, Germany). Methanol (HPLC gradient grade, MeOH) was purchased from Honyewell (Seelze, Germany). 2.4. Sampling and Analysis At 42 days of age, blood samples (3 mL/bird, 3 birds/pen) were collected by car- diac puncture in non-heparinized tubes for the hematological and serological analysis. Blood samples (3 mL/bird, 3 birds/pen) were collected also into heparinized tubes for in- terleukin 8 (IL-8), corticosterone, DON and DON-3S determination (Table 2). Blood serum of each bird was separated by centrifugation at 4500× g for 10 min. Plasma was separated by centrifugation at 1000× g for 15 min for IL-8 and corticosterone determination and at 2851× g for 10 min for DON and DON-3S determination. Samples were stored at −20 ◦C until further analysis. Mortality rates were 13%, 20% and 13% for the control treatment, and treatments 2 and 3, respectively. Twelve birds in each treatment were idividually weighed and humanely euthanized according to IRTA ethics instructions at 42 days. Immediately, the entire intestine was carefully removed and the distal part of the jejunum (5 cm taken from Meckel’s diverticulum) was collected for each bird, rinsed in PBS, and subsequently stored in RNAlater (Vidra Foc, Barcelona, Spain) for 24 h at ambient temperature. Then, samples were stored at −80 ◦C without RNAlater until quantitative real-time PCR (qRT- PCR) analysis. Liver samples were excised, weighed, and stored at −20 ◦C until lyophiliza- tion. Fresh excreta samples from each cage were collected daily (day 1 to day 42) and stored at −20◦C until lyophilization. One cage sample was a pool of excreta of three birds. Lyophilized liver and excreta samples were stored at darkness at ambient temperature until further analysis. Table 2. Collection of biological sample. Sample Analysis 1 Blood Hematology Serum Response to common vaccines Plasma IL-8 Corticosterone DON and DON-3S Small intestine (Jejunum) IL-6, IL-1β, IL-10, IFN-γ Liver DON and DON-3S Excreta DON and DON-3S 1 IL-8,interleukin 8; DON, deoxynivalenol; DON-3S, deoxynivalenol 3-sulphate; IL-6, interleukin 6; IL-1β interleukin- 1β, IL-10, interleukin 10; IFN-γ; interferon gamma. Table 2. Collection of biological sample. Hematology Response to common vaccines IL-8 Corticosterone DON and DON-3S IL-6, IL-1β, IL-10, IFN-γ DON and DON-3S DON and DON-3S 1 IL-8,interleukin 8; DON, deoxynivalenol; DON-3S, deoxynivalenol 3-sulphate; IL-6, interleukin 6; IL-1β interleukin- 1β, IL-10, interleukin 10; IFN-γ; interferon gamma. Animals 2021, 11, 147 5 of 15 2.5. DON and DON-3S Determination in Different Biological Matrices (Plasma, Liver, and Excreta) 2.5.1. Chemicals, Products and Reagents 2.5. DON and DON-3S Determination in Different Biological Matrices (Plasma, Liver, and Excreta) 2.5.1. 2.7. Response to Common Vaccines (NDV and IBV) Antibody titers against NDV or IBV were determined by the hemagglutination inhibi- tion (HI) test using standard protocols by the World Organisation for animal health (OIE). Serial twofold serum dilutions were made in PBS and 0.025 mL was added to the wells. Four hemagglutination (HA) units of the test antigen were added to each dilution and incubated at room temperature for 30 min. An equal volume of 1% chicken red blood cells (RBCs) in PBS was then added to the wells until agglutination occurred in the negative control sample. All plates included NDV or IBV-negative and NDV or IBV-positive control sera. The highest dilution of serum displaying the inhibition of agglutination was desig- nated as the reciprocal log2 HI titer for that serum sample. Moreover, IBV was determined in serum using an ELISA test kit (Idexx®, Westbrook, ME, USA) according to the protocols speciﬁed by the supplier. Brieﬂy, 96-well plates were coated with viral antigen; after the incubation of the test sample (100 µL) in the coated well, an IBV-speciﬁc antibody formed a complex with the coated viral antigens. After washing away unbound material from the wells, a conjugate (100 µL) was added which bound to any attached chicken antibody in the wells. Unbound conjugate was washed away and 100 µL of substrate solution (TMB) was added to the wells and incubated for 15 min at ambient temperature. Subsequent color development was directly related to the amount of IBV antibody present in the test sample. The color development was stopped with a stop solution (100 µL) and the absorbance values were measured and recorded at 650 nm. Then, the amount of antibody to IBV present in the test sample was calculated. 2.5.4. LC-MS/MS Analysis The HPLC-MS/MS analysis was carried out with a Transcend 600 LC (Thermo Scien- tiﬁc TranscendTM, Thermo Fisher Scientiﬁc, San Jose, CA, USA) coupled to an Orbitrap (ExactiveTM, Thermo Fisher Scientiﬁc, Bremen, Germany) with an electrospray ionization (ESI) source (HESI-II, Thermo Fisher Scientiﬁc, San Jose, CA, USA). Chromatographic separation was achieved using a Zorbax Plus C18 (1.8 µm × 2.1 × 100 mm) column from Agilent (San Jose, CA, USA). Gradient elution was established with a mobile phase con- sisting of 5 mM ammonium formate in water (eluent A) and methanol (eluent B) at a ﬂow rate of 0.2 mL/min. The gradient elution started at 95% B at 1 min and was decreased to 0% B at 8 to 12 min afterwards; it increased to 95% at 12.5 min, which was maintained up to 14 min. The column temperature was set at 25 ◦C and the injection volume was 10 µL. MS analyses were performed using a selected reaction monitoring (SRM) mode with positive and negative electrospray ionization (ESI±). The settings on the spectrometer were as follows: compounds were ionized by electrospray ionization in the positive and negative mode, measured ﬁrst in full scan, and then in targeted MS/MS mode at a collision Animals 2021, 11, 147 6 of 15 energy of 30 eV (both in the range from m/z 50–500). ESI parameters were as follows: spray voltage, 4 kV; sheath gas (N2, >95%), 35 (adimensional); auxiliary gas (N2, >95%), 10 (adimensional); skimmer voltage, 18 V; capillary voltage, 35 V; tube lens voltage, 95 V; heater temperature, 305 ◦C; capillary temperature, 300 ◦C. The capillary and nozzle voltage were 4000 V and 95 V (−95 V in ESI-), respectively. Finally, the data were processed using XcaliburTM version 3.0 (Quanbrowser and Qualbrowser) and Mass FrontierTM 7.0. 2.6. Blood Hematology Hemoglobin (HGB, g/dL) and erythrocytes (Red Blood Cells, RBC/µL) were mea- sured using a CELL-DYN 3700 hematology analyzer (Abbott, Chicago, IL, USA). A blood sample was collected in microcentrifuge (Haematokrit 200, Helltich Zentrifugen, Tuttlingen, Germany) capillary tubes for hematocrit (HCT, %) determination, which was performed in a Neubauer chamber (Brand, Germany). The mean corpuscular volume (MCV, fL) and mean corpuscular hemoglobin (MCH, pg) were determined by the hematologic analyzer CELL- DYN 3700 (Abbott, Chicago, IL, USA), and mean corpuscular hemoglobin concentrations (MCHC, g/dL) were calculated as MCHC = hemoglobin/hematocrit. Leukocytes per µL and the differential leukocyte count (heterophils, lymphocytes, monocytes, eosinophils and basophils, %) were also measured using a hematologic analyzer CELL-DYN 3700 (Abbott, Chicago, IL, USA). 2.8. Plasma IL-8 Determination IL-8 was determined in plasma using a commercially available ELISA kit (chicken IL-8 ELISA kit) according to the manufacturer’s instructions (MyBioSource, San Diego, CA, USA). Brieﬂy, 96-well plates were pre-coated with anti-IL-8 antibody, and the biotin conjugated anti-IL-8 antibody was used for the detection of antibodies. The standards, test samples, and biotin-conjugated detection antibody (50 µL each) were subsequently added to the wells and washed with wash buffer. Streptavidin–horseradish peroxidase enzyme (HRP) (50 µL) was added and unbound conjugates were washed away with wash buffer. TMB (3, 3′, 5, 5′ tetramethylbenzidine) substrates were used to visualize the HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue-color product that changed to yellow after adding an acidic stop solution. As the density of yellow was proportional to the IL-8 amount of the sample captured on the plate, the optical density absorbance at Animals 2021, 11, 147 7 of 15 450 nm in a microplate reader (Anthos, Labtec instruments, Salzburg, Austria) was read, and the concentration of IL-8 was calculated. 2.10.1. Stress Index (Heterophil to Lymphocyte Ratio) The heterophil to lymphocyte ratio (H/L), considered as an indicator of stress, was calculated by dividing the number of heterophils by the number of lymphocytes [13,20]. 2.10.2. Plasma Corticosterone Determination 2.10.2. Plasma Corticosterone Determination The plasma level of corticosterone was measured with a commercially available ELISA kit (chicken corticosterone ELISA kit) according to the manufacturer’s instructions (Cusabio, Houston, TX, USA). Brieﬂy, the microtiter plate provided in this kit was pre- coated with an antigen. Standards, samples, antibody speciﬁc for corticosterone (CORT) (50 µL each), and HRP-conjugate (100 µL) were added to the appropriate microtiter plate wells. The competitive inhibition reaction was launched between pre-coated CORT and CORT in samples. Then, 100 µL of substrate solution (TMB) was added to the wells, incubated for 15 min at 25 ◦C, and protected from light. The color development was stopped with an acid solution (50 µL) and the intensity of the developed yellow color was measured. A microplate reader (SkanIt, Thermo Fisher Scientiﬁc, Madrid, Spain) capable of measuring absorbance at 450 nm was used. 2.11. Statistical Analysis Statistical analysis was performed by SAS software (SAS 9.4, SAS Institute, Cary, NC, USA). After the determination of normality and variance homogeneity, data were evaluated as a completely randomized design by a one-way analysis of variance (ANOVA) using the General Linear Model Procedure to test the effect of different treatments. Response to common vaccine parameters were logarithmically transferred to maintain the homogeneity of variance. Each cage was considered to be an experimental unit. Results were shown as means ± standard error of the means (SEM). Orthogonal polynomials were used to determine linear and quadratic dose responses. To test the normal distribution of data, a Kol–Mogrov–Smirnov test was used. The signiﬁcance level was set at p ≤0.05. A trend was deﬁned as p-value between 0.05 and 0.10 (0.05 < p ≤0.10). 2.10. Physiological Stress Related-Parametrs 2.10. Physiological Stress Related-Parametrs 2.10.1. Stress Index (Heterophil to Lymphocyte Ratio) 2.9. Gene Expression by Quantitative Real-Time PCR (qRT-PCR) Total RNA from the tissue samples of the distal jejunum (20 mg) was extracted using an RNeasy mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted into 50 µL of Rnase free water and stored at −80 ◦C. The yield of RNA was determined by spectrophotometry (BioPhotometer, Eppendorf, Hamburg, Germany). Cytokine gene expression (IL-6, IL-1β, IFN-γ and IL-10) were evaluated as previously described by Reid et al. [18]. The mRNA quantiﬁcation of cytokines was determined by qRT-PCR using QuantiTect™SYBR® Green one-step RT-PCR Kit (QIAGEN, Hilden, Germany). The PCR ampliﬁcation was performed using 7500-Fast Real-time PCR (Applied Biosystems, CA, USA) [18].The threshold cycle values (Ct) were t normalized to the reference gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)). The average ∆Ct of the control samples was used to calculate the target gene expression according to the 2−∆∆Ct method [19]. This relative quantiﬁcation related the PCR signal of the target transcript gene in a treatment group to the average signal of untreated control. Duplicate samples were used. 3.1. Dietary Mycotoxin Concentrations The DON levels found in control diets were 65 and 73 µg/kg in the starter and grower diets respectively. The concentrations of DON in contaminated feeds were close to 5 and Animals 2021, 11, 147 8 of 15 15 mg/kg, as expected. Diets also included lesser amounts of ZEN, FBs and OTA. AFB1 was not detected in all experimental diets (Table 3). 15 mg/kg, as expected. Diets also included lesser amounts of ZEN, FBs and OTA. AFB1 was not detected in all experimental diets (Table 3). 15 mg/kg, as expected. Diets also included lesser amounts of ZEN, FBs and OTA. AFB1 was not detected in all experimental diets (Table 3). Table 3. Mycotoxin analysis of experimental feeds. Mycotoxin 1 (µg/kg) Control Group DON Group (5000 µg/kg) DON Group (15,000 µg/kg) Starter Grower Starter Grower Starter Grower DON 65 73 4760 4650 14,390 15,120 ZEN <LOD <LOD 84.4 85.9 242 259 FBs 142 225 257 216 216 275 OTA 0.94 1.59 0.90 1.11 1.21 1.10 AFB1 <LOD <LOD <LOD <LOD <LOD <LOD 1 DON = deoxynivalenol; ZEN = zearalenone; FBs = fumonisins; OTA = ochratoxin; AFB1 = aﬂatoxin B1; limit of detection (LOD) of DON, ZEN, FBs, OTA, and AFB1: 50, 1.75, 25, 0.5, 0.3 µg/kg, respectively. Table 3. Mycotoxin analysis of experimental feeds. Table 3. Mycotoxin analysis of experimental feeds. 3.2. DON and DON-3S Determination in Plasma, Liver, and Excreta After a chronic DON feeding of broilers at a low (5 mg/kg) or high level (15 mg/kg) for 42 days, DON and DON-3S were analyzed in plasma, liver and excreta. Results showed that, in the non-contaminated control treatment, DON was below the limit of quantiﬁcation (LOQ) (5 ng/mL) and DON-3S could not be identiﬁed. Similarly, DON was below the LOQ (5 ng/mL) in plasma and liver in broilers fed a contaminated diet at both levels but was detected only in excreta. DON-3S was detected in plasma and excreta at both levels and was signiﬁcantly lower in the DON low dosage group. Interestingly, DON-3S was also detected in liver but only at the highest level assayed (Table 4). Table 4. Average concentrations of DON and DON-3S (peak area) in plasma, liver and excreta of broilers fed low DON level (5 mg/kg feed) and high DON level (15 mg/kg feed). Table 4. 3.1. Dietary Mycotoxin Concentrations Average concentrations of DON and DON-3S (peak area) in plasma, liver and excreta of broilers fed low DON level (5 mg/kg feed) and high DON level (15 mg/kg feed). Table 4. Average concentrations of DON and DON-3S (peak area) in plasma, liver and excreta of broilers fed low DON level (5 mg/kg feed) and high DON level (15 mg/kg feed). Dietary Treatment/Biological Matrix DON 1 DON-3S 2 (×106) Plasma (ng/mL) Control ND 3 ND DON low level (5 mg/kg) ND 0.27 ± 0.01 b DON high level (15 mg/kg) ND 0.62 ± 0.15 a SEM - 0.11 p-Value - 0.01 Liver (ng/g) Control ND ND DON low level (5 mg/kg) ND ND DON high level (15 mg/kg) ND 0.70 ± 0.37 Excreta (ng/g) Control ND ND DON low level (5 mg/kg) 22.0 110 b DON high level (15 mg/kg) 24.1 295 a SEM 11.9 22.2 p-Value 0.81 0.0001 1 DON, deoxynivalenol; 2 DON-3S, deoxynivalenol 3-sulphate; a,b within the same column, different superscripts are signiﬁcantly different (p value < 0.05);3 ND = not detectable (limit of detection (LOD) = 1.5 ng/mL. 3.3. Hematological Indices Response to Common Vaccines (NDV and IBV) No signiﬁcant effects of dieatray treatments were observed on titers against NDV and IBV in broilers at 42 days (p > 0.05) (Table 6) Table 6. Effects of DON-contaminated feed (5 and 15 mg/kg) on antibody titers against NDV and IBV in broiler chickens. Dietary Treatment 1 Item 2 Control DON (5 mg/kg) DON (15 mg/kg) SEM p-Value Linear Quadratic Titers against NDV (HA) 0.38 0.30 0.25 0.15 0.82 0.56 0.84 Titers against IBV (HA) 2.92 3.20 3.00 0.30 0.81 0.95 0.52 Titers against IBV (ELISA) 874 851 786 120 0.31 0.13 0.76 1 DON, deoxynivalenol; SEM, standard error of mean (n = 5); 2 NDV, Newcastle Disease Virus; HA, haemagglutination inhibition; IBV, infectious bronchitis virus. of DON-contaminated feed (5 and 15 mg/kg) on antibody titers against NDV and IBV in broiler chickens. Table 6. Effects of DON-contaminated feed (5 and 15 mg/kg) on antibody titers against NDV and 3.5. Plasma IL-8 Production and Realtive mRNA Expression of Immune Genes In plasma, IL-8 was signiﬁcantly up-regulated in all broiler chickens receiving DON (5 and 15 mg/kg) compared to the control group (p = 0.001) (Figure 1). DON feeding at 5 mg/kg signiﬁcantly stimulated the mRNA relative expression of IL-6, IFN-γ, and IL-1β in the jejunal tissues of broiler chickens (p < 0.05). However, the mRNA expression of these genes was comparable to the control group when the diet was contaminated with 15 mg/kg feed (Figure 2). 3.3. Hematological Indices The results of blood hematology paramters are reported in (Table 5). DON contami- nated feed decreased the HGB level in a dose dependent mannner (p = 0.0002). Furthermore, the presence of DON at 5 mg/kg did not affect RBC, MCV, and MCHC (p > 0.05). However, 15 mg/kg DON in broiler diets signiﬁcantly reduced RBC and MCHC and increased MCV Animals 2021, 11, 147 9 of 15 blood level (p < 0.05). Leukogram data were not affected by the different dietary treatments (p > 0.05). le 5. Effects of DON-contaminated feed (5 and 15 mg/kg) on hematological parameters of broiler chickens. Table 5. Effects of DON-contaminated feed (5 and 15 mg/kg) on hematological parameters of broiler chickens. Dietary Treatment 1 Item 2 Control DON (5 mg/kg) DON (15 mg/kg) SEM p-Value Linear Quadratic HCT (%) 31.9 30.1 30.2 0.74 0.16 0.15 0.20 HGB (g/dL) 12.1 a 11.1 b 10.1 c 0.29 0.0002 <0.0001 0.54 RBC (×106/µL) 2.3 a 2.3 a 1.9 b 0.38 <0.0001 <0.0001 0.06 MCV (fL) 132 b 133 b 151 a 4.26 0.004 0.001 0.29 MCH (pg) 50.1 49.4 50.5 0.41 0.14 0.22 0.12 MCHC(g/dL) 37.8 a 37.3 a 34.1 b 0.93 0.004 0.001 0.50 Leukocyte (×103/µL) 15.8 18.3 19.2 1.85 0.37 0.20 0.55 Eosinophil (%) 6.00 4.66 7.07 0.93 0.33 0.27 0.32 Basophil (%) 7.84 7.08 6.61 1.32 0.51 0.26 0.76 Lymphocyte (%) 41.2 37.7 37.1 2.81 0.52 0.33 0.55 Monocyte (%) 2.15 2.75 0.92 0.70 0.19 0.17 0.21 Total heterophils (%) 44.5 48.7 48.3 3.17 0.64 0.43 0.61 1 DON, deoxynivalenol; SEM, standard error of mean (n = 5); a,b,c: means values with different superscripts with the same row differ (p ≤ 0.05); 2 HCT, hematocrit; RBC, red blood cell; HGB, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. 1 DON, deoxynivalenol; SEM, standard error of mean (n = 5); a,b,c: means values with different superscripts with the same row differ (p ≤ 0.05); 2 HCT, hematocrit; RBC, red blood cell; HGB, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration. 3.4. Response to Common Vaccines (NDV and IBV) No signiﬁcant effects of dieatray treatments were observed on titers against NDV and IBV in broilers at 42 days (p > 0.05) (Table 6) 3.4. 3.6. Physiological Stress Parameters The effects of experimental treatments on welfare-related indicators are presented in Table 7. No signiﬁcant differences among the diet groups were detected for the stress index (H/L ratio). However, birds fed DON at both levels (5 and 15 mg/kg) showed lower plasma corticosterone level than birds fed a control diet (p = 0.03). 10 of 15 Animals 2021, 11, 147 Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with dif- ferent superscripts for each cytokine differ (p ≤ 0.05). b a a 0 20 40 60 80 100 120 control 5 mg/kg 15 mg/kg plasma IL-8 (pg/mL) Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with different superscripts for each cytokine differ (p ≤ 0.05). mg/kg feed (Figure 2). Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with dif- ferent superscripts for each cytokine differ (p ≤0.05). b a a 0 20 40 60 80 100 120 control 5 mg/kg 15 mg/kg plasma IL-8 (pg/mL) Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with dif- ferent superscripts for each cytokine differ (p ≤ 0.05). Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with different superscripts for each cytokine differ (p ≤ 0.05). Figure 1. Effects of DON-contaminated feed (5 and 15 mg/kg) on plasma IL-8 levels in broiler chickens. Bars show the means and the standard error of mean (SEM) (n = 5); a,b,: values with dif- ferent superscripts for each cytokine differ (p ≤0 05) Figure 2. Effects of DON-contaminated feed (5 and 15 mg/kg) on the relative mRNA expression of immune genes (IL-6, IL-10, IFN-ᵞ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. 3.6. Physiological Stress Parameters Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤ 0.05). 3 6 Physiological Stress Parameters b ab b b a a a a b b b ab −0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 IL-6 IL-10 IFN-ᵞ IL-1β Fold change control DON (5mg/kg) DON (15mg/kg) Figure 2. Effects of DON-contaminated feed (5 and 15 mg/kg) on the relative mRNA expression of immune genes (IL-6, IL-10, IFN-ᵞ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤ 0.05). b ab b b a a a a b b b ab −0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 IL-6 IL-10 IFN-ᵞ IL-1β Fold change control DON (5mg/kg) DON (15mg/kg) Figure 2. Effects of DON-contaminated feed (5 and 15 mg/kg) on the relative mRNA expression of immune genes (IL-6, IL-10, IFN-γ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤0.05). Fi 2 Eff f DON i d f d (5 d 15 /k ) h l i RNA i f b ab b b a a a a b b b ab −0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 IL-6 IL-10 IFN-ᵞ IL-1β Fold change control DON (5mg/kg) DON (15mg/kg) b ab b b a a a a b b b ab −0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 IL-6 IL-10 IFN-ᵞ IL-1β Fold change control DON (5mg/kg) DON (15mg/kg) immune genes (IL-6, IL-10, IFN-ᵞ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤ 0.05). h l l Figure 2. Effects of DON-contaminated feed (5 and 15 mg/kg) on the relative mRNA expression of immune genes (IL-6, IL-10, IFN-ᵞ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. 4. Discussion In practice, it is almost impossible to ﬁnd feed that does not have a basal DON contamination, given the frequent contamination of cereals with this mycotoxin. Thus, the dietary concentration of DON was 65 and 73 µg/kg in the starter and grower control feed, respectively. This level was too low to cause adverse effects in broiler chickens [3]. Dietary ZEN concentrations ranged between below the LOQ and 259 µg/kg and increased as DON increased in the diet. DON and ZEN can be found alone or together [21]. According to literature reports, the content of ZEN in the present study was not enough to produce synergism with the amounts of DON that negatively affect the health of broiler chickens [22]. Besides, FBs and OTA levels were below the guidance value established for poultry feed (20 and 0.1 mg/kg for FBs and OTA, respectively) [3]. Furthermore, the levels of AFB1 were below the limit of detection in all feeds. Therefore, the effects observed in the evaluated indicators in the current study were not attributable to AFB1, ZEN, FBs, and OTA, and thus, only DON was responsible for these effects. y p Although other authors have stated that the concentrations of DON chosen for this study should not have major effects on performance [4,5], our results revealed that 15 mg/kg reduced body weight gain and altered the feed conversion ratio of broiler chickens at 42 days [15]. Regarding the pathway of metabolization, DON was not detected in the plasma of birds even at 15 mg/kg feed. This result could be explained by the low absorption of this mycotoxin into plasma. The absolute oral bioavailability at the guidance value in chickens fed orally is poor, amounting to only 19.3% [23]. Similarly, DON was not detected in the liver. This result could be related to its rapid metabolism and excretion. Other researchers explain the lack of quantiﬁcation of DON in plasma and liver by the protective effect of the hepatic/renal ﬁrst pass effect. In fact, poultry are characterized by the protective hepatic/renal ﬁrst pass effect. This effect is controlled by gut and liver enzymes, which induce the oxidation, reduction or hydrolysis (phase I reactions), and/or conjugation (phase II reactions) of toxins [24]. However, our results revealed that DON could be quantiﬁed in excreta of birds fed DON-contaminated diets. 3.6. Physiological Stress Parameters Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤ 0.05). Figure 2. Effects of DON-contaminated feed (5 and 15 mg/kg) on the relative mRNA expression of immune genes (IL-6, IL-10, IFN-γ, and IL-1β) in the jejunal tissues of broiler chickens determined by qRT-PCR. Bars show the means and the standard error of means (SEM) (n = 5); a,b: values with different superscripts for each cytokine differ (p ≤0.05). 3.6. Physiological Stress Parameters The effects of experimental treatments on welfare-related indicators are presented in Table 7. No significant differences among the diet groups were detected for the stress in- dex (H/L ratio). However, birds fed DON at both levels (5 and 15 mg/kg) showed lower plasma corticosterone level than birds fed a control diet (p = 0.03). 3.6. Physiological Stress Parameters The effects of experimental treatments on welfare-related indicators are presented in Table 7. No significant differences among the diet groups were detected for the stress in- dex (H/L ratio). However, birds fed DON at both levels (5 and 15 mg/kg) showed lower plasma corticosterone level than birds fed a control diet (p = 0.03). Table 7. Effects of DON-contaminated feed (5 and 15 mg/kg) on the H/L ratio and plasma corticosterone level of broiler chickens. Dietary Treatment 1 Item 2 Control DON (5 mg/kg) DON (15 mg/kg) SEM p-Value Linear Quadratic H/L ratio 1.1 1.3 1.3 0.15 0.47 0.30 0.50 Plasma corticosterone (ng/mL) 2.93 a 2.34 b 2.44 b 0.15 0.03 0.07 0.04 1 DON, deoxynivalenol; SEM, standard error of mean (n = 5); a,b: values with different superscripts with the same row differ (p ≤0.05); 2 H/L ratio, heterophil to lymphocyte ratio. ysio ogi a S ess a a e e s The effects of experimental treatments on welfare-related indicators are presented in Table 7. No significant differences among the diet groups were detected for the stress in- 3.6. Physiological Stress Parameters The effects of experimental treatments on welfare-related indicators are presented in Table 7. Effects of DON-contaminated feed (5 and 15 mg/kg) on the H/L ratio and plasma corticosterone level of broiler chickens. 1 DON, deoxynivalenol; SEM, standard error of mean (n = 5); a,b: values with different superscripts with the same row differ (p ≤0.05); 2 H/L ratio, heterophil to lymphocyte ratio. 3.6. Physiological Stress Parameters 11 of 15 11 of 15 Animals 2021, 11, 147 4. Discussion In fact, bone marrow is an immune organ that is susceptible to DON mycotoxin because cells rapidly divide in this organ. On the other hand, the values of hematological parameters observed were still within the range of reference values, and no anemia was induced [31]. The feeding of chickens with contaminated diets containing 9 or 18 mg of DON/kg of feed signiﬁcantly decreased the hemoglobin concentration and the RBC count [32,33]. Moreover, it has been reported that DON could affect the humoral immune response by reducing the antibody titers against NDV and IBV [13,34]. However, in the current research, neither 5 nor 15 mg/kg of DON in broilers feed affected the vaccinal immune response after regular vaccination with NDV and IBV in broilers (p > 0.05). Yegani et al. [35] observed no effect of feeding broiler breeder hens with grains naturally contaminated with Fusarium mycotoxins based on DON (12.6 mg/kg) for 12 weeks on antibody titers against NDV. This result was also in agreement with those of Harvey et al. [33], who reported no effect on NDV antibody titers in White Leghorn chicks fed 18 mg of DON/kg of feed for 9 weeks. Regarding the antibody titers against IBV, Swamy et al. [22] found no effect on IBV titers with different concentrations of DON (4.7 and 8.3 mg/kg of feed) in broiler chickens exposed for 21 days and 42 days. Similar ﬁndings were reported by Yegani et al. [35], after feeding broiler breeder hens a concentration of 12.6 mg DON/kg for 28 and 56 days. Furthermore, no effects were found with a concentration of 12.2 mg DON/kg on antibodies against IBV titers in broilers chickens after 14 and 28 days [36]. The failure to observe signiﬁcant results in response to common vaccines and the variability between literature reports suggests that those parameters could not be a relevant biomarker for DON toxicity in poultry. p y p y IL-8 is a proinﬂammatory cytokine involved in pathogen defense and immune regula- tion, and it is considered as an early biomarker of the inﬂammation process [37]. In this study, DON presence in chicken feed at 5 and 15 mg/kg resulted in an increase of IL-8 production in plasma, suggesting that DON could have an effect on the innate immune response and inﬂammation process. 4. Discussion The detection of DON in excreta may be attributed to the rapid clearance of this toxin into excreta. The mentioned ﬁndings were previously also described when broilers were fed DON at 5 mg/kg by Awad et al. [25]. p y g g y Toxicokinetic studies performed in chickens (after single intravenous or oral bolus in- jection of labeled or synthetic DON) have demonstrated that DON-3S is the most abundant metabolite in plasma and excreta [6,8,26,27]. In addition, only one chronic study evaluated DON-3S in excreta of broiler chickens after chronic feeding of DON at a lower concentra- tion than the maximum recommended (1.7 mg/kg) [28]. In the current study, DON-3S was detected in plasma and excreta of broilers exposed to recommended levels and at 15 mg/kg, suggesting the extensive metabolization of DON to DON-3S in broiler chickens. Interestingly, DON-3S was detected in liver, but only in broilers exposed to the highest concentration tested. This result could be associated to the bird’s cholesterol metabolism, as the blood cholesterol level was signiﬁcantly affected in birds fed 15 mg DON/kg [15]. This result could correlate with the adverse effect of 15 mg DON/kg on performance found in a recent research work from our group [15], and may conﬁrm that, if feed contamination is lower (5 mg/kg), birds continue to have sufﬁcient capacity for excretion and a limited deposition into the liver. In conclusion, DON-3S is a suitable biomarker for DON exposure in broilers; this biotransformation could be considered as a detoxiﬁcation pathway [29] and may explain the low susceptibility of broilers to DON at guidance levels [26]. It has been suggested that the impairment of the immune system is the most important outcome of DON toxicity [10]. Consequently, it was expected that DON might affect blood hematological parameters, response to common vaccines (NDV and IBV), and cytokine production. In fact, the results of the current study showed that DON at higher levels (15 mg/kg) slightly more prominently affected hematological indices than 5 mg/kg feed. A signiﬁcant dose-dependent decrease of hemoglobin concentration and a signiﬁcant effect of a dietary dose of 15 mg DON/kg feed on RBC values, MCV, and MCHC were Animals 2021, 11, 147 12 of 15 12 of 15 observed. The loss of HGB concentration and RBC count induced by DON mycotoxicosis could be a marker of bone marrow malfunction [30]. 4. Discussion The up-regulation of IL-8 has been shown in in vitro and in vivo studies in humans, rodents and farm animals [38,39]. To our knowledge, the up-regulation observed of the plasma IL-8 of broiler chickens after DON exposure is new information. The duration of exposure of DON is an important variable, as with a shorter duration (35 days), no signiﬁcant differences between control and contaminated feeding groups were observed in a previous feeding trial with broiler chickens [40]. In the actual study, the effects of DON on the gene expression of cytokines in jejunum tissues of broilers were evaluated as markers of the intestinal immune system. The addition of 5 mg DON/kg feed signiﬁcantly up-regulated the mRNA expression of the proinﬂam- matory cytokines IL-6, IFN-γ, and IL-1β, suggesting that DON at the guidance level is immunostimulatory in broilers aged 42 days. These results are in agreement with previous results indicating that DON exposure from 2 to 5 mg/kg up-regulated the proinﬂammatory cytokines in broiler chickens, such as IL-6 in jejunum, IL6 and IL-1B in spleen, and IFN-γ in cecal tonsils [41–43]. The up-regulation of the mRNA gene expression of cytokines is due to the ability of DON, as a protein-synthesis inhibitor, to impair the synthesis of high turnover proteins and, as a result, to induce a transient expression of speciﬁc mRNAs [44]. In a similar manner, it has been suggested that this induction of immune-related genes by DON mycotoxin is due to the increasing of the binding activity of transcription factors in leukocytes such as the nuclear factor κB (NF-κB) at the transcription level, and to the increasing stability of the mRNA at the post-transcription level [11,42]. However, mRNA expression of IL-6, IFN-γ, and IL-1β was comparable to control group when broilers fed 15 mg/kg, suggesting that this dose did not induce immune-suppression in broilers aged 42 days. The mRNA level of the anti-inﬂammatory IL-10 was not statistically affected, suggesting that DON did not affect anti-inﬂammatory cytokines, as previously reported in some studies [42,43]. IL-10 is involved in the cross-regulation of IFN-γ gene expression [45]. The up-regulation of IFN-γ gene expression, therefore, may not be necessary to correlate the changes in IL-10 gene expression [42]. 4. Discussion Animals 2021, 11, 147 13 of 15 13 of 15 The physiological stress includes the elevation of the stress index (H/L ratio), deﬁned as a result of the impairment of the number of circulating heterophils and lymphocytes, and an elevation of the circulating levels of corticosterone [46], although the effect of DON on the H/L ratio and plasma corticosterone level in poultry was not extensively documented [14,47]. As no signiﬁcant effect was observed on blood heterophils and lymphocytes, therefore, dietary DON did not affect the stress index H/L ratio. Similarly, Danicke et al. [34] did not ﬁnd signiﬁcant differences in the H/L ratio of broilers fed a contaminated diet with a concentration of 14 mg/kg of DON per kg. Corticosterone is the primary glucocorticoid secreted by the adrenal glands in birds and is involved in immune reactions and stress responses [48]. 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[Cr mycotoxins induces a corticosterone stress response in broiler chickens. Poult. Sci. 2017, 96, 14–17. [CrossRef] [PubMed] 15. Riahi, I.; Marquis, V.; Ramos, A.J.; Brufau, J.; Esteve-Garcia, E.; Pérez-Vendrell, A.M. Effects of deoxynivalenol contaminated-diets on productive morphological and physiological indicators in broiler chickens Animals 2020 10 1795 [CrossRef] [PubMed] 15. Riahi, I.; Marquis, V.; Ramos, A.J.; Brufau, J.; Esteve-Garcia, E.; Pérez-Vendrell, A.M. Effects of deoxynivalenol contaminated-diets on productive, morphological, and physiological indicators in broiler chickens. Animals 2020, 10, 1795. [CrossRef] [PubMed] on productive, morphological, and physiological indicators in broiler chickens. Animals 2020, 10, 1795. [CrossRef] [PubMed] 16. References 1. Creppy, E.E. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicol. Lett. 2002, 127, 19–28. [CrossRef] 2. Gruber-Dorninger, C.; Jenkins, T.; Shatzmayr, G. 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Individual and combined effects of fumonisin B1 present in fusarium moniliforme culture material and T-2 Toxin or deoxynivalenol in broiler chicks. Poult. Sci. 1997, 76, 1239–1247. [CrossRef] [PubMed] 5. Harvey, R.B.; Kubena, L.F.; Rottinghaus, G.E.; Turk, J.R.; Casper, H.H.; Buckley, S.A. Moniliformin from Fusarium fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks. Avian Dis. 1997, 41, 957. [CrossRef] [PubMed] 6. Broekaert, N.; Devreese, M.; van Bergen, T.; Schauvliege, S.; De Boevre, M.; De Saeger, S.; Vanhaecke, L.; Berthiller, F.; Michlmayr, H.; Malachová, A.; et al. In vivo contribution of deoxynivalenol-3-β-d-glucoside to deoxynivalenol exposure in broiler chickens and pigs: Oral bioavailability, hydrolysis and toxicokinetics. Arch. Toxicol. 2017, 91, 699–712. [CrossRef] [PubMed] H.; Malachová, A.; et al. In vivo contribution of deoxynivalenol-3-β-d-glucoside to deoxynivalenol exposure in broiler chickens and pigs: Oral bioavailability, hydrolysis and toxicokinetics. Arch. Toxicol. 2017, 91, 699–712. [CrossRef] [PubMed] p g y y y 7. Dänicke, S.; Brezina, U. Kinetics and metabolism of the fusarium toxin deoxynivalenol in farm diagnosis of exposure and intoxication and carry over. Food Chem. Toxicol. 2013, 60, 58–75. [CrossR 7. Dänicke, S.; Brezina, U. Kinetics and metabolism of the fusarium toxin deoxynivalenol in farm animals: Consequences for diagnosis of exposure and intoxication and carry over. Food Chem. Toxicol. 2013, 60, 58–75. [CrossRef] [PubMed] g p y 8. 5. Conclusions In conclusion, DON and DON-3S in excreta are suitable metabolites of DON exposure in broilers fed the guidance level (5 mg/kg). Moreover, DON at this level induced proin- ﬂammatory cytokine production, suggesting that DON is immunostimulatory in broiler chickens at 5 mg/kg for 42 days. The effect of this high dose (15 mg/kg) was observed on the deposition of DON-3S in the liver and on the reduction in hematological parameters, suggesting that DON affects the health status of birds. Further studies are required to directly elucidate DON and DON-3S in excreta as relevant end-points for the efﬁcacy testing of detoxiﬁers, and further investigations will need to pay closer attention to the most important immune system indicators that could be affected by DON mycotoxicosis in poultry. Author Contributions: Conceptualization, I.R., V.M., J.B. and A.M.P.-V.; methodology, I.R., V.M., J.B. and A.M.P.-V.; software, I.R. and A.M.P.-V.; validation, V.M., A.J.R., E.E.-G., J.B. and A.M.P.-V.; formal analysis, I.R. and E.E.-G.; investigation, I.R., V.M. and A.M.P.-V.; resources, I.R., V.M., A.J.R. and A.M.P.-V.; data curation, I.R. and E.E.-G.; writing—original draft preparation, I.R.; writing— review and editing, I.R., V.M., A.J.R., E.E.-G., J.B. and A.M.P.-V. visualization, I.R., V.M., A.J.R., J.B. and A.M.P.-V.; supervision, V.M., A.J.R. and A.M.P.-V.; project administration, I.R.,V.M. and A.M.P.-V.; funding acquisition, V.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Phileo by Lesaffre Company. Institutional Review Board Statement: The study was conducted according to the current regula- tions on the use and handling of experimental animals (Decree 214/97, Generalitat de Catalunya, Catalonia, Spain) and was approved by the Ethical Committee for Animal Experimentation of INSTITUTE OF AGRIFOOD RESEARCH AND TECHNOOGY (IRTA) (Number 10625 /03/09/2019). Informed Consent Statement: Not applicable for studies not involving humans. Data Availability Statement: All data sets collected and analyzed during the current study are available from the corresponding author on fair request. Acknowledgments: Insaf Riahi gratefully acknowledges Phileo by Lesaffre for their pre-doctoral grant. Acknowledgments: Insaf Riahi gratefully acknowledges Phileo by Lesaffre for their pre-doctoral grant. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W4312880808	https://mirtr.elpub.ru/jour/article/download/1523/2920	Russian	null	CORRELATION OF TECHNICAL SOLUTIONS AND CONSTRUCTION COSTS OF HSR	Mir transporta	2,018	cc-by	4,629	110 ECONOMICS 110 ECONOMICS 110 ECONOMICS ЭКОНОМИКА УДК 625.111:69.003.12 ECONOMICS Корреляция технических решений и стоимости строительства ВСМ Екатерина РЫЖИК Ekaterina A. RYZHIK КАРЕВ UKAREV Correlation of Technical Solutions and Construction Costs of HSR (текст статьи на англ. яз.– English text of the article – p. 122) ЮКАРЕВ YUKAREV Correlation of Technical Solutions and Construction Costs of HSR (текст статьи на англ. яз.– English text of the article – p. 122) Дюкарев Леонид Александрович – кандидат технических наук, главный специалист Управления проверки сметной документации и экспертизы проектов организации строительства ФАУ «Главгосэкспертиза России», Москва, Россия. Рыжик Екатерина Александровна – кандидат технических наук, доцент кафедры проектирования и строительства железных дорог Российского университета транспорта (МИИТ), Москва, Россия. Р Р азвитие высокоскоростного транс- порта невозможно без поддержки государства, что подтверждено мировым опытом строительства высоко- скоростных железнодорожных магистра- лей. Correlation of Technical Solutions and Construction Costs of HSR (текст статьи на англ. яз.– English text of the article – p. 122) Решение о строительстве первой в мире высокоскоростной магистрали (Tōkaidō Shinkansen) на другом конце континента – в Японии было принято в 1956 году. Пред- ложенный проект ВСМ нормальной колеи встретил значительное сопротивление, в том числе в среде консерваторов-желез- нодорожников, а также со стороны авто- мобильного и авиационного лобби. И очень мало кто из специалистов верил, что в ре- гулярной коммерческой эксплуатации можно обеспечить движение поездов со скоростью 250 км/ч [1]. В статье обозначены актуальность и специфика высокоскоростных железнодорожных магистралей в России. Указаны особенности учёта стоимости строительства ВСМ. Обоснована необходимость оценки проектных решений на основании технико-экономического сравнения вариантов. Приведены существующие и перспективные технические решения и технологии строительства ВСМ, рассмотрены возможности для дальнейшего перехода на эстакадный метод их возведения. Представлен сравнительный анализ сметной стоимости строительства ВСМ‑2 Москва– Казань и близких по характеру объектов- аналогов, проекты которых реализованы в международной практике. В 1960-е годы в ответ на начало строи- тельства ВСМ в Японии французское правительство провело масштабные ис- следования по созданию новых технологий на железнодорожном транспорте (поездов на магнитной и воздушной подушке, а так- же скоростного поезда для обычных желез- ных дорог). В 1976 году власти выделили деньги на крупномасштабную реализацию Ключевые слова: железная дорога, высокоскоростная магистраль, строительство, технико-экономическое сравнение, стоимость линии, стоимость технических решений, земляное полотно, безбалластное верхнее строение пути, ВСМ в мире. • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Рис. 1. Типы конструкции безбалластного верхнего строения пути. Рис. 1. Типы конструкции безбалластного верхнего строения пути. Будущая магистраль Москва–Казань объединяет существующие в мире, а так- же перспективные технические решения и технологии в области высокоскорост- ного железнодорожного транспорта. Корреляция технических решений и стоимости строительства ВСМ Проект ВСМ‑2 в РФ разрабатывается с привлечением специалистов из Китая (корпорация «Эр Юань») в полном соот- ветствии с требованиями нормативных документов. Проектная документация и результаты инженерных изысканий являются объектом анализа государ- ственной экспертизы ФАУ «Главгосэкс пертиза России», кроме того, проект проходит ведомственную экспертизу ОАО «РЖД», научно-методологическое сопро- вождение осуществляется ведущими транспортными университетами страны (РУТ (МИИТ), ПГУПС), технологиче- ский и ценовой аудит проводится с при- проекта TGV. Пассажирское сообщение на линиях TGV было открыто в 1981 году, что ознаменовало начало эксплуатации первых ВСМ в Европе. В Китае к 1993 году средняя скорость движения пассажирских поездов состав- ляла 48 км/ч, железные дороги стали уступать место в рейтинге популярности авиасообщению и автомобильному транспорту. Учитывая это, министерство железнодорожного транспорта разрабо- тало стратегию повышения скорости движения поездов за счёт создания высо- коскоростных линий. Строительство ВСМ Китая началось спустя 40 лет после пуска в эксплуатацию первой японской линии, однако уже сегодня страна обла- дает наиболее протяжённой сетью высо- коскоростных железнодорожных маги- стралей на планете (более 20 000 км). 111 111 • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) к Е. А. Корреляция технических решений и стоимости строительства ВСМ 112 Рис. 2. Системы рельсовых скреплений БВСП. Рис. 2. Системы рельсовых скреплений БВСП. технологии MIP (mixed-in-place), RDV – виброуплотнение, FDP (full displacement pile), ROB – вибробетонные колонны); влечением зарубежных компаний (Фран- ции, Германии, Италии). влечением зарубежных компаний (Фран- ции, Германии, Италии). Таблица 1 • территориальный – связан со слож- ными геологическими (неблагоприятная физико-геологическая среда), географиче- скими (рельеф местности, близость круп- ных городов, что приводит к увеличению затрат по аренде или выкупу территорий для отвода) и климатическими (вечная мерзлота, погребённые льды, термокарсты) условиями строительства; • транспортный – характеризуется от- далённостью объекта строительства от сырьевых баз, что приводит к увеличению транспортных расходов на доставку мате- риалов и конструкций; Таблица 1 Таблица 1 Основные технические параметры ВСМ‑2 Москва–Казань № п/п Наименование основных технических параметров Ед. изм. Значение 1 Эксплуатационная длина главных путей в двухпутном измерении: км 790 1.1. 1, 2 этапы: ст. Москва-Техническая Курская–ст. Железнодорожная 23 км км 27 1.2. 3, 4 этапы: ст. Железнодорожная 23 км–ст. Владимир ВСМ (вкл.) км 172 1.3. 5, 6 этапы: ст. Владимир ВСМ (искл.)–ст. Аэропорт (вкл.) км 224 1.4. 7, 8 этапы: вход в Нижний Новгород (блок-пост 410 км (вкл.)–ст. Н. Нов- город ВСМ (вкл.)–ст. Аэропорт ВСМ (искл.) км 20 1.5. 9, 10 этапы: ст. Аэропорт ВСМ (искл.)–ст. Чебоксары ВСМ (вкл.) км 229 1.6. 11, 12 этапы: ст. Чебоксары ВСМ (искл.)–ст. Казань ВСМ (вкл.) км 118 1.7. 15 этап: строительство административно-технического здания для раз- мещения (ДЦУ) на станции Владимир ВСМ объект 1 2 Безбалластное верхнее строение пути (БВСП) с шириной колеи 1520 мм км 712 3 Верхнее строение пути на балласте (ВСП) с шириной колеи 1520 мм км 78 4 Максимальная скорость движения высокоскоростных пассажирских поездов км/ч 400 5 Величина наибольшего уклона продольного профиля главных путей ‰ 24 6 Минимальный радиус кривой в плане для скоростей не менее 400 км/ч м 10 000 7 Величина междупутного расстояния между осями главных путей при скорости до 400 км/ч мм 5 000 8 Количество остановочных пунктов шт. 16 9 Внеклассные мосты через крупные реки (Клязьма, Ока, Сура, Волга) шт. 5 10 Новые тяговые подстанции шт. 14 11 Время хода между Москвой и Казанью не более час 3 часа 30 мин. Основные технические параметры ВСМ‑2 Москва–Казань Основные технические параметры ВСМ‑2 Москва–Казань Основные технические параметры ВСМ‑2 Москва–Казань • организационный – в формировании цены строительной продукции одновре- менно участвуют заказчик, проектиров- щик, подрядчик, поставщики материалов и оборудования, в случае финансирования строительства объекта из средств федераль- ного бюджета – экспертиза (эксперт); технических и технологических вариан- тов осуществлялось с помощью технико- экономического сравнения вариантов. технических и технологических вариан- тов осуществлялось с помощью технико- экономического сравнения вариантов. Скорость совершенствования совре- менных технологий в строительстве не уступает темпам роста информационных и компьютерных технологий. Лучшим по- казателем тенденции является инноваци- онный высокоскоростной железнодорож- ный транспорт. Проектирование ВСМ идёт по пути накопления и использования лучшего опыта зарубежных стран и при- менения инновационных технологий, обе- спечивающих безопасность эксплуатации в сложных климатических условиях строи тельства. ТЕХНИКО-ЭКОНОМИЧЕСКОЕ СРАВНЕНИЕ • определение марки стрелочных пере- водов; • типы пролётных строений железно- дорожных мостов; Постоянное совершенствование в ми- ровой практике технических решений и технологий создания инфраструктуры ВСМ способствовали появлению множе- ства конкурентоспособных вариантов, в том числе удовлетворяющих требованиям ВСМ‑2 Москва–Казань. Например: • типы скреплений (рис. 2) (WJ‑8, DFF300 Vossloh, SFC Pandrol, СМ‑1). В «Положении о составе разделов про- ектной документации…» (утв. постанов- лением правительства РФ от 16 февраля 2008 г. № 87) [2] не закреплена обязатель- ность вариантной проработки проектных решений. Однако отдельные норматив- ные документы (своды правил) указыва- ют, что принятие основных технических решений должно быть обосновано раз- работкой вариантов путём сравнения технико-экономических показателей [3–5]. В соответствии с требованиями технического задания при проектирова- нии ВСМ‑2 Москва–Казань выполнена вариантная проработка проектных реше- ний, то есть принятие конструктивных, В «Положении о составе разделов про- ектной документации…» (утв. постанов- лением правительства РФ от 16 февраля 2008 г. № 87) [2] не закреплена обязатель- ность вариантной проработки проектных решений. Однако отдельные норматив- ные документы (своды правил) указыва- ют, что принятие основных технических решений должно быть обосновано раз- работкой вариантов путём сравнения технико-экономических показателей [3–5]. В соответствии с требованиями технического задания при проектирова- нии ВСМ‑2 Москва–Казань выполнена вариантная проработка проектных реше- ний, то есть принятие конструктивных, • выбор типа конструкции безбалласт- ного верхнего строения пути (рис. 1) (двух- секционная монолитная RHEDA 2000, LVT, Zublin; на сплошном подрельсовом осно- вании неразрезного типа Bögl, CRTS II, PORR; на сплошном подрельсовом осно- вании блочного типа CRTS I, III, Shinkansen); • выбор типа конструкции безбалласт- ного верхнего строения пути (рис. 1) (двух- секционная монолитная RHEDA 2000, LVT, Zublin; на сплошном подрельсовом осно- вании неразрезного типа Bögl, CRTS II, PORR; на сплошном подрельсовом осно- вании блочного типа CRTS I, III, Shinkansen); • выбор технологии укрепления осно- вания земляного полотна (укрепление грунтов бетонными сваями по типу CFA (CFG), буронабивные сваи под защитой обсадных труб, укрепление грунтов по • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ щественному удорожанию 1 км проек- тируемой линии; щественному удорожанию 1 км проек- тируемой линии; Пересечения ВСМ‑2 с существующи- ми и проектируемыми автомобильными дорогами, железными дорогами и комму- никациями предусматриваются только в разных уровнях. Защита пересекаемых трубопроводов и подземных коммуника- ций устраивается на всю ширину полосы отвода. • технологический – применение инно- вационных решений требует использова- ния высокопроизводительной строитель- ной техники (грузоподъёмное крановое оборудование, фронтальные машины для погрузки балок пролётного строения, бал- ковозы, импортные буровые установки, рельсоукладочный состав на пневмоколёс- ном ходу), которая позволяет не только сократить сроки строительства, но и суще- ственно повысить качество строительной продукции. СТОИМОСТЬ И НОРМИРОВАНИЕ ПРОЕКТНЫХ РЕШЕНИЙ Сметная стоимость строительства (да- лее – стоимость) является одним из основ- ных моментов анализа проектных реше- ний. Все чаще вопрос «сколько?» транс- формируется в вопрос «почему столько?». При этом ответ на последний вопрос суще- ственно зависит от многих факторов, ос- новными из которых остаются [6]: 113 • технический – наличие внекласс- ных мостов, тоннелей, слабых основа- ний под земляное полотно, необходи- мость дополнительных мероприятий по инженерной защите территории от карстовых явлений могут привести к су- • технический – наличие внекласс- ных мостов, тоннелей, слабых основа- ний под земляное полотно, необходи- мость дополнительных мероприятий по инженерной защите территории от карстовых явлений могут привести к су- • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 3. Прохождение трассы ВСМ‑2 Москва–Казань. Рис. 3. Прохождение трассы ВСМ‑2 Москва–Казань. РАЗДЕЛ «ЗЕМЛЯНОЕ ПОЛОТНО» Земляное полотно ВСМ‑2, за исключе- нием раздельных пунктов, проектируется под два пути и должно удовлетворять сле- дующим требованиям СТУ [7], которые оказывают определяющее влияние на стоимость: В ходе проектирования ВСМ‑2 Мо- сква–Казань выполнен многофакторный анализ проектных решений с территори- альной привязкой к месту строительства. В рамках статьи будет рассмотрена стои- мость принятых в проектной документации технических решений по основным цено- образующим разделам: «Земляное полот- но», «Искусственные сооружения» и «Без- балластное верхнее строение пути». При этом делается попытка ответить на озву- ченный ранее вопрос: «Почему столько?». • максимальная накопленная остаточ- ная деформация основной площадки зем- ляного полотна при безбалластной кон- струкции верхнего строения пути за весь срок её полезного использования должна обеспечить возможность устранения про- садок регулировкой креплений и не пре- вышать 15 мм; • в зависимости от влажности, проч- ностных и деформативных свойств грун- тов, однородности их залегания основания земляного полотна следует подразделять на прочные, недостаточно прочные и слабые; Основные технические параметры про- ектируемой высокоскоростной железнодо- рожной магистрали Москва–Казань пред- ставлены в таблице 1, прохождение трассы ВСМ‑2 отражено на рис. 3. • в соответствии с п. 2.3.1 и таблицей 3.2 СТУ «Земляное полотно» на участках недостаточно прочных и слабых оснований необходимо их укрепление для соблюдения требований по осадке насыпи; Зона тяготения ВСМ‑2 Москва–Казань объединяет территории семи субъектов Российской Федерации: Москва, Москов- ская область, Владимирская область, Ни- жегородская область, Чувашская Респу- блика, Республика Марий Эл и Республи- ка Татарстан. • разница в осадках земляного полотна и искусственного сооружения (мост, водо- пропускная труба, тоннель и т. д.) в зоне их сопряжения не должна превышать 5 мм; • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 4. Стоимость элементов типовой конструкции земляного полотна ВСМ‑2 Москва–Казань. Рис. 4. Стоимость элементов типовой конструкции земляного полотна ВСМ‑2 Москва–Казань. Рис. 5. Вариантная проработка логистических схем доставки грунта. Рис. 5. Вариантная проработка логистических схем доставки грунта. полотна ВСМ‑2: коэффициент уплотнения грунтов 1-го и 2-го защитных слоёв должен составлять не менее 1,00, коэффициент уплотнения грунтов насыпи – не менее 0,98. РАЗДЕЛ «ЗЕМЛЯНОЕ ПОЛОТНО» • требования к грунтам насыпей и за- щитных слоёв представлены в разделе 3.1 СТУ «Земляное полотно», соответственно в проектной документации предусматри- вается отсыпка насыпи дренирующим грунтом; Конструкция земляного полотна по проектной документации ВСМ‑2 Москва– Казань состоит из: • устанавливаются повышенные требо- вания к уплотнению грунтов земляного • устанавливаются повышенные требо- вания к уплотнению грунтов земляного • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) , Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 6. Противодеформационные мероприятия в зоне основания ЗП ВСМ. 116 Рис. 6. Противодеформационные мероприятия в зоне основания ЗП ВСМ. ормационные мероприятия в зоне основания ЗП ВСМ • основания земляного полотна (ЗП), естественного или укреплённого на участ- ках слабых грунтов; В связи с отсутствием в пределах про- ектируемой трассы карьеров готовых грунтовых смесей первого защитного слоя (ЩПГС) в проектной документации предусмотрена доставка компонентов смеси до временных грузовых дворов и приготовление ЩПГС в построечных условиях. • основания земляного полотна (ЗП), естественного или укреплённого на участ- ках слабых грунтов; • тела насыпи (отсыпается дренирую- щим грунтом); • второго защитного слоя высотой 2,1–2,3 м (отсыпается из крупнозернистых песков, песчано-гравийных смесей (ПГС), обогащённых песчано-гравийных смесей (ОПГС)); В границах прохождения ВСМ‑2 рас- пространены отрицательные физико- геологические явления: заболочен- ность, карст, овражная эрозия, подмыв берегов, пучение грунтов, затопление. Из опасных экзогенных геологических процессов наиболее проявляются карст и оползнеобразование. В соответствии с отчётами инженерных изысканий для выполнения требований СТУ намечены следующие противодеформационные мероприятия в зоне основания земля- ного полотна (рис. 6): • первого защитного слоя высотой 0,28 м (устраивается из щебёночно-песча- но-гравийных смесей (ЩПГС) в соответ- ствии с гранулометрическим составом, определённым СТУ); • асфальтобетонного покрытия высо- той 0,12 м. Типовой поперечный профиль земля- ного полотна представлен на рис. 4. Типовой поперечный профиль земля- ного полотна представлен на рис. 4. На этапе выполнения инженерных изысканий по разведке грунтовых строи- тельных материалов в пределах трассы было выявлено отсутствие пригодных грунтов, отвечающих требованиям СТУ для отсыпки первого и второго защитных сло- ёв и тела насыпи земляного полотна. По каждому этапу проекта ВСМ‑2 сделана вариантная проработка логистических схем доставки грунта от карьера поставщика до места производства работ (рис. 5). Опти- мальные, экономически целесообразные решения отражены в транспортных схемах, согласованных с заказчиком. • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) РАЗДЕЛ «ЗЕМЛЯНОЕ ПОЛОТНО» 7), а именно: РАЗДЕЛ «ЗЕМЛЯНОЕ ПОЛОТНО» • укрепление основания призматиче- скими сваями на участках со скоростями движения до 250 км/ч; • буронабивные неармированные бе- тонные сваи с применением технологии непрерывного полого шнека (CFG), с устройством гибкого ростверка (диаметр свай 0,5–0,6 м, расстояние между ними – 3–5 диаметра сваи); • буронабивные железобетонные сваи, с устройством железобетонного ростверка, диаметр свай 1,25 м; • буронабивные железобетонные сваи, • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 7. График жизненного цикла различных конструкций БВСП. Рис. 7. График жизненного цикла различных конструкций БВСП. • струйная цементация грунтов (Jet grouting) на участках распространения карста. тических условиях, схожих с трассой Москва–Казань, надёжна исключительно безбалластная конструкция. При технико-экономическом сравне- нии вариантов противодеформационных мероприятий наиболее востребованным оказываются применение бетонных свай, технологии непрерывного полого шнека CFA (CFG). Таким способом планируется укреплять более 90 % основания земляного полотна на протяжении всей трассы. В соответствии с СТУ‑2 «Верхнее стро- ение пути» [7] для главных путей ВСМ‑2 с максимальной скоростью более 200 км/ч выбор типа конструкции БВСП в проект- ной документации определён с учётом следующих особенностей: • эксплуатационные параметры БВСП должны обеспечиваться в диапазоне тем- ператур рельсов от –48°C до +67°C; Основные проектные решения по раз- делу «Земляное полотно» приняты с учётом технико-экономического сравнения вариан- тов, возможное количество которых ограни- чено необходимостью соблюдения требова- ний нормативных документов для обеспече- ния безопасности движения поездов с мак- симальными установленными скоростями. При этом соблюдение указанных требований оказывает существенное влияние на стои- мость сооружения земляного полотна, кото- рая составляет более 19 % от общей стоимо- сти строительства ВСМ‑2 (рис. 12). р ур р • для устройства несущей конструкции следует применять бетон классом не ниже В40, маркой по водопроницаемости не ниже W8, по морозостойкости – не ниже F300; • необходимо сохранить высокие тем- пы укладки для сокращения сроков строи тельства с соблюдением требований по безопасности, прочности, надёжности и ремонтопригодности. • для устройства несущей конструкции следует применять бетон классом не ниже В40, маркой по водопроницаемости не ниже W8, по морозостойкости – не ниже F300; б • необходимо сохранить высокие тем- пы укладки для сокращения сроков строи тельства с соблюдением требований по безопасности, прочности, надёжности и ремонтопригодности. Решение о применении конструктив- ного типа БВСП принималось на основа- нии технико-экономического обоснования с учётом оптимизации стоимости жизнен- ного цикла конструкции (п. 4.1.2 СТУ‑2) [7]. В рамках такого обоснования в составе проектной документации представлен рас- чёт жизненного цикла для различных кон- струкций БВСП (рис. Таблица 2 Таблица 2 Стоимость верхнего строения пути на перегонах для участков ВСМ‑2 Наименование работ и затрат на участках Ед. изм. Длина участка Сметная стои- мость в базис- ном уровне цен, тыс. руб. Расчётный показатель единич- ной стоимости, тыс. руб. в базис- ном уров- не цен 2000 г. в уровне цен I кв. 2017 г. Итого в том числе на один путь ст. Железнодорожная (искл.)– ст. Ногинск (искл.) ВСП на балласте для скоростей до 250 км/ч 1 км 29,01 175 710 6 057 48 334 24 167 ст. Ногинск (искл.)–ст. Орехо- во-Зуево (искл.) Безбалласт- ное верхнее строение пути 1 км 32 650 023 20 313 162 100 81 050 ст. Орехово-Зуево (искл.)– км 97+580 Безбалластное верхнее строение пути 1 км 7,88 158 035 20 055 160 041 80 020 км 97+580 – ст. Петушки (искл.) Безбалластное верхнее строение пути 1 км 28,02 559 518 19 969 146 570 73 285 ст. Петушки (искл.)–ст. Владимир (искл.) Безбалластное верхнее строение пути 1 км 61,9 1 269 060 20 502 150 483 75 242 Стоимость верхнего строения пути на перегонах для участков ВСМ‑2 конструкции БВСП и графическое моде- лирование этапов строительства позволили рассчитать стоимость укладки безбалласт- ного верхнего строения пути ВСМ‑2 и по- лучить положительные заключения ведом- ственной экспертизы ОАО «РЖД» и ФАУ «Главгосэкспертиза России». Основные показатели стоимости представлены в таб лице 2 как для участка с балластным ВСП для скоростей движения до 250 км/ч, так и для безбалластной конструкции. конструкции БВСП и графическое моде- лирование этапов строительства позволили рассчитать стоимость укладки безбалласт- ного верхнего строения пути ВСМ‑2 и по- лучить положительные заключения ведом- ственной экспертизы ОАО «РЖД» и ФАУ «Главгосэкспертиза России». Основные показатели стоимости представлены в таб лице 2 как для участка с балластным ВСП для скоростей движения до 250 км/ч, так и для безбалластной конструкции. Таким образом, исходя из условий дви- жения высокоскоростных поездов со скоро- стью до 400 км/ч, работы в холодной зоне, смешанного движения с разной нагрузкой от колёсной пары на рельсы, а также с учётом оптимизации стоимости жизненного цикла, в проектной документации ВСМ‑2 Москва– Казань предусмотрено модернизированное БВСП на сплошном подрельсовом основа- нии типа CRTS III RUS (рис. 8). Выбор имеющихся в Российской Феде- рации сметных расценок для определения сметной стоимости БВСП весьма ограни- чен, однако анализ существующей в мире технологии производства работ, 3D-модели Проектная документация ВСМ‑2 Мо- сква–Казань также получила положитель- ные заключения по результатам проведе- ния обязательного технологического • МИР ТРАНСПОРТА, том 16, № 5, С. РАЗДЕЛ «ВЕРХНЕЕ СТРОЕНИЕ ПУТИ» В мировой практике для строительства высокоскоростных магистралей применяют- ся как балластное, так и безбалластное верх- нее строение пути. Балластное предпочитают в европейских странах с относительно мяг- ким климатом, небольшой амплитудой температур (Франция, Испания, Италия) и скоростями движения поездов до 320 км/ч. Безбалластное больше в доверии для скоро- стей движения до 350 км/ч в азиатских стра- нах – Япония, Корея, Китай, а также в Гер- мании. Опыт проектирования и строитель- ства ВСМ в Китае показывает, что в клима- • двухсекционной монолитной (RHEDA 2000, LVT, Zublin); • на сплошном подрельсовом основа- нии неразрезного типа (Bögl, CRTS II, PORR); • на сплошном подрельсовом основа- нии блочного типа (CRTS I, Shinkansen); • на сплошном подрельсовом основа- • на сплошном подрельсовом основа- нии блочного типа (CRTS III, CRTS III RUS). • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ к Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 8. Принятая конструкция БВСП – CRTS III RUS. Рис. 8. Принятая конструкция БВСП – CRTS III RUS. • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Таблица 2 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ Рис. 9. Анализ-сравнение стоимости плит БВСП и БМП. Рис. 9. Анализ-сравнение стоимости плит БВСП и БМ и ценового аудита (далее – ТЦА) и научно- методологического сопровождения (РУТ (МИИТ), ПГУПС). го расстояния транспортировки конструк- ций по подготовленному земляному по- лотну ВСМ; го расстояния транспортировки конструк- ций по подготовленному земляному по- лотну ВСМ; • в проектное положение пролётные строения устанавливаются специальными консольно-шлюзовыми агрегатами на пневмоколёсном ходу грузоподъёмностью 900 т. В рамках подготовки сметных расчётов выполнен конъюнктурный анализ рынка, а также представлено сравнение стоимости плиты безбалластного верхнего строения пути типа CRTS III RUS с широко приме- няемой в мостостроении конструкцией плиты безбалластного мостового полотна (БМП) (рис. 9). Разница в стоимости от- носительно плит БМП – повод к модерни- зации производства для изготовления без- балластных плит верхнего строения пути. В процессе разработки проектной до- кументации был выполнен сравнительный анализ удельной стоимости строительства одного погонного метра мостов/эстакад на стадии обоснования инвестиций, по про- ектной документации ВСМ‑2, объектов- аналогов в Китае и Франции (рис. 10). РАЗДЕЛ «ИСКУССТВЕННЫЕ СООРУЖЕНИЯ» Предложенные в проектной докумен- тации организационно-технологические схемы производства работ позволяют со- кратить сроки строительства и оптимизи- ровать сметную стоимость железнодорож- ных мостов/эстакад относительно стадии обоснования инвестиций. Дальнейшая доводка на стадии строительства техноло- гии производства работ может способство- вать переходу на инновационный «эстакад- ный метод» строительства ВСМ. Это основной ценообразующий раздел проекта ВСМ‑2 – его доля в сметной стои- мости строительства составляет более 21 % (рис. 12). Наиболее частые на трассе ис- кусственные сооружения – эстакадные мосты. Они используются не только в ме- стах водотоков, но и заменяют высокие насыпи (выше 8–10 м). В проекте ВСМ‑2 Москва–Казань при- меняются нетривиальные для России ре- шения по строительству искусственных сооружений, которые позволили типизи- ровать технологические процессы и опти- мизировать сметную стоимость: В проекте ВСМ‑2 Москва–Казань при- меняются нетривиальные для России ре- шения по строительству искусственных сооружений, которые позволили типизи- ровать технологические процессы и опти- мизировать сметную стоимость: ЛИТЕРАТУРА 1. Киселёв И. П. Полвека высокой скорости // Железные дороги мира. – 2015. – № 2. – С. 70–77. 1. Киселёв И. П. Полвека высокой скорости // Железные дороги мира. – 2015. – № 2. – С. 70–77. 2. Постановление правительства Российской Федерации от 16 февраля 2008 г. № 87 «О составе раз- делов проектной документации и требованиях к их содержанию» // Минстрой России. [Электронный ресурс]: http://www.minstroyrf.ru/docs/535/. Доступ 01.06.2018. 2. Постановление правительства Российской Федерации от 16 февраля 2008 г. № 87 «О составе раз- делов проектной документации и требованиях к их содержанию» // Минстрой России. [Электронный ресурс]: http://www.minstroyrf.ru/docs/535/. Доступ 01.06.2018. 2. Постановление правительства Российской Федерации от 16 февраля 2008 г. № 87 «О составе раз- делов проектной документации и требованиях к их содержанию» // Минстрой России. [Электронный ресурс]: http://www.minstroyrf.ru/docs/535/. Доступ 01.06.2018. 3. СП 238.1326000.2015 Железнодорожный путь // Сервисный центр железнодорожного транспорта. [Электронный ресурс]: http://sczdt.ru/normativnye- dokumenty/. Доступ 01.06.2018. 3. СП 238.1326000.2015 Железнодорожный путь // Сервисный центр железнодорожного транспорта. [Электронный ресурс]: http://sczdt.ru/normativnye- dokumenty/. Доступ 01.06.2018. На сегодняшний день определить окон- чательную совокупную стоимость реализа- ции проекта невозможно (не определены затраты на подвижной состав, эксплуата- ционные расходы), но уже сейчас можно достоверно ответить «почему столько?». Представленные данные подтверждают, что стоимость проектных решений при повышении скорости движения поездов более чем на 200 км/ч взаимозависима от повышения требований к безопасности и бесперебойности движения высокоско- ростного железнодорожного транспорта. На сегодняшний день определить окон- чательную совокупную стоимость реализа- ции проекта невозможно (не определены затраты на подвижной состав, эксплуата- ционные расходы), но уже сейчас можно достоверно ответить «почему столько?». Представленные данные подтверждают, что стоимость проектных решений при повышении скорости движения поездов более чем на 200 км/ч взаимозависима от повышения требований к безопасности и бесперебойности движения высокоско- ростного железнодорожного транспорта. y/ Д у 4. СП 41.13330.2012 Бетонные и железобетонные конструкции гидротехнических сооружений. Актуа- лизированная редакция СНиП 2.06.08-87 // Минстрой России. Федеральный центр нормирования, стандар- тизации и технической оценки соответствия в строи тельстве. [Электронный ресурс]: https://www.faufcc. ru/technical-regulation-in-constuction/formulary- list/?s=41. Доступ 15.06.2018. / 4. СП 41.13330.2012 Бетонные и железобетонные конструкции гидротехнических сооружений. Актуа- лизированная редакция СНиП 2.06.08-87 // Минстрой России. Федеральный центр нормирования, стандар- тизации и технической оценки соответствия в строи тельстве. [Электронный ресурс]: https://www.faufcc. ru/technical-regulation-in-constuction/formulary- list/?s=41. Доступ 15.06.2018. 5. СП 35.13330.2011 Мосты и трубы. Актуализи- рованная редакция СНиП 2.05.03-84 // Минстрой России. Федеральный центр нормирования, стандар- тизации и технической оценки соответствия в строи тельстве. [Электронный ресурс]: https://www.faufcc. ru/technical-regulation-in-constuction/formulary- list/?s=34. Доступ 15.06.2018. Проект ВСМ‑2 определённо «вызов» и для строительного комплекса страны. Координаты авторов: Дюкарев Л. А. – DjukarevLA@yandex.ru, Рыжик Е. А. – CatRyzhik@yandex.ru. Статья поступила в редакцию 01.07.2018, принята к публикации 09.08.2018. • «тянет» за собой прогресс всего промыш- ленного комплекса страны и доказывает коммерческую перспективу движения поездов со скоростями свыше 250 км/ч. выше доля основных строительных объ- ектов (более чем на 36 %) (рис. 11, 12). Удельная стоимость строительства 1 км по проектной документации ВСМ‑2 соиз- мерима и сопоставима с мировыми объ- ектами-аналогами (рис. 13). При этом су- ществует объективная и обоснованная возможность снижения стоимости строи- тельства мостов и уменьшения размера затрат на устройство безбалластного верх- него строения пути (в частности, относи- тельно аналогичных объектов Китая). Редакция напоминает, что вся интерпретация фактов и выводов относится исключительно к сфере ответственности авторов. ЗАКЛЮЧЕНИЕ Высокоскоростная железнодорожная магистраль Москва–Казань уникальный, технически сложный, первый в России инфраструктурный проект такого уровня. Соблюдение требований технического за- дания, нормативных документов и при- менение передового опыта зарубежных стран оказывают определяющее влияние на сметную стоимость строительства. В сравнении с железными дорогами общей сети со скоростями движения поездов до 200 км/ч в проекте ВСМ‑2 значительно • разработаны унифицированные кон- струкции балочных пролётных строений коробчатого типа под два железнодорож- ных пути (расстояние между ними 5000 мм) из преднапряжённого железобетона пол- ной длиной 23,6 и 34,2 м; • предусмотрено создание полигонов для изготовления балок, исходя из наличия естественных препятствий и рационально- • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Дюкарев Л. А., Рыжик Е. А. Корреляция технических решений и стоимости строительства ВСМ 120 Рис. 10. Средняя стоимость строительства 1 погонного метра железнодорожных мостов ВСМ. 120 Рис. 10. Средняя стоимость строительства 1 погонного метра железнодорожных мостов ВСМ. Рис. 11. Структура стоимости строительства железных дорог общей сети и ВСМ‑2 в разрезе сводного сметного расчёта. Рис. 11. Структура стоимости строительства железных дорог общей сети и ВСМ‑2 в разрезе сводного сметного расчёта. Рис. 12. Стоимость строительства основных объектов ВСМ‑2. Рис. 12. Стоимость строительства основных объектов ВСМ‑2. . 12. Стоимость строительства основных объектов ВС • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018) Рис. 13. Сопоставительный анализ стоимости 1 км ВСМ. Рис. 13. Сопоставительный анализ стоимости 1 км ВСМ. ЛИТЕРАТУРА Применение современных инновационных технологий в строительстве невозможно без повышения уровня механизации и ав- томатизации производства и, как след- ствие, ускоренного его развития и расши- рения. Международный опыт показывает, что строительство и эксплуатация ВСМ 6. Бучкин В. А., Дюкарев Л. А. Методика расчёта строительной стоимости // Мир транспорта. – 2012. – № 6. – С. 86–92. 7. Специальные технические условия «Проекти- рование участка Москва–Казань высокоскоростной железнодорожной магистрали Москва–Казань–Ека- теринбург со скоростями движения до 400 км/ч. Ак- туализированные в 2017 году» // АО «Скоростные магистрали». [Электронный ресурс]: http://www.hsrail. ru/information/documents/docs/. Доступ 11.06.2018. Координаты авторов: Дюкарев Л. А. – DjukarevLA@yandex.ru, Рыжик Е. А. – CatRyzhik@yandex.ru. Статья поступила в редакцию 01.07.2018, принята к публикации 09.08.2018. • Редакция напоминает, что вся интерпретация фактов и выводов относится исключительно к сфере ответственности авторов. • МИР ТРАНСПОРТА, том 16, № 5, С. 110–129 (2018)
https://openalex.org/W2805958520	https://europepmc.org/articles/pmc6018402?pdf=render	English	null	Measuring Women's Empowerment in Sub-Saharan Africa: Exploratory and Confirmatory Factor Analyses of the Demographic and Health Surveys	Frontiers in psychology	2,018	cc-by	7,666	ORIGINAL RESEARCH published: 19 June 2018 doi: 10.3389/fpsyg.2018.00994 Ibitola O. Asaolu 1, Halimatou Alaofè 1, Jayleen K. L. Gunn 2, Akosua K. Adu 3, Amanda J. Monroy 4, John E. Ehiri 1, Mary H. Hayden 5 and Kacey C. Ernst 2 Ibitola O. Asaolu 1, Halimatou Alaofè 1, Jayleen K. L. Gunn 2, Akosua K. Adu 3, Amanda J. Monroy 4, John E. Ehiri 1, Mary H. Hayden 5 and Kacey C. Ernst 2 1 Department of Health Promotion Sciences, Mel and Enid Zuckerman College of Public Health, University of Arizona, Tucson, AZ, United States, 2 Department of Epidemiology and Biostatistics, Mel and Enid Zuckerman College of Public Health, University of Arizona, Tucson, AZ, United States, 3 Department of Epidemiology, College of Public Health, University of Kentucky, Lexington, KY, United States, 4 School of Geography and Development, University of Arizona, Tucson, AZ, United States, 5 Climate Science and Applications Program, National Center for Atmospheric Research, Boulder, CO, United States Edited by: Jin Eun Yoo, Korea National University of Education, South Korea Reviewed by: Kátia Bones Rocha, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil Jose D. Perezgonzalez, Massey University Business School, New Zealand Edited by: Jin Eun Yoo, Korea National University of Education, South Korea Background: Women’s status and empowerment inﬂuence health, nutrition, and socioeconomic status of women and their children. Despite its beneﬁts, however, research on women’s empowerment in Sub-Saharan Africa (SSA) is limited in scope and geography. Empowerment is variably deﬁned and data for comparison across regions is often limited. The objective of the current study was to identify domains of empowerment from a widely available data source, Demographic and Health Surveys, across multiple regions in SSA. Reviewed by: Kátia Bones Rocha, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil Jose D. Perezgonzalez, Massey University Business School, New Zealand Methods: Demographic and Health Surveys from nineteen countries representing four African regions were used for the analysis. A total of 26 indicators across different dimensions (economic, socio-cultural, education, and health) were used to characterize women’s empowerment. Pooled data from all countries were randomly divided into two datasets—one for exploratory factor analysis (EFA) and the other for Conﬁrmatory Factor Analysis (CFA)—to verify the factor structure hypothesized during EFA. *Correspondence: Ibitola O. Asaolu ibitola@email.arizona.edu Specialty section: This article was submitted to Quantitative Psychology and Measurement, a section of the journal Frontiers in Psychology Results: Four factors including attitudes toward violence, labor force participation, education, and access to healthcare were found to deﬁne women’s empowerment in Central, Southern, and West Africa. However, in East Africa, only three factors were relevant: attitudes toward violence, access to healthcare ranking, and labor force participation. There was limited evidence to support household decision-making, life course, or legal status domains as components of women’s empowerment. Received: 06 February 2018 Accepted: 28 May 2018 Published: 19 June 2018 Received: 06 February 2018 Accepted: 28 May 2018 Keywords: women’s empowerment, gender equality, exploratory factor analysis, conﬁrmatory factor analysis, Sub-Saharan Africa INTRODUCTION country and fail to demonstrate disparities that may be occurring at the sub-national level. Women’s empowerment and gender equality concepts are important in fostering health and human development. Empowerment describes the process of change wherein an individual with prior inability to choose has the access and freedom to make choices (Kabeer, 2005). Gender equality is achieved when both men and women enjoy the same socio- economic rights and opportunities and have equal access to education, health care, decent work, and representation in political and economic decision-making processes (World Bank, 2012). Gender equality can be eﬀectively achieved through empowerment, comprising three broad categories, namely (1) agency, which describes the ability to make decisions regardless of existing power relations; (2) resources—including health, education, and physical assets—are the channels through which agency is exercised; and (3) achievements—such as economic opportunities and improved socio-political status—the outcomes of agency (Kabeer, 2005). Over the years, there has been steady improvement in the status of African girls and women, (United Nations, 1979; African Union, 2016) particularly in agency and achievement categories of empowerment (United Nations, 2015). However, identifying ways to track and compare this progress is complicated by the lack of a validated standard method to measure women’s empowerment across countries, subnational, and individual levels. Individual-level studies have primarily focused on determining the associations between women’s empowerment and health care access and outcomes including antenatal care, contraceptive use, child mortality, and nutritional outcomes (Jennings et al., 2014; Heaton, 2015; Phan, 2016; Alaofè et al., 2017; Asaolu et al., 2017). However, studies generally have not incorporated multidimensional indicators of women’s empowerment or used an appropriate validation methodology that scientiﬁcally corroborates their proposed measures. For example, Jennings et al.’s (2014) study used Demographic and Health Survey (DHS) data from eight African countries to characterize women’s empowerment (Jennings et al., 2014). However, the study did not apply a statistical methodology— such as exploratory factor analysis (EFA) or conﬁrmatory factor analysis (CFA)—to identify and validate the latent construct of women’s empowerment (Costello and Osborne, 2005; Schreiber et al., 2006). Similarly, the women’s empowerment measure developed by Phan (2016) used EFA to describe four domains of women’s empowerment across four Southeast Asian countries (Phan, 2016), but the measure was not validated. INTRODUCTION Furthermore, since the study used data from Southeast Asia, Phan’s measure cannot be generalized to African countries given the diﬀerences between the continents as exempliﬁed by variations in fertility, educational attainment, and access to improved water and sanitation (World Health Organization, 2015). There are currently several indicators that use country-level factors to obtain a measure of empowerment at regional and global levels. Two globally-used indices include the Gender Development Index (GDI)—examining gender diﬀerences in human development (health, knowledge, and living standards)—and Gender Inequality Index (GII) assessing gender gaps in reproductive health, empowerment, and labor force participation1. Speciﬁc indices have also been created for Africa including the African Gender Equality Index (AGEI) and the African Gender and Development Index (UNECA, 2011; African Development Bank, 2015). The AGEI measures gender diﬀerences in economic opportunities, human development, and legal rights; the index emphasizes the role of gender equality in advancing agricultural and business productivity (African Development Bank, 2015). Furthermore, the AGDI considers three domains: social power (capabilities), economic power (opportunities), and political power (agency). The AGDI builds upon other indices with the inclusion of the African women’s progress scoreboard (AWPS) that details the status of the African woman and girl by considering global treaties that support empowerment (UNECA, 2011). While these indicators have been useful in tracking general progress toward women’s empowerment and equality goals, (UNECA, 2011; African Development Bank, 2015)2 the data sources limit the ability to examine what individual women are experiencing within the g Most recently, Ewerling et al. (2017) used DHS data from 34 African countries to create the Survey-Based Women’s Empowerment (SWPER) Index which has three domains: attitude to violence, social independence, and decision making (Ewerling et al., 2017). The SWPER indicator is the ﬁrst developed for broad use across Africa. SWEPR index correlated well with other indicators using national level data, but it has several key limitations. The SWPER’s precision may be limited by the application of principal component analysis, and external and convergent validity. The index lacks factor loadings of each domain for individual countries, making it diﬃcult to ascertain the order of domains of women’s empowerment across countries. External validation was assessed by correlating SWEPR with the GDI, but these measures of empowerment are distinct. GDI assesses gender equality by calculating the female to male ratio of the human development index (HDI) that comprise speciﬁc indicators (i.e. 1UNDP, (n.d.)###UNDP (n.d.)United Nations Development Programme. Gender Development Index. http://hdr.undp.org/en/content/gender-development-index- gdi.v. (Accessed January 17). 2UNDP, (n.d.)###UNDP (n.d.)United Nations Development Programme. Gender Inequality Index. http://hdr.undp.org/en/content/gender-inequality-index-gii. (Accessed January 17). Citation: Asaolu IO, Alaofè H, Gunn JKL, Adu AK, Monroy AJ, Ehiri JE, Hayden MH and Ernst KC (2018) Measuring Women’s Empowerment in Sub-Saharan Africa: Exploratory and Conﬁrmatory Factor Analyses of the Demographic and Health Surveys. Front. Psychol. 9:994. doi: 10.3389/fpsyg.2018.00994 Conclusion: This foremost study advances scholarship on women’s empowerment by providing a validated measure of women’s empowerment for researchers and other stakeholders in health and development. Keywords: women’s empowerment, gender equality, exploratory factor analysis, conﬁrmatory factor analysis, Sub-Saharan Africa June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org Measuring Women’s Empowerment in Africa Asaolu et al. 1UNDP, (n.d.)###UNDP (n.d.)United Nations Development Programme. Gender Development Index. http://hdr.undp.org/en/content/gender-development-index- gdi.v. (Accessed January 17). 2 Indicators for Economic Dimension Indicators for Economic Dimension This dimension includes labor force participation domain, created from the following indicators: respondent’s occupation, type of earning from respondent’s work, seasonality of respondent’s occupation, and income ratio. Respondents’ occupation was described by the following scores: 1—if they worked for a family member; 2—if they worked for someone else; and 3—if they were self-employed. Women’s earnings were depicted using the following scores: 1—if they were paid in-kind only; 2—if they were paid cash and in-kind; 3—if they were paid in cash only. The seasonality of a woman’s job was coded as follows: 1—if the woman worked occasionally or seasonally; 2—if the woman worked all year. Women’s incomes were compared to their partners’, and they were assigned the following scores: 1—if their partner did not bring in any income; 2—if they earned less than their partner; 3—if they earned about the same as their partner; 4—if they earned more than their partner. Women were scored 0 if they were unemployed. INTRODUCTION health, knowledge, and living standards), while SWPER used only women’s data to describe diﬀerent indicators of empowerment (i.e. attitudes to violence, social independence, and decision-making). Convergent validity was also conducted by associating the SWPER index with three outcomes: modern contraceptive use, institutional delivery, and stunting (Ewerling et al., 2017). Convergent validity is a type of construct validity used to demonstrate signiﬁcant correlation between a new scale and existing validated scale; both scales should measure the same construct, thus making them theoretically identical (Netemeyer et al., 2003; Dmitrienko et al., 2007). Although associated with these three outcomes, women’s empowerment is theoretically diﬀerent from modern contraceptive use, institutional delivery, and stunting. Furthermore, the three outcomes do not fully June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 2 Measuring Women’s Empowerment in Africa Asaolu et al. describe women’s empowerment and may not adequately validate the SWPER index. describe women’s empowerment and may not adequately validate the SWPER index. Empowerment Indicators Previous studies have suggested four important dimensions of women’s empowerment in developing nations at the household level: economic, socio-cultural, education, and health (Jennings et al., 2014; Pratley, 2016). We identiﬁed several variables within each of these four dimensions and scores were assigned, with higher values reﬂecting greater level of empowerment. The scoring process was developed using evidence from previous literature (Kishor and Gupta, 2009; Jennings et al., 2014; Shimamoto and Gipson, 2015; Phan, 2016). Appendix A summarizes the aggregation rules used to code all variables and create the four broad dimensions and 10 overall domains. Therefore, available measures of women’s empowerment in sub-Saharan Africa (SSA) have several drawbacks: they lack external validity, have few domains of empowerment, and/or are not generalizable to sub-Saharan Africa. Building upon Kabeer’s (2005) deﬁnition, research has identiﬁed diﬀerent multidimensional and contextually relevant indicators of women’s empowerment. Indeed, empirical research lists several measures of women’s empowerment such as agency, autonomy, capacity for action, self-determination, and self-conﬁdence (e.g., Cheston and Kuhn, 2001; Malhotra et al., 2002; Narayan, 2005; Hansen, 2015). These deﬁnitions stress that women’s empowerment is a multifaceted concept and propose that empowerment is a process from being un-empowered to becoming empowered. Combining these views, we propose that women’s empowerment is a multifaceted process of change that involves individual and collective awareness, behavior, institutions, and outcomes embedded in distinct social and cultural contexts. The goal of this study was therefore to develop a valid measure of women’s empowerment both generalizable to sub-Saharan Africa and robust enough to develop region-speciﬁc indicators. ndicators for Socio-Cultural Dimension Indicators for Socio-Cultural Dimension This dimension includes: domains of household decision- making, attitude toward violence, life course indicator, and land or home ownership. Participation in decision-making was assessed by three items, namely: (1) person who decides respondent’s healthcare; (2) person who decides large household purchases; and (3) person who decides whether respondent can visit her family or relatives. Women were assigned the following scores: 0—if the decision was made by husband/partner alone, someone else, or other; 1—if the decision was jointly made by respondent and her husband/partner; 2—if the respondent alone made the decision. Attitudes toward violence were assessed using ﬁve variables describing whether beating was justiﬁed if the wife: goes out without telling her husband; neglects the children; argues with her husband; refuses sex with her husband; burns food. Women who answered “Yes” and “Don’t know” were scored 0 while women who responded “No” were scored 1. The life course domain which was measured by two indicators—age at ﬁrst birth and age at ﬁrst cohabitation—were scored as follows: 0, <15- years-old; 1, between 15- and 17-years-old; 2, between 18- and 20-years-old; 3, 21-years-old and older. Finally, two indicators— home and land ownership—described the legal status of women in possessing properties. Women were assigned the following scores: 0—if they did not own a home or land; 1—if they owned a MATERIALS AND METHODS Data were extracted from the DHS in June of 2015 from countries with data available from the previous 4 years in sub-Saharan Africa. All data were from Phases 5 and 6 of the survey. The DHS characteristics and administration procedures have been previously described (ICF International, 2015). There were DHS data for 42 countries, but only 23 countries recent surveys were conducted between 2011 and 2014. DHS Data from the Republic of Congo, Guinea, Liberia, and Senegal were excluded because of missing values. Single, widowed, divorced, and separated women were excluded as most questions on women’s empowerment were asked only of married women and women living with a partner. Of the remaining 167,163 partnered-women, those with missing entries on variables included in this study were excluded from the study using listwise deletion. The prevalence of women with complete data after listwise deletion was 66.6%. Finally, 19 countries were retained and classiﬁed into one of the following regions: (1) Central Africa: Cameroon, Democratic Republic of Congo, and Gabon; (2) East Africa: Comoros, Ethiopia, and Uganda; (3) Southern Africa: Mozambique, Namibia, Zambia, and Zimbabwe; and (4) West Africa: Benin, Cote d’Ivoire, Gambia, Ghana, Mali, Nigeria, Sierra Leone, and Togo. Overall, the ﬁnal analysis was limited to 111,368 partnered-women with complete information on the variables used in the study (Table 1). Indicators for Education Dimension This dimension comprised three domains—literacy, highest educational level, and spousal diﬀerence in educational attainment. Women’s literacy was described by the following values: 0—if they could not read at all; 1—if they were able to read part of a sentence; 2—if they were able to read an entire sentence; 3—if they did not need a reading card to assess their literacy. Women’s highest educational level was measured using the following scores: 0—No education; 1—primary education, 2—secondary education; and 3—higher education. Spousal/partner diﬀerence in educational level was measured by comparing the educational attainment of respondent and her spouse/partner. Women with less educational attainment than their spouse/partner were scored 0 while those who had equal or greater educational attainment than their spouse were given 1 or 2, respectively. Data Access and Ethical Considerations Data Access and Ethical Considerations Access to the data sets was oﬃcially granted by the DHS program after submitting a request outlining the purpose of the analyses (https://dhsprogram.com/data/new-user-registration.cfm). The DHS contains de-identiﬁed secondary data and is considered exempt under University of Arizona’s human subjects review. June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 3 Measuring Women’s Empowerment in Africa Asaolu et al. TABLE 1 \| Distribution of respondents by region and country. Region in Africa Country/survey Total women sample (15–49 years) Selected women sample Central (n = 16,047) Cameroon 2011 15,426 3,270 Congo (Democratic Republic) 2014 10,819 9,233 Gabon 2012 8,422 3,544 East (n = 11,993) Comoros 2012 5,329 1,733 Ethiopia 2011 16,515 6,742 Uganda 2011 8,674 3,518 Southern (n = 19,683) Mozambique 2011 13,745 5,529 Namibia 2013 10,018 2,801 Zambia 2014 16,411 6,538 Zimbabwe 2011 9,171 4,815 West (n = 63,645) Benin 2012 16,599 7,525 Cote d’Ivoire 2012 10,060 4,277 Gambia 2013 10,233 5,441 Ghana 2014 9,396 3,822 Mali 2013 10,424 6,397 Niger 2012 11,160 7,158 Nigeria 2013 38,948 20,358 Sierra Leone 2013 16,658 4,628 Togo 2014 9,480 4,039 All countries Sub-Saharan Africa 247,888 111,368 The number in bold represents the total number of participants included in this study. All countries The number in bold represents the total number of participants included in this study. medical help; (2) having money for healthcare; (3) distance to health facility; (4) not wanting to go healthcare facility alone. Women were assigned a 0 score if they reported problems accessing healthcare; otherwise, respondents were scored 1. home or land jointly; 2—if they owned home or land alone only or “both alone and jointly.” Data Analysis y Data analysis was conducted in four main steps using STATA Version 13.1 (Stata Corporation, College Station, TX). First, the 26 retained variables in the present study were operationalized to make them eligible for factor analysis. Before running any analysis procedure, the correlation matrix was examined to justify undertaking the factor analysis. The χ2 for the Bartlett test of sphericity was signiﬁcant at alpha = 0.01, and the Kaiser– Meyer–Olkin test showed a score of 0.80, indicating that the correlation among the variables was suﬃciently strong for a factor analysis. Next, we randomly split the data (by region) into two datasets: one for EFA and the other for CFA within each region, as recommended by Worthington and Whittaker (2006), Cabrera-Nguyen (2010) and Fokkema and Greiﬀ(2017). Therefore, we conducted exploratory and conﬁrmatory analyses on two separate datasets in each region. Given the contextual nature of women’s empowerment (Shimamoto and Gipson, 2015), all the factor analyses (EFA and CFA) were performed to identify the possible underlying factors and verify the factor structure for each region of sub-Saharan Africa. Indicators for Health Dimension This dimension includes the sex negotiation and access to healthcare domains. Women’s ability to negotiate sex was measured by indicators describing if they could refuse sex or ask their partner to use a condom. Women were scored 0 if they could not refuse sex or ask their partner to use a condom; otherwise, women were scored 1. Access to healthcare was classiﬁed by four indicators examining the diﬃculty in getting medical help, namely: (1) receiving permission before getting Because our data includes categorical variables, a factor analysis was performed using a polychoric correlation matrix (Holgado-Tello et al., 2010). Afterwards, we used the “factormat” command to conduct an EFA using the matrix as input rather June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 4 Measuring Women’s Empowerment in Africa Asaolu et al. than raw variables. The number of factors retained was based on three criteria: (i) the Kaiser criterion (eigenvalues >1); (ii) inﬂection point of the screen plot; and (iii) interpretability of factors (Costello and Osborne, 2005; Suhr, 2006). Furthermore, an oblique rotation was used over orthogonal rotation because of observed correlation among factors Holgado-Tello et al. (2010). women’s empowerment. However, individual factor loadings for attitude toward violence, labor force participation, and access to healthcare varied between regions. In Central and Southern Africa, attitude toward violence consisted of four items (goes out with telling partner, neglects children, argues with husband, and burns foods) while in East Africa it comprised the ﬁrst three items. In West Africa, attitude toward violence consists of four items: refuses sex, neglects children, argues with husband and burns food. Similarly, labor force participation includes type of earning, seasonality of occupation, and type of occupation for Southern Africa while it consisted of income ratio for other regions. Subsequently, we conducted a CFA on the remaining half of the randomly-split data to validate the hypothesized domains from the EFA. The CFA provided ﬁt indices about the appropriateness of the model based on the covariance structure of the observed data such as root mean square error of approximation (RMSEA), standardized root mean square (SRMS), Bentler comparative ﬁt index (CFI), and Tucker-Lewis index (TLI) (Schreiber et al., 2006). These modiﬁcation indices explored how the model might be adjusted to improve its ﬁt. Finally, we used the Lagrange test to derive a precise model by considering co-variance between variable in the same domain. Indicators for Health Dimension When the co-variance was >100, we removed the items with lower EFA factor loading, resulting in the ﬁnal set of variables (West Africa, n = 14; Central Africa, n = 13; East Africa, n = 10; and Southern Africa, n = 12) of included questions. The basis of such techniques is to explicitly penalize overly complex models and/or to test the model’s ability to generalize by evaluating its performance on a set of data not used for EFA, which is assumed to approximate the typical unseen data that a model will encounter. The initial and ﬁnal models with ﬁt-statistics are described in Appendix B. DISCUSSION This is the ﬁrst study to use exploratory and conﬁrmatory factor analyses to identify and validate domains of women’s empowerment across the extensive sociocultural and demographic diverse regions of sub-Saharan Africa. The study emerged three valid factors of women’s empowerment in East Africa, and four valid factors in Central, Southern, and West Africa. This study builds upon existing studies (Jennings et al., 2014; Phan, 2016; Ewerling et al., 2017) by employing an extensive number of variables to describe a region-speciﬁc and validated measure of women’s empowerment. In particular, this study expands on the SWEPR index by using a comprehensive list of indicators and domains of empowerment; Ewerling et al. (2017) used 15 variables in the principal component analysis in contrast with 26 variables used in our exploratory factor analysis (Ewerling et al., 2017). Although there is some overlap between the SWEPR index and this study’s measure of empowerment (i.e., variables describing attitudes toward violence and education), the current study presents “access to healthcare” and “labor force participation” as validated indicators of women’s empowerment. In addition this study provides region-speciﬁc indicators of women’s empowerment across sub-Saharan Africa. East Africa had three indicators of women’s empowerment, with “labor force participation” loading as the last indicator. In Central, Southern, and West Africa, however, there were four indicators of women’s with “labor force participation” loading as the second factor. Furthermore, this study used EFA to reﬁne the speciﬁc indicators of women’s empowerment from 10 to 4 domains. Finally, this study used an appropriate validation methodology, CFA, to validate the proposed measures of women’s empowerment across diﬀerent regions of sub-Saharan Africa. Frontiers in Psychology \| www.frontiersin.org June 2018 \| Volume 9 \| Article 994 RESULTS Scree plots showing the eigenvalues of the underlying factors derived from EFA are shown in Figure 1, with initial EFA by region shown on the initial EFA by region shown on the top and ﬁnal structure (after CFA and model reﬁnement) on the bottom. The 10 underlying factors submitted to EFA were reﬁned down to four factors in Central Africa, Southern Africa, and West Africa and three factors in East Africa. In all regions, the ﬁrst two factors accounted for most of the variation in the sample (53.4–61.2%, data not shown). Based on factor loadings from ﬁnal EFA analyses in Figure 2, the underlying domains that contribute to women’s empowerment include attitude toward violence, labor force participation, education, and access to healthcare. Speciﬁcally, the ﬁrst factor loaded on attitude toward violence indicators in all four regions. In Central, Southern, and West Africa, labor force participation items lined up in the second factor, items describing education loaded on the third factor, and variables representing access to healthcare items clustered in the fourth factor. In East Africa, however, the second and third factors loaded on access to healthcare indicator and labor force participation respectively. Education did not emerge as a factor of women’s empowerment in East Africa. The domain describing women’s attitudes toward violence emerged as the ﬁrst and persistent factor of women’s empowerment across all four regions. A substantial proportion of African women and men alike justify violence against women as evidenced in a systematic review of intimate partner violence studies conducted in three of the four African regions: Ghana, Kenya, Nigeria, Uganda, Zambia, and Zimbabwe. The review demonstrated that both sexes justiﬁed violence if woman burns food, neglects child, agues with or insults, or accuses partner of inﬁdelity (Waltermaurer, 2012). With a high prevalence of violence justiﬁcation, it is not surprising that many women experience intimate partner violence in African nations. In a Within the individual factors, loadings stayed consistent between regions, but a few changed ranks within a given factor, resulting in diﬀerent set of variables in the ﬁnal model (Table 2). For example, education consists of two variables (literacy and highest level of education) in Central, Southern and West Africa—the regions where the domain contributed to June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 5 Asaolu et al. Frontiers in Psychology \| www.frontiersin.org RESULTS Measuring Women’s Empowerment in Africa FIGURE 1 \| Scree plots of the initial EFA and the ﬁnal model (after CFA and revision). recent publication, prevalence estimates of physical or sexual intimate partner violence ranged from 11.5% in Burkina b d including HIV (Li et al., 2014). Therefore, the inﬂuence of violence intersects with another empowerment domain, h l h FIGURE 1 \| Scree plots of the initial EFA and the ﬁnal model (after CFA and revision). including HIV (Li et al., 2014). Therefore, the inﬂuence of violence intersects with another empowerment domain, health. recent publication, prevalence estimates of physical or sexual intimate partner violence ranged from 11.5% in Burkina Faso, 25.9% in Ivory Coast, 33.1% in Mozambique, and up to 35.2% in Zimbabwe (Peterman et al., 2015). Violence against women and girls can impede their health and socio-economic development. For instance, child brides are less likely to receive antenatal care and more likely to live in poor households (UNICEF, 2015). Women who experience intimate partner violence are at risk of poor pregnancy outcomes (Hill et al., 2016), depression, anxiety, and posttraumatic stress disorder (Lagdon et al., 2014), and sexually transmitted infections Labor force participation also emerged as a prominent indicator, suggesting that women’s economic empowerment remains salient to development. These results are consistent with a similar study of empowerment in Southeast Asia (Phan, 2016). Across sub-Saharan Africa, women’s economic empowerment is stalled by low educational attainment among women, cultural practices that place the burden of domestic work on women and girls, customs that inhibit women from owning lands June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 6 Asaolu et al. Measuring Women’s Empowerment in Africa FIGURE 2 \| Factor loadings for independent EFA analyses. FIGURE 2 \| Factor loadings for independent EFA analyses. FIGURE 2 \| Factor loadings for independent EFA analyses. June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 7 Measuring Women’s Empowerment in Africa Asaolu et al. TABLE 2 \| The four domains for empowerment as determined by exploratory factor analysis (latent variables analysis), with ﬁnal composition after conﬁrmatory factor analysis and model reﬁnement by regions. RESULTS Speciﬁcally, microﬁnance, local savings groups, and community banks can support poor African women who may not possess collateral for loans from advanced ﬁnancial institutions (van Rooyen et al., 2012). Also, the scaling of successful small and medium scale businesses should be supported by advanced ﬁnancial institutions and favorable trade policies. Supporting women’s economic empowerment through microﬁnance, international trade ventures, and eﬀective policies result in increased “achievements”, the last arm of empowerment (UN, 2016; Ouida et al., 2017). Kenya, and Rwanda, have been noted for reducing barriers to healthcare through the provision of national health insurance scheme, increased governmental for healthcare, or utilizing community health workers in expanding health services (EIU, 2017). Nonetheless, many African countries cannot ensure accessible healthcare for their citizens, causing most Africans to still pay out-of-pocket for their medical expenses (World Health Organization, 2013). It is estimated that 11 million Africans become impoverished because of exorbitant out-of- pocket expenses (EIU, 2017). It is therefore important for African governments to foster health and healthcare through increased spending on health, training of health workers, and implementation of universal health coverage. Removing barriers to healthcare empowers women by providing resources for which women can exercise their agency. Kenya, and Rwanda, have been noted for reducing barriers to healthcare through the provision of national health insurance scheme, increased governmental for healthcare, or utilizing community health workers in expanding health services (EIU, 2017). Nonetheless, many African countries cannot ensure accessible healthcare for their citizens, causing most Africans to still pay out-of-pocket for their medical expenses (World Health Organization, 2013). It is estimated that 11 million Africans become impoverished because of exorbitant out-of- pocket expenses (EIU, 2017). It is therefore important for African governments to foster health and healthcare through increased spending on health, training of health workers, and implementation of universal health coverage. Removing barriers to healthcare empowers women by providing resources for which women can exercise their agency. Surprisingly, there were regional diﬀerences in four domains of women’s empowerment. First, the justiﬁcation of spousal violence “when women refuse sex” was pertinent to only West Africa. Despite evidence for African women condoning spousal violence pertaining to women’s refusal of sex (Mugweni et al., 2015), many West African women still justify this form of violence (Uthman et al., 2009; Dako-Gyeke, 2013). RESULTS Domains Central Africa East Africa Southern Africa West Africa Attitude toward violence Goes out with telling partner Goes out with telling partner Goes out with telling partner Neglects children +Neglects children + Argues with husband +Burns food + Neglects children + Argues with husband + Neglects children + Argues with husband + Burns food + Argues with husband + Refuses sex + Burns food Work/labor force participation Type of earning + Seasonality + Type of occupation + Income ratio Type of earning + Seasonality + Type of occupation + Income ratio Type of earning + Seasonality + Income ratio Type of earning + Seasonality + Type of occupation + Income ratio Education Literacy + Level of education Literacy + Level of education Literacy + Level of education Access to healthcare Cannot go alone + Needs permission + Distance Cannot go alone + Needs Permission + Distance Cannot go alone + Needs money + Distance Cannot go alone + Needs permission + Needs money + Distance and properties, and workplace sexual harassment (Ouida et al., 2017). These challenges put women at a disadvantage in terms of career advancement, skill acquisition, access to markets, and scaling up successful jobs. Consequently, African women are less represented in international trade ventures such as exporting cash-crops despite the fact they are over-represented in small-scale farming (UN, 2016). Thus, to promote women’s economic empowerment, there must be concerted eﬀorts promoting business enterprise. Speciﬁcally, microﬁnance, local savings groups, and community banks can support poor African women who may not possess collateral for loans from advanced ﬁnancial institutions (van Rooyen et al., 2012). Also, the scaling of successful small and medium scale businesses should be supported by advanced ﬁnancial institutions and favorable trade policies. Supporting women’s economic empowerment through microﬁnance, international trade ventures, and eﬀective policies result in increased “achievements”, the last arm of empowerment (UN, 2016; Ouida et al., 2017). and properties, and workplace sexual harassment (Ouida et al., 2017). These challenges put women at a disadvantage in terms of career advancement, skill acquisition, access to markets, and scaling up successful jobs. Consequently, African women are less represented in international trade ventures such as exporting cash-crops despite the fact they are over-represented in small-scale farming (UN, 2016). Thus, to promote women’s economic empowerment, there must be concerted eﬀorts promoting business enterprise. Frontiers in Psychology \| www.frontiersin.org RESULTS The only available study on the diverse forms of spousal violence justiﬁcation showed that 27.8, 29.7, and 37.6% of women in Liberia, Nigeria, and Burkina-Faso justiﬁed wife beating when a woman refuses sex with her husband (Uthman et al., 2009). Second, although education was a prominent factor of women’s empowerment across other regions, it did not emerge for East Africa. This discrepancy may be explained by the fact that indicators of education in Comoros, Ethiopia, and Uganda vary vastly with 42, 49, and 73% of women aged 15– 24 completing their primary education in Ethiopia, Uganda, and Comoros respectively (UNEESCO, 2017). A similar trend was also observed for average years of education; women in Ethiopia have lower years (5.1) of education than women in Ugandan (6.1 years) and Comoros (8.1 years) (UNEESCO, 2017). Therefore, with Ethiopia performing poorer than Comoros and Uganda, there may be a poor correlation between education variables Access to healthcare was also a signiﬁcant contributor of women’s empowerment. The variables describing the access to healthcare domain—distance, money, and permission—embody the crux of empowerment, i.e., whether women have the “access” to make beneﬁcial health choices. Financial constraints can cause all three forms of delay highlighted by Thaddeus and Maine, i.e., delay in seeking care, getting to a medical facility, and receiving care. Furthermore, farther distance, poor road conditions, or unreliable/no transportation could result in delayed or no care (Thaddeus and Maine, 1994). Improved health outcomes can be predicted by national healthcare ﬁnancing. Unfortunately, public health expenditure is <4% of gross domestic product across several African countries (World Health Organization, 2013). In general, nations with limited governmental investment in health tend to have high out-of-pocket expenses, constituting an impediment to receiving healthcare. The impact of health ﬁnancing on health outcomes is evident by the inverse association between total health expenditure per capita and maternal mortality ratio and under-5 mortality rate (World Health Organization, 2013). East African countries including, Ethiopia, June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 8 Measuring Women’s Empowerment in Africa Asaolu et al. Three important factors were identiﬁed commonly across the regions; attitude toward violence, economics, and access to healthcare. Third, it explored regional level diﬀerences important to the cultural and economic context of sub-Saharan Africa. These regional level analyses revealed important variation; education emerged as a factor of empowerment in Central, Southern, and West Africa. AUTHOR CONTRIBUTIONS IA, HA, JG, and KE: Conceptualized the research. IA and HA: Research methodology was developed, Data analysis was conducted. IA, HA, and KE: Interpretation of Results was done. IA, HA, AA, and AM: The original manuscript was drafted. JG, JE, MH, and KE: Review and edits of manuscripts were prepared. IA, HA, JG, and KE: Conceptualized the research. IA and HA: Research methodology was developed, Data analysis was conducted. IA, HA, and KE: Interpretation of Results was done. IA, HA, AA, and AM: The original manuscript was drafted. JG, JE, MH, and KE: Review and edits of manuscripts were prepared. REFERENCES Dako-Gyeke, P. (2013). “Safe Sex Talk:” negotiating safe sex practices in heterosexual relationships. Medit. J. Soc. Sci. 4, 309–318. doi: 10.5901/mjss.2013.v4n2p309 African Development Bank. (2015). African Gender Equality Index. African Union (2016). 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The SWPER index for women’s empowerment in Africa: development and validation of an index based on survey data. Lancet Global Health 5, E916–E923. doi: 10.1016/S2214-109X(17)30292-9 Asaolu, I. O., Okafor, C. T., Ehiri, J. C., Dreifuss, H. M., and Ehiri, J. E. (2017). Association between measures of women’s empowerment and use of modern contraceptives: an analysis of Nigeria’s Demographic and Health Surveys. Front. Public Health 4:293. doi: 10.3389/fpubh.2016.00293 Fokkema, M., and Greiﬀ, S. (2017). How performing PCA and CFA on the same data equals trouble: overﬁtting in the assessment of internal structure and some editorial thoughts on it. Eur. J. Psychol. Assess. 33, 399–402. doi: 10.1027/1015-5759/a000460 Cabrera-Nguyen, P. (2010). Author guidelines for reporting scale development and validation results in the Journal of the Society for Social Work and Research. J. Soc. Soc. Work Res. 1, 99–103. doi: 10.5243/jsswr.2010.8 Hansen, N. (2015). The development of psychological capacity for action: the empowering eﬀect of a microﬁnance programme on women in Sri Lanka. J. Soc. Issues 71, 597–613. doi: 10.1111/josi.12130 Cheston, S., and Kuhn, L. (2001). Engendering Developme Cheston, S., and Kuhn, L. (2001). Engendering Development: Through Gender Equality in Rights, Resources, and Voice. Washington, DC: World Bank. Heaton, T. B. (2015). Are improvements in child health due to increasing status of women in developing nations? Biodemogr. Soc. Biol. 61, 252–265. doi: 10.1080/19485565.2015.1047487 Costello, A. LIMITATIONS There are two primary limitations to this study. First, due to the DHS’s study design, this study could not assess women’s cultural perception of empowerment. It is possible that women represented in these surveys have a diﬀerent perception of (un)empowerment than results presented in this study. Second, because most of the indicators of women were applicable to only partnered women, this study could not explore empowerment among single, widowed, divorced, or separated women. Another limitation is our inability to include more nations in the analyses. Precisely, East Africa had only three countries, which limits the generalizability of these ﬁndings to the region. RESULTS This study has overcome the shortcomings of current indices that mainly focus on developed countries, and recently on Asian countries where the perceptions of attitude toward violence and labor force participation may diﬀer from that of sub-Saharan Africa. This validated measure is a useful tool for global health researchers in assessing the impact of women’s empowerment on health outcomes. The measure also allows for comparisons across diﬀerent countries and regions of sub-Saharan Africa while accounting for unique characteristics of each context. and the latent construct of women’s empowerment in East Africa. Third, in West Africa, the reﬁned model excluded “age at ﬁrst cohabitation” and “age at ﬁrst birth,” leaving behind only indicators of education. These variables may have dropped out due to their high correlation with education which is associated with marriage and childbirth; West African girls often stop attending school once they are married and/or pregnant while those in school are less likely to be married and pregnant (UNICEF, 2015; Wodon et al., 2017). FUNDING This research eﬀort on this manuscript was not supported by any grant. Therefore, we did not include any grant or funding information. This work builds upon the growing body of literature using the DHS datasets to deﬁne indicators for women’s empowerment. This study improved upon previous indicators in several key ways. First, it is the ﬁrst to utilize exploratory and conﬁrmatory factor analyses in describing and validating the structure of women’s empowerment across sub-Saharan Africa. Second, it expanded the number of domains used in previous studies (Jennings et al., 2014; Alaofè et al., 2017; Ewerling et al., 2017). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2018.00994/full#supplementary-material REFERENCES B., and Osborne, J. W. (2005). Best practices in exploratory factor analysis: four recommendations for getting the most from your analysis. Pract. Assess. Res. Eval. 10, 1–9. June 2018 \| Volume 9 \| Article 994 Frontiers in Psychology \| www.frontiersin.org 9 Asaolu et al. Measuring Women’s Empowerment in Africa Hill, A., Pallitto, C., McCleary-Sills, J., and Garcia-Moreno, C. (2016). 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Age and intimate partner violence: an analysis of global trends among women experiencing victimization in 30 developing Countries. J. Adolesc. Health 57, 624–630. doi: 10.1016/j.jadohealth.2015.08.008 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Phan, L. (2016). Measuring women’s empowerment at household level using DHS data of four Southeast Asian Countries. Soc. Indic. Res. 126, 359–378. doi: 10.1007/s11205-015-0876-y Copyright © 2018 Asaolu, Alaofè, Gunn, Adu, Monroy, Ehiri, Hayden and Ernst. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W1661815949	https://link.springer.com/content/pdf/10.1007/s00432-015-2030-2.pdf	English	null	Modal variety of microsatellite instability in human endometrial carcinomas	Journal of cancer research and clinical oncology	2,015	cc-by	10,615	J Cancer Res Clin Oncol (2016) 142:353–363 DOI 10.1007/s00432-015-2030-2 ORIGINAL ARTICLE – CANCER RESEARCH Abstract Purpose Microsatellite instability (MSI) in human endo- metrial cancer (EC) was analysed using a unique fluores- cent technique. MSI is associated with various human neo- plasms. However, the reported frequency of MSI differs widely in each malignancy. Methodological difficulties have in fact been pointed out in its assay techniques. Methods We previously established a sensitive fluorescent technique in which the major methodological problems are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. In the present study, we have applied this tech- nique to 94 ECs. Results Significant microsatellite alterations were observed in 38 (40.4 %) tumours of the panel. The two modes, Type A and Type B, were indeed observed in this malignancy. More importantly, we found that the modes more closely correlated with the molecular and Keywords Microsatellite instability · DNA mismatch repair · Endometrial cancer · KRAS mutation · Familial predisposition Takako Eto1 · Yan Zhao2 · Akiko Maruyama1 · Kaname Miyashita3 · Aiko Yasui4 · Seiki Nakao4 · Kenichi Taguchi4 · Mototsugu Shimokawa4 · Shinya Oda4 · Toshiaki Saito1 Takako Eto1 · Yan Zhao2 · Akiko Maruyama1 · Kaname Miyashita3 · Aiko Yasui4 · Seiki Nakao4 · Kenichi Taguchi4 · Mototsugu Shimokawa4 · Shinya Oda4 · Toshiaki Saito1 Received: 12 May 2015 / Accepted: 7 August 2015 / Published online: 23 August 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com clinicopathological backgrounds of the tumours than the established and widely used MSI grades, MSI-H and MSI- L. Type B MSI widely correlated with family history of hereditary non-polyposis colorectal cancer-associated can- cers, whereas MSI-H only did with that of colorectal can- cer. Furthermore, mutation in the KRAS oncogene, which has been regarded as generally infrequent in microsatellite- unstable tumours, was clearly associated with Type A MSI. Conclusions Our observations may suggest a biologi- cal relevance and a potential utility of the modal classifi- cation of MSI and, furthermore, added complexities to genomic instability underlying tumourigenesis in human endometrium. 1 Gynecology Service, National Kyushu Cancer Center, Fukuoka 811‑1395, Japan 3 Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan 4 Clinical Research Institute, National Kyushu Cancer Center, Fukuoka 811‑1395, Japan * Shinya Oda soda@nk‑cc.go.jp Background Electronic supplementary material The online version of this article (doi:10.1007/s00432-015-2030-2) contains supplementary material, which is available to authorized users. Somatic instability of repetitive DNA sequences compris- ing minimal reiterative motifs, i.e. microsatellite instabil- ity (MSI), has initially been reported in tumours arising in Lynch syndrome [LS, alias hereditary non-polyposis colo- rectal cancer (HNPCC)] patients (Aaltonen et al. 1993), in which germline mutations in the genes functioning in DNA mismatch repair (MMR) are often found. MMR is an important cellular system that counteracts replication errors caused by DNA polymerases and, consequently, guarantees the high fidelity of DNA replication on the genome. Repeti- tive sequences such as microsatellites are particularly prone to replication errors, because DNA polymerases often slip on the repetitive sequences, and strand misalignment is formed. These replication errors, if uncorrected, are fixed 1 Gynecology Service, National Kyushu Cancer Center, Fukuoka 811‑1395, Japan 2 The Third Surgery Department, Liaoning Cancer Hospital and Institute, Shenyang 110042, The People’s Republic of China 2 The Third Surgery Department, Liaoning Cancer Hospital and Institute, Shenyang 110042, The People’s Republic of China 3 Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan 4 Clinical Research Institute, National Kyushu Cancer Center, Fukuoka 811‑1395, Japan 1 3 1 3 3 J Cancer Res Clin Oncol (2016) 142:353–363 354 oversimplification (Goel et al. 2003; Hawkins et al. 2001; Jass et al. 2002; Young et al. 2001). Attention has been drawn to the classification of the MSI phenotypes in human cancer. oversimplification (Goel et al. 2003; Hawkins et al. 2001; Jass et al. 2002; Young et al. 2001). Attention has been drawn to the classification of the MSI phenotypes in human cancer. during subsequent replication as addition or deletion of one or more repeat units. Thus, the phenomenon of MSI has been considered to reflect MMR deficiency in tumour cells. As MSI is frequently observed in various human neoplasms (Arzimanoglou et al. 1998), analyses of MSI have been prevalent in the fields of oncology or pathology. Numerous studies have been done on a wide variety of human malig- nancies and addressed the characteristics of MSI+ tumours. Microsatellite alterations are most frequently observed in tumours occurring in LS kindred. More than 90 % of LS tumours are MSI+ (Liu et al. 1996). In the sporadic setting, endometrial carcinoma (EC) is one of the most microsat- ellite-unstable human neoplasms. Background EC indeed occurs in LS patients and is the second most frequent malignancy in LS. Numerous data have been collected on the microsatellite alterations observed in EC, and similarly to colorectal can- cer, the MSI-H phenotype is now regarded as characteristic of a subset of ECs (Yeramian et al. 2013). However, also in EC, the data accumulated in the literature are diverse (see Discussion). In addition, the Type A/B classification has not been applied in assessing MSI in ECs. Using our unique dual-colour fluorescent technique (Oda et al. 1997), we analysed microsatellite alterations in detail in a rela- tively large panel of tumours from EC patients and found that the two modes of MSI, i.e. Type A and Type B, are indeed observed in this malignancy. More importantly, the modes more closely correlated with the clinicopathological and molecular backgrounds of the tumours than the MSI- H/L grades, which suggests that, in addition to the estab- lished classification according to the frequencies of micro- satellite changes, the modal classification may also be of potential use. Here, we report the modal variety of MSI in human endometrial cancer and its biological significance. The reported frequency for MSI+ tumours in each malignancy, however, differs widely in the literature. In order to manage the confusion raised in the field, the National Cancer Institute (NCI) sponsored the workshop, ‘Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition’ (Boland et al. 1998) in 1997, which concluded that the variety of microsatel- lites used was a major cause of discrepancies among data from various laboratories and, consequently, recommended a panel of five microsatellites as a ‘working reference panel’. In addition, the NCI workshop recommended that the MSI+ phenotype should be classified into two different grades, i.e. MSI-H (high) and MSI-L (low), according to the frequencies of changes in a defined set of microsatel- lite markers. However, the diversity of data in the literature has not improved since then. Analysis of MSI is now com- monplace, but several methodological problems have in fact been pointed out in the conventional assay techniques (Maehara et al. 2001) and may also account for the vari- ability in results. We previously established a unique fluo- rescent technique designated as high-resolution fluorescent microsatellite analysis (HRFMA), in which products of polymerase chain reaction (PCR) are precisely and quan- titatively resolved (Oda et al. 1997). Patients and tissue specimens Samples of cancer and the corresponding normal tissues were collected from consecutive 94 EC patients who under- went surgery in Gynaecology Division of National Kyushu Cancer Center (NKCC) from 1997 to 2003. According to histopathological diagnosis, non-endometrioid-type tumours were excluded from the patient panel. This panel includes one LS patient who fulfilled Amsterdam Criteria II (Vasen et al. 1999). Specimens, taken immediately after resection, were placed in liquid nitrogen, and then stored at −80 °C. Background In this technique, (a) electrophoretic profiles of microsatellites PCR products are simplified enzymatically or by primer sequence modifica- tions, (b) each DNA fragment is detected quantitatively by use of an automated DNA sequencer, and (c) two differ- ently labelled PCR products derived from tumour (red) and the corresponding normal tissues (green) are co-electro- phoresed (see Fig. 1), in order to exclude migration errors. Application of this technique has revealed a number of previously unrecognised aspects of MSI in human cancer. In particular, we found two qualitatively distinct patterns of microsatellite alterations, i.e. Type A and Type B (Oda et al. 2005). Although this distinction has not widely been discussed, our previous data suggest that different molec- ular abnormalities may underlie these two modes of MSI (Oda et al. 2005). It is widely known that tumours exhibit- ing the MSI-H phenotype form a distinct entity with unique clinicopathological and molecular characteristics, particu- larly in colorectal cancer (Jass et al. 2002), and accord- ingly, two mutually exclusive pathways are hypothesised in colorectal tumourigenesis (Lengauer et al. 1998). Several reports however suggest that this distinction might be an DNA extraction Tissue specimens were lysed in digestion buffer (10 mM Tris-Cl pH 8.0, 0.1 M EDTA pH 8.0, 0.5 % SDS, 20 µg/ ml pancreatic RNase). After treatment with proteinase K and extraction with phenol, DNA was precipitated with ethanol, then dissolved in 1X TE (10 mM Tris-Cl pH 7.5, 1 3 3 355 J Cancer Res Clin Oncol (2016) 142:353–363 1 mM EDTA). The concentration of DNA was determined by OD260 using a spectrophotometer. The quality of DNA was checked by agarose gel electrophoresis. Microsatellite instability Microsatellite analysis using fluorescence-labelled prim- ers and an automated DNA sequencer has been described in detail (Oda et al. 1997). Briefly, five selected human dinucleotide microsatellites, D2S123, D5S107, D10S197, D11S904 and D13S175 (Oki et al. 1999), were ampli- fi d b l h i ti (PCR) M l Discussion). Forward primers were labelled with the fluo- rescent compound, 6-FAM (6-carboxyfluorescein) or HEX (6-carboxy-2′, 4′, 7′, 4, 7, hexachloro-fluorescein). TaKaRa Taq (Takara Bio Inc., Otsu, Japan) was used as a thermo- stable polymerase. To compare the electrophoretic profiles between two samples, 6-FAM-labelled products and HEX- labelled products were mixed and co-electrophoresed in the ABI310 sequencer (Life Technologies, Carlsbad, CA, USA). The data were processed using the GeneScan soft- ware (Life Technologies). DNA i Fig. 1 Microsatellite alterations observed in human endome- trial cancer. Using genomic DNA samples prepared from tumour and the corresponding normal tissues, microsatel- lite sequences, indicated at the right top of each panel, were independently amplified by PCR with differentially labelled primers, then mixed and co-electrophoresed in an automated DNA sequencer. Representative results are shown and detected microsatel- lite alterations are indicated by arrows: red lines cancer, green lines normal tissues, a negative cases, b Type A MSI, c Type B MSI, d cases suspected for LOH. Data in cases of separate and inseparable heterozygous microsatellites are shown in left and right columns, respectively. DNA extraction To compare the electrophoretic profiles tween two samples, 6-FAM-labelled products and HEX- belled products were mixed and co-electrophoresed in e ABI310 sequencer (Life Technologies, Carlsbad, CA, SA). The data were processed using the GeneScan soft- re (Life Technologies). 0 690 460 230 D10S197 37 160 0 0 2 0 2 1 D2S123 61 0 5 2 140 180 120 160 140 0 8 1 0 0 1 (-bp) 0 2400 1600 800 D5S107 32 0 8 1 (-bp) D5S107 6 (-bp) 140 180 160 0 0 2 0 2 1 0 3600 2400 1200 D10S197 48 (-bp) 0 5 2 D2S123 52 (-bp) 0 3600 2400 1200 D13S175 47 80 120 100 0 4 1 0 6 (-bp) 200 240 220 0 6 2 0 8 1 0 840 560 280 D2S123 7 (-bp) 0 7 2 D2S123 1 120 160 140 0 8 1 0 0 1 (-bp) 0 2100 1400 700 D5S107 6 B B (-bp) 210 250 230 0 7 2 0 9 1 0 840 560 280 D2S123 1 C D (-bp) 190 230 210 0 5 2 0 7 1 0 2100 1400 700 D2S123 52 D Discussion). Forward primers were labelled with the fluo- rescent compound, 6-FAM (6-carboxyfluorescein) or HEX (6-carboxy-2′, 4′, 7′, 4, 7, hexachloro-fluorescein). TaKaRa Taq (Takara Bio Inc., Otsu, Japan) was used as a thermo- stable polymerase. To compare the electrophoretic profiles between two samples, 6-FAM-labelled products and HEX- labelled products were mixed and co-electrophoresed in the ABI310 sequencer (Life Technologies, Carlsbad, CA, USA). The data were processed using the GeneScan soft- ware (Life Technologies). 1 mM EDTA). The concentration of DNA was determined by OD260 using a spectrophotometer. The quality of DNA was checked by agarose gel electrophoresis. 1 mM EDTA). The concentration of DNA was determined by OD260 using a spectrophotometer. The quality of DNA was checked by agarose gel electrophoresis. Microsatellite instability Microsatellite analysis using fluorescence-labelled prim- ers and an automated DNA sequencer has been described in detail (Oda et al. 1997). Briefly, five selected human dinucleotide microsatellites, D2S123, D5S107, D10S197, D11S904 and D13S175 (Oki et al. 1999), were ampli- fied by polymerase chain reaction (PCR). Mononucleo- tide microsatellites were not used in order to exclude the influence of the terminal deoxynucleotidyl transferase (TDT) activity of thermostable DNA polymerases (see DNA extraction Patient codes corresponding to those used in Table 1 are also indicated at the right of each panel (-bp) 0 690 460 230 D10S197 37 160 0 0 2 0 2 1 (-bp) 0 1500 1000 500 D2S123 61 190 230 210 0 5 2 0 7 1 A 140 180 120 160 140 0 8 1 0 0 1 (-bp) 0 2400 1600 800 D5S107 32 120 160 140 0 8 1 0 0 1 (-bp) 0 2100 1400 700 D5S107 6 B D (-bp) 140 180 160 0 0 2 0 2 1 0 3600 2400 1200 D10S197 48 (-bp) 190 230 210 0 5 2 0 7 1 0 2100 1400 700 D2S123 52 (-bp) 0 3600 2400 1200 D13S175 47 80 120 100 0 4 1 0 6 (-bp) 200 240 220 0 6 2 0 8 1 0 840 560 280 D2S123 7 (-bp) 210 250 230 0 7 2 0 9 1 0 840 560 280 D2S123 1 C J Cancer Res Clin Oncol (2016) 142:353–363 355 Fig. 1 Microsatellite alterations observed in human endome- trial cancer. Using genomic DNA samples prepared from tumour and the corresponding normal tissues, microsatel- lite sequences, indicated at the right top of each panel, were independently amplified by PCR with differentially labelled primers, then mixed and co-electrophoresed in an automated DNA sequencer. Representative results are shown and detected microsatel- lite alterations are indicated by arrows: red lines cancer, green lines normal tissues, a negative cases, b Type A MSI, c Type B MSI, d cases suspected for LOH. Data in cases of separate and inseparable heterozygous microsatellites are shown in left and right columns, respectively. DNA extraction Patient codes corresponding to those used in Table 1 are also indicated at the right of each panel (-bp) 0 690 460 230 D10S197 37 160 0 0 2 0 2 1 (-bp) 0 1500 1000 500 D2S123 61 190 230 210 0 5 2 0 7 1 A 140 180 120 160 140 0 8 1 0 0 1 (-bp) 0 2400 1600 800 D5S107 32 120 160 140 0 8 1 0 0 1 (-bp) 0 2100 1400 700 D5S107 6 B D (-bp) 140 180 160 0 0 2 0 2 1 3600 (-bp) 190 230 210 0 5 2 0 7 1 0 2100 1400 700 D2S123 52 (-bp) 0 3600 2400 1200 D13S175 47 80 120 100 0 4 1 0 6 (-bp) 200 240 220 0 6 2 0 8 1 0 840 560 280 D2S123 7 (-bp) 210 250 230 0 7 2 0 9 1 0 840 560 280 D2S123 1 C (-bp) 0 1500 1000 500 D2S123 61 190 230 210 0 5 2 0 7 1 A 120 160 140 0 8 1 0 0 1 (-bp) 0 2100 1400 700 D5S107 6 B (-bp) 210 250 230 0 7 2 0 9 1 0 840 560 280 D2S123 1 C (-bp) 0 1500 1000 500 D2S123 61 190 230 210 0 5 2 0 7 1 A Fig. 1 Microsatellite alterations observed in human endome- trial cancer. Using genomic DNA samples prepared from tumour and the corresponding normal tissues, microsatel- lite sequences, indicated at the right top of each panel, were independently amplified by PCR with differentially labelled primers, then mixed and co-electrophoresed in an automated DNA sequencer. Representative results are shown and detected microsatel- lite alterations are indicated by arrows: red lines cancer, green lines normal tissues, a negative cases, b Type A MSI, c Type B MSI, d cases suspected for LOH. Data in cases of separate and inseparable heterozygous microsatellites are shown in left and right columns, respectively. Patient codes corresponding to those used in Table 1 are also indicated at the right of each panel scussion). Forward primers were labelled with the fluo- cent compound, 6-FAM (6-carboxyfluorescein) or HEX carboxy-2′, 4′, 7′, 4, 7, hexachloro-fluorescein). TaKaRa q (Takara Bio Inc., Otsu, Japan) was used as a thermo- ble polymerase. Results 3′ exonuclease activity, TaKaRa Ex Taq™ (Takara Bio Inc.). The sequencing strategy used is the same as the one reported by Kolodner Kolodner et al. (1994, 1995), except that the sequence complementary for M13 universal primer was deleted from each of the primer sequences, and that one-step PCR was employed. PCR products were used as a template for cycle sequencing reactions using BigDye Terminator Cycle Sequencing Kit (Life Technologies). Mutations found in one PCR product were verified by reverse sequencing and finally confirmed in two independently amplified PCR products. High‑resolution fluorescent microsatellite analysis (HRFMA) reveals the modal variety of microsatellite instability in endometrial cancer In MSI+ tumours, changes in microsatellite lengths are sometimes minor and as small as loss or gain of a sin- gle repeat unit, and cells carrying microsatellite changes are not always major in a given sample. However, using HRFMA, such minor and subtle alterations are sensi- tively and quantitatively detected, and results are highly reproducible in several independent experiments. We have applied this technique for MSI analyses in a panel of 94 ECs, in order to analyse microsatellite alterations in this malignancy in detail. The system sensitively detected vari- ous microsatellite changes. Examples are shown in Fig. 1. The basic electrophoretic profile of PCR-amplified dinu- cleotide microsatellite sequences is a cluster of three two base-pitched peaks. In human populations, microsatellites are highly polymorphic and, therefore, the parental alleles are different in length in many cases. In such cases, two peak clusters are separate (Fig. 1a, left) or, sometimes, partially overlap (Fig. 1a, right). Abnormal microsatel- lite length changes in cancer cells are detected as appear- ance of new peaks or changes in the peak heights in the electrophoretic profiles (Fig. 1, red lines). In the analyses of ECs, microsatellite length alterations were relatively small and within 6-bp in some cases (Fig. 1b). In the other, more drastic changes involving longer than 6-bp were also observed (Fig. 1c). We have designated the former type of microsatellite alterations as Type A and the latter as Type B in our previous observations (Oda et al. 2005). As Type B alterations involve large and, sometimes, discontinu- ous differences in microsatellite length, it can appear as if ‘new’ alleles, in addition to the parental alleles, are present (Fig. 1c). In EC, this type of microsatellite changes were frequently observed (Table 1), which is in clear contrast to other malignancies including colorectal cancer (Ikeda et al. 2001). In DNA fragment analyses using an automated sequencer, loss of heterozygosity (LOH) is also detectable (Fig. 1d, left), but some patterns of peak clusters are theo- retically indistinguishable between MSI and LOH (Fujii et al. 2009) (Fig. 1d, right). These cases are indicated as ‘LOH’’ in Table 1. Despite minimised false positives, sig- nificant microsatellite alterations were observed in a con- siderable number of the subjects of the panel, and, conse- quently, the frequency of MSI+ tumours has been estimated to be 40.4% (38/94). Immunohistochemistry Tissue specimens were fixed in buffered 10 % formaldehyde and embedded in paraffin. Prior to the assay, the specimens were sectioned at 4 µm and deparaffinised using xylene. Antigen retrieval was done by heating the sections in cit- rate buffer. Endogenous peroxidase activity was blocked by incubation in 3.0 % H2O2. Non-specific protein binding was inhibited by incubation in 10 % normal goat serum. The sections were reacted with an adequately diluted anti- MSH2 mouse monoclonal antibody, clone GB12 (Calbio- chem Novabiochem-Oncogen, Oncogen Research Products, Cambridge, MA, USA), or an anti-MLH1 antibody, clone 14 (Calbiochem Novabiochem-Oncogen, Oncogen Research Products) at 4 °C overnight. The sections were next reacted with biotinylated goat anti-mouse IgG antibody (Dako Den- mark A/S, Glostrup, Denmark) and then with peroxidase- labelled streptavidin (Dako Denmark A/S). Finally, the sections were incubated with diaminobenzidine and H2O2. Counterstaining was done using Mayer’s haematoxylin. Negative control experiments were also performed by replac- ing the primary antibodies with a non-specific mouse IgG. DNA sequencing All the exons and exon-intron junctions of MSH2 and MLH1 were amplified by PCR using Taq polymerase with 1 3 3 356 J Cancer Res Clin Oncol (2016) 142:353–363 High‑resolution fluorescent microsatellite analysis (HRFMA) reveals the modal variety of microsatellite instability in endometrial cancer According to the established NCI guideline, the MSI+ ECs were also classified into MSI-H and MSI-L (Table 1). The frequencies for MSI-H and MSI-L were 37.2 % (35/94) and 3.2 % (3/94), respectively. The 107-bp region in the exon 2 of the KRAS gene that encompasses the mutation hotspots, codons 12 and 13, was amplified by PCR using ‘c-Ki-ras/12 primer set’ (‘c-Ki-ras/12 forward’, 5′-GACTGAATATAAACTT- GTGG; ‘c-Ki-ras/12 reverse’, 5′-CTATTGTTGGATCAT- ATTCG) (Takara Bio Inc.) and TaKaRa Ex Taq™ (Takara Bio Inc.). PCR products were directly used as a template for cycle sequencing reactions using BigDye termina- tor cycle sequencing kit (Life Technologies). The reverse primer, ‘c-Ki-ras/12 reverse’, was used for cycle sequenc- ing reactions. Mutations found in one PCR product were verified by sequencing using our original inner primer that is 5′-adjacent to ‘c-Ki-ras/12 reverse’, KRAS2EX2R2 (5′-TCCACAAAATGATTCTGAATTAGC), and finally confirmed in three independently amplified PCR products. Statistical analysis Fisher’s exact probability test was used in all the statistical analyses. Statistical analysis 1 3 1 3 J Cancer Res Clin Oncol (2016) 142:353–363 357 1 3 Patient code Microsatellite MSI A/B MSI-H/L D2S D5S D10S D11S D13S 1 B B B – B B H 2 B B B A – Ba H 3 B B B A A B H 4 B B B A B B H 5 A B B A B B H 6 B A B – B B H 7 B B B A B B H 8 B B B A B B H 9 B A B – B B H 10 A A B A B B H 11 A B B – A B H 12b B B A A A B H 13 B A B – A B H 14 A B B – A B H 15 A B B A – B H 16 B A B A – B H 17 B – A – B B H 18 A B B A A B H 19 A A B A B B H 20 B A A – A B H 21 A A B A A B H 22 A A B A A B H 23 A A B A A B H 24 A B A A A B H 25 B A A A A B H 26 B A A A – B H 27 B A A – A B H 28 A A A – B B H 29 A A B – – B H 30 A B A – A B H 31 A A A – A A H 32 A A A A – A H 33 A A LOH’c A A A H 34 A – – A A A H 35 A – A – – A H 36 A – – – – A L 37 A – – LOH’ – A L 38 LOH A – – – A L 39 LOH’ – – – N S 40 – – – – LOH’ N S 41 – LOH’ LOH’ – – N S 42 – – – LOH’ – N S 43 LOH’ – – – – N S 44 LOH’ LOH’ – – LOH’ N S 45 – LOH’ – LOH – N S 46 – LOH – – LOH’ N S 47 – LOH LOH’ LOH LOH’ N S 48 LOH – LOH’ – – N S 49 – – LOH LOH LOH’ N S 50 LOH – LOH’ – – N S 51 LOH – LOH’ – – N S 52 LOH – – LOH LOH’ N S Table 1 Microsatellite alterations observed n 94 endometrioid endometrial cancer patients Patient code Microsatellite MSI A/B MSI-H/L D2S D5S D10S D11S D13S 1 B B B – B B H 2 B B B A – Ba H 3 B B B A A B H 4 B B B A B B H 5 A B B A B B H 6 B A B – B B H 7 B B B A B B H 8 B B B A B B H 9 B A B – B B H 10 A A B A B B H 11 A B B – A B H 12b B B A A A B H 13 B A B – A B H 14 A B B – A B H 15 A B B A – B H 16 B A B A – B H 17 B – A – B B H 18 A B B A A B H 19 A A B A B B H 20 B A A – A B H 21 A A B A A B H 22 A A B A A B H 23 A A B A A B H 24 A B A A A B H 25 B A A A A B H 26 B A A A – B H 27 B A A – A B H 28 A A A – B B H 29 A A B – – B H 30 A B A – A B H 31 A A A – A A H 32 A A A A – A H 33 A A LOH’c A A A H 34 A – – A A A H 35 A – A – – A H 36 A – – – – A L 37 A – – LOH’ – A L 38 LOH A – – – A L 39 LOH’ – – – N S 40 – – – – LOH’ N S 41 – LOH’ LOH’ – – N S 42 – – – LOH’ – N S 43 LOH’ – – – – N S 44 LOH’ LOH’ – – LOH’ N S 45 – LOH’ – LOH – N S 46 – LOH – – LOH’ N S 47 – LOH LOH’ LOH LOH’ N S 48 LOH – LOH’ – – N S 358 J Cancer Res Clin Oncol (2016) 142:353–363 MSI microsatellite instability, A Type A MSI, B Type B MSI, H MSI-high, L MSI-low, IHC immunohisto- chemistry, + expressed, − no change/not expressed, ND not done, LOH loss of heterozygosity a Tumours are classified as Type B when Type B alterations are observed in at least one marker b The patient fulfilled Amsterdam Criteria II (Vasen et al. a Tumours are classified as Type B when Type B alterations are observed in at least one marker MSI microsatellite instability, A Type A MSI, B Type B MSI, H MSI-high, L MSI-low, IHC immunohisto- chemistry, + expressed, − no change/not expressed, ND not done, LOH loss of heterozygosity b The patient fulfilled Amsterdam Criteria II (Vasen et al. 1999), and a deleterious mutation (I586delT) of MLH1 was found in the tumour. Sequence alterations were not detected in the other tumours c Changes theoretically indistinguishable between MSI and LOH are indicated as ‘LOH’’ (see text) MSI microsatellite instability, A Type A MSI, B Type B MSI, H MSI-high, L MSI-low, IHC immunohisto- chemistry + expressed no change/not expressed ND not done LOH loss of heterozygosity Statistical analysis 1999), and a deleterious mutation (I586delT) of MLH1 was found in the tumour. Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds Table 3 Clinicopathological variables and the NCI classification of MSI in endometrial cancer We next examined whether the observed microsatellite-unsta- ble phenotypes correlated with common clinicopathological variables of the tumours. In EC, the MSI+ phenotype, particu- larly the MSI-H phenotype, has been reported to correlate with tumour grade, stage and patient survivals (see Supplementary Table). However, none of these parameters correlated with the MSI-H phenotype in this study (Table 3). Instead, we found a significant correlation with patient age and, more importantly, family history of colorectal cancer. This tendency was also confirmed in the Type A/B classification. The more important finding is that Type B MSI correlated not only with family his- tory of colorectal cancer but also with that of ‘HNPCC-asso- ciated cancers’ (Vasen et al. 1999) (Table 4), which frequently arise in LS patients and includes, in addition to colorectal can- cer and EC, carcinomas in the stomach, pancreas, small intes- tine, ovary and the biliary or urinary tracts and the specific types of tumours in the brain and the skin. The MSI+ phenotype is observed in more than 90 % of LS tumours (Liu et al. 1996) and now regarded as a molecular hallmark of LS. The strong association between Type B MSI and HNPCC- associated cancers may suggest that the Type B phenotype may better reflect the biological backgrounds of the tumours. The MSI+ phenotype in EC has also been reported to correlate with various gene mutations or gene expression changes in cancer cells. We therefore tested whether Type A/B phenotypes correlated with these molecular abnor- malities. The KRAS gene is one of the most frequently mutated oncogenes in various human malignancies and is indeed known to be mutated in EC. Several early stud- ies have reported that KRAS mutation was significantly associated with the MSI+ phenotype (Duggan et al. 1994; Lagarda et al. 2001). We therefore sequenced the genomic region of the KRAS gene that encompasses the most fre- quently mutated codons, i.e. codons 12 and 13, in the MSI+ tumours of our panel. KRAS mutation was found in five tumours. Intriguingly, KRAS mutation was more closely associated with Type A MSI than with MSI-H/L (Tables 5 and 6). This finding is consistent with our previous obser- vation that KRAS mutation was frequently found in Type A colorectal tumours (Zhao et al. 2008). Statistical analysis KRAS mutation was also frequent in tumours without family history of malig- nancies, although this tendency was not statistically signifi- cant (p = 0 07 data not shown) Table 2 Relationship between MSI-H/L and Type A/B MSI p = 0.01 Type A Type B Subtotal MSI-H 5 30 35 MSI-L 3 0 3 Subtotal 8 30 38 Table 3 Clinicopathological variables and the NCI classification of MSI in endometrial cancer a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). The MSI grade p value MSI-L MSI-H MSS Number of cases 3 35 56 Age ≤55 3 23 24 0.02 ≥56 0 12 32 Stage 1 2 24 39 0.79 2 0 0 2 3 1 9 14 4 0 2 1 Grade 1 0 14 23 0.45 2 2 12 24 3 1 9 9 Survival Alive 3 33 47 0.31 Dead 0 2 9 Family history Any cancer Yes 1 22 30 0.54 No 2 13 26 Colorectal cancer Yes 1 7 3 0.03 No 2 28 53 Gastric cancer Yes 0 11 16 0.70 No 3 24 40 HNPCC-associated cancersa Yes 1 16 16 0.20 No 2 19 40 Double cancer Yes 0 4 12 0.43 No 3 31 44 Menopause Before 2 12 19 0.58 After 1 23 37 J Cancer Res Clin Oncol (2016) 142:353–363 359 J Cancer Res Clin Oncol (2016) 142:353 363 Table 2 Relationship between MSI-H/L and Type A/B MSI p = 0.01 Type A Type B Subtotal MSI-H 5 30 35 MSI-L 3 0 3 Subtotal 8 30 38 Table 3 Clinicopathological variables and the NCI classification of MSI in endometrial cancer a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). Statistical analysis The other HNPCC-related tumours were not found in the patients’ kindred MSI grade p value MSI-L MSI-H MSS Number of cases 3 35 56 Age ≤55 3 23 24 0.02 ≥56 0 12 32 Stage 1 2 24 39 0.79 2 0 0 2 3 1 9 14 4 0 2 1 Grade 1 0 14 23 0.45 2 2 12 24 3 1 9 9 Survival Alive 3 33 47 0.31 Dead 0 2 9 Family history Any cancer Yes 1 22 30 0.54 No 2 13 26 Colorectal cancer Yes 1 7 3 0.03 No 2 28 53 Gastric cancer Yes 0 11 16 0.70 No 3 24 40 HNPCC-associated cancersa Yes 1 16 16 0.20 No 2 19 40 Double cancer Yes 0 4 12 0.43 No 3 31 44 Menopause Before 2 12 19 0.58 After 1 23 37 Table 2 Relationship between MSI-H/L and Type A/B MSI p = 0.01 Type A Type B Subtotal MSI-H 5 30 35 MSI-L 3 0 3 Subtotal 8 30 38 The relationships between MSI-H/L and Type A/B MSI are expressed in Table 2, which are highly parallel to the figure obtained in our previous observations of colorectal cancer (Ikeda et al. 2001). Statistical analysis Sequence alterations were not detected in the other tumours c Changes theoretically indistinguishable between MSI and LOH are indicated as ‘LOH’’ (see text) Patient code Microsatellite MSI A/B MSI-H/L D2S D5S D10S D11S D13S 53 LOH – – – – N S 54 – LOH LOH – N S 55 – – LOH – LOH N S 56 – – – – LOH N S 57 – LOH – – N S 58 – – LOH LOH – N S 59 – – LOH – – N S 60 – LOH – – N S 61 – – – – – N S 62 – – – – – N S 63 – – – – – N S 64 – – – – – N S 65 – – – – – N S 66 – – – – – N S 67 – – – – – N S 68 – – – – – N S 69 – – – – – N S 70 – – – – – N S 71 – – – – – N S 72 – – – – – N S 73 – – – – – N S 74 – – – – – N S 75 – – – – – N S 76 – – – – – N S 77 – – – – – N S 78 – – – – – N S 79 – – – – – N S 80 – – – – – N S 81 – – – – – N S 82 – – – – – N S 83 – – – – – N S 84 – – – – – N S 85 – – – – – N S 86 – – – – – N S 87 – – – – – N S 88 – – – – – N S 89 – – – – – N S 90 – – – – – N S 91 – – – – – N S 92 – – – – – N S 93 – – – – – N S 94 – – – – – N S Table 1 continued – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – N – – – – – N – – – – – N – – – – – – – – – – – – – – – – – – – – 1 1 3 3 359 J Cancer Res Clin Oncol (2016) 142:353–363 The relationships between MSI-H/L and Type A/B MSI are expressed in Table 2, which are highly parallel to the figure obtained in our previous observations of colorectal cancer (Ikeda et al. Statistical analysis 2001). Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds We next examined whether the observed microsatellite-unsta- ble phenotypes correlated with common clinicopathological variables of the tumours. In EC, the MSI+ phenotype, particu- larly the MSI-H phenotype, has been reported to correlate with tumour grade, stage and patient survivals (see Supplementary Table). However, none of these parameters correlated with the MSI-H phenotype in this study (Table 3). Instead, we found a significant correlation with patient age and, more importantly, family history of colorectal cancer. This tendency was also confirmed in the Type A/B classification. The more important finding is that Type B MSI correlated not only with family his- tory of colorectal cancer but also with that of ‘HNPCC-asso- ciated cancers’ (Vasen et al. 1999) (Table 4), which frequently arise in LS patients and includes, in addition to colorectal can- cer and EC, carcinomas in the stomach, pancreas, small intes- tine, ovary and the biliary or urinary tracts and the specific types of tumours in the brain and the skin. The MSI+ phenotype is observed in more than 90 % of LS tumours (Liu et al. 1996) and now regarded as a molecular hallmark of LS. The strong association between Type B MSI and HNPCC- associated cancers may suggest that the Type B phenotype may better reflect the biological backgrounds of the tumours. The MSI+ phenotype in EC has also been reported to correlate with various gene mutations or gene expression changes in cancer cells. We therefore tested whether Type A/B phenotypes correlated with these molecular abnor- malities. The KRAS gene is one of the most frequently mutated oncogenes in various human malignancies and is indeed known to be mutated in EC. Several early stud- ies have reported that KRAS mutation was significantly associated with the MSI+ phenotype (Duggan et al. 1994; Lagarda et al. 2001). We therefore sequenced the genomic region of the KRAS gene that encompasses the most fre- quently mutated codons, i.e. codons 12 and 13, in the MSI+ tumours of our panel. KRAS mutation was found in five tumours. Intriguingly, KRAS mutation was more closely associated with Type A MSI than with MSI-H/L (Tables 5 and 6). This finding is consistent with our previous obser- vation that KRAS mutation was frequently found in Type A colorectal tumours (Zhao et al. 2008). Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds The other HNPCC-related tumours were not found in the patients’ kindred MSI mode p value Type A Type B negative Number of cases 8 30 56 Age <55 8 18 24 0.01 >56 0 12 32 Stage 1 5 21 39 0.76 2 0 0 2 3 3 7 14 4 0 2 1 Grade 1 2 12 23 0.65 2 3 11 24 3 3 7 9 Survival Alive 8 28 47 0.36 Dead 0 2 9 Family history Any cancer Yes 3 20 30 0.26 No 5 10 26 Colorectal cancer Yes 1 7 3 0.04 No 7 23 53 Gastric cancer Yes 0 11 16 0.14 No 8 19 40 HNPCC-associated cancersa Yes 1 16 16 0.03 No 7 14 40 Double cancer Yes 0 4 12 0.33 No 8 26 44 Menopause Before 3 11 19 0.95 After 5 19 37 Table 5 KRAS mutation and MSI-H/L P = 0.35 MSI-L MSI-H Subtotal KRAS Mutant 1 4 5 Wild type 2 31 33 Subtotal 3 35 38 Table 4 Clinicopathological variables and the MSI mode of MSI in endometrial cancer Expression of one of the essential MMR genes, MLH1, is known to be often lost in microsatellite-unstable ECs, primarily due to silencing by promoter methylation (Her- man et al. 1998), and another essential MMR gene, MSH2, has also been reported to be expressed at low levels in some previous studies (Hardisson et al. 2003; Ju et al. 2006; Peiro et al. 2002). Loss of expression of these essen- tial MMR genes causes defective MMR in cells and, con- sequently, leads to a destabilisation of microsatellites on the genome. Using immunohistochemistry, we examined expression of MSH2 and MLH1 proteins in the EC tissues of our panel. Although both antigens were not detected in several cases, we found that loss of MLH1 expression was observed in the majority of MSI+ tumours (22/36), whereas being significantly less frequent in microsatellite-stable tumours (4/51, p < 0.01). We further examined whether MLH1 silencing is more closely associated with any of the MSI subcategories. However, MLH1 expression loss was similarly frequent both in Type B and in MSI-H tumours, and therefore, the correlations were not significantly dif- ferent between the two classifications (data not shown). Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds KRAS mutation was also frequent in tumours without family history of malig- nancies, although this tendency was not statistically signifi- cant (p = 0.07, data not shown). a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). The other HNPCC-related tumours were not found in the patients’ kindred 1 3 3 360 J Cancer Res Clin Oncol (2016) 142:353–363 Table 4 Clinicopathological variables and the MSI mode of MSI in endometrial cancer a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). The other HNPCC-related tumours were not found in the patients’ kindred MSI mode p value Type A Type B negative Number of cases 8 30 56 Age <55 8 18 24 0.01 >56 0 12 32 Stage 1 5 21 39 0.76 2 0 0 2 3 3 7 14 4 0 2 1 Grade 1 2 12 23 0.65 2 3 11 24 3 3 7 9 Survival Alive 8 28 47 0.36 Dead 0 2 9 Family history Any cancer Yes 3 20 30 0.26 No 5 10 26 Colorectal cancer Yes 1 7 3 0.04 No 7 23 53 Gastric cancer Yes 0 11 16 0.14 No 8 19 40 HNPCC-associated cancersa Yes 1 16 16 0.03 No 7 14 40 Double cancer Yes 0 4 12 0.33 No 8 26 44 Menopause Before 3 11 19 0.95 After 5 19 37 Table 5 KRAS mutation and MSI-H/L MSI-L MSI-H Subtotal KRAS Mutant 1 4 5 Wild type 2 31 33 Subtotal 3 35 38 1 Expression of one of the essential MMR genes, MLH1, is known to be often lost in microsatellite-unstable ECs, primarily due to silencing by promoter methylation (Her- man et al. 1998), and another essential MMR gene, MSH2, has also been reported to be expressed at low levels in some previous studies (Hardisson et al. 2003; Ju et al. 2006; Peiro et al. 2002). Loss of expression of these essen- tial MMR genes causes defective MMR in cells and, con- sequently, leads to a destabilisation of microsatellites on the genome. Using immunohistochemistry, we examined expression of MSH2 and MLH1 proteins in the EC tissues of our panel. Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds Although both antigens were not detected in several cases, we found that loss of MLH1 expression was observed in the majority of MSI+ tumours (22/36), whereas being significantly less frequent in microsatellite-stable tumours (4/51, p < 0.01). We further examined whether MLH1 silencing is more closely associated with any of the MSI subcategories. However, MLH1 expression loss was similarly frequent both in Type B and in MSI-H tumours, and therefore, the correlations were not significantly dif- ferent between the two classifications (data not shown). Contrary to the results of the MLH1 immunohistochemis- try, MSH2 expression loss was similarly infrequent both in MSI+ tumours and in those with stable microsatellites and, consequently, not associated with the MSI+ phenotype (data not shown). Discussion Numerous studies have been done on the microsatellite alterations observed in human ECs to date. Supplementary Table provides a summary of the literature. Studies of vari- ous sizes have been conducted on various subjects of dif- ferent histological subtypes and genetic backgrounds, and the reported frequency for MSI+ tumours varies from 10 to 100 %, even if confined to the studies about endometri- oid-type tumours in the sporadic setting. This variability in results may not be explicable merely from the variety of the subjects (Supplementary Table). As mentioned above, the reported frequency of MSI+ tumours in each malig- nancy is indeed diverse in the literature, and this confusion in the field has continued ever since. The variety of targets for analysis, i.e. microsatellites, will also undoubtedly lead to the variability in results. Although the NCI workshop in 1997 (Boland et al. 1998) recommended the ‘working ref- erence panel’ comprising two mononucleotide and three dinucleotide microsatellites, various microsatellites, includ- ing tetranucleotide microsatellites, have in fact been used in the field (see Supplementary Table). Moreover, the MSI frequency differs widely even within the studies using only mononucleotide and dinucleotide microsatellites (Supple- mentary Table). We believe that, in addition to selection of Table 4 Clinicopathological variables and the MSI mode of MSI in endometrial cancer a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). Type A/B MSI characterises endometrial carcinomas with unique clinicopathological and molecular backgrounds Contrary to the results of the MLH1 immunohistochemis- try, MSH2 expression loss was similarly infrequent both in MSI+ tumours and in those with stable microsatellites and, consequently, not associated with the MSI+ phenotype (data not shown). Discussion We previously established a sensitive fluorescent technique in which all of these methodologi- cal problems are overcome, and, in the present study, this technique was applied to address microsatellite altera- tions in ECs. The overall frequency of MSI+ tumours was 40 %, and several previous studies in the literature indeed reported similar frequencies (Furlan et al. 2006; Hardisson et al. 2003; Ohwada et al. 2002; Risinger et al. 2005) (Sup- plementary Table). p y The MSI+ phenotype is now connected to specific clin- icopathological characteristics of tumours. In colorectal cancer, MSI+ tumours more frequently occur in the proxi- mal colon and often exhibit characteristic histopathological features such as poor/signet ring cell differentiation, mucin secretion and lymphocyte infiltration (Jass et al. 2002), which are currently known as ‘MSI-H histology’ (Umar et al. 2004). Patient outcomes are in general believed to be more favourable in MSI+ colorectal carcinomas (Popat et al. 2005). Accordingly, various clinicopathological features of tumours have been examined and are regarded as associ- ated with the MSI+ phenotype in other human malignan- cies. In EC, tumour histology, grade, location, stage and patient survivals have been reported (Supplementary Table). These parameters did not correlate with MSI in this study (Tables 3, 4). The clinical and histopathological phenotypes of tumours are determined by highly complicated systems. Their relationships to MSI may therefore be more complex than hitherto suspected. MSI is now regarded as a molecular hallmark of LS. Indeed, more than 90% of LS tumours are MSI+ (Liu et al. 1996), which implies that MSI well reflects Using HRFMA, we confirmed that endometrioid-type EC is the most microsatellite-unstable neoplasm among the human malignancies thus far tested. In general, the frequen- cies for MSI-H and MSI-L in colorectal cancer are 5–10 and 10–20 %, respectively. The MSI-H phenotype is very rare in the other human neoplasms, and the most frequently observed microsatellite-unstable phenotypes are generally MSI-L. Nevertheless, the MSI-H phenotype predominates in our panel of ECs (Table 1). The frequency for MSI-H exceeded 30%. The MSI-H phenotype is typical of LS (Liu et al. 1996). The high frequency of this phenotype in endometrioid-type EC may suggest that the majority of this subset of ECs arises from molecular backgrounds similar to those of LS or, in other words, via the MIN pathway. Discussion The tight connection between Type B MSI and LS tumourigenesis may suggest a biological importance of Type B alterations and, in addition, a potential advantage of the Type A/B classification. The MSI+ phenotype is also connected to various other genomic changes. Genetic insta- bility observed in colorectal cancer has been regarded as deriving two mutually exclusive pathways, the chromosomal instability (CIN) pathway frequently associated with muta- tions in various oncogenes or tumour suppressors such as TP53 and the microsatellite instability (MIN) pathway, in which TP53 mutations are rare and, instead, mutations are found in genes harbouring mononucleotide repeats within their ORFs (Schwartz et al. 1999). The frequency for KRAS mutations in MSI+ tumours has been controversial. Muta- tions in the KRAS oncogene were initially regarded as infre- quent in MSI+ tumours (Ionov et al. 1993; Salahshor et al. 1999; Samowitz et al. 2001). However, in our previous study, we have shown that KRAS mutations are indeed observed in microsatellite-unstable tumours and, more importantly, are relatively frequent in tumours exhibiting Type A MSI (Zhao et al. 2008). Also in this study, KRAS mutation was closely associated with Type A instability. We have previously dem- onstrated that Type A MSI is a direct consequence of defec- tive MMR (Oda et al. 2005). A close association of Type A MSI with point mutations is highly consistent with the mutator phenotype in cells deficient in MMR (de Wind et al. 1995; Reitmair et al. 1997). These observations may thus suggest that the modal classification is biologically relevant. U i HRFMA fi d h d i id targets for analysis, methodological problems at least partly account for the variability in results. Microsatellite length changes are sometimes as small as one or two base pairs in mononucleotide or dinucleotide microsatellites. Such small sequence alterations are not detectable in electrophoresis with significant migration errors, or under the influence of TDT activity of thermostable DNA polymerases such as Taq, which adds one additional base to PCR products in a sequence-dependent manner. In addition, cell populations carrying microsatellite alterations are not always predomi- nant in a given sample. However, it is difficult to detect less abundant PCR products in an assay system using autora- diography or silver staining, due to their nonlinear detec- tion characteristics. Discussion Numerous studies have been done on the microsatellite alterations observed in human ECs to date. Supplementary Table provides a summary of the literature. Studies of vari- ous sizes have been conducted on various subjects of dif- ferent histological subtypes and genetic backgrounds, and the reported frequency for MSI+ tumours varies from 10 to 100 %, even if confined to the studies about endometri- oid-type tumours in the sporadic setting. This variability in results may not be explicable merely from the variety of the subjects (Supplementary Table). As mentioned above, the reported frequency of MSI+ tumours in each malig- nancy is indeed diverse in the literature, and this confusion in the field has continued ever since. The variety of targets for analysis, i.e. microsatellites, will also undoubtedly lead to the variability in results. Although the NCI workshop in 1997 (Boland et al. 1998) recommended the ‘working ref- erence panel’ comprising two mononucleotide and three dinucleotide microsatellites, various microsatellites, includ- ing tetranucleotide microsatellites, have in fact been used in the field (see Supplementary Table). Moreover, the MSI frequency differs widely even within the studies using only mononucleotide and dinucleotide microsatellites (Supple- mentary Table). We believe that, in addition to selection of a Carcinomas in the colorectum, endometrium, ovary and stomach were scored as ‘HNPCC-associated cancers’ (Vasen et al. 1999). The other HNPCC-related tumours were not found in the patients’ kindred 1 3 Table 5 KRAS mutation and MSI-H/L P = 0.35 MSI-L MSI-H Subtotal KRAS Mutant 1 4 5 Wild type 2 31 33 Subtotal 3 35 38 Table 5 KRAS mutation and MSI-H/L Table 5 KRAS mutation and MSI-H/L J Cancer Res Clin Oncol (2016) 142:353–363 361 Table 6 KRAS mutation and Type A/B MSI P = 0.05 Type A Type B Subtotal KRAS Mutant 3 2 5 Wild type 5 28 33 Subtotal 8 30 38 molecular abnormalities underlying LS tumourigenesis. One important finding of the present study is that Type B MSI did significantly correlate with family history of HNPCC-asso- ciated cancers, and that, on the other hand, MSI-H did not. This may be partly because the MSI-H phenotype sometimes includes Type A tumours (see Table 1) and inevitably tends to be heterogeneous in terms of the mode of microsatellite alterations. Discussion This hypothesis may be consistent with the consensus that mutations in TP53 are rela- tively infrequent in endometrioid-type EC (Yeramian et al. 2013), which are similarly regarded as rare in the MIN path- way of colorectal cancer (Ionov et al. 1993; Salahshor et al. 1 3 3 362 J Cancer Res Clin Oncol (2016) 142:353–363 1999; Samowitz et al. 2001; Simms et al. 1998). Tumouri- genesis in this pathway has not yet been well understood. Destabilisation of repetitive motifs may disrupt the genes harbouring repeats such as TGFBR2, IGF2R, BAX, CASP5 etc (Schwartz et al. 1999). On the other hand, mutations in established oncogenes and tumour suppressor genes and chromosomal instability leading to LOH in tumour suppres- sor loci are not hypothesised in the MIN pathway. Tumouri- genesis without this classical model is still enigmatic. Type B MSI is observed in tumours exhibiting the MSI-H pheno- type (Ikeda et al. 2001). This has been confirmed also in the present study (Table 2). Type B MSI indeed predominates in microsatellite-unstable ECs (Table 1). Our previous study suggests that defective MMR may be a promoting and, con- sequently, highly coincidental (as typical in Lynch syndrome patients), but insufficient factor for Type B changes, whereas being necessary and sufficient for Type A instability. In other words, whereas Type A MSI is a direct consequence of defec- tive MMR, molecular abnormalities in addition to MMR defi- ciency may contribute to Type B alterations of microsatellites (Oda et al. 2005). Frequent Type B instability in endometrioid ECs suggests the possibility that this malignancy may provide a clue to Type B mechanisms and also serve as a good model for the MIN pathway tumourigenesis. 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https://openalex.org/W4255931800	https://www.researchsquare.com/article/rs-49199/latest.pdf	English	null	Are weight status and weight perception associated with academic performance among youth?	Research Square (Research Square)	2,020	cc-by	8,063	Are weight status and weight perception associated with academic performance among youth? Maram Livermore Brock University Faculty of Applied Health Sciences Markus J Duncan Brock University Faculty of Applied Health Sciences Scott T. Leatherdale University of Waterloo Faculty of Applied Health Sciences Karen Allison Patte (  kpatte@brocku.ca ) Brock University Faculty of Applied Health Sciences https://orcid.org/0000-0002-5214-1943 Research article Keywords: Obesity, overweight, academic achievement, weight perception, education, youth Posted Date: September 23rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-49199/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 26th, 2020. See the published version at https://doi.org/10.1186/s40337-020-00329-w. Are weight status and weight perception associated with academic performance among youth? Maram Livermore Brock University Faculty of Applied Health Sciences Markus J Duncan Brock University Faculty of Applied Health Sciences Scott T. Leatherdale University of Waterloo Faculty of Applied Health Sciences Karen Allison Patte (  kpatte@brocku.ca ) Brock University Faculty of Applied Health Sciences https://orcid.org/0000-0002-5214-1943 Research article Keywords: Obesity, overweight, academic achievement, weight perception, education, youth Posted Date: September 23rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-49199/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 26th, 2020. See the published version at https://doi.org/10.1186/s40337-020-00329-w. Research article Version of Record: A version of this preprint was published on October 26th, 2020. See the published version at https://doi.org/10.1186/s40337-020-00329-w. Page 1/18 Page 1/18 Page 1/18 Abstract Background: Emerging evidence suggests perceptions of being overweight account for many of the psychosocial consequences commonly associated with obesity. Previous research suggests an obesity achievement gap, yet limited research has explored weight perception in association with academic performance. Moreover, underweight perceptions have typically been excluded from research. The current study examined how BMI classification and weight perception relate to academic performance in a large cohort of youth. Methods: We used cross-sectional survey data from 61,866 grade 9-12 students attending the 122 Canadian schools that participated in Year 6 (2017/2018) of the COMPASS study. Mixed effect regression models were used to examine associations between students’ BMI classification and weight perceptions and their math and English/French course grades. All models were stratified by sex and adjusted for sociodemographic covariates and school clustering. Results: For English/French grades, males and females with overweight or underweight perceptions were less likely to achieve higher grades than their peers with perceptions of being at “about the right weight”, controlling for BMI and covariates. For math grades, females with overweight perceptions, and all students with underweight perceptions, were less likely to achieve higher grades than their peers with “about the right weight” perceptions. All students with BMIs in the obesity range were less likely to report grades of 60% or higher than their peers with “normal-weight” BMIs, controlling for weight perception and covariates. Overweight BMIs were predictive of lower achievement in females for English/French grades, and in males for math grades, relative to “normal-weight” BMIs. Results for students that did not respond to the weight and weight perception items resembled those for obesity BMI and overweight/underweight perceptions, respectively. Conclusions: Overall, this study demonstrates that an obesity achievement gap remains when controlling for students’ perceptions of their weight, and that both underweight and overweight perceptions predict lower academic performance, regardless of BMI classification. Results suggest barriers to academic success exist among youth with larger body sizes, and those with perceptions of deviating from “about the right weight”. Introduction About 35% of Canadian children and adolescents are at risk of having overweight or obesity [1]. Numerous studies have shown that childhood obesity is associated with various physical health concerns [2]. Additionally, larger-bodied adolescents are at increased risk of adverse psychosocial outcomes [3]. Strauss and Pollack reported that children and adolescents face many challenges but “few problems in childhood have as significant an impact on emotional development as being overweight” (p.747) [4]. In fact, children with overweight or obesity report lower quality of life scores than children diagnosed with cancer [5]. Previous research also indicates the presence of an obesity achievement gap Page 2/18 for children and adolescents [6,7]. More specifically, some evidence suggests students with obesity have poorer academic achievement, more absenteeism, higher dropout rates [6,8], and are less likely to pursue and attain post-secondary education [9,10]. A recent meta-analysis by He et al. included 60 studies of weight status and academic performance and found a pooled correlation (r=-.111) between higher Body Mass Index (BMI) and lower grades [11]. The researchers concluded that the relationship between weight status and academic achievement was moderated by geographical region, with lower grades more likely to be associated with high BMI in North America than other cultures, possibly due to differing weight norms and given the stigma associated with overweight and obesity. Emerging evidence suggests weight perception—individuals’ subjective appraisal of their body weight—accounts for many of the psychosocial consequences commonly associated with obesity [13-15]. That is, the perception of being overweight, rather than body weight itself, may account for risks of lower self-concept and poor mental health. One of the potential factors linking obesity and the perception of overweight to adverse outcomes is the experience of stigma and bias. An individual may need to perceive themselves as overweight, in order to internalize the bias associated with overweight/obesity. To date, the relationship between self-perceptions of weight and academic achievement has been largely overlooked. To our knowledge, only one study has examined weight perception as a predictor of academic achievement [28]. Among US adolescents (ages 14 to 17) participating in the 2003 Youth Risk Behavior Study, Florin, Shultz, and Stettler found that perceived overweight status was associated with lower grades, regardless of BMI classification, and that obesity was no longer associated with grades when controlling for weight perception [28]. Introduction Replication is necessary, particularly in more recent samples, given potential shifts in social weight norms (e.g., related to obesity prevalence, body positive movements, and anti-obesity stigma and weight bias efforts). Furthermore, consideration of underweight perceptions in addition to overweight, and exploration of sex or gender differences, are warranted. Related to thinness and muscularly sociocultural body ideals, girls/women are more likely to report perceptions of underweight than boys/men, while boys/men tend to be split between perceptions of underweight and overweight [16]. Regardless of body size, both overweight and underweight perceptions appear detrimental to mental and physical health relative to perceptions of being “about the right weight” [13-25]; however, the latter has received relatively limited attention. Further examination of a potential weight-related achievement gap is critical to inform a learning environment that will enable all youth to thrive. Deterrents to academic achievement in adolescence have critical implications for future career opportunities and successful transitions to adulthood, with school failure and dropout increasing the risk of later unemployment, poverty, lower quality life, criminality, violence, and various health risk behaviors [6,7,29]. We sought to determine if larger-bodied students report lower grades in secondary school than their peers with “normal-weight” BMIs. In addition, we explored whether weight perceptions predicted grades, while controlling for weight status. It was hypothesized that perceptions of being at “about the right weight” would provide a protective effect, and any relationship between BMI classification and academic achievement would be reduced when controlling for weight perception. Page 3/18 Design and Participants The COMPASS study is a prospective cohort study designed to collect longitudinal and hierarchical health data from a large sample of grade 9 through 12 students (ages 14-19) enrolled in Canadian secondary schools. Schools and school boards were purposely selected based on whether they permitted active-information passive-consent parental permission protocols, which are critical for collecting robust data among youth [30]. The COMPASS student questionnaire (Cq), a self-report paper-and-pencil survey, is completed once annually by full school samples during one classroom period. All grade 9 through 12 students attending participating schools were eligible to participate and could decline at any time. A full description of recruitment methods [31] and the COMPASS study are available in print [32] and online (www.compass.uwaterloo.ca). The COMPASS study received ethics approval from the University of Waterloo and Brock University Human Research Ethics Committee and all participating school boards. We used cross-sectional data from Year 6 (2017-2018 school year) of the COMPASS study, which included 66,434 students at 122 secondary schools in Ontario (n=61), British Columbia (n=16), Alberta (n=8) and Quebec (n=37). The overall student response rate in Year 6 was 81.85% of eligible students. Student non-participation primarily resulted from absences or scheduled study-periods during data collection. Participants with missing outcome, sex, or covariate data were removed, leaving a final sample of 61,866 adolescents. Methods Design and Participants Design and Participants Descriptive Statistics Descriptive statistics for all variables are described in Table 1. In this sample, 49.4% of participants identified as male and 50.6% as female. About two-thirds (66.6%) of the sample identified as white and one-third as non-white, mixed, or other race/ethnicity. In terms of weight status, 5.7%, 12.0%, 54.7% and 3.6% of the total sample had BMIs in the obesity, overweight, “normal-weight”, and underweight Descriptive statistics for all variables are described in Table 1. In this sample, 49.4% of participants identified as male and 50.6% as female. About two-thirds (66.6%) of the sample identified as white and one-third as non-white, mixed, or other race/ethnicity. In terms of weight status, 5.7%, 12.0%, 54.7% and 3.6% of the total sample had BMIs in the obesity, overweight, “normal-weight”, and underweight categories, respectively, while 19.6% of students did not report their weight, and the remaining proportion were missing sex, age, or height data to categorize BMI. Additionally, a total of 23.8% and 58.7% students reported overweight and “about the right weight” perceptions respectively, while 1.4% did not respond to the weight perception item. More males reported perceptions of “underweight” than females (21.2% versus 11.1%), while more females reported perceptions of overweight (26.5% versus 20.6%) and “about the right weight” (60.7% versus 56.2%) than males. Just over half of students (51.0-51.3%) reported math and English/French grades above 60%. 3.6% of the total sample had BMIs in the obesity, overweight, normal weight , and underweight categories, respectively, while 19.6% of students did not report their weight, and the remaining proportion were missing sex, age, or height data to categorize BMI. Additionally, a total of 23.8% and 58.7% students reported overweight and “about the right weight” perceptions respectively, while 1.4% did not respond to the weight perception item. More males reported perceptions of “underweight” than females (21.2% versus 11.1%), while more females reported perceptions of overweight (26.5% versus 20.6%) and “about the right weight” (60.7% versus 56.2%) than males. Just over half of students (51.0-51.3%) reported math and English/French grades above 60%. The concordance between weight perception and BMI category is presented in Figure 1. The weighted Kappa was 0.392 among females and 0.370 among males. The majority of males (74.9%) and females (81.3%) with BMIs in the obesity category reported perceptions of overweight. Measures Weight Status. Student weight status was defined by Body Mass Index (BMI; kg/m²) classification determined based on student-reported height and weight [33], and the World Health Organization [34] age- and sex-adjusted cut points (underweight, normal weight, overweight, obesity). A previous study found the weight status measure to be reliable, valid, and valuable for use when objective methods are not feasible [33]. Given the prevalence of missing BMI data, and as missing self-reported weight data may not be missing at random [35], two separate categories were created for missing weight status based on which variables were missing to determine BMI classification: missing BMI classification due to weight not being reported, and missing BMI due to missing age, sex, or height. Weight Perception. Subjective perception of weight status was determined using the question, “How do you describe your weight?” Response options included: “very underweight”, “slightly underweight”, “about the right weight”, “slightly overweight” and “very overweight”. Responses were collapsed into three categories: underweight, about right, and overweight. In addition, missing weight perception responses were included as a fourth category. Covariates. Participant-reported race/ethnicity (categorized into white and, non-white minority, multiethnic, or other) and school grade (9, 10, 11, 12, other [Secondary I- II in Quebec]) were entered into the model as covariates. Also, student weekly spending/saving money (categorized into $1-$20, Page 4/18 $21-$100, >$100, don’t know) was included as an indicator of part-time employment and/or allowance, as proxy for student-level SES in the absence of data on parental income or education data. $21-$100, >$100, don’t know) was included as an indicator of part-time employment and/or allowance, as proxy for student-level SES in the absence of data on parental income or education data. Academic Performance. Academic performance was assessed using student-reported grades. Participants reported their approximate overall mark in their current or most recent math and English (in Ontario, Alberta and BC schools) or French (in Quebec) courses. Grades were dichotomized as ≥ 60% or <60% for both math and English/French grade models. ce. Academic performance was assessed using student-reported grades. Statistical Analysis R software [36] was used to conduct frequency descriptive statistics and mixed effect logistic regression models. Separate models explored the association between BMI status and weight perception with math grades and English/French course grades, stratified by sex and controlling for weekly spending money, school grade, and race/ethnicity. Mixed models were used to account for school clustering by adding a random intercept at the school level. No interaction effect between weight status and weight perception was indicated when tested (results not reported). Descriptive Statistics Among those with overweight BMIs, most females reported perceptions of overweight (62.2%); whereas more males reported “about right” weight perceptions (54.5%) than overweight perceptions (42.4%). Most males (64.2%) and females (61.5%) in the underweight BMI category reported underweight perceptions. In the “normal weight” BMI category, more males reported underweight perceptions (27.5%) than females (12.2%), while females were more likely to perceive their weight as “about right” (71.6%) or overweight (15.4%) than males (65.4%; 5.9%). Page 5/18 Page 5/18 Page 5/18 Students with missing BMI data resembled those with “normal weight” BMIs, except a higher proportion reported overweight perceptions. About half of students with missing BMI data reported “about right” weight perceptions (51.1% males; 55.1% females), over a quarter reported overweight perceptions (25.1% males; 30.8% females), and 9.7% of females and 19.0% of males reported underweight perceptions. Students with missing BMI data resembled those with “normal weight” BMIs, except a higher proportion reported overweight perceptions. About half of students with missing BMI data reported “about right” weight perceptions (51.1% males; 55.1% females), over a quarter reported overweight perceptions (25.1% males; 30.8% females), and 9.7% of females and 19.0% of males reported underweight perceptions. Math Course Grades See Table 2 for model results testing BMI classification and weight perception as predictors of math grades, after stratifying by sex and controlling for covariates. In males only, having over $100 a week available for spending or saving and identifying as nonwhite, mixed, or other race/ethnicity were associated with lower odds of reporting math grades over 60%, relative to males without any weekly spending money and of white race/ethnicity, respectively. Students with BMIs in the obesity range were less likely to report a math grade above 60% compared to those with BMIs considered “normal weight”. In males only, overweight BMIs were associated with a lower likelihood of report a math grade above 60% compared to those with “normal-weight” BMIs. No effect resulted for underweight BMI relative to “normal- weight” BMI. In females only, weight perceptions of overweight predicted a lower likelihood of reporting math grades above 60% when compared to perceptions of being “about the right weight”, controlling for BMI classification and covariates. In both males and females, perceptions of underweight were associated with lower likelihood of higher math grades than perceptions of being “about the right weight”. Student with missing BMI data either due to not reporting weight or missing height, age or sex data were more likely to have lower math grades when compared to those in the “normal-weight” BMI category. Similarly, students with missing weight perception data were more likely to report lower math grades when compared to those with “about the right weight” perceptions. Discussion The current study examined whether weight status and student perceptions of their weight were associated with academic grades in selected secondary school courses (math and English/French) in a large population study of Canadian youth. Results support the links between obesity, as determined by BMI, and lower academic performance in both males and females. Overweight BMI classifications were also associated with lower odds of high grades in English/French courses among females, and in math classes among male students, relative to BMIs considered “normal-weight”. Similar to previous research [28], females with overweight perceptions were less likely to achieve higher course grades in math, and both males and females with overweight perceptions had lower odds of grades above 60% in their English/French courses, relative to their peers with perceptions of being at “about the right weight”, controlling for BMI classification and covariates. While no effect was found for underweight relative to “normal-weight” BMI, females and males with perceptions of being underweight had lower odds of high grades in math and English/French courses relative to students with “about the right weight” perceptions. Results for youth with missing BMI or weight perception data resembled those for obesity BMI classifications and perceptions of overweight or underweight, respectively. Overall, this study demonstrates that an obesity achievement gap remains when controlling for students’ perceptions of their weight, and that weight perceptions—both underweight and overweight—predict lower academic performance, regardless of BMI classification. Further research is needed to determine the mechanisms underlying these relationships, in order to remove barriers to academic success among youth with larger body sizes, and those with perceptions of deviating from “about the right weight”. To the best of our knowledge, only one previous study has examined weight perception as a predictor of academic grades. The current study provides necessary replication and builds on existing literature, by examining these relationships in a more recent and larger sample of youth. In a sample of approximately 11,000 US adolescents participating in the 2003 Youth Risk Behavior Study, perceptions of overweight were found to be a stronger predictor of academic outcomes than BMI, and obesity was no longer a significant predictor of academic performance when accounting for overweight perceptions [28]. Overweight perceptions have been linked to poor mental health, psychosocial distress, and low self- esteem in adolescents [29,38-40], factors that have also been associated with lower academic achievement [37]. English/French Course Grades Table 3 summarizes the resultant model testing BMI classification, weight perception, and covariates as predictors of English/French grades by sex. Both males and females identifying as nonwhite, mixed, or other race/ethnicity had lower odds of English/French grades above 60% than their counterparts identifying as white. No differences in English/French grades resulted by spending money or grade. Both male and female adolescents with BMIs in the obesity range, and females with overweight BMIs, were less likely to report higher grades than their peers with BMIs considered “normal weight”. Both males and females with weight perceptions of overweight and underweight were less likely to report higher grades (above 60%) in their English/French classes when compared to those with perceptions of being at “about the right weight”. Students with missing BMI data due to either not reporting weight, or because of missing height, age, or sex data, had significantly lower odds of reporting English/French grades above 60%, when compared to those with “normal-weight” BMIs. Similarly, students with missing weight perception data were less likely to have English/French grades above 60% when compared to those with “about the right weight” perceptions. Page 6/18 Discussion In fact, researchers have reported perceiving oneself as overweight to be a stronger predictor of behavioural issues and mental distress than actual weight status [13,27,40]. Similarly, the current results indicate that perceptions of overweight significantly predicted poorer academic outcomes independent of BMI classification, with the exception of math grades in males. However, unlike Florin et al. [28], obesity BMI classifications remained predictive of lower grades when controlling for weight perception. The current study suggests other factors appear to contribute to the obesity achievement gap, such as parental education, mental health, or external weight bias. Students with obesity are more likely to experience weight-based bullying within the school context [28,39]. Also, weight bias has been documented in physical education teachers [9,43,44,54] and given its pervasiveness across the Page 7/18 Page 7/18 population [9,43], may contribute to educators’ perceptions of students’ academic abilities. Several studies have indicated that the association between BMI and academic performance was no longer significant when models adjusted for parental/familial characteristics [12,47]. For instance, Datar et al. concluded that overweight status is not a causal factor of lower academic performance, as weight-related differences in test scores became insignificant when social and behavioural variables, such as SES and parental time spent with the child were considered [47]. The authors cautioned that higher weight students may be labeled as lower achievers, as weight is a more obvious marker than sociodemographic characteristics. population [9,43], may contribute to educators’ perceptions studies have indicated that the association between BMI an significant when models adjusted for parental/familial cha concluded that overweight status is not a causal factor of l differences in test scores became insignificant when social parental time spent with the child were considered [47]. The students may be labeled as lower achievers, as weight is a characteristics. Adolescents who perceive themselves as overweight may be at risk of internalizing weight stigma. Despite the high prevalence of obesity, weight stigma continues to be problematic. Stereotypes that individuals living with obesity are lazy, unintelligent, or lack willpower contribute to stigma and discrimination [43]. Internalized stigma, or self-stigma, occurs when individuals apply negative stereotypes to themselves and believe that the stigma is deserved [44], leading to low self-esteem and psychological distress [43]. Research indicates that bias toward individuals with overweight and obesity persists in health care, employment, and home settings [9]. Education, however, has received less research attention, particularly at the secondary school level. Discussion It is plausible that adolescents who perceive themselves as overweight have lower self-concepts related to internalized weight stigma, which in turn, contributes to poorer academic engagement and performance. That is, students who feel their weight is “about right” may be more likely to succeed because they have not internalized negative stereotypes. Students with underweight perceptions also reported lower grades than those with “about right” perceptions. Interestingly, the effect of underweight perceptions was more consistent than overweight perceptions in predicting lower grades in both females and males, and across Math and English/French grades. To our knowledge, no previous study has examined underweight perceptions in relation to academic performance. While less studied, underweight perceptions have been associated with depressive and anxiety symptoms in males [21,41,42] and suicidality and lower health-related quality of life in all youth [25,27]. Based on this result, it is plausible that links between weight perception and academic performance relate more to deviations from the social norm or sociocultural body ideals than to weight stigma. Results are consistent with sociocultural body ideals of thinness for women and muscularity for men, with more males reporting underweight perceptions than females. About one-fifth of males and one-tenth of females reported underweight perceptions; yet only 1.5% and 2.1% of female and males had BMIs classified as underweight, respectively. Another plausible explanation is that youth with underweight perceptions were experiencing weight restrictions (e.g., due to food insecurity) and/or had lost weight, while “normal-weight” by BMI, which in turn contributed to reduced ability to perform in school. Results highlight the importance of including weight perceptions across the spectrum in future research. Interestingly, students that did not report their body weight or weight perception tended to have the lowest likelihood of achieving higher grades, comparable to the odds for obesity BMI classification. Far more males and females with missing BMI data reported perceptions of overweight than their peers with Page 8/18 Page 8/18 “normal-weight” BMIs. Adolescents are less likely to report their weight as BMI increases and if they have poor body image [35,45]. Missing self-reported weight status and perceptions may be influenced by an awareness of societal norms of thinness/muscularity ideals and weight bias attitudes [45,46]. Discussion Hence, if adolescents with larger body sizes did not report their weight due to concerns of judgment by others, results lend support to the theory that negative perceptions regarding body weights outside of “about right” contribute to lower academic performance. Future research should explore both internalized and externalized weight bias, and associated lower self- concept and mental distress, as possible mechanisms explaining links between higher weight status, and perceptions of overweight and underweight, with academic achievement. Upstream strategies targeting the negative connotations of varying body sizes may prove valuable, to prevent the adverse psychosocial outcomes associated with perceptions of being overweight or underweight. Enhanced efforts to prevent weight-based bullying and promote weight acceptance are also advised. Previous research suggests bullying victimization predicts changes from perceptions of being at “about the right weight” to underweight and overweight perceptions among youth [55]. Limitations Several limitations require consideration. First, while the large sample supports generalizability, the COMPASS study was not designed to be representative. Second, cross-sectional data was used to explore relationships. Future longitudinal analysis of COMPASS data will assist in establishing temporality and testing potential mechanistic contributors (e.g., mental health, self-concept, bullying victimization, school connectedness). Third, the use of self-reported data carries risks of recall and social desirability bias. For instance, lower achieving students may over report their achievement. However, a review of 37 independent samples found strong response validity of self-reported grades in high school students [48]. Similarly, weight status was based on student-reported height and weight, and as such, results likely reflect greater concordance between weight perception and weight status than exists. However, time and cost constraints preclude the feasibility of obtaining objective height and weight measures, not to mention the potential harm of weight measurements in a school-based study. Also, a strong correlation between measured and self-reported BMI has been shown in youth [33,49]. Fourth, the reference point that youth used to answer the weight perception question is not entirely clear. That is, it is not known whether respondents were comparing their weight to their ideal body, their peers, a medical standard, or some other alternative. For instance, responses of ‘about the right weight’ may indicate weight satisfaction rather than youths’ perception of how their weight compared to an external reference point. Lastly, as discussed, this study did not assess the contribution of self-esteem or mental health. Mental health may confound the relationship between weight status or perception and academic achievement. Several studies have linked poor mental health with lower grades [37], and a recent intervention promoting positive mental health and wellbeing significantly improved academic grades in participating schools [50]. Future studies, using longitudinal designs, standardized test grades, and including mental health, self-concept, and stigma measures should be considered. Several limitations require consideration. First, while the large sample supports generalizability, the COMPASS study was not designed to be representative. Second, cross-sectional data was used to explore relationships. Future longitudinal analysis of COMPASS data will assist in establishing temporality and testing potential mechanistic contributors (e.g., mental health, self-concept, bullying victimization, school connectedness). Third, the use of self-reported data carries risks of recall and social desirability bias. For instance, lower achieving students may over report their achievement. However, a review of 37 connectedness). Conclusion Results support the existence of an achievement gap by both weight status and weight perception. This study has widespread implications with over 2 million adolescents at risk of having overweight or obesity in Canada [1], and over 40% of youth reporting weight perceptions other than “about right”. Research is needed to further examine the mechanisms underlying these associations. Academic achievement sets a lifelong trajectory of health and wellbeing. Lower academic achievement is linked to increased rates of unemployment, poverty, criminality, and negative future health outcomes [6,7,29,51]. The obesity achievement gap has been suggested as an early contributor to later SES disparities found by weight status [52]. The present study contributes to a body of research that encourages the consideration of both overweight and underweight perceptions and their potential impact on adolescent emotional and physical health. Upstream strategies to prevent negative connotations associated with body sizes divergent from “about right” and to promote weight acceptance merit consideration. Further exploration is necessary to inform policies and interventions that foster a learning environment that will enable all youth to thrive. Limitations Third, the use of self-reported data carries risks of recall and social desirability bias. For instance, lower achieving students may over report their achievement. However, a review of 37 independent samples found strong response validity of self-reported grades in high school students [48]. Similarly, weight status was based on student-reported height and weight, and as such, results likely reflect greater concordance between weight perception and weight status than exists. However, time and cost constraints preclude the feasibility of obtaining objective height and weight measures, not to mention the potential harm of weight measurements in a school-based study. Also, a strong correlation between measured and self-reported BMI has been shown in youth [33,49]. Fourth, the reference point that youth used to answer the weight perception question is not entirely clear. That is, it is not known whether respondents were comparing their weight to their ideal body, their peers, a medical standard, or some other alternative. For instance, responses of ‘about the right weight’ may indicate weight satisfaction rather than youths’ perception of how their weight compared to an external reference point. Lastly, as discussed, this study did not assess the contribution of self-esteem or mental health. Mental health may confound the relationship between weight status or perception and academic achievement. Several studies have linked poor mental health with lower grades [37], and a recent intervention promoting positive mental health and wellbeing significantly improved academic grades in participating schools [50]. Future studies, using longitudinal designs, standardized test grades, and including mental health, self-concept, and stigma measures should be considered. Page 9/18 Page 9/18 Page 9/18 Declarations Funding: The COMPASS study has been supported by a bridge grant from the CIHR Institute of Nutrition, Metabolism and Diabetes (INMD) through the “Obesity – Interventions to Prevent or Treat” priority funding awards (OOP-110788; awarded to SL), an operating grant from the CIHR Institute of Population and Public Health (IPPH) (MOP-114875; awarded to SL), a CIHR project grant (PJT-148562; awarded to SL), a CIHR bridge grant (PJT-149092; awarded to KP/SL), a CIHR project grant (PJT-159693; awarded to KP), and by a research funding arrangement with Health Canada (#1617-HQ-000012; contract awarded to SL). Acknowledgements: The authors would like to thank the schools, school boards, and students that have participated in the COMPASS study, and all COMPASS staff and team members. Acknowledgements: The authors would like to thank the schools, school boards, and students that have participated in the COMPASS study, and all COMPASS staff and team members. Competing interests: The authors declare that they have no competing interests/conflicts of interests. Ethics approval and consent to participate: The University of Waterloo Office of Research Ethics (ORE#17264) and participating school boards approved all procedures. All students attending participating schools were invited to participate using active-information passive-consent parental permission protocols. Students could withdraw from the study at any time. Consent for publication: N/A Authors’ contributions: ML lead the writing of the manuscript. MJD conducted all analysis. STL is the PI of the COMPASS Study. ML and KAP conceptualized the manuscript study. All authors (ML, MJD, STL, KAP) contributed to manuscript revisions, results interpretation, and approved the final version. Page 10/18 Page 10/18 Availability of data and material: COMPASS study data is available upon request through completion and approval of an online form: https://uwaterloo.ca/compass-system/information-researchers/data-usage- application. The datasets used during the current study are available from the corresponding author on reasonable request. Code availability: R software was used for all analysis. Further details available upon request. Code availability: R software was used for all analysis. Further details available upon request. ode availability: R software was used for all analysis. Further details availab References 1. Shentow-Bewsh R, Zuberi D. Reducing the prevalence of obesity in Canada: a call to action. Soc Work Public Health. 2018;33(6):329-341. doi:10.1080/19371918.2018.1482252 2. Must A, Anderson SE. Effects of obesity on morbidity in children and adolescents. Nutr Clin Care. 2003;6(1):4-48. 3. Erickson SJ, Robinson TN, Haydel KF, Killen JD. 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Mixed-effect model testing BMI category and weight perception as predictors of Math grades ≥60% among youth Tables Table 1: Descriptive statistics for secondary school students in Year 6 (2017/2018) of the COMPASS study (N=61,886) Table 1: Descriptive statistics for secondary school students in Year 6 (2017/2018) of the COMPASS study (N=61,886) Female (n = 31,334) Male (n =30,552) % (n) % (n) Grade 9 10 11 12 Othera 24.3 (7614) 24.5 (7677) 23.3 (7301) 14.9 (4669) 13.0 (4073) 24 (7332) 24.3 (7424) 23.1 (7058) 15.7 (4797) 12.9 (3941) Race/ethnicity White Non-white, multiethnic, or other 66.7 (20889) 33.3 (10445) 66.6 (20355) 33.4 (10197) BMI Classification Underweight “Normal Weight” Overweight Obesity Missing weight Missing age, sex, or height 1.5 (470) 56.8 (17798) 10.3 (3227) 4.1 (1285) 21.7 (6799) 5.6 (1755) 2.1 (642) 50.6 (15459) 13.3 (4063) 7.4 (2261) 19.4 (5927) 7.2 (2200) Weight Perception Underweight “About the right weight” Overweight Missing 11.1 (3478) 60.7 (19019) 26.5 (8304) 1.7 (533) 21.2 (6477) 56.2 (17170) 20.6 (6294) 2.0 (611) Weekly Spending Money None $1-$20 14.2 (4449) 25.9 (8116) 17.9 (5469) 24.7 (7546) $21-$100 >$100 25.5 (7990) 16.9 (5295) 22.0 (6721) 20.4 (6233) Don’t know 17.5 (5484) 15.0 (4583) Math Grades 60-100% 0-59% 51.0 (15980) 49.0 (15354) 51.2 (15643) 48.8 (14909) English/French Grades 60-100% 0-59% 51.3 (16074) 48.7 (15260) 51.1 (15612) 48.9 (14940) a Secondary I-II in Quebec schools. BMI = body mass index. Table 1: Descriptive statistics for secondary school students in Year 6 (2017/2018) of the COMPASS study (N=61,886) a Secondary I-II in Quebec schools. BMI = body mass index. a Secondary I-II in Quebec schools. BMI = body mass index. Table 2. Tables Mixed-effect model testing BMI category and weight perception as predictors of Math grades ≥60% among youth Page 15/18 Females (N=31,334) Males (N=30,552) AOR 95% CI AOR 95% CI BMI Category (Reference: “Normal weight”) Obesity 0.70 0.59-0.82 0.62 0.55-0.71 Overweight 0.90 0.80-1.02 0.87* 0.79-0.97 Underweight 0.93 0.70-1.25 0.85 0.68-1.07 Missing weight data 0.56 0.51-0.61 0.62 0.57-0.68 Missing sex, age, or height data 0.64 0.55-0.74 0.65 0.57-0.74 Weight Perception (Reference: “About the right weight”) Overweight 0.82 0.75- 0.89 0.91 0.83- 1.00 Underweight 0.77 0.69- 0.86 0.90* 0.82- 0.97 Missing weight perception Race/ethnicity (Reference: White) Nonwhite, multiethnic, or other Grade (Reference: 9) 10 11 12 Othera Weekly Spending Money (Reference: None) $1-$20 $21-$100 >$100 Don’t know 0.64 0.92 0.82 0.96 0.91 1.10 0.94 0.94 0.94 1.05 0.50- 0.83 0.85- 1.00 0.74- 0.90 0.87- 1.07 0.81- 1.02 0.95- 1.26 0.84- 1.05 0.84- 1.05 0.83- 1.07 0.93- 1.19 0.59 0.87 0.80 0.83 0.88 1.14 0.93 0.89 0.86* 1.02 0.47- 0.73 0.81- 0.94 0.73- 0.88 0.75- 0.91 0.79- 0.99 0.99- 1.31 0.84- 1.02 0.80- 0.99 0.77- 0.95 0.91- 1.14 Note: Cross-sectional data from Year 6 (2017/2018) of the COMPASS study (N=61,886). The outcome reference category is math grades <60%. Mixed models were used to account for school clustering by adding a random intercept at the school level. * p < .01; ** p < .001 * p < .01; p < .001 a Secondary I-II in Quebec schools. Table 3. Mixed-effect model testing BMI category and weight perception as predictors of English/French grades ≥60% among youth Page 16/18 Page 16/18 Females (N=31,334) Males (N=30,552) AOR 95% CI AOR 95% CI BMI Category (Reference: “Normal weight”) Obesity 0.51 0.41-0.64 0.63 0.55-0.73 Overweight 0.71 0.60-0.85 0.87 0.77-0.99 Underweight 1.04 0.68-1.59 0.93 0.71-1.21 Missing weight data 0.45 0.40-0.50 0.54 0.49-0.60 Missing sex, age, or height data 0.49 0.40-0.59 0.58 0.51-0.66 Weight Perception (Reference: “About the right weight”) Overweight 0.76 0.68-0.85 0.83 0.75-0.92 Underweight 0.59** 0.51-0.69 0.87* 0.79-0.95 Missing weight perception Race/ethnicity (Reference: White) Nonwhite, multiethnic, or other Grade (Reference: 9) 10 11 12 Othera Weekly Spending Money (Reference: None) $1-$20 $21-$100 >$100 Don’t know 0.50 0.67 0.96 0.87 1.07 1.19 0.88 0.95 0.88 0.99 0.36-0.68 0.60-0.75 0.83-1.10 0.75-1.00 0.90-1.27 0.98-1.44 0.75-1.03 0.80-1.11 0.74-1.06 0.84-1.18 0.66 0.74 0.94 0.94 1.06 1.12 1.08 1.05 0.93 1.09 0.51-0.84 0.68-0.80 0.85-1.05 0.84-1.05 0.93-1.21 0.97-1.29 0.97-1.21 0.93-1.18 0.83-1.05 0.96-1.23 Note: Cross-sectional data from Year 6 (2017/2018) of the COMPASS study (N=61,886). The outcome reference category is English/French grades <60%. Mixed models were used to account for school clustering by adding a random intercept at the school level. * p < 01 ** p < 001 a Secondary I-II in Quebec schools. Figures Page 17/18 Figure 1 Weight perception and BMI category concordance among female and male secondary school students participating in Year 6 (2017/2018) of the COMPASS study Figure 1 Weight perception and BMI category concordance among female and male secondary school students participating in Year 6 (2017/2018) of the COMPASS study Weight perception and BMI category concordance among female and male secondary school students participating in Year 6 (2017/2018) of the COMPASS study Page 18/18
https://openalex.org/W4308304314	https://malariajournal.biomedcentral.com/counter/pdf/10.1186/s12936-022-04338-9	English	null	Improved adherence to test, treat, and track (T3) malaria strategy among Over-the-Counter Medicine Sellers (OTCMS) through interventions implemented in selected rural communities of Fanteakwa North district, Ghana	Malaria journal	2,022	cc-by	7,648	Soniran et al. Malaria Journal (2022) 21:317 https://doi.org/10.1186/s12936-022-04338-9 Soniran et al. Malaria Journal (2022) 21:317 https://doi.org/10.1186/s12936-022-04338-9 Malaria Journal Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Improved adherence to test, treat, and track (T3) malaria strategy among Over‑the‑Counter Medicine Sellers (OTCMS) through interventions implemented in selected rural communities of Fanteakwa North district, Ghana Olajoju Temidayo Soniran1,2, Benedicta Ayiedu Mensah1, Ndong Ignatius Cheng1,3, Benjamin Abuaku1† and Collins Stephen Ahorlu1† Abstract Background: Prompt diagnosis and treatment of malaria prevents a mild case from developing into severe disease and death. Unfortunately, parasitological testing of febrile children is greater in the public and formal private sector than in the informal private sector where many patients with malaria-like symptoms first seek treatment. This study was aimed at improving implementation of the T3 policy among OTCMS using some interventions that could be scaled-up easily at the national level. Methods: Interventions were evaluated using a two-arm, cluster randomized trial across 8 rural communities (4 clusters per arm), in two adjacent districts of Ghana. A total of 7 OTCMS in the intervention arm and 5 OTCMS in the control arm in the selected communities participated in the study. Five interventions were implemented in the intervention arm only. These were acquisition of subsidized malaria rapid diagnostic test (RDT) kits, training of OTCMS, supportive visits to OTCMS, community sensitization on malaria, and introduction of malaria surveillance tool. The pri- mary outcome was the proportion of children under 10 years with fever or suspected to have malaria visiting OTCMS and getting tested (using RDT) before treatment. Secondary outcomes included OTCMS adherence to national malaria treatment guidelines and the recommended RDT retail price. Outcomes were measured using mystery client (an adult who pretends to be a real patient) surveys supplemented by a household survey. Proportions were com- pared using chi-square test or Fisher exact test. Results: Following deployment of interventions, mystery client survey showed that OTCMS’ adherence to malaria protocol in the intervention arm increased significantly (p < 0.05) compared to the control arm. Household surveys in the intervention arm showed that caregivers self-treating their children or visiting drug vendors significantly decreased in favour of visits to OTCMS shops for treatment (p < 0.001). End-line malaria testing rate was higher © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International Licen permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriat original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Th other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise to the material. Background Malaria continues to account for high mortality and morbidity globally especially in sub-Saharan African countries. In 2020, global malaria deaths was estimated at 627,000 and World Health Organization (WHO) Afri- can Region accounted for 96% of these deaths. Children under 5 years and pregnant women are the most affected [1]. Most malaria related deaths can be averted if cases are detected on time, diagnosed promptly, and treated according to recommended malaria management guide- lines [2]. Methods Study areah The study was conducted in Fanteakwa North and Fan- teakwa South districts in the eastern region of Ghana. The two districts were previously one (Fanteakwa dis- trict) until March 2018 when the southern part of the district was split off to create Fanteakwa South district and the remaining part renamed Fanteakwa North dis- trict. The area has been described elsewhere [10]. Briefly, Fanteakwa North district has 1 hospital, no health centre, 1 clinic, 31 community health-based planning services (CHPS) compounds, and 28 OTCMS, while the Fan- teakwa South has no hospital, 2 health centres, 1 clinic, 15 CHPS compounds, and 19 OTCMS. Begoro, the capi- tal town of Fanteakwa North district acted as a buffer between the two districts in this study. The WHO recommends a confirmatory blood test for all suspected malaria cases and a prescription of arte- misinin-based combination therapy (ACT) for those who test positive [3]. The deployment of malaria rapid diagnostic tests (RDTs) is a useful measure in the man- agement of uncomplicated malaria particularly in highly endemic rural settings, where microscopy is a challenge [4]. In the public health care sector, procurement of RDTs has increased significantly across sub-Saharan Africa [5, 6]. Sadly, the availability and use of RDTs is low in the private medicine retail (PMR) sector where most patients with malaria-like symptoms seek treatment [7]. In Ghana, Over-the-Counter Medicine Sellers (OTCMS) a subsidiary of the PMR, are usually the first point of call for patients because there are no consulta- tion fees, little or no waiting times, and patients’ prefer- ence for self-medication. OTCMS are regulated by the Pharmacy Council of Ghana and limited by the Phar- macy Act of Ghana to selling only class C (over the coun- ter) medicines which includes antimalarial drugs. As of 2012, out of over 10,000 OTCMS in Ghana, 233 had been accredited by the National Health Insurance Authority [8, 9]. Although, previous studies in Ghana and across sub-Saharan Africa had reported varying uptake of RDT among OTCMS, adherence to RDT-negative test result is very low [10] and community members still held the view that RDT-negative results did not mean ‘no malaria ill- ness’ and would therefore use ACT [11, 12].i Abstract Conclusion: Interventions targeting OTCMS in rural communities have the potential of improving adherence to the T3 malaria policy and subsequently improving management of uncomplicated malaria in Ghana. Trial registration: ISRCTN registry ISRCTN77836926. Registered on 4 November 2019. Keywords: Malaria, Private medicine retailers, Over-the-counter medicine sellers, Test, treat, and track (T3), Implementation research Keywords: Malaria, Private medicine retailers, Over-the-counter medicine sellers, Test, treat, and track (T3), Implementation research However, evidence to guide decisions on how and where to scale up RDTs amongst OTCMS is currently lack- ing [7]. Hence, objective of this study was to evaluate the combined effectiveness of provider and community interventions on RDT testing rates in the study areas. Abstract If material is not included in the article’s Creative Commons licence and your intended use is not permitt regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (ht mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit †Benjamin Abuaku and Collins Stephen Ahorlu have contributed equally to this work Correspondence: temidayoolajoju@yahoo.com 1 Department of Epidemiology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana Full list of author information is available at the end of the article †Benjamin Abuaku and Collins Stephen Ahorlu have contributed equally to this work Correspondence: temidayoolajoju@yahoo.com Correspondence: temidayoolajoju@yahoo.com 1 Department of Epidemiology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana Full list of author information is available at the end of the article 1 Department of Epidemiology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Soniran et al. Malaria Journal (2022) 21:317 Page 2 of 10 compared with the baseline rate, though not statistically significant (30.8% vs 10.5%; p = 0.1238). OTCMS in the inter- vention arm also adhered to the subsidized RDT retail price of GHc2.40. Baseline phaseh The baseline phase involved conducting community entry and household surveys in each of the intervention clusters, and in-depth interviews of OTCMS in both the intervention and control clusters. (v) Introduction of malaria surveillance tool for use by OTCMS: the OTCMS were enlightened and trained on how to keep accurate record of all suspected malaria cases attended to using this tool. The commu- nities were also sensitized on the surveillance tool. Baseline household survey: The survey was conducted in the intervention arm to document preintervention malaria testing rates among children under 10 years vis- iting OTCMS for malaria treatment in the past 1 month preceding the survey. In-depth interviews: In-depth interviews with OTCMS in the selected clusters/communities in both study arms were conducted to determine possible factors preventing the effective management of malaria at their level.hi Evaluation phase Th The primary outcome was measured using mystery client surveys and end-line household survey conducted in the evaluation phase. f The findings of the baseline phase had been reported elsewhere [13]. Mystery client survey: Mystery client surveys were used to evaluate OTCMS conduct in implementing the T3 strategy. Mystery client data collection covered 9th to 11th months of the intervention period. The mystery clients also assessed the process of RDT use in the two study arms. A total of 13 mystery clients were recruited and given intensive training (including practical sessions) over 3 days on the clinical scenario, how to conduct and interpret a malaria blood test using RDT, and how to fill the assessment checklist/form. Each OTCMS was visited twice a month by a different mystery client for 3 months (August 2020–October 2020). Two different clinical sce- narios were presented by the mystery clients during the visits to the OTCMS: Study proceduresh (b) OTCMS conducts a malaria blood test on patients suspected of uncomplicated malaria before pre- scription of medicine. The study had 4 phases. These are preparatory, baseline, intervention, and evaluation. Preparatory phase In the preparatory phase, meetings were scheduled with relevant stakeholders including the district and regional health directorates, the National Malaria Control Pro- gramme (NMCP), relevant non-governmental organiza- tions such as Strengthening Health Outcomes through the Private Sector (SHOPS), OTCMS, and traditional leaders in the selected communities. Houses in the selected clusters were mapped and lists of households with children under 10 years old were generated with corresponding GPS coordinates. (iii) Quarterly supportive visits to OTCMS: the OTCMS in the intervention arm were visited quarterly during the implementation phase to monitor and assess their malaria management practices. The skills acquired during the earlier training workshop was reinforced, and technical guidance provided on challenges experi- enced. (iv) Community sensitization on malaria focusing on the T3 strategy: the intervention communities were sensi- tized on malaria and importance of demanding malaria testing before treatment. Community health workers and town criers (‘Gongong’) were engaged to carry out this activity at churches, mosques, community durbars and on market days. (iv) Community sensitization on malaria focusing on the T3 strategy: the intervention communities were sensi- tized on malaria and importance of demanding malaria testing before treatment. Community health workers and town criers (‘Gongong’) were engaged to carry out this activity at churches, mosques, community durbars and on market days. Study design Thi i l This implementation research study was conducted between September 2019 and November 2020. A quan- titative approach using household questionnaire surveys targeting caregivers of children under 10 years in the intervention arm only and mystery clients visiting the OTCMS in both intervention and control arms. Interven- tions were evaluated using a two-arm (intervention and control), cluster randomized trial across 8 rural clusters (4 clusters per arm), in two adjacent districts of Ghana. This study evaluated the combined effectiveness of dif- ferent interventions. The intervention arm has 8 clus- ters, and out of these, 4 were randomly selected using a computer-generated list. The control arm had 4 clus- ters, and all these were included in the study. An urban sub-district (Begoro) in the intervention district acted as a buffer between the two arms. A total of 7 OTCMS in the intervention arm and 5 OTCMS in the control arm participated in the study. The aim of the study was to evaluate the combined effectiveness of provider and Given the importance of OTCMS as a first source of care and antimalarial treatment in Ghana, scaling up RDTs in these outlets will achieve universal access to prompt parasite-based diagnosis prior to treatment. Soniran et al. Malaria Journal (2022) 21:317 Soniran et al. Malaria Journal (2022) 21:317 Page 3 of 10 vention clusters on malaria management protocol, appropriate treatment, and follow-ups on their cli- ents. Malaria management protocol in this study is referred to when: community interventions on the proportion of children under 10 years who receive treatment for malaria without testing at OTCMS as well as the level of service provider (OTCMS) adherence to malaria case management guide- lines. This was accomplished by comparing malaria RDT testing rates between pre-intervention and post-inter- vention periods. (a) (a) OTCMS sight every patient suspected of malaria for examination and diagnosis. This includes requesting caregivers of febrile children (visiting the OTCMS for prescription without the child physi- cally present) to bring the child to his/her outlet. (i) Pretending to have fever in the past 24 h. about 10 years of experience (50%); married (58.7%); with Senior High School educational background (75%); Christians (91.7%); and practiced farming as an additional occupation (58.3) (Table 1). (ii) A caregiver seeking medical care on behalf of his/ her febrile child (who is not physically present at the OTCMS shop at the time of the visit). Caregivers of children under 10 years that partici- pated in the household survey were 291 and 346 in the intervention and control arms, respectively. Major- ity were females (94.7%); aged 18–30 years; married (79.1%); with primary educational background; Chris- tians (94.5%); and practiced either petty trading or farming (35.3% and 34.2%, respectively). There was a significant difference (p < 0.05) in the proportions of socio-demographic variables considered in the study between the two arms (Table 2). The mystery client then observed the OTCMS’ response whether RDT will be proposed or not before treatment. The mystery client filled the assessment checklist based on outcome of his/her visit, but when out of sight of the OTCMS. A total of 72 visits (42 and 30 visits in the intervention and control arms respectively) were conducted in the mystery client survey.h End-line household survey: The survey was conducted in the intervention arm to document post-intervention malaria testing rates among children under 10 years vis- iting OTCMS for malaria treatment in the past 1 month preceding the survey. A semi-structured questionnaire was developed, pre-tested for validity and administered by trained data collectors to respondents (caregivers/ mothers of children under 10 years old). The question- naire covered topics such as: Socio-demographics of respondent; knowledge of malaria and its transmission; history of fever among children under 10 years in the past 1 month; caregiver’s treatment-seeking behaviour; and insecticide-treated bed net usage. A total of 1,225 children under 10 years were under the care of the caregivers. These included 574 and 651 chil- dren in the intervention and control arms respectively. Data collection ll Data collection team The study employed data collectors (school teachers) from each community who could con- duct the mystery client survey. The data collectors were trained on the purpose of the study and questionnaire administration. Following the training, the data collec- tion tools were pretested, and adjustments were made where necessary. (i) Pretending to have fever in the past 24 h. Table 1 Demographic characteristics of OTCMS service providers that participated in the study Table 1 Demographic characteristics of OTCMS service providers that participated in the study Characteristics Both arms n (%) Intervention arm n (%) Control arm n (%) Total OTCMS 12 (100) 7 (100) 5 (100) Gender Male 9 (75) 6 (85.7) 3 (60) Female 3 (25) 1 (14.3) 2 (40) Age (years) 20–30 6 (50.0) 2 (28.6) 4 (80) 31–40 1 (8.3) 1 (14.3) 0 (0) 41–50 1 (8.3) 1 (14.3) 0 (0) 51–60 2 (16.7) 1 (14.3) 1 (20) > 60 2 (16.7) 2 (28.6) 0 (0) Years of experience 1–10 6 (50.0) 2 (28.6) 4 (80) 11–20 5 (41.7) 4 (57.1) 1 (20) 21–30 1 (8.3) 1 (14.3) 0 (0) Marital status Single 5 (41.7) 2 (28.6) 3 (60) Married 7 (58.3) 5 (71.4) 2 (40) Level of education Junior high school 1 (8.3) 0 (0) 1 (20) Senior high school 9 (75.0) 5 (71.4) 4 (80) Tertiary 2 (16.7) 2 (28.6) 0 (0) Religion Christianity 11 (91.7) 6 (85.7) 5 (100) Islam 1 (8.3) 1 (14.3) 0 (0) Additional occupation None 4 (33.3) 2 (28.6) 2 (40) Farming/fishing 7 (58.3) 4 (57.1) 3 (60) Petty trader 1 (8.3) 1 (14.3) 0 (0) Characteristics Both arms n (%) Intervention arm n (%) Control arm n (%) Data analysis Data were double entered and cleaned using Microsoft Access 2010 (Microsoft Inc., Redmond, Washington) and analysed using STATA version 11.0. Variables of interest were summarized using descriptive statistics. Propor- tions between groups were compared using chi-square test or fisher exact test (p ≤ 0.05 considered statistically significant). The interventions implemented in this study included: The interventions implemented in this study included: The interventions implemented in this study included: (i) Provision of subsidized RDT kits for OTCMS- The RDT kits were obtained from the National Malaria Control Programme and supplied to OTCMS at no cost. The OTCMS were instructed to test their febrile clients at a subsidized rate of GH¢2.40/kit (~ $0.44), a means of providing incentive for the OTCMS. (ii) Training of OTCMS: A 2 day training workshop was conducted for OTCMS in the selected inter- Soniran et al. Malaria Journal (2022) 21:317 Soniran et al. Malaria Journal (2022) 21:317 Page 4 of 10 (i) Pretending to have fever in the past 24 h. (i) Pretending to have fever in the past 24 h. Socio‑demographic characteristics of study OTCMS, caregivers and children under 10 years old A total of 80 of the children sent to a govern- ment health facility (87.9%; 95% CI 88.1–98.1) were reported to have received a malaria test before treatment while 16 of the children sent to OTCMS (30.8%; 95% CI 1.8–34.5) were reported to have received a malaria test Caregivers’ report on management of fever in children In the intervention arm only, the proportion of children reported to have had fever within 30 days prior to the sur- vey was 26.8% (154/ 574). Many of these febrile children (59.1%) were reported to have been sent to a government health facility whilst 33.8% (52/154) were sent to OTCMS for care. A total of 80 of the children sent to a govern- ment health facility (87.9%; 95% CI 88.1–98.1) were reported to have received a malaria test before treatment while 16 of the children sent to OTCMS (30.8%; 95% CI 1.8–34.5) were reported to have received a malaria test Caregivers report on management of fever in children In the intervention arm only, the proportion of children reported to have had fever within 30 days prior to the sur- vey was 26.8% (154/ 574). Many of these febrile children (59.1%) were reported to have been sent to a government health facility whilst 33.8% (52/154) were sent to OTCMS for care. A total of 80 of the children sent to a govern- ment health facility (87.9%; 95% CI 88.1–98.1) were reported to have received a malaria test before treatment while 16 of the children sent to OTCMS (30.8%; 95% CI 1.8–34.5) were reported to have received a malaria test Socio‑demographic characteristics of study OTCMS, caregivers and children under 10 years old A total of 12 OTCMS service providers participated in the study. These included 7 OTCMS and 5 OTCMS in the intervention and control arms, respectively. Major- ity were males (75%); aged 20–30 years (50%); had Soniran et al. Malaria Journal (2022) 21:317 Page 5 of 10 Table 2 Demographic characteristics of caregivers of children under 10 years in the end-line household survey Characteristics n (%) Intervention Arm Control Arm X2, p value Total caregivers 637 (100) 291 (100) 346 (100) Sex n (%) 29.95, < 0.00001 Males 34 (5.3) 31 (10.7) 3 (0.9) Females 603 (94.7) 260 (89.3) 343 (99.1) Age (years) n (%) 15.09, 0.0045 < 18 3 (0.5) 2 (0.7) 1 (0.3) 18–30 264 (41.4) 105 (36.1) 159 (46.0) 31–40 214 (33.6) 97 (33.3) 117 (33.8) 41–50 106 (16.6) 53 (18.2) 53 (15.3) > 50 50 (7.8) 34 (11.7) 16 (4.6) Marital status n (%) 10.07, 0.039 Single 79 (12.4) 29 (10.0) 50 (14.5) Married 504 (79.1) 238 (81.8) 266 (76.8) Separated 18 (2.8) 5 (1.7) 13 (3.8) Divorced 17 (2.7) 6 (2.1) 11 (3.2) Widowed 19 (3.0) 13 (4.5) 6 (1.7) Educational level, n (%) 54.85. < 0.00001 None 111 (17.4) 79 (27.2) 32 (9.2) Primary 148 (23.2) 81 (27.8) 67 (19.4) Junior High 317 (49.8) 114 (39.2) 203 (58.7) Senior High 43 (6.7) 10 (3.4) 33 (9.5) Vocational training 1 (0.2) 0 (0.0) 1 (0.3) University 17 (2.7) 7 (2.4) 10 (2.9) Religion, n (%) 9.9463, 0.0069 None 8 (1.3) 6 (2.1) 2 (0.6) Christianity 602 (94.5) 266 (91.4) 336 (97.1) Islam 27 (4.2) 19 (6.5) 8 (2.3) Primary occupation, n (%) 114.70, < 0.00001 Unemployed 66 (10.4) 36 (12.4) 30 (8.7) Farming/ Fishing 218 (34.2) 158 (54.3) 60 (17.3) Petty trading 225 (35.3) 55 (18.9) 170 (49.1) Civil servant/ Government official 23 (3.6) 10 (3.4) 13 (3.8) Artisan 100 (15.7) 31 (10.7) 69 (19.9) Other 5 (0.7) 1 (0.3) 4 (1.2) Majority were males (53.2); aged 6–10 years (54.3%); and biological son/daughters of their caregivers (91.1%) (Table 3). Caregivers’ report on management of fever in children In the intervention arm only, the proportion of children reported to have had fever within 30 days prior to the sur- vey was 26.8% (154/ 574). Many of these febrile children (59.1%) were reported to have been sent to a government health facility whilst 33.8% (52/154) were sent to OTCMS for care. OTCMS’ adherence to malaria protocol Mystery client surveys in the two study arms showed that the total adherence to malaria protocol by OTCMS was 55.6%. However, adherence to malaria protocol by OTCMS (66.7%) in the intervention arm was significantly (p < 0.05) higher compared to the control arm (40%) (Table 6). Caregivers’ treatment‑seeking behaviour In the intervention arm only, the proportion of caregivers self-treating their febrile children or visiting drug ven- dors (drug peddlers) during the pre-intervention period (baseline) significantly decreased in favour of visits to OTCMS shops after the deployment of interventions (endline) (p < 0.001) (Fig. 1). Soniran et al. Malaria Journal (2022) 21:317 Page 6 of 10 Table 3 Demographic characteristics of children ≤ 10 years under the caregivers in the end-line household survey Characteristics Both arms n (%) Intervention Arm n (%) Control Arm n (%) X2, p value Total children 1,225 (100) 574 (100) 651 (100) Gender n (%) Males 652 (53.2) 312 (54.4) 340 (52.2) 0.47, 0.491 Females 573 (46.8) 262 (45.6) 311 (47.8) Age (years) n (%) 0.03, 0.872 < 5 560 (45.7) 261 (45.5) 299 (45.9) 6–10 665 (54.3) 313 (54.5) 352 (54.1) Age (mean + SD) 5.03 ± 2.83 4.79 ± 2.72 t = −1.4810, 0.9306 Caregiver-child relationship, n (%) 16.14, 0.001 Son/daughter 1116 (91.1) 508 (88.6) 608 (93.5) Grandchild 91 (7.4) 60 (10.5) 31 (4.8) Niece/nephew 16 (1.3) 6 (0.9) 10 (1.5) Not related 1 (0.1) 0 (0.0) 1 (0.2) Table 3 Demographic characteristics of children ≤ 10 years under the caregivers in the end-line household survey Post-intervention, malaria testing rate by OTCMS was higher compared to pre-intervention period, though not statistically significant (30.8% vs 10.5%; p = 0.1238). Pre- scription of ACT to children not diagnosed also reduced after the intervention but not statistically significant (60% vs 80%; p = 0.2385) (Table 5). 0 20 40 60 80 100 120 Self Drug vendor OTCMSHealth facility Self Drug vendor OTCMSHealth facility Proportion (%) Source of healthcare Not tested Tested Before intervention Aer intervention Fig. 1 Treatment-seeking behaviour of caregivers and proportion of febrile children tested before treatment Discussionh The OTCMS are often the first source of care for health- care seekers in the communities and hence, have the potential for increased universal access to prompt para- site-based diagnosis prior to treatment, if RDTs are intro- duced and scaled-up in this sector. However, evidence to guide decisions on introduction and scaling-up of RDTs among OTCMS is lacking [14]. This study presents find- ings indicating the possibility of scaling-up RDTs among the OTCMS. Health-seeking behaviour for malaria treatment is important in the success of preventing malaria related mortality [15, 16]. Before implementation of interven- tions, caregivers of febrile children practiced self-treat- ment and patronized drug peddlers [13]. But this attitude changed because of interventions implemented which included sensitization on malaria and increased malaria testing using RDT before prescription of medicine by OTCMS.h (ACT) prescribed to clients ‘not tested’ in the interven- tion arm was 64.3% compared to the control arm (75%) though not statistically significant (p > 0.05) (Table 7). (ACT) prescribed to clients ‘not tested’ in the interven- tion arm was 64.3% compared to the control arm (75%) though not statistically significant (p > 0.05) (Table 7). Malaria testing rate and prescription of medicine by OTCMS’ Mystery client survey also showed that malaria testing rate in the intervention arm (38.1%) was higher than in the control arm (23.3%) though not statistically signifi- cant (p = 0.1853). The proportion of anti-malarial drug before treatment (Table 4). Children who received an ACT without a malaria test were 10(11.6%) at the gov- ernment health facility and 24(60%) at the OTCMS. Table 4 Caregivers’ report on diagnosis and treatment of febrile children < 10 years old (intervention arm only) ACT Artemisinin-based Combination Therapy; () malaria testing/non-testing rate Treatment N (%) Sources of treatment and diagnosis Self n (%) Drug vendor n (%) OTCMS n (%) Health centre n (%) Tested Not tested Tested Not tested Tested Not tested Tested Not tested Herbs 4 (2.6) 0 (0.0) 3 (100) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100) 0 (0.0) 0 (0.0) ACT 132 (85.7) 0 (0.0) 1 (100) 2 (40) 3 (60) 16 (40) 24 (60) 76 (88.4) 10 (11.6) Non-antimalarials 18 (7.1) 0 (0.0) 2 (100) 0 (0.0) 0 (0.0) 0 (0.0) 11 (100) 4 (80) 1 (20) 0 (0.0) 6 (100) 2 (40) 3 (60) 16 (30.8) 36 (69.2) 80 (87.9) 11 (12.1) Grand Total 154 (100) 6 (3.9) 5 (3.2) 52 (33.8) 91 (59.1) Table 4 Caregivers’ report on diagnosis and treatment of febrile children < 10 years old (intervention arm only Soniran et al. Malaria Journal (2022) 21:317 Page 7 of 10 Table 5 Caregivers’ reports on malaria testing rate and treatment of their febrile children by OTCMS ACT Artemisinin-based Combination Therapy Before intervention n (%) After intervention n (%) Tested Not tested Total Tested Not tested Total Herbs 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (2.7) 1 (100) ACT 2 (20) 8 (80) 10 (100) 16 (40) 24 (60) 40 (100) Non-antimalarials 0 (0.0) 9 (100) 9 (100) 0 (0.0) 11 (30.6) 11 (100) Total 2 (10.5) 17 (89.5) 19 (100) 16 (30.8) 36 (69.2) 52 (100) Table 5 Caregivers’ reports on malaria testing rate and treatment of their febrile children by OTCMS Table 6 OTCMS’ adherence to malaria protocol during mystery client visits Adherence to protocol Number of visits X2, p value Intervention arm Control arm Total Yes 28 (66.7) 12 (40) 40 (55.6) 5.04, 0.0247 No 14 (33.3) 18 (60) 32 (44.4) Total 42 (100) 30 (100) 72 (100) Assessment of OTCMS’ conduct of RDT and interpretation of results Majority of OTCMS in the intervention arm followed the recommended steps while conducting the malaria test for their clients as 75% wore a glove before conducting the malaria test; labelled client’s RDT cassette correctly (75%); used the recommended finger (68.8%); disposed of the lancet correctly (68.8%); wiped the first blood using cotton wool (87.5%); allowed an average of 17.1 min to lapse before reading the test result; and interpreted the results correctly (100%) (Table 8). The interventions implemented also improved OTCMS’ adherence to malaria protocol. The exist- ing practice of OTCMS on management of malaria was observed in the control arm. Prescription of antimalari- als by OTCMS to caregivers of febrile children without the child physically present for a malaria blood test to be conducted on him or her was a prevalent practice. For febrile adults visiting the OTCMS, the recommended Table 7 Malaria testing rate and prescription of medicine to mystery clients by OTCMS Intervention Control Tested Not tested Total Tested Not tested Total Positive Negative Positive Negative ACT 3 (21.4) 2 (14.3) 9 (64.3) 14 (100) 3 (18.8) 1 (6.3) 12 (75) 16 (100) Quinine 0 (0) 0 (0) 1 (100) 1 (100) 0 (0) 0 (0) 2 (100) 2 (100) Non-antimalarial 1 (7.7) 8 (61.5) 4 (30.8) 13 (100) 0 (0) 3 (42.9) 4 (57.1) 7 (100) No drug prescribed 0 (0) 2 (14.3) 12 (85.7) 14 (100) 0 (0) 0 (0) 5 (100) 5 (100) Grand total 16 (38.1) 26 (61.9) 42 (100) 7 (23.3) 23 (76.7) 30 (100) Table 7 Malaria testing rate and prescription of medicine to mystery clients by OTCMS Soniran et al. Assessment of OTCMS’ conduct of RDT and interpretation of results Malaria Journal (2022) 21:317 Page 8 of 10 Table 8 Assessment of OTCMS’ conduct of RDT and interpretation of results Parameters Intervention arm n (%) Control arm n (%) Conducted a malaria blood test 16 (100) 7 (100) Wore hand gloves before conducting test 12 (75) 0 (0) Indicated the right labelling on cassette 12 (75) 3 (42.9) Used recommended finger 11 (68.8) 2 (28.6) Disinfected finger 16 (100) 7 (100) Lancet disposed correctly 11 (68.8) 5 (71.4) First blood wiped using cotton wool 14 (87.5) 3 (42.9) Used the right amount of blood 11 (68.8) 6 (85.7) Blood placed in the appropriate well of RDT kit 16 (100) 7 (100) Blood collecting device disposed correctly 10 (62.5) 4 (57.1) Buffer placed in appropriate well 16 (100) 7 (100) Recommended amount of buffer 15 (93.8) 7 (100) Checked time immediately after adding buffer 5 (31.3) 0 (0) Disposed other waste materials correctly 12 (75) 6 (85.7) Average time elapsed before reading test results (minutes) 17.1 9.4 Interpreted the results correctly 16 (100) 5 (71.4) Table 8 Assessment of OTCMS’ conduct of RDT and interpretation of results malaria test before prescription of medicine was optional based on patient’s ability to afford the cost of testing and sometimes availability of RDT kit in stock. However, with the introduction and regular supply of RDT kits as part of the interventions implemented in the present study, malaria testing rate increased, and most clients could afford at least 1 Ghanaian cedi ($0.17) for a malaria test (unpublished report). The malaria testing rate reported in the present study is between 30.8% and 38.1%, but in a similar study (except for monthly supervision of health- care providers and no promotional activities) conducted in Myanmar, RDT uptake was 59% [17]. Generally, previ- ous studies with more intensive interventions produced better outcomes, but it is unclear whether such efforts could be maintained or scaled up to national level [7]. It is also noteworthy that the OTCMS in the inter- vention arm applied the skills acquired in their training while conducting RDT compared to their counterparts. They were conscious of protecting themselves and their clients against health risks, hence hand gloves were worn before conducting the malaria test. A higher proportion of the OTCMS also labelled individual patient’s cassettes to avoid confusion and misdiagnosis especially when attending to more than one patient at a time. Ethics approval and consent to participate This study was approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana (NMIMR-IRB CPN 086/18–19). All participants (caregivers of children and over the counter medicine sellers) signed an informed consent. All methods were performed in accordance with the relevant guidelines and regulations. Assessment of OTCMS’ conduct of RDT and interpretation of results The recommended finger (closest to the index finger) was pricked for collection of blood samples as recommended by the health authorities. Majority of the trained OTCMS also wiped off the first blood after finger prick before collecting the blood sample for the test, placed the right amount of blood and buffer in the appropriate places of the device and waited for an average time of 17 min though the manufacturer’s recommended time was 20 min. Majority of them also disposed the lancets inside improvised empty beverage cans which were buried under the ground or burnt with fire, along with other waste materials (like used cotton wool and used kits). They acted this way because of the knowledge that such materials constitute hazards to the environment. Interpretation of RDT results and adher- ence was also encouraging as majority of the patients that tested negative were not given any anti-malarials but directed to the nearest health facility. Observa- tion also showed that OTCMS who did not engage in other businesses outside their profession at their out- let/shop location conducted better malaria blood tests. The ability of the OTCMS to adhere to these guidelines p During mystery client survey, the increased malaria testing rate reported though not statistically significant compared to the control arm may be due to the outbreak of coronavirus disease 2019 (COVID-19) pandemic at the middle of the study period. In two communities, anec- dotal reports showed that client patronage of OTCMS reduced during this period because of misconception that malaria blood test is being used to conduct COVID- 19 surveillance. However, it is noteworthy that the quantity of anti-malarials prescribed by OTCMS in the intervention arm reduced (though not significantly) com- pared to the control arm. This shows that if malaria RDT is scaled up among OTCMS along with proper train- ing and monitoring, a lot of anti-malarials prescribed to patients not having malaria in previous practice will be saved for confirmed malaria positive patients. Page 9 of 10 Soniran et al. Malaria Journal (2022) 21:317 Soniran et al. Malaria Journal (2022) 21:317 improved the quality of the RDT conducted compared to their counterpart in the control arm. Funding This study was funded by the WHO Special Programme for Research and Training in Tropical Diseases (TDR) Postdoctoral Fellowship programme in Implementation Research. The TDR had no role in the design, data collection, analysis, and interpretation of data for this study. 10. Soniran OT, Abuaku B, Ahorlu C. Evaluating interventions to improve test, treat and track (T3) malaria strategy among over the counter medicine sellers (OTCMS) in some rural communities of Fanteakwa North district, Ghana: study protocol for a cluster randomized controlled trial. Trials. 2020;21:623. 10. Soniran OT, Abuaku B, Ahorlu C. Evaluating interventions to improve test, treat and track (T3) malaria strategy among over the counter medicine sellers (OTCMS) in some rural communities of Fanteakwa North district, Ghana: study protocol for a cluster randomized controlled trial. Trials. 2020;21:623. Acknowledgements 4. Amoah LE, Abankwa J, Oppong A. Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2 based malaria rapid diagnostic tests in Ghana. Malar J. 2016;15:101. 4. Amoah LE, Abankwa J, Oppong A. Plasmodium falciparum histidine rich protein-2 diversity and the implications for PfHRP 2 based malaria rapid diagnostic tests in Ghana. Malar J. 2016;15:101. We appreciate the Director, Eastern Region Health Directorate and mem- bers of staff of Fanteakwa North and South Districts Health Directorates for technically supporting this study. We appreciate the Chiefs, elders and all subjects who participated in the study; Mr. Daniel Okyere, Ms. Rosemond Kaye (National Service Personnel) as well as other members of the research team and staff of the Epidemiology Department, Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Ghana. 5. WHO. World Malaria Report 2015. Geneva, World Health Organization. 2015. https://apps.who.int/iris/handle/10665/200018. Accessed 03 Mar 2019. 5. WHO. World Malaria Report 2015. Geneva, World Health Organization. 2015. https://apps.who.int/iris/handle/10665/200018. Accessed 03 Mar 2019. 6. UNITAID. Malaria diagnostics landscape update. Geneva. 2015. www. unitaid.org/assets/Malaria_Diagnostics_Landscape_Update_Fe_2015.pdf Accessed 02 Jan 2022. 6. UNITAID. Malaria diagnostics landscape update. Geneva. 2015. www. unitaid.org/assets/Malaria_Diagnostics_Landscape_Update_Fe_2015.pdf Accessed 02 Jan 2022. Abbreviations OTCMS O h OTCMS: Over-the-counter medicine sellers; T3: Test, treat, and track; RDT: Rapid diagnostic test; WHO: Word Health Organization; ACT: Artemisinin-based combination therapy; PMR: Private medicine retailers; CHPS: Community health-based planning services; NMCP: National malaria control programme; SHOPS: Strengthening health outcomes through the private sector; COVID-19: Coronavirus disease 2019. 2. WHO. Malaria: diagnostic testing; microscopy. Geneva, World Health Organization. 2018. https://www.who.int/malaria/areas/treatment/overv iew/en/. Accessed 18 July 2019. 2. WHO. Malaria: diagnostic testing; microscopy. Geneva, World Health Organization. 2018. https://www.who.int/malaria/areas/treatment/overv iew/en/. Accessed 18 July 2019. y 3. WHO. World Malaria Report 2017. Geneva, World Health Organiza- tion. 2017. https://www.who.int/publications/i/item/9789241565523. Accessed 21 Nov 2021. 3. WHO. World Malaria Report 2017. Geneva, World Health Organiza- tion. 2017. https://www.who.int/publications/i/item/9789241565523. Accessed 21 Nov 2021. Author details 1 1 Department of Epidemiology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana. 2 Department of Science Labora- tory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Afikpo, Ebonyi State, Nigeria. 3 Department of Biochemistry, Faculty of Science, Catholic University of Cameroon, Bamenda, Cameroon. With proper supervision and monitoring, scalable interventions targeting OTCMS in rural communities have the potential of improving adherence to the T3 malaria policy and subsequently improving management of uncomplicated malaria in Ghana. Received: 29 June 2022 Accepted: 23 October 2022 Received: 29 June 2022 Accepted: 23 October 2022 Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. References 1. WHO. World malaria report 2021. Geneva, World Health Organization. 2021. https://www.who.int/teams/global-malaria-programme/reports/ world-malaria-report-2021. Accessed 07 Dec 2021. 1. WHO. World malaria report 2021. Geneva, World Health Organization. 2021. https://www.who.int/teams/global-malaria-programme/reports/ world-malaria-report-2021. Accessed 07 Dec 2021. Author contributions 7. Visser T, Bruxvoort K, Maloney K, Leslie T, Barat LM, Allan R, et al. Introduc- ing malaria rapid diagnostic tests in private medicine retail outlets: a systematic literature review. PLoS ONE. 2017;12:e0173093. 7. Visser T, Bruxvoort K, Maloney K, Leslie T, Barat LM, Allan R, et al. Introduc- ing malaria rapid diagnostic tests in private medicine retail outlets: a systematic literature review. PLoS ONE. 2017;12:e0173093. OTS conceived the study; OTS, NIC, BA, and CSA contributed to experimental design; OTS, BA, and CSA played significant parts in data collection; and OTS, BAM, BA, and CSA contributed to sample size calculation and analysis of data. OTS wrote the main manuscript text including tables and figures. OTS, BAM, NIC, BA, and CSA reviewed the manuscript. All authors read and approved the manuscript. 8. Ashigbie PG, Azameti D, Wirtz VJ. Challenges of medicines management in the public and private sector under Ghana’s National Health Insurance Scheme—a qualitative study. J Pharm Policy Pract. 2016;9:6. 8. Ashigbie PG, Azameti D, Wirtz VJ. Challenges of medicines management in the public and private sector under Ghana’s National Health Insurance Scheme—a qualitative study. J Pharm Policy Pract. 2016;9:6. 9. Ghana National Health Insurance Authority. 2012 Annual Report. http:// www.nhis.gov.gh/files/2012%20NHIA%20ANNUAL%20REPORT.pdf. Accessed 13 May 2022. 9. Ghana National Health Insurance Authority. 2012 Annual Report. http:// www.nhis.gov.gh/files/2012%20NHIA%20ANNUAL%20REPORT.pdf. Accessed 13 May 2022. Conclusions Currently, the proposed scale up of RDTs among OTCMS is limited by controversies and lack of evidence to guide decisions on how and where to scale up RDTs [7]. Some of the concerns of public officials include fear that OTCMS may not treat patients according to malaria guidelines, may not adhere to test results, and handle hazardous wastes improperly which may lead to the spread of infectious illnesses [18–20]. It is notewor- thy that the present study provided evidence on some of these gaps which might help policy makers in making informed decision. Availability of data and materials Improving uptake and use of malaria rapid diagnostic tests in the context of artemisinin drug resistance containment in eastern Myanmar: an evaluation of incentive schemes among informal private healthcare providers. Malar J. 2015;14:105. 18. Baiden F, Webster J, Owusu-Agyei S, Chandramohan D. Would rational use of antibiotics be compromised in the era of test-based management of malaria? Trop Med Int Health. 2011;16:142–4. 18. Baiden F, Webster J, Owusu-Agyei S, Chandramohan D. Would rational use of antibiotics be compromised in the era of test-based management of malaria? Trop Med Int Health. 2011;16:142–4. 19. Poyer S, Shewchuk T, Tougher S, Ye Y, Group AC, Mann AG, et al. Avail- ability and price of malaria rapid diagnostic tests in the public and private health sectors in 2011: results from 10 nationally representative cross- sectional retail surveys. Trop Med Int Health. 2015;20:744–56. 19. Poyer S, Shewchuk T, Tougher S, Ye Y, Group AC, Mann AG, et al. Avail- ability and price of malaria rapid diagnostic tests in the public and private health sectors in 2011: results from 10 nationally representative cross- sectional retail surveys. Trop Med Int Health. 2015;20:744–56. 20. USAID DELIVER PROJECT Task Order 3. Health care waste management of malaria rapid diagnostic tests in health clinics. 2011. https://pdf.usaid. gov/pdf_docs/PA00KV1Z.pdf. Accessed 02 Feb 2022. 20. USAID DELIVER PROJECT Task Order 3. Health care waste management of malaria rapid diagnostic tests in health clinics. 2011. https://pdf.usaid. gov/pdf_docs/PA00KV1Z.pdf. Accessed 02 Feb 2022. Availability of data and materials 11. Danquah DA, Buabeng KO, Asante KP, Mahama E, Bart-Plange C, Owusu- Dabo E. Malaria case detection using rapid diagnostic test at the commu- nity level in Ghana: consumer perception and practitioners’ experiences. Malar J. 2016;15:34. 11. Danquah DA, Buabeng KO, Asante KP, Mahama E, Bart-Plange C, Owusu- Dabo E. Malaria case detection using rapid diagnostic test at the commu- nity level in Ghana: consumer perception and practitioners’ experiences. Malar J. 2016;15:34. The datasets supporting the conclusions of this article are available in the Department of Epidemiology, Noguchi Memorial Institute for Medical Research and are available through the corresponding author on reasonable request. Page 10 of 10 Soniran et al. Malaria Journal (2022) 21:317 Soniran et al. Malaria Journal (2022) 21:317 12. Audu R, Anto BP, Koffuor GA, Abruquah AA, Buabeng KO. Malaria rapid diagnostic test evaluation at private retail pharmacies in Kumasi. Ghana J Res Pharm Pract. 2016;5:175–80. 13. Soniran OT, Abuaku B, Anang A, Opoku-Afriyie P, Ahorlu C. Factors impacting test-based management of suspected malaria among caregivers of febrile children and private medicine retailers within rural communities of Fanteakwa North district, Ghana. BMC Public Health. 2021;21:1899. 14. Roll Back Malaria Partnership Case Management Working Group. Key learnings for malaria programme managers from AFMm Phase 1. 2013. https://www.theglobalfund.org/media/6845/rbm_amfmkeylearnings_ report_en.pdf. Accessed 12 Mar 2019. 14. Roll Back Malaria Partnership Case Management Working Group. Key learnings for malaria programme managers from AFMm Phase 1. 2013. https://www.theglobalfund.org/media/6845/rbm_amfmkeylearnings_ report_en.pdf. Accessed 12 Mar 2019. p p 15. Workineh B, Mekonnen FA. Early treatment-seeking behaviour for malaria in febrile patients in northwest Ethiopia. Malar J. 2018;7:406. 15. Workineh B, Mekonnen FA. Early treatment-seeking behaviour for malaria in febrile patients in northwest Ethiopia. Malar J. 2018;7:406. 16. Gerald M. Assessing factors influencing health seeking behaviour for malaria treatment in children under five years in Rwimi Town Council Kabarole District. Int J Sch Cogn Psychol. 2015;2:151. g y 17. Aung T, White C, Montagu D, McFarland W, Hlaing T, Khin HS, et al. Improving uptake and use of malaria rapid diagnostic tests in the context of artemisinin drug resistance containment in eastern Myanmar: an evaluation of incentive schemes among informal private healthcare providers. Malar J. 2015;14:105. 17. Aung T, White C, Montagu D, McFarland W, Hlaing T, Khin HS, et al. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W2972369460	https://www.aclweb.org/anthology/W19-4811.pdf	English	null	Character Eyes: Seeing Language through Character-Level Taggers	null	2,019	cc-by	4,998	Abstract character-word correspondence in very different patterns, and yet the bi-directional LSTM appears to be, or is assumed to be, capable of capturing them all. In large multilingual settings, it is not uncommon to tune hyperparameters on a handful of languages, and apply them to the rest (e.g., Pin- ter et al., 2017). Character-level models have been used exten- sively in recent years in NLP tasks as both supplements and replacements for closed- vocabulary token-level word representations. In one popular architecture, character-level LSTMs are used to feed token representations into a sequence tagger predicting token-level annotations such as part-of-speech (POS) tags. In this work, we examine the behavior of POS taggers across languages from the perspective of individual hidden units within the charac- ter LSTM. We aggregate the behavior of these units into language-level metrics which quan- tify the challenges that taggers face on lan- guages with different morphological proper- ties, and identify links between synthesis and afﬁxation preference and emergent behavior of the hidden tagger layer. In a comparative ex- periment, we show how modifying the balance between forward and backward hidden units affects model arrangement and performance in these types of languages. Character-level models have been used exten- sively in recent years in NLP tasks as both supplements and replacements for closed- vocabulary token-level word representations. In one popular architecture, character-level LSTMs are used to feed token representations into a sequence tagger predicting token-level annotations such as part-of-speech (POS) tags. In this work, we challenge this implicit gener- alization. We train character-based sequence tag- gers on a large selection of languages exhibiting various strategies for word formation, and sub- ject the resulting models to a novel analysis of the behavior of individual units in the character- level Bi-LSTM hidden layer. This reveals dif- ferences in the ability of the Bi-LSTM architec- ture to identify parts-of-speech, based on typolog- ical properties: hidden layers trained on agglutina- tive languages ﬁnd more regularities on the char- acter level than in fusional languages; languages that are sufﬁx-heavy give a stronger signal to the backward-facing hidden units, and vice versa for preﬁx-heavy languages. In short, character-level recurrent networks function differently depending on how each language expresses morphosyntactic properties in characters. In this work, we examine the behavior of POS taggers across languages from the perspective of individual hidden units within the charac- ter LSTM. 1https://github.com/ruyimarone/ character-eyes ∗Work done while at Georgia Institute of Technology. Abstract We aggregate the behavior of these units into language-level metrics which quan- tify the challenges that taggers face on lan- guages with different morphological proper- ties, and identify links between synthesis and afﬁxation preference and emergent behavior of the hidden tagger layer. In a comparative ex- periment, we show how modifying the balance between forward and backward hidden units affects model arrangement and performance in these types of languages. 1 Introduction Subword vector representations are now a stan- dard part of neural architectures for natural lan- guage processing (e.g., Bojanowski et al., 2017; Peters et al., 2018). In particular, charac- ter representations have been shown to handle out-of-vocabulary words in supervised tagging tasks (Ling et al., 2015; Lample et al., 2016). These advantages generalize across multiple lan- guages, where morphological formation may dif- fer greatly but the character composition of words remains a relatively reliable primitive (Plank et al., 2016). These empirical results motivate a novel Bi- LSTM architecture, in which the number of hid- den units is unbalanced across the forward and backward directions. We ﬁnd empirical corre- spondence between the analytical ﬁndings above and performance of such unbalanced Bi-LSTM models, allowing us to translate the typological properties of a language into concrete recommen- dations for model selection. 1 Proceedings of the Second BlackboxNLP Workshop on Analyzing and Interpreting Neural Networks for NLP, pages 95–102 Florence, Italy, August 1, 2019. c⃝2019 Association for Computational Linguistics Character Eyes: Seeing Language through Character-Level Taggers Marc Marone ∗ Microsoft mmarone6@gatech.edu Yuval Pinter School of Interactive Computing Georgia Institute of Technology uvp@gatech.edu Marc Marone ∗ Microsoft Jacob Eisenstein Facebook AI Research jacobeisenstein@fb.com 3 Tagging Task We train a set of LSTM tagging models, follow- ing the setup of Ling et al. (2015). A word rep- resentation trained from a character-level LSTM submodule is fed into a word-level bidirectional LSTM, with each word’s hidden state subse- quently fed into a two-layer perceptron producing tag scores, which are then softmaxed to produce a tagging distribution. For languages with addi- tional morphosyntactic attribute tagging, we fol- low the architecture in Pinter et al. (2017) where the same word-level Bi-LSTM states are used to predict each attribute’s value using its own per- ceptron+softmax scaffolding. In order to pro- duce character models which would be as infor- mative as possible to our subsequent analysis, we do not include word-level embeddings, pre-trained or otherwise, in our setup. Our analysis assumes a characterization of unit roles, where each hidden unit is observed to have some speciﬁc function. Findings from Linzen et al. (2016) and others suggest that a single hid- den unit can learn to track complex syntactic rules. Radford et al. (2017) found that a character-level language model can implicitly assign a single unit to track sentiment, without being directly super- vised. Kementchedjhieva and Lopez (2018) also examined individual units in a character model and found complex behavior by inspecting acti- vation patterns by hand. Most recently, Dalvi et al. (2019) performed post-hoc tuning of neu- rons trained in language model and machine trans- lation components, and examined their ability to predict grammatical functions. Like them, we per- form an aggregative analysis of individual units to reach measurable quantities of models at a whole, but apply our method to taggers trained directly on supervised grammatical tasks, and focus on cross- lingual variation as the main object of investiga- tion. 2 Related Work †Afﬁxation: S/s is strongly/weakly sufﬁxing; P/p is strongly/weakly preﬁxing; = is equally preﬁx- ing/sufﬁxing; ∅is little afﬁxation. ‡Morphological syn- thesis: agglutinative, fusional, introﬂexive, isolating. 2 Related Work While the advantages of character-level models are readily apparent, existing evaluation methods fail to explain the mechanism by which these mod- els encode linguistic knowledge about morphol- ogy and orthography. Different languages exhibit Several recent papers attempt to explain neural network performance by investigating hidden state activation patterns on auxiliary or downstream 95 Language Afﬁx† Morph POS Accuracy % synth‡ Dev Test Arabic S int 96.11 95.93 Bulgarian S fus 97.91 97.80 Coptic p agg 92.54 92.51 Danish S fus 95.59 95.46 Greek S fus 96.13 96.46 English S fus 93.65 93.30 Spanish S fus 95.75 95.00 Basque = agg 92.99 92.43 Persian s fus 96.07 96.10 Irish = fus 89.35 Hebrew s int 95.71 94.60 Hindi S fus 95.03 94.91 Hungarian S agg 94.14 92.00 Indonesian S iso 92.55 92.68 Italian S fus 96.82 96.95 Latvian s fus 94.70 93.09 Russian S fus 95.29 95.25 Swedish S fus 95.80 95.73 Tamil S agg 86.46 87.58 Thai ∅ fus 91.37 Turkish S agg 92.08 92.48 Ukrainian S fus 95.68 95.26 Vietnamese ∅ iso 88.51 86.58 Chinese S iso 93.05 93.11 tasks. On the word level, Linzen et al. (2016) trained LSTM language models, evaluated their performance on grammatical agreement detection, and analyzed activation patterns within speciﬁc hidden units. We build on this analysis strategy as we aggregate (character-) sequence activation pat- terns across all hidden units in a model into quan- titative measures. Substantial prior work exists on the character level as well (Karpathy et al., 2015; Vania and Lopez, 2017; Kementchedjhieva and Lopez, 2018; Gerz et al., 2018). Smith et al. (2018) examined the character component in multilingual parsing models empirically, comparing it to the contribu- tion of POS embeddings and pre-trained embed- dings. Chaudhary et al. (2018) leveraged cross- lingual character-level correspondence to train NER models for low-resource languages. Godin et al. (2018), compared CNN and LSTM charac- ter models on a type-level prediction task on three languages, using the post-network softmax values to see which models identify useful character se- quences. Unlike their analysis, we examine a more applied token-level task (POS tagging), and focus on the hidden states within the LSTM model in or- der to analyze its raw view of word composition. Table 1: Attributes and tagging accuracy by lan- guage (Irish and Thai do not have both dev and test sets). 2We used 8 as our frequency threshold, and deﬁne unam- biguous forms as ones tagged at least 60% of the time with a single POS. 3.1 Language Selection As our goal is to examine the relationship between character-level modeling and linguistic properties, we drove language selection based on two mor- phological properties deemed relevant to the archi- 96 acter embedding size is set to 256, initialized us- ing the method of Glorot and Bengio (2010). The word-level bidirectional LSTM has two layers and a hidden state size of 128, with 50% dropout ap- plied in the style of Gal and Ghahramani (2016). Each attribute-prediction MLP has a single hidden layer that is the same size as the tagset size for that attribute, and includes a tanh nonlinearity. Mod- els were trained for up to 80 epochs, and we select the model with the highest POS tagging accuracy on the dev set. Training used SGD with 0.9 mo- mentum, and all models were implemented using DyNet 2.0 (Neubig et al., 2017). tectural effects examined. All 24 datasets were ob- tained from Universal Dependencies (UD) version 2.3 (Nivre et al., 2018), and linguistic properties were found in the World Atlas of Language Struc- tures (Bickel and Nichols, 2013; Dryer, 2013). The selected languages and their properties are presented in Table 1. We note that eleven of the 24 languages selected are not Indo-European. tectural effects examined. All 24 datasets were ob- tained from Universal Dependencies (UD) version 2.3 (Nivre et al., 2018), and linguistic properties were found in the World Atlas of Language Struc- tures (Bickel and Nichols, 2013; Dryer, 2013). The selected languages and their properties are presented in Table 1. We note that eleven of the 24 languages selected are not Indo-European. Afﬁxation. To evaluate the role of forward and backward units in a bidirectional model, we se- lected all languages available in UD which are not classiﬁed as either weakly or strongly sufﬁx- ing in inﬂectional morphology (the vast majority of UD languages). This includes a single preﬁx- ing language (Coptic), two equally sufﬁxing and preﬁxing languages (Basque and Irish), and two languages with little afﬁxation (Thai and Viet- namese). 3.3 Results In our initial setup, we represent words using a concatenation of the ﬁnal states from a bidirec- tional character-level LSTM with 64 forward and backward hidden units each. The results for POS tagging, presented in Table 1, are on par with simi- lar models (Plank et al., 2016, for example) despite not including a word-level type embedding com- ponent. We attribute this success to our large char- acter embedding size of 256, corroborating ﬁnd- ings reported by Smith et al. (2018). Morphological Synthesis. Linguistically func- tional features vary between being expressed as distinct tokens (isolating languages), detectable unique character substrings (agglutinative), fused together but still distinguishable from the stem (fusional), and non-linearly represented within the word form (introﬂexive). This property has previously been found to affect performance in character-level models (Pinter et al., 2017; Gerz et al., 2018; Chaudhary et al., 2018), and thus we select representatives of each group, including most available non-fusional languages. 3.2 Technical Setup Most of our selected languages have only a sin- gle UD 2.3 treebank. For languages with mul- tiple treebanks we selected the largest, except in the cases of Spanish and Indonesian, where we selected the GSD treebanks. The Irish IDT tree- bank has only a train and test split, so we used the test set for early stopping. The Thai PUD treebank only provided a single dataset with 1000 instances, which we shufﬂed and partitioned into a 850/150 split. Tokens were normalized to remove noisy data: tokens containing ‘http’ were replaced with ‘URL’ and tokens containing ‘@’ were replaced with ‘EMAIL’. This was most relevant (293 re- placements) for the English treebank, which con- tained many long URLs. 4 Analysis We next analyze the models trained on the tagging task in an attempt to see how their character-level hidden states encode different manifestations of linguistic information. We suggest that individual hidden units in the character-level sequence model attune to track patterns in the words which would indicate their linguistic roles (POS and morpho- logical properties), and so patterns in character- role regularity across typologically different lan- guages would manifest themselves in an observ- able form at the individual unit activation level. This motivates us to devise metrics which would characterize languages through aggregation of in- dividual unit behaviour. The max absolute diff base measure is deﬁned as: The max absolute diff base measure is deﬁned as: bmad(w, i) = \|w\|−1 max c=1 \|hc+1 i −hc i\|. This metric aims to quantify the relative impor- tance of forward and backward units in discrimi- nating POS for L. We use only the top units for the metric as a de-noising heuristic, under the as- sumption that all units end up with some minimal amount of mass even without performing a func- tion. Figure 1 demonstrates these two metrics for a sample (word, unit) pair, showing how the former captures the general level of activation the word caused on the unit, while the latter captures the lo- cal character pattern deemed most important by it. We intentionally did not consider metrics based on the ﬁnal activation values, the direct signals used by the later layers in the model, as these bear no insight into the effect of a word’s composition on the learned model. 3This consideration also motivated our choice of UD data, which is tokenized to separate syntactic fusion such as He- brew and Arabic function words, or Spanish del. 4We omit the following ‘character-simple’ part-of-speech tags: INTJ, NUM, PROPN, PUNCT, SYM, X. 4.1 Metrics For each language, we run the character-level BiLSTM from the trained tagger on POS- unambiguous word types occurring frequently in the training set, grouped into their parts of speech.2 This ﬁltering was done in order to focus Hyperparameters. For the initial bidirectional character-level LSTM, we used a total hidden state size of 128 (64 units in each direction). The char- 97 Figure 1: Activations of the English model’s unit 42 (forward) on the word characterizing. bavg\|·\| is 0.42, and bmad is 0.96 (the drop from the second i to n). puted as: T X t=1 B X b=1 P(t, b)[ ln P(t, b) −ln P(t) −ln P(b)], and we call the resulting number the POS- Discrimination Index, or PDI. Intuitively, a higher PDI implies that the unit activates differently on words of different parts of speech, i.e. it is a better discriminator for the task. Figure 1: Activations of the English model’s unit 42 (forward) on the word characterizing. bavg\|·\| is 0.42, and bmad is 0.96 (the drop from the second i to n). At this point a language produces a set of dh PDI scores, one for each unit. We sort them from high to low, and deﬁne two language-level metrics: The mass is the sum of PDI values for all units, M(L) := Pdh i=1 PDI(L, i), intuitively meant to quantify the degree of success the model has in assigning hidden units to discriminate POS in this language. The head forwardness is the propor- tion of forward-directional units (under the sorted ordering) before the point at which half of the mass accumulates (in a random setup, this num- ber would tend to 0.5): on the more consistent generalizations found by the taggers during training, as our goal is to qual- ify properties of languages.3 On each word w, we observe each hidden unit hi’s activation level (out- put) on each character hc i. We obtain a base mea- sure b(w, i) based on the activation pattern. For example, an average absolute base measure is de- ﬁned as the average of absolute value activations: bavg\|·\|(w, i) = 1 \|w\| \|w\| X c=1 \|hc i\|. n k : Pk i=1 PDI(L, i) ≤M(L) 2 ∧hk is forward o n k : Pk i=1 PDI(L, i) ≤M(L) 2 o 4.2 PDI Patterns The PDI patterns on the bavg\|·\| base measure with B = 16 bins on all 24 languages are presented in Table 2. We see that agglutinative languages, where we can expect a better discrimination sig- nal to emerge from the consistently-formed mor- phemes, cluster mostly at the top of the PDI mass scale, suggesting more individual character-level units extract these signals successfully. Introﬂex- ive languages, where character sequences seldom correspond to useful indications of POS or mor- phosyntactic attributes, cluster towards the bot- tom. Next, we derive a language-level metric for each hidden unit, based on the principle of Mutual In- formation (MI). The base metric’s range ([0, 1) for bavg\|·\|, [0, 2) for bmad) is divided into B bins of equal size, and base activations from each word are summed across each of the T POS tag cate- gories4, then normalized to produce a joint proba- bility distribution. The mutual information is com- We present the full unit-level PDI value distri- butions for Coptic, a preﬁxing agglutinative lan- guage, and English, a sufﬁxing fusional language, 98 Figure 2: Distribution of PDI values (bavg\|·\|) across hidden units in Coptic and English, shown in ordered PDI values from largest to smallest, with blue (orange) bars indicating forward (backward) units. The black line demarcates the median point of mass accumulation. Figure 2: Distribution of PDI values (bavg\|·\|) across hidden units in Coptic and English, shown in ordered PDI values from largest to smallest, with blue (orange) bars indicating forward (backward) units. The black line demarcates the median point of mass accumulation. in Figure 2 (trends for bmad are similar). Con- sistent with other agglutinative languages, Cop- tic’s cumulative mass is very large (M(cop) = 58.1), suggesting the predictive qualities of the sequence-based LSTM allows good discrimina- tion from the character signal, as one might ex- pect from an agglutinative language. Conversely, M(eng) = 16, demonstrating the difﬁculty pre- sented by fusional languages. The accumulation of 71% forward (80% backward) units in the head of the Coptic (English) value ranking suggests an interesting relationship between afﬁxation and LSTM direction: LSTM units are likely to hone in on POS-indicative signals, which often occur as afﬁxes, in the beginning of their run, causing acti- vation values to rise (in absolute value) and stay large throughout the subsequent traversal of the stem. 4.2 PDI Patterns Unfortunately, since no other preﬁxing lan- guages are available in UD, we were not able to pursue this hypothesis further. Language Mass Mass % of forward median units until index median Tamil 71.0 55 49.1 Irish 62.0 56 42.9 Coptic 58.1 56 71.4 Hungarian 47.9 55 50.9 Greek 31.2 55 45.5 Turkish 30.1 54 57.4 Russian 25.9 54 40.7 Thai 25.9 55 47.3 Ukrainian 25.0 54 37.0 Vietnamese 24.2 55 36.4 Chinese 23.8 47 42.6 Danish 21.7 54 44.4 Swedish 20.8 53 34.0 Basque 20.6 51 64.7 Indonesian 20.3 45 71.1 Latvian 17.0 52 42.3 Spanish 16.1 45 33.3 English 16.0 50 20.0 Bulgarian 15.6 52 46.2 Italian 14.1 48 56.2 Arabic 12.6 46 58.7 Hebrew 11.4 51 74.5 Persian 10.3 50 46.0 Hindi 8.4 51 41.2 Table 2: PDI statistics for UD 2.3 models, bavg\|·\| metric, sorted by the mass metric (sum of PDIs). Agglutinative languages in bold, introﬂexive in italics. 4.3 Asymmetric Directionality Based on these observations, we conduct a direc- tionality balance study, where we vary the num- ber of hidden units in the forward and backwards dimensions. In addition to the models analyzed above, which use 64 forward and 64 backward units (denoted hereafter 64/64), we trained mod- els with imbalanced directionality (128/0, 96/32, 32/96, 0/128). We test the hypothesis that imbal- anced models affect languages differently based on their linguistic properties and statistical met- rics. We note that these settings do not maintain parameter set size: intra-direction transition oper- ations are quadratic in that direction’s hidden layer size, and so this adds a possible advantage in favor of direction-imbalanced models. Table 2: PDI statistics for UD 2.3 models, bavg\|·\| metric, sorted by the mass metric (sum of PDIs). Agglutinative languages in bold, introﬂexive in italics. Table 2: PDI statistics for UD 2.3 models, bavg\|·\| metric, sorted by the mass metric (sum of PDIs). Agglutinative languages in bold, introﬂexive in italics. 99 Language 128/0 96/32 64/64 32/96 0/128 Arabic 96.29 96.08 96.06 96.09 96.16 Bulgarian 97.95 97.86 97.84 97.74 97.71 Coptic 93.10 92.80 92.58 92.98 92.91 Danish 95.93 95.68 95.61 95.60 95.70 Greek 96.19 96.07 96.01 96.00 95.93 English 93.86 93.74 93.65 93.80 93.87 Spanish 95.74 95.63 95.64 95.64 95.77 Basque 93.52 93.13 92.89 92.59 92.90 Persian 96.31 96.20 96.11 96.02 96.20 Irish 89.54 89.35 88.95 89.11 89.07 Hebrew 95.76 95.72 95.60 95.50 95.57 Hindi 95.35 95.22 95.12 95.11 95.25 Hungarian 94.25 94.29 94.20 93.97 94.00 Indonesian 92.42 92.34 92.49 92.53 92.55 Italian 97.00 96.78 96.87 96.88 97.01 Latvian 95.10 94.84 94.69 94.58 94.61 Russian 95.51 95.39 95.32 95.31 95.36 Swedish 95.93 95.69 95.64 95.52 95.85 Tamil 87.54 87.28 86.88 86.28 85.99 Thai 91.52 91.27 91.38 91.47 91.32 Turkish 93.14 92.45 92.06 92.03 92.09 Ukrainian 95.72 95.76 95.63 95.68 95.66 Vietnamese 87.98 87.92 88.23 87.83 87.85 Chinese 93.01 93.17 93.12 93.03 93.04 Table 4: Full scores for the directionality balance ex- periment, each point averaged over three random seed runs. Language 128/0 96/32 64/64 32/96 0/128 Type (base) Inﬂectional Afﬁxation Categories S. sufﬁx +0.22 +0.07 94.50 -0.06 -0.02 W. sufﬁx +0.26 +0.12 95.46 -0.07 -0.01 Equal p/s +0.61 +0.32 90.99 -0.07 +0.06 Little aff. -0.06 -0.21 89.59 -0.16 -0.22 W. preﬁx +0.52 +0.22 92.91 +0.40 +0.33 Morphological Synthesis Categories Introﬂex. +0.17 +0.05 95.87 -0.06 +0.01 Fusional +0.22 +0.07 94.95 +0.01 +0.06 Agglutina. 5 Conclusion While character-level Bi-LSTM models compute meaningful word representations across many lan- guages, the way they do it depends on each lan- guage’s typological properties. These observa- tions can guide model selection: for example, in agglutinative languages we observe a strong preference for a single direction of analysis, mo- tivating the use of unidirectional character-level LSTMs for at least this type of language. In future work, we plan to introduce further control into our metrics by incorporating dataset attributes such as tag distribution and number of instances, as well as learning-related properties like convergence rate and effect of initialization. 4.3 Asymmetric Directionality +0.59 +0.27 91.58 -0.16 -0.15 Isolating -0.14 -0.13 91.15 -0.15 -0.13 Overall +0.25 +0.08 93.85 -0.05 -0.01 Table 3: Imbalanced models’ mean POS accuracy on UD development data (differences between three aver- aged random runs in all models; boldfaced when sig- niﬁcant at p < 0.05 using a paired two-tailed t-test). Table 3: Imbalanced models’ mean POS accuracy on UD development data (differences between three aver- aged random runs in all models; boldfaced when sig- niﬁcant at p < 0.05 using a paired two-tailed t-test). Table 3: Imbalanced models’ mean POS accuracy on UD development data (differences between three aver- aged random runs in all models; boldfaced when sig- niﬁcant at p < 0.05 using a paired two-tailed t-test). The results for this study are presented in Ta- ble 3 as averages for the language categories listed in Table 1 (the full, raw results are available in Ta- ble 4). Table 4: Full scores for the directionality balance ex- periment, each point averaged over three random seed runs. One trend which emerges is the preference of agglutinative languages for imbalanced models, whereas the other languages are little affected by this change. This could be explained by the in- crease in inter-unit interaction in the larger direc- tion of an imbalanced model – contiguous char- acter sequences consistently code reliable linguis- tic features in these languages. A second ﬁnd- ing is the slight bias of sufﬁxing languages to- wards more forward units and of the preﬁxing lan- guage to more backward units, indicating that hid- den LSTM units are better in detecting formations close to their ﬁnal state. Coupled with the ﬁnd- ings regarding PDI mass distribution in the dif- ferent directional units in § 4.2, we suggest that a subtle relation exists between morphological in- formation and model directionality: units which end their run on the afﬁx are more important for detecting the POS signal, so having more of them helps the model. We also note the stability of iso- lating and little-afﬁxing languages to directional- ity balance, possibly owing to the relatively small signiﬁcance of contiguous character sequences in detecting word role. 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https://openalex.org/W2507324130	https://europepmc.org/articles/pmc5007310?pdf=render	English	null	Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from<i>Bothrops marajoensis</i>(Marajó Lancehead) Snake Venom	Biochemistry research international	2,016	cc-by	7,425	Hindawi Publishing Corporation Biochemistry Research International Volume 2016, Article ID 2053459, 10 pages http://dx.doi.org/10.1155/2016/2053459 Hindawi Publishing Corporation Biochemistry Research International Volume 2016, Article ID 2053459, 10 pages http://dx.doi.org/10.1155/2016/2053459 Hindawi Publishing Corporation Biochemistry Research International Volume 2016, Article ID 2053459, 10 pages http://dx.doi.org/10.1155/2016/2053459 1Multidisciplinary Research Laboratory, S˜ao Francisco University (USF), Avenida S˜ao Francisco de Assis 218, Jardim S˜ao Jos´e, 12916 350 B P li SP B il g ¸ 2Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, Cidade Universit´aria Zeferino Vaz, 13083-365 Campinas, SP, Brazil 3Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tess´alia Vieira de Camargo 126, Cidade Universit´aria Zeferino Vaz, 13083-881 Campinas, SP, Brazil 3Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tess´alia Vieira de Camargo 126, Cidade Universit´aria Zeferino Vaz, 13083-881 Campinas, SP, Brazil Correspondence should be addressed to Thalita Rocha; thalita.rocha@usf.edu.br Received 13 November 2015; Revised 2 June 2016; Accepted 3 July 2016 Academic Editor: Tzi Bun Ng Academic Editor: Tzi Bun Ng Academic Editor: Tzi Bun Ng Copyright © 2016 Antonio Lisboa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal. Antonio Lisboa,1 Rodolfo Melaré,1 Junia R. B. Franco,1 Carolina V. Bis,1 Marta Gracia,1 Luis A. Ponce-Soto,2 Sérgio Marangoni,2 Léa Rodrigues-Simioni,3 Maria Alice da Cruz-Höfling,2 and Thalita Rocha1,2 Antonio Lisboa,1 Rodolfo Melaré,1 Junia R. B. Franco,1 Carolina V. Bis,1 Marta Gracia,1 Luis A. Ponce-Soto,2 Sérgio Marangoni,2 Léa Rodrigues-Simioni,3 Maria Alice da Cruz-Höfling,2 and Thalita Rocha1,2 1Multidisciplinary Research Laboratory, S˜ao Francisco University (USF), Avenida S˜ao Francisco de Assis 218, Jardim S˜ao Jose 12916-350 Braganc¸a Paulista, SP, Brazil 1. Introduction hemorrhagic activities attributed to the phospholipases A2 contained in their venoms. One dominant effect of Bothrops snakebites is marked myotoxicity without any clinical sign of neurotoxicity. However, in vitro studies in amphibian, avian, and mammalian nerve-muscle preparations have shown neurotoxicity, with total and/or irreversible neuromuscular blockade, at very low concentrations of venom [7, 8] or toxins [8–10] from different species. Hence, it is likely that a direct action of phospholipase A2 (PLA2) myotoxins plus an indirect action caused by tissue anoxia has key roles In Brazil, the accidents caused by Bothrops snakes are close to 74% according to the National System of Accidents Notifica- tion (SINAN) [1]; they constitute a major public health prob- lem in Brazil. Great part of these accidents occurs at remote country areas far from appropriate first aid intervention [2]. Generally, the envenomation picture is characterized by pronounced myonecrosis [3–5] and systemic effects [6]. Such effects result from proteolytic, myotoxic, blood-clotting, and 2 Biochemistry Research International (a) (b) 1: 9.000.000 (c) Figure 1: (a) Image of an adult male specimen of Bothrops marajoensis (photo: Silvia Cardoso, Ph.D.; Museu Biol´ogico/Instituto Butantan, S˜ao Paulo, SP, Brazil). (b) The Brazilian map with Par´a state in gray and (c) an enlarged detail of the Maraj´o Island and costal drainage at the Amazon estuary. (a) (b) 1: 9.000.000 (c) 1: 9.000.000 (b) (c) (a) Figure 1: (a) Image of an adult male specimen of Bothrops marajoensis (photo: Silvia Cardoso, Ph.D.; Museu Biol´ogico/Instituto Butantan, S˜ao Paulo, SP, Brazil). (b) The Brazilian map with Par´a state in gray and (c) an enlarged detail of the Maraj´o Island and costal drainage at the Amazon estuary. in the effects of Bothrops venom on the muscle and nerve fibres, including immediate destruction of motor axons and complete depletion of intramuscular motor nerve trunks [11]. integral protein of the synaptic vesicle, binds to synaptobrevin and may act to prevent vesicle docking at the axolemma of the terminal bouton; SNAP25 is a plasma membrane protein (t-SNARE), which in association with synaptobrevin forms a stable complex responsible for vesicle docking, priming, and fusion [17]. Alterations in this complex can affect the neurotransmitter release and induce a presynaptic-blocking effect. Brazilian species of the Bothrops genus are close to 50. 1. Introduction Geographically, same species can be distributed all over the country while others are endemic, for example, Bothrops marajoensis, which is only found in the Maraj´o Island, Par´a State, in the very North of Brazil [12, 13] (Figure 1).ii f In this communication we describe our observations on the cellular pathology of the motor nerve terminal and skeletal muscle fibres exposed in vitro to two major toxins isolated from the venom of B. marajoensis. Our hypothesis is that BmjeTX-I and BmjeTX-II affect neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal. Notified accidents with B. marajoensis are rare; a first case of hemorrhagic stroke in a child bitten by B. marajoensis in Anaj´as city, Maraj´o Island, was recently reported; the permanent hemiplegia sequela suffered by the victim was attributed to delayed medical intervention [14]. Also, an accident occurred with a specimen in captivity that bit the right hand thumb of the snakes’ caretaker in the Instituto Butantan; the bite produced immediate excruciating pain and edema, which spread rapidly to her hand and part of the forearm leading to ecchymosis and blister formation. The intense pain and throbs persisted for 48 hours despite rapid medical care at the Hospital Vital Brazil (Silvia Cardoso, Ph.D., personal communication, Instituto Butantan). 2.2. Toxins and Reagents. BmjeTX-I and BmjeTX-II isolated from B. marajoensis snake venom were purified as described in full detail by Ponce-Soto et al. [17]. 2.2. Toxins and Reagents. BmjeTX-I and BmjeTX-II isolated from B. marajoensis snake venom were purified as described in full detail by Ponce-Soto et al. [17]. 2. Material and Methods The preparations were allowed to stabilize for at least 20 min before addition of a single concentration of BmjeTX-I or BmjeTX-II PLA2 toxins (10 𝜇g/mL). Such concentration was the same used by Ponce- Soto et al. [17]. and in 0.1% Triton X-100 in phosphate buffered saline (PBS) (15 min, room temperature), rinsed with PBS, and incubated overnight in a moist chamber at 4∘C with appropriate primary antibodies (diluted to a final concentration of 1/150 synap- tophysin, 1/250 SNAP25, and 1/300 synaptobrevin). Control of the immunoreaction was done by omitting the primary antibodies. On the following day, the slides were allowed to return to room temperature, washed in PBS, and incubated with the appropriate FITC-conjugated secondary antibody (goat anti-rabbit 1/80, rabbit anti-goat 1/800, and goat anti- mouse 1/100) or TRITC-conjugated 𝛼-BgTX (diluted to a half secondary antibody concentration) for 2 h at room temperature and then mounted in glycerin jelly. The positive immunolabelling was accessed, using a fluorescence BX51TF microscope (Olympus Optical Co. Ltd., Tokyo, Japan), from 6 serial sections and the positive reactions were counted for statistical purposes (morphometry). 2.3. Chick Biventer Cervicis (CBC) Preparation. Chicks (𝑛= 12) were killed by halothane inhalation and immediately the biventer cervicis muscles were removed and mounted under a tension of 1 g in a 5 mL organ bath containing Krebs solution (composition in mM: NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4 1.17, MgSO4 1.17, NaHCO3 25, and glucose 11.65), pH 7.5 at 37∘C, and carbogen aeration (95%/5% O2/CO2, v/v) [18] for twitch tension recordings. The preparations were allowed to stabilize for at least 20 min before addition of a single concentration of BmjeTX-I or BmjeTX-II PLA2 toxins (10 𝜇g/mL). Such concentration was the same used by Ponce- Soto et al. [17]. A bipolar platinum ring electrode was placed around the muscle and coupled to a Grass S48 stimulator (0.1 Hz, 0.2 ms duration, 4–8 V). Isometric muscle contractions and con- tractures were recorded via a force displacement transducer (Load Cell BG-50 Grams, Kulite Semiconductor Products, Inc.) coupled to a physiograph (Gould, Model RS 3400). Contractures to exogenous acetylcholine (ACh, 110 𝜇M) and potassium chloride (KCl, 40 mM) were obtained in the absence of field stimulation prior to toxins addition and by the end of the experiment (120 min). 2.7. Western Blotting (WB). 2. Material and Methods CBC nerve-muscle preparation (𝑛 = 4), maintained in carbogen-aerated Krebs solu- tion (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were homogenized in 1 mL of antiprotease cocktail. Aliquots of 15 𝜇g proteins were used for 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (120 V, 90 min). After electrophoretic proteins transfer to nitrocellulose membrane (400 mA, 90 min), the samples were blocked overnight at 4∘C in PBS containing 5% w/v dried milk and probed with primary antibodies (diluted in PBS containing 3% w/v dried milk to a final concentration of 1/500 synaptophysin, 1/500 synaptobrevin, and 1/1000 SNAP25) for 4 h. Blots were washed in PBS and incubated with a corresponding HRP- conjugated secondary antibody diluted in PBS containing 1% w/v dried milk to a final concentration of 1/1000 goat anti- rabbit, 1 : 5000 rabbit anti-mouse, and 1 : 3000 rabbit anti- goat, respectively.hi 2.4. Morphological Analysis by Light Microscopy. Longitudi- nal cryosections of CBC (𝑛= 4), maintained in carbogen- aerated Krebs solution (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, perme- abilized in ethanol and methanol (−20∘C, 10 min each bath), rinsed with distilled water, and stained with haematoxylin and eosin (HE) for histological analyses. 2.4. Morphological Analysis by Light Microscopy. Longitudi- nal cryosections of CBC (𝑛= 4), maintained in carbogen- aerated Krebs solution (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, perme- abilized in ethanol and methanol (−20∘C, 10 min each bath), rinsed with distilled water, and stained with haematoxylin and eosin (HE) for histological analyses. 2.5. Morphological Analysis by Transmission Electron Microscopy (TEM). Samples containing end-plate regions of CBC (𝑛= 4), maintained in aerated Krebs solution (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were pinned side by side to wax under slight longitudinal tension, immersion- fixed in Karnovsky’s solution for 30 minutes at room temperature and overnight at 4∘C. 2. Material and Methods Afterwards, the samples were washed in 0.1 M sodium cacodylate buffer, post-fixed in 1% OsO4 solution for 2 h, washed in the same buffer followed by distilled water, contrasted with 5% uranyl acetate for 1 h, washed in distilled water, dehydrated in acetone series, and embedded in Epon resin (Epon : acetone 2 : 1, 1 : 1, and 1 : 2 and pure EPON). Ultrathin sections (60 nm thick) stained with uranyl acetate followed by lead citrate were examined in a Leo 906 transmission electron microscope (Zeiss, Oberkochen, Germany). The ultrastructure of the muscle fibres and neuromuscular junctions and morphometry of synaptic vesicles were provided. The blots were scanned, stored as TIFF files, and quan- tified using Image J 1.45s software (Wayne Rasband, NIH, Bethesda, MD, USA). Densitometric data of endogenous control were generated by incubating blots with GAPDH (1/1000, followed by 1/1000 goat HRP-conjugated anti-rabbit). After rinsing in PBS, the immunoreactive bands were detected by chemiluminescence (Super Signal, Pierce West Pico Chemiluminescent Substrate, USA) using X-ray film (BioMax XAR Film Kodak, USA). Experimental data were expressed in terms of relative optical density. 2.8. Statistics. Quantitative data were expressed as mean ± standard deviation (SD). Statistical significance was deter- mined by one-way ANOVA followed by the Bonferroni post hoc test with 𝑝< 0.05 indicating significance. All analyses were done using Prism software (GraphPad Inc., San Diego, CA, USA). 2.6. Immunofluorescence (IF) Analysis. Transversal cryosec- tions of CBC (𝑛= 4 animals), maintained in carbogen- aerated Krebs solution (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, per- meabilized in ethanol and methanol (−20∘C, 10 min each), 2.6. Immunofluorescence (IF) Analysis. Transversal cryosec- tions of CBC (𝑛= 4 animals), maintained in carbogen- aerated Krebs solution (control) or incubated with 10 𝜇g/mL of BmjeTX-I or BmjeTX-II for 120 minutes under indirect stimulation, were collected onto subbed glass slides, per- meabilized in ethanol and methanol (−20∘C, 10 min each), 2. Material and Methods 2.1. Animals. Male HY-line W36 chicks (4–8-day-old) were supplied by Globo Aves Agrovicultura Ltda (Campinas, SP, Brazil). Animals were housed at 25∘C under a 12 h light/dark cycle with free access to food and water. All procedures were approved by the Institutional Committee for Ethics in Animal Use (CEUA/S˜ao Francisco University, protocol number 000.11.10) and are in accordance with the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA) guidelines. In neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND), concen- trations as low as 1 or 5 𝜇g/mL of B. marajoensis crude venom elicited increases in the frequency of miniature end-plate potentials (MEPPs) which occurs concomitantly with twitch tension facilitation followed by presynaptic neuromuscular paralysis but without evidences of muscle damage [15].h p y g Three basic Asp-49 phospholipases A2 (PLA2) have been isolated from B. marajoensis venom: Bmaj-9 (13679.33 Da) [16], BmjeTX-I (13808.89 Da), and BmjeTX-II (13863.97 Da) PLA2s [17] and were shown to act presynaptically in CBC preparations. At appropriate concentrations, BmjeTX-I and BmjeTX-II induce neuromuscular junction blockade at presynaptic sites with no response elicited in proper muscle and also myonecrosis with systemic interleukin-6 response, moderate marked paw, and cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes [17]. All reagents were obtained from Sigma-Aldrich (MO, USA) if not stated otherwise. Rabbit anti-glyceraldehyde-3- phosphate dehydrogenase (GAPDH-FL335) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); OCT-Tissue Tek was from Sakura Finetek. EPON EMBed-812 Kit, uranyl acetate, osmium tetroxide, lead citrate, and glutaraldehyde were from Electron Microscopy Sciences (Hatfield, PA, USA). Western blotting reagents were purchased from Sigma- Aldrich, Biorad, Amresco, and Kodak. All salts for the physiological solution were of analytical or sequencing grade. y y Venom’s toxins that act on presynaptic sites interfere in complex machinery where proteins, such as synaptophysin, synaptobrevin, and SNAP25, undertake specific roles at precise steps of neurotransmitter release. Synaptophysin, an Biochemistry Research International 3 2.3. Chick Biventer Cervicis (CBC) Preparation. Chicks (𝑛= 12) were killed by halothane inhalation and immediately the biventer cervicis muscles were removed and mounted under a tension of 1 g in a 5 mL organ bath containing Krebs solution (composition in mM: NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4 1.17, MgSO4 1.17, NaHCO3 25, and glucose 11.65), pH 7.5 at 37∘C, and carbogen aeration (95%/5% O2/CO2, v/v) [18] for twitch tension recordings. 3. Results 3.1. Myography. CBC preparations incubated with BmjeTX- I or BmjeTX-II (10 𝜇g/mL) displayed total and irreversible 4 Biochemistry Research International ∗ ∗∗ KCl ACh KCl ACh 1 5 12 16 KCl addition ACh addition Toxins addition Washes 0min 10min 20min 120 min −2 0 2 4 (a) ∗ ∗∗ KCl ACh KCl ACh 2 6 13 17 KCl addition ACh addition Toxins addition Washes 0min 10min 20min 120 min −1 0 1 2 3 4 5 (b) ∗ ∗∗ KCl addition ACh addition Toxins addition Washes KCl ACh KCl ACh 3 7 14 18 0min 10min 20min 120 min 0 1 2 3 4 (c) Figure 2: Chick biventer cervicis preparations in Krebs (control, (a)), or incubated with 10 𝜇g/mL of BmjeTX-I (b) and with 10 𝜇g/mL of BmjeTX-II (c) toxins isolated from B. marajoensis crude venom. Notice, in (b) and (c), the irreversible neuromuscular blockade induced after 31.2 ± 3.5 min and 30 ± 8.1 min (𝑝< 0.05 from Krebs) of toxins (∗) addition, respectively. ACh and KCl addition; ∗∗: washes (𝑛= 12 for each treatment). 4 Biochemistry Research International Figure 2: Chick biventer cervicis preparations in Krebs (control, (a)), or incubated with 10 𝜇g/mL of BmjeTX-I (b) and with 10 𝜇g/mL of BmjeTX-II (c) toxins isolated from B. marajoensis crude venom. Notice, in (b) and (c), the irreversible neuromuscular blockade induced after 31.2 ± 3.5 min and 30 ± 8.1 min (𝑝< 0.05 from Krebs) of toxins (∗) addition, respectively. ACh and KCl addition; ∗∗: washes (𝑛= 12 for each treatment). neuromuscular blockade after 31.2±3.5 min and 30±8.1 min (𝑝< 0.05 from Krebs), respectively, similarly to previous report [16]. The neuromuscular effects of both PLA2 toxin isoforms resulted from increase in the twitch response of indirectly stimulated neuromuscular preparations. None of the toxins interfered significantly with the contractures to exogenously applied ACh and KCl after 120 min incubation (𝑛= 12) (Figure 2). any damage. Intramuscular axons showed well organized myelin sheath and normal neurofilaments and mitochondria (Figures 4(a)–4(c)). Opposed to controls, CBC preparations incubated either with BmjeTX-I or BmjeTX-II presented hypercontracted fibres and clusters of swollen mitochondria and/or deprived of cristae, blurred Z line with loss of myofilaments, and sar- comeres disorganization. Diffuse swelling of the sarcotubular system was also observed and some fibres exhibited myonu- clei with chromatin and nucleoli alterations. Intramuscular motor nerve fibres displayed multishaped alterations of the myelin sheath and axons (Figures 4(d)–4(i)). 3. Results Western Blotting of Presynaptic Proteins. Control CBC preparation homogenate showed differential proportion among the three presynaptic proteins examined. The baseline of synaptophysin was double that of SNAP25’s which by its turn doubles the baseline of synaptobrevin. In contrast, no expression of synaptophysin, synaptobrevin, or SNAP25 protein was found in homogenate of CBC incubated with BmjeTX-I or BmjeTX-II. Significant differences (𝑝< 0.01) were observed between control and PLA2s-incubated prepa- rations (Figure 7). length were due to intramembranous presence of postsynap- tic receptors (Figure 5(a)). By contrast, NMJs in the muscles were exposed to BmjeTX-I and BmjeTX-II, despite a number of them exhibit- ing morphology similar to controls; many others exhibited a clear and significant (𝑝< 0.05) reduction in the density of synaptic vesicles inside the terminal bouton while the remaining vesicles were clumped together in small aggre- gates; a massive mitochondrial damage and absence of presynaptic membrane were also observed (Figures 5(b) and 5(c)). The terminal bouton area and the quantity of synaptic vesicles per area were significantly reduced as compared to control (𝑝< 0.05) (Figures 5(d)–5(f)). 3. Results Regions with normal ultrastructure were also present (not shown).i 3.2. Muscle Morphology and Ultrastructural Analysis. Light microscopy showed that control CBC (incubated for 120 min in Krebs solution) presented muscle fibres with normal mor- phology while CBC incubated with BmjeTX-I or BmjeTX-II presented regions with fibres with different pathologic states, including vacuolated or swollen fibres, presenting loosely and/or densely clumping of myofibrils (Figure 3) but also regions with normality (not shown). In order to support the twitch tension findings which showed that BmjeTX-I and BmjeTX-II PLA2 caused a presynaptic neuromuscular blockade, we further investigated the neuromuscular junction (NMJ) ultrastructure by TEM. Control samples incubated with Krebs solution showed no abnormal pre- and postsynaptic ultrastructure. The terminal bouton was clearly defined by a continuous axolemma; it was adjusted into a well-delineated synaptic gutter and covered by processes of the Schwann cell. As usual, the synaptic vesicles and mitochondria were polarized to presynaptic axolemma and Schwann cell, respectively; typically, the postsynaptic sarcolemma was unfolded in chick muscle; densities along its Transmission electron microscopy was used to assess whether BmjeTX-I and BmjeTX-II affect the subcellular integrity of muscle fibres and neuromuscular junction (NMJ) of biventer cervicis. Control muscles showed typical ultra- structure with paralleled myofibrils and organized sarcom- eres. Longitudinal- and cross-sectioned myofibrils were typi- cally separated by profiles of sarcoplasmic reticulum; subsar- colemmal nuclei and mitochondria were observed without Biochemistry Research International 5 ∗ (a) ∗∗ ∗ (b) ∗∗ ∗ 10𝜇m (c) Figure 3: Light micrographs of chick biventer cervicis preparations incubated with Krebs solution (control, (a)) or incubated with BmjeTX- I (b) and BjmeTX-II (c) (10 𝜇g/mL each). All images were obtained from longitudinal sections. Notice that in (a) the fibres are normal in appearance, while in (b) and (c) several fibres appear with disrupted myofibrils (white arrows) and hypercontracted zones throughout their length (∗∗) and nucleus (∗); HE, 𝑛= 4 per treatment. ∗∗ ∗ (b) ∗ (a) ∗∗ ∗ 10𝜇m (c) (c) (a) (b) Figure 3: Light micrographs of chick biventer cervicis preparations incubated with Krebs solution (control, (a)) or incubated with BmjeTX- I (b) and BjmeTX-II (c) (10 𝜇g/mL each). All images were obtained from longitudinal sections. Notice that in (a) the fibres are normal in appearance, while in (b) and (c) several fibres appear with disrupted myofibrils (white arrows) and hypercontracted zones throughout their length (∗∗) and nucleus (∗); HE, 𝑛= 4 per treatment. 3.4. 4. Discussion mi S 1𝜇m (a) m N 2𝜇m (b) ∗∗ n m 2𝜇m (c) m mi # 1𝜇m (d) m N mi 2𝜇m (e) N n ∗∗ 500nm (f) mi m 2𝜇m (g) N 2𝜇m (h) N n ∗∗ 2𝜇m (i) Figure 4: Electron micrographs of chick biventer cervicis preparations incubated in Krebs solution (control, (a)–(c)), BmjeTX-I ((d)–(f)), and BmjeTX-II ((g)–(i)) toxins. No alterations were observed in control ((a)–(c)). Treated preparations ((d)–(i)) displayed hypercontracted myofilaments (mi) and loss of typical sarcomeres (S) organization, edema (#), mitochondria (m), swelling, and cristae deprivation. Myonuclei (N) of some fibres showed apoptotic-like phenotype and intramuscular nerve fibres were affected (∗∗); n: neurofilaments; arrows: blurred Z line (𝑛= 4 per treatment) ∗∗ n m 2𝜇m (c) ∗∗ n m 2𝜇m (c) mi S 1𝜇m (a) m N 2𝜇m (b) (b) (c) (a) N n ∗∗ 500nm (f) m mi # 1𝜇m (d) m N mi 2𝜇m (e) (d) (f) (e) mi m 2𝜇m (g) N n ∗∗ 2𝜇m (i) N 2𝜇m (h) (i) (h) (g) Figure 4: Electron micrographs of chick biventer cervicis preparations incubated in Krebs solution (control, (a)–(c)), BmjeTX-I ((d)–(f)), and BmjeTX-II ((g)–(i)) toxins. No alterations were observed in control ((a)–(c)). Treated preparations ((d)–(i)) displayed hypercontracted myofilaments (mi) and loss of typical sarcomeres (S) organization, edema (#), mitochondria (m), swelling, and cristae deprivation. Myonuclei (N) of some fibres showed apoptotic-like phenotype and intramuscular nerve fibres were affected (∗∗); n: neurofilaments; arrows: blurred Z line (𝑛= 4 per treatment). blockade. The KCl- and ACh-evoked contracture was unaf- fected by either of the PLA2s isoforms indicating that at the concentration used the effect in the nicotinic receptors was absent; instead it indicates that BmjeTX-I and BmjeTX-II (10 𝜇g/mL) action was primarily at the motor nerve terminal, as suggested elsewhere [17]. Bmaj-9, another presynaptic- acting PLA2 from B. marajoensis venom, likewise induced a total and irreversible blockade after 70 ± 5 min at same concentration and same nerve-muscle preparation; likewise, the contracture evoked by KCl and ACh remained unchanged by the Bmaj-9 [16]. blocking postsynaptic acetylcholine receptors or interfering with the muscle contractile mechanisms [15]. Nevertheless, concentration as high as 20 𝜇g/mL produced total blockade at around 100 min [15] and promoted significant reduction of KCl- and ACh-induced contractures. The data show the importance of concentration to disclose myotoxic and neurotoxic effect of venom. 4. Discussion In the present study we have used two PLA2s purified from B. marajoensis venom, BmjeTX-I and BmjeTX-II [17], which as for the venom [15] act presynaptically and possess neuromuscular blocking effect [17]. Our goal was to evaluate the involvement of proteins of the presynaptic apparatus in such effect by comparing their content in CBC control preparations with their content in matched preparations incubated with the two PLA2s toxins at a same concentration.h 3.3. Immunofluorescence of Presynaptic Proteins. Control terminals of biventer cervicis nerve-muscle preparation showed immunolabelling of synaptophysin, synaptobrevin, and SNAP25 proteins (Figures 6(a)–6(c)) while their coun- terparts incubated with BmjeTX-I or BmjeTX-II showed very weak expression for all three proteins. Figures 6(d)– 6(i) illustrate regions of neuromuscular contacts in which labelling is very faint.h The proteins examined showed a 100% basal expression in control, whereas they showed a scale proportion where synaptophysin > SNAP25 > synaptobrevin in preparations incubated with BmjeTX-I and synaptophysin ≥synapto- brevin = SNAP25 in preparations incubated with BjmeTX- II (Figure 6(j)). The ACh receptors into the postsynaptic membrane, marked with TRITC-conjugated 𝛼-bungarotoxin (𝛼-BgTX), were well preserved for all experimental groups (data not shown). The study was undertaken in in vitro preparations, which typically provide faster and reliable information on the effects on neuromuscular junction components. Besides, in muscle- nerve preparation incubation muscle and motor nerve fibres were in closer contact with the toxin through their length instead of a limited tissue portion as when the toxin is injected intramuscularly. Chick biventer cervicis (CBC) preparation incubated with either of the toxins showed irreversible twitch tension Biochemistry Research International 6 mi S 1𝜇m (a) m N 2𝜇m (b) ∗∗ n m 2𝜇m (c) m mi # 1𝜇m (d) m N mi 2𝜇m (e) N n ∗∗ 500nm (f) mi m 2𝜇m (g) N 2𝜇m (h) N n ∗∗ 2𝜇m (i) Figure 4: Electron micrographs of chick biventer cervicis preparations incubated in Krebs solution (control, (a)–(c)), BmjeTX-I ((d)–(f)), and BmjeTX-II ((g)–(i)) toxins. No alterations were observed in control ((a)–(c)). Treated preparations ((d)–(i)) displayed hypercontracted myofilaments (mi) and loss of typical sarcomeres (S) organization, edema (#), mitochondria (m), swelling, and cristae deprivation. Myonuclei (N) of some fibres showed apoptotic-like phenotype and intramuscular nerve fibres were affected (∗∗); n: neurofilaments; arrows: blurred Z line (𝑛= 4 per treatment). 4. Discussion M m # ∗ 1𝜇m (c) m M ∗ sv 500nm (a) M m ∗ sv ∗ 1𝜇m (b) (b) (a) (c) (b) Synaptic vesicle (SV) D E D E 0 50 100 150 200 Number of SV per bouton BmjeTX-I BmjeTX-II Control (e) Number of SV per TB F G F G 0 1 2 3 Number of SV × area (mm2) BmjeTX-I BmjeTX-II Control (f) Number of SV per TB Terminal bouton (TB) Terminal bouton (TB) A B A C B C 0 20 40 60 80 Area (𝜇m2) BmjeTX-I BmjeTX-II Control (d) (e) (d) (f) Figure 5: Electron micrographs of neuromuscular junction (NMJ) and synaptic morphometry of control (a), BmjeTX-I-treated (b), and BmjeTX-II-treated (c) chick biventer cervicis preparations. (a) Observe the bouton filled with synaptic vesicles (sv), intact mitochondria (m), and presynaptic membrane (arrow) associated with an unfolded but highly contrasted postsynaptic membrane due to the presence of acetylcholine receptors (double arrow). (b, c) Distorted terminal button, undefined axolemma (arrow), reduction of synaptic vesicles (#), and swollen mitochondria are present in the terminals; M: muscle fibre. (d)–(f) Graphical demonstration of terminal bouton area (d), mean number of synaptic vesicles per bouton (e), and relative number of synaptic vesicles per terminal bouton area (f) for control, BmjeTX-I, and BmjeTX-II groups. The bars with same uppercase letters indicate that there was significant difference (A,C,D,E𝑝< 0.0001; B,F𝑝< 0.001; G𝑝< 0.05); data are mean ± SD one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). this was not supported by our findings; we can just speculate that other venom components probably antagonize the effects of the PLA2 toxins.h (tethering) and docks to the presynaptic membrane and an ATPase-dependent process facilitates the SV priming. Once primed the SV can fuse and release the neurotransmitter. This process is mediated by vesicle-associated, cytosolic, and membranous proteins [19].i The blockade of the twitch tension in CBC preparations incubated with BmjeTX-I or BmjeTX-II was found to occur in connection with the shift of synaptophysin, synaptobrevin, and SNAP25 proteins from the biventer cervicis end-plates relative to the control counterparts which have shown total expression of the three proteins (IHC and WB data). 4. Discussion f Based on the data, it can be concluded that the presynaptic action of the venom could be on account of Bmaj-9 [16], BmjeTX-I, and BmjeTX-II [17] and that the presynaptic- blocking effect is higher with BmjeTX-I and BmjeTX-II than with Bmaj-9. Interestingly, the presynaptic blockade is achieved more fastly whenever each of the three toxins is used than with the whole venom. A substantiated explanation for In agreement, 1 𝜇g/mL low concentration of B. mara- joensis venom was able to induce neuromuscular blockade without depressing the responses to exogenous ACh and KCl, 7 Biochemistry Research International m M ∗ sv 500nm (a) M m ∗ sv ∗ 1𝜇m (b) M m # ∗ 1𝜇m (c) Terminal bouton (TB) A B A C B C 0 20 40 60 80 Area (𝜇m2) BmjeTX-I BmjeTX-II Control (d) Synaptic vesicle (SV) D E D E 0 50 100 150 200 Number of SV per bouton BmjeTX-I BmjeTX-II Control (e) Number of SV per TB F G F G 0 1 2 3 Number of SV × area (mm2) BmjeTX-I BmjeTX-II Control (f) Figure 5: Electron micrographs of neuromuscular junction (NMJ) and synaptic morphometry of control (a), BmjeTX-I-treated (b), and BmjeTX-II-treated (c) chick biventer cervicis preparations. (a) Observe the bouton filled with synaptic vesicles (sv), intact mitochondria (m), and presynaptic membrane (arrow) associated with an unfolded but highly contrasted postsynaptic membrane due to the presence of acetylcholine receptors (double arrow). (b, c) Distorted terminal button, undefined axolemma (arrow), reduction of synaptic vesicles (#), and swollen mitochondria are present in the terminals; M: muscle fibre. (d)–(f) Graphical demonstration of terminal bouton area (d), mean number of synaptic vesicles per bouton (e), and relative number of synaptic vesicles per terminal bouton area (f) for control, BmjeTX-I, and BmjeTX-II groups. The bars with same uppercase letters indicate that there was significant difference (A,C,D,E𝑝< 0.0001; B,F𝑝< 0.001; G𝑝< 0.05); data are mean ± SD one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). 4. Discussion In con- trol CBC incubated with Krebs solution the immunolabelling of presynaptic proteins delineated their distribution in the synaptic contacts over the myofibres indicating an operative biventer cervicis contractile structure which was responsive to the indirect electric nerve stimulation; these data and the colabelling of acetylcholine receptors (AChR) as proved by 𝛼-bungarotoxin labelling even in treated preparations (figure not shown) confirmed the normal and functional pattern of the postsynaptic machinery in controls and postsynaptic intactness in treated preparations (not shown). Another important finding of this study was that the area of the terminals was significantly reduced in preparations incubated with Bjme-TX-I and Bjme-TX-II with reduction significantly higher with the former than with the latter. Moreover, the number of synaptic vesicles per terminal was significantly decreased when CBC preparations were incubated with either of the PLA2 toxins. In contrast, the number of synaptic vesicles relative to the area of the terminal was significantly reduced in preparations incubated with Bjme-TX-II but not with Bjme-TX-I. Synaptophysin expression is related to SV integrity [19] and its absence in preparations incubated with both toxins is in conformity with the reduction of vesicles in the terminal endings and the blockade of twitch tension in preparations treated with the toxins. On the other side, the reduction of the area of the nerve terminal seems to indicate that the presynaptic action of the two toxins is beyond the effect on the expression of the presynaptic proteins, the exact nature of which is obscure. Complex presynaptic machinery is necessary to release the neurotransmitter at the synaptic cleft of the neuromuscu- lar junction. Initially the synaptic vesicles (SV) are attached to a fine, filamentous actin cytoskeletal network in the presy- naptic portion of the nerve terminal. 4. Discussion The neurotransmitter vesicle binds to an active target zone near calcium channels Snake presynaptic PLA2 neurotoxins (SPANs) paralyze the neuromuscular junctions (NMJs) in vertebrate skeletal Biochemistry Research International 8 Control Synaptophysin (Syf) (a) Control Synaptobrevin (Syb) (b) Control SNAP25 (S25) (c) BmjeTX-I Synaptophysin (Syf) (d) BmjeTX-I Synaptobrevin (Syb) (e) BmjeTX-I SNAP25 (S25) (f) BmjeTX-II Synaptophysin (Syf) (g) BmjeTX-II Synaptobrevin (Syb) (h) BmjeTX-II SNAP25 (S25) 50𝜇m (i) ∗ ∗ ∗ ∗ ∗ ∗ ∗ BmjeTX-I BmjeTX-II NMJ proteins 0 50 100 150 % of labelling Syf Syf S25 S25 Syb Syb 𝛼BgTx Control (j) re 6: Immunofluorescence for synaptophysin, Syf (a, d, g), synaptobrevin, Syb (b, e, h), and SNAP25, S25 (c, f, i) in neurom ion (NMJ) of chick biventer cervicis. In controls ((a)–(c)) all proteins were expressed indicating active presynaptic machinery. H mjeTX-I-treated ((d)–(f)) and BmjeTX-II-treated (g)–(i) preparations no immunolabelling was detected indicating inactivity naptic machinery. (j) Percentage of Syf, Syb, and S25 protein showed 100% expression in control preparations and represent e control bar, whereas a remarkable reduction was observed for BmjeTX-I- and BmjeTX-II-treated samples. 𝛼-Bungarotoxin (𝛼 used to indicate the ACh receptor. Data were expressed as mean ± SD (∗𝑝< 0.0001 relative to control), one-way ANOVA plus Bon est (𝑛= 4 per treatment). Synaptobrevin (Syb) (b) Synaptobrevin (Syb) Control Synaptophysin (Syf) (a) Control Synaptophysin (Syf) (a) Control Synaptobrevin (Syb) (b) Control SNAP25 (S25) (c) BmjeTX-I Synaptophysin (Syf) (d) BmjeTX-I Synaptobrevin (Syb) (e) BmjeTX-I SNAP25 (S25) (f) BmjeTX-II Synaptophysin (Syf) (g) BmjeTX-II Synaptobrevin (Syb) (h) BmjeTX-II SNAP25 (S25) 50𝜇m (i) ∗ ∗ ∗ ∗ ∗ ∗ ∗ BmjeTX-I BmjeTX-II NMJ proteins 0 50 100 150 % of labelling Syf Syf S25 S25 Syb Syb 𝛼BgTx Control (j) igure 6: Immunofluorescence for synaptophysin, Syf (a, d, g), synaptobrevin, Syb (b, e, h), and SNAP25, S25 (c, f, i) in neurom unction (NMJ) of chick biventer cervicis. In controls ((a)–(c)) all proteins were expressed indicating active presynaptic machinery. H n BmjeTX-I-treated ((d)–(f)) and BmjeTX-II-treated (g)–(i) preparations no immunolabelling was detected indicating inactivi resynaptic machinery. (j) Percentage of Syf, Syb, and S25 protein showed 100% expression in control preparations and represen ingle control bar, whereas a remarkable reduction was observed for BmjeTX-I- and BmjeTX-II-treated samples. 𝛼-Bungarotoxin was used to indicate the ACh receptor. Data were expressed as mean ± SD (∗𝑝< 0.0001 relative to control), one-way ANOVA plus Bo osttest (𝑛= 4 per treatment). 4. Discussion 25kDa 37kDa (c) (d) Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II 38kDa 18kDa (a) (b) Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II 18kDa (b) Control BmjeTX-I BmjeTX-II 38 18 25 37 (a) (b) (c) (d) Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Pixels density A D B E C F Control BmjeTX-I BmjeTX-II Syf Syb S25 Syf Syb S25 Syf Syb S25 F E D C B A 0 50000 100000 150000 (arbitrary units, AU) Figure 7: Western blotting analysis for synaptophysin ((a), Syf), synaptobrevin ((b), Syb), and SNAP25 ((c), S25) protein expression in control, BmjeTX-I, and BmjeTX-II chick biventer cervicis preparations normalized to GAPDH (d). The bars with same uppercase letters indicate that there was significant difference: A,D𝑝< 0.001; C,F𝑝< 0.001; B,E𝑝< 0.05. Data were expressed as mean ± SD; one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). Pixels density A D B E C F Control BmjeTX-I BmjeTX-II Syf Syb S25 Syf Syb S25 Syf Syb S25 F E D C B A 0 50000 100000 150000 (arbitrary units, AU) Figure 7: Western blotting analysis for synaptophysin ((a), Syf), synaptobrevin ((b), Syb), and SNAP25 ((c), S25) protein expression in control, BmjeTX-I, and BmjeTX-II chick biventer cervicis preparations normalized to GAPDH (d). The bars with same uppercase letters indicate that there was significant difference: A,D𝑝< 0.001; C,F𝑝< 0.001; B,E𝑝< 0.05. Data were expressed as mean ± SD; one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). muscles by reducing the content of SV. Studies using primary neuronal cultures show synaptic swelling, with surface expo- sure of the luminal domain of the synaptic vesicle protein synaptotagmin I, and exocytosis of neurotransmitters. Other studies using an equimolar mixture of lysophospholipids and fatty acids, which mimics the biological effects of SPANs, indicate a possible role of local lipid changes in SV release [19]. by Rodrigues-Simioni et al. [20] in frog nerve-muscle prepa- rations incubated with B. jararacussu venom. Moreover, the changes in the intramuscular motor nerve axons are additional evidence of the neurotoxic action of the two PLA2s. Competing Interests The authors declare that they have no competing interests. 5. Conclusion In this study, the 10 𝜇g/mL concentration of the toxins also induced alterations in the muscle fibres. It is likely that the onset of changes was triggered by damage of the phospholipid bilayer of the fibre membrane as inferred by histological and ultrastructural findings. Neuromuscular blockade and/or muscle damage, as induced by BmjeTX-I and BmjeTX-II in CBC preparations, was also reported in nerve-muscle preparations treated with other toxins, such as BthTX-I [8, 10] and BthTX-II [5] from B. jararacussu venom and BnpTX-I and BnpTX-II toxins [9] from B. (neuwiedi) pauloensis venom. Muscle damage inferred by rapid elevation of plasma CK activity in mice was also observed by Ponce- Soto et al. [17] in vivo with the two toxins here investigated. These results revealed that BmjeTX-I and BmjeTX-II PLA2s displayed a myotoxic effect, as the majority of venoms and toxins were isolated from Bothrops snakes, which is not associated with impairment of the nicotinic receptors of the postsynaptic sarcolemma. The absence of proteins involved in synaptic vesicles integrity (synaptophysin), vesicle docking, and transmitter exocytosis (synaptophysin, synaptobrevin, and SNAP25), in prepara- tions incubated with BmjeTX-I and BmjeTX-II, is direct and strong evidence supporting our hypothesis that BmjeTX-I and BmjeTX-II affect neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal. The study reinforces the hypothesis that the lack of these proteins was responsible for the neurotoxic- ity caused by both toxins from Bothrops marajoensis venom. 4. Discussion Control Control Control (b) Synaptobrevin (Syb) Synaptobrevin (Syb) Synaptophysin (Syf) y p ( y ) (e) S t b i (S b) BmjeTX-I BmjeTX-I (e) Synaptobrevin (Syb) ( ) SNAP25 (S25) BmjeTX-II Synaptophysin (Syf) (g) Synaptobrevin (Syb) (h) NMJ proteins Synaptophysin (Syf) (g) (h) NMJ proteins (i) (g) (h) (i) ∗ ∗ ∗ ∗ ∗ ∗ ∗ BmjeTX-I BmjeTX-II NMJ proteins 0 50 100 150 % of labelling Syf Syf S25 S25 Syb Syb 𝛼BgTx Control (j) (j) Figure 6: Immunofluorescence for synaptophysin, Syf (a, d, g), synaptobrevin, Syb (b, e, h), and SNAP25, S25 (c, f, i) in neuromuscular junction (NMJ) of chick biventer cervicis. In controls ((a)–(c)) all proteins were expressed indicating active presynaptic machinery. However, in BmjeTX-I-treated ((d)–(f)) and BmjeTX-II-treated (g)–(i) preparations no immunolabelling was detected indicating inactivity of the presynaptic machinery. (j) Percentage of Syf, Syb, and S25 protein showed 100% expression in control preparations and represented as a single control bar, whereas a remarkable reduction was observed for BmjeTX-I- and BmjeTX-II-treated samples. 𝛼-Bungarotoxin (𝛼BgTx) was used to indicate the ACh receptor. Data were expressed as mean ± SD (∗𝑝< 0.0001 relative to control), one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). Biochemistry Research International 9 38kDa 18kDa 25kDa 37kDa (a) (b) (c) (d) Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Control BmjeTX-I BmjeTX-II Pixels density A D B E C F Control BmjeTX-I BmjeTX-II Syf Syb S25 Syf Syb S25 Syf Syb S25 F E D C B A 0 50000 100000 150000 (arbitrary units, AU) Figure 7: Western blotting analysis for synaptophysin ((a), Syf), synaptobrevin ((b), Syb), and SNAP25 ((c), S25) protein expression in control, BmjeTX-I, and BmjeTX-II chick biventer cervicis preparations normalized to GAPDH (d). The bars with same uppercase letters indicate that there was significant difference: A,D𝑝< 0.001; C,F𝑝< 0.001; B,E𝑝< 0.05. Data were expressed as mean ± SD; one-way ANOVA plus Bonferroni posttest (𝑛= 4 per treatment). References [1] SINAN, 2015, http://sinan.saude.gov.br/. [2] R. Milani Jr., M. T. Jorge, F. P. Ferraz De Campos et al., “Snake bites by the jararacucu (Bothrops jararacussu): clinicopathologi- cal studies of 29 proven cases in Sao Paulo State, Brazil,” Monthly Journal of the Association of Physicians, vol. 90, no. 5, pp. 323– 334, 1997. [17] L. A. Ponce-Soto, D. Martins-de-souza, and S. Marangoni, “Neurotoxic, myotoxic and cytolytic activities of the new basic PLA2 isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (Maraj´o Lancehead) snake venom,” Protein Journal, vol. 29, no. 2, pp. 103–113, 2010. [3] D. Mebs and C. L. Ownby, “Myotoxic components of snake ven- oms: their biochemical and biological activities,” Pharmacology & Therapeutics, vol. 48, no. 2, pp. 223–236, 1990. [18] B. L. Ginsborg and J. Warriner, “The isolated chick biventer cervicis nerve-muscle preparation,” British Journal of Pharma- cology and Chemotherapy, vol. 15, pp. 410–411, 1960. [4] B. Lomonte and J. M. Guti´errez, “Phospholipases A2 from viperidae snake venoms: how do they induce skeletal muscle damage?” Acta Chimica Slovenica, vol. 58, no. 4, pp. 647–659, 2011. [19] D. Dumitru and A. A. Amato, “Neuromuscular junction disor- ders,” in Neuromuscular Disorders, A. A. Amato and J. Russell, Eds., McGraw Hill Education, New York, NY, USA, 2008. [20] L. Rodrigues-Simioni, N. Borgese, and B. Ceccarelli, “The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation,” Neuroscience, vol. 10, no. 2, pp. 475–489, 1983. [5] J. M. Guti´errez, J. N´u˜nez, A. C. O. Cintra, M. I. Homsi- Brandeburgo, and J. R. Giglio, “Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phos- pholipase A2 isolated from the venom of the snake Bothrops jararacussu,” Experimental and Molecular Pathology, vol. 55, no. 3, pp. 217–229, 1991. [6] J. M. Guti´errez and C. L. Ownby, “Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mecha- nisms of local and systemic myotoxicity,” Toxicon, vol. 42, no. 8, pp. 915–931, 2003. [7] L. de Souza Queir´oz, M. J. Marques, and H. Santo Neto, “Acute local nerve lesions induced by Bothrops jararacussu snake venom,” Toxicon, vol. 40, no. 10, pp. 1483–1486, 2002. [8] M. C. Ferraz, E. H. Yoshida, R. V. S. Tavares et al., “An isoflavone from Dipteryx alata vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis,” Molecules, vol. 19, no. 5, pp. 5790–5805, 2014. [9] V. M. Acknowledgments The authors thank Mr. Gildo B. Leite for technical assis- tance and Dr. Silvia Cardoso from Museu Biol´ogico/Instituto Also, muscle ultrastructural alterations as those induced by BmjeTX-I and BmjeTX-II are similar to those described Biochemistry Research International 10 Butantan for providing the picture of B. marajoensis main- tained in captivity. This work was supported by Fundac¸˜ao de Amparo `a Pesquisa do Estado de S˜ao Paulo (FAPESP) Grants 2011/00001-1 and 2005/53625-1. Antonio Lisboa and Rodolfo Melar´e are Master’s students. Junia R. B. Franco, Carolina V. Bis, and Marta Gracia are undergraduate stu- dents supported by solcharships from Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq) (to Junia R. B. Franco) and FAPESP (to Marta Gracia). Maria Alice da Cruz-H¨ofling is 1A research fellow from CNPq. [13] J. A. Campbell and W. W. Lamar, The Venomous Reptiles of Latin America, vol. 1, Comstock Publishing Associates/Cornell, New York, NY, USA, 1989. [14] P. P. de Oliveira Pardal, A. C. J. D. S. Pinheiro, C. T. C. Silva, P. R. S. G. Santos, and M. A. D. C. Gadelha, “Hemorrhagic stroke in children caused by Bothrops marajoensis envenoming: a case report,” Journal of Venomous Animals and Toxins Including Tropical Diseases, vol. 21, article 53, 2015. [15] W. L. G. Cavalcante, S. Hernandez-Oliveira, C. Galbiatti et al., “Biological characterization of Bothrops marajoensis snake venom,” Journal of Venom Research, vol. 2, pp. 37–41, 2011. [16] C. Galbiatti, T. Rocha, P. Randazzo-Moura et al., “Pharmacolog- ical and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom,” Journal of Venomous Animals and Toxins Including Tropical Diseases, vol. 18, no. 1, pp. 62–72, 2012. References Rodrigues, S. Marcussi, R. S. Cambraia et al., “Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom,” Toxicon, vol. 44, no. 3, pp. 305–314, 2004. [10] Y. Oshima-Franco, G. B. Leite, C. A. Belo et al., “The presy- naptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom,” Basic & Clinical Pharmacology & Toxicology, vol. 95, no. 4, pp. 175–182, 2004. [11] R. Hern´andez, C. Cabalceta, P. Saravia-Otten, A. Chaves, J. M. Guti´errez, and A. Rucavado, “Poor regenerative outcome after skeletal muscle necrosis induced by bothrops asper venom: alterations in microvasculature and nerves,” PLoS ONE, vol. 6, no. 5, Article ID e19834, 2011. [12] A. R. Hoge and A. S. Romano, “Sinopse das serpentes pec¸onhentas do Brasil. Serpentes, Elapidae e Viperidae,” Mem´o- rias do Instituto Butantan, vol. 36, pp. 109–207, 1973.
https://openalex.org/W4305072365	https://www.jmir.org/2022/10/e43220/PDF	English	null	Correction: A Teleconsultation Device, Consult Station, for Remote Primary Care: Multisite Prospective Cohort Study	JMIR. Journal of medical internet research/Journal of medical internet research	2,022	cc-by	582	Correction: A Teleconsultation Device, Consult Station, for Remote Primary Care: Multisite Prospective Cohort Study Géraldine Falgarone1,2, MD, PhD; Guilhem Bousquet1,3, MD, PhD; Arnaud Wilmet4, MD; Albert Brizio4, MD; Valérie Faure4, MD; Celestin Guillouet4, MD; Franck Baudino4, MD; Isabelle Roque5, MD; Samuel Mayol6, PhD; Frederic Pamoukdjian1,7, MD, PhD 1UMR_S942 MASCOT, INSERM, Université Sorbonne Paris Nord, Bobigny, France 2Unité de Médecine Ambulatoire, Hôpital Avicenne, Assistance Publique–Hôpitaux de Paris, Bobigny, France 3Service de Cancérologie, Hôpital Avicenne, Assistance Publique–Hôpitaux de Paris, Bobigny, France 4H4D (Health for Development), Paris, France 5Université Paris Cité, Paris, France 6Institut Universitaire de Technologie, Université Sorbonne Paris Nord, St-Denis, France 7Service de Médecine Gériatrique, Hôpital Avicenne, Assistance Publique–Hôpitaux de Paris, Bobigny, France *these authors contributed equally Corresponding Author: Géraldine Falgarone, MD, PhD Unité de Médecine Ambulatoire Hôpital Avicenne Assistance Publique–Hôpitaux de Paris 125 Rue de Stalingrad Bobigny, 93009 France Phone: 33 148026870 Fax: 33 148955258 Email: g.falgarone@aphp.fr Related Article: Correction of: https://www.jmir.org/2022/5/e33507 JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Falgarone et al Corrigenda and Addenda Frederic Pamoukdjian In “A Teleconsultation Device, Consult Station, for Remote Primary Care: Multisite Prospective Cohort Study” (J Med Internet Res 2022;24(5):e33507), the authors noted one error. The correction will appear in the online version of the paper on the JMIR Publications website on October 13, 2022, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories. In the originally published article, author Frederic Pamoukdjian’s name incorrectly appeared as: Frederic Pamoukjian It has now been corrected to: Frederic Pamoukjian It has now been corrected to: This is a non–peer-reviewed article. Submitted 04.10.22; accepted 04.10.22; published 13.10.22. Please cite as: Falgarone G, Bousquet G, Wilmet A, Brizio A, Faure V, Guillouet C, Baudino F, Roque I, Mayol S, Pamoukdjian F Correction: A Teleconsultation Device, Consult Station, for Remote Primary Care: Multisite Prospective Cohort Stud J Med Internet Res 2022;24(10):e43220 URL: https://www.jmir.org/2022/10/e43220 doi: 10.2196/43220 PMID: J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e43220 \| p. 1 (page number not for citation purposes) https://www.jmir.org/2022/10/e43220 XSL•FO RenderX ©Géraldine Falgarone, Guilhem Bousquet, Arnaud Wilmet, Albert Brizio, Valérie Faure, Celestin Guillouet, Franck Baudino, Isabelle Roque, Samuel Mayol, Frederic Pamoukdjian. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 13.10.2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. J Med Internet Res 2022 \| vol. 24 \| iss. 10 \| e43220 \| p. 2 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH Falgarone et al ©Géraldine Falgarone, Guilhem Bousquet, Arnaud Wilmet, Albert Brizio, Valérie Faure, Celestin Guillouet, Franck Baudino, Isabelle Roque, Samuel Mayol, Frederic Pamoukdjian. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 13.10.2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on https://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2022/10/e43220 XSL•FO RenderX
https://openalex.org/W2942724779	https://europepmc.org/articles/pmc6686783?pdf=render	English	null	Gross Primary Productivity of Four European Ecosystems Constrained by Joint CO<sub>2</sub> and COS Flux Measurements	Geophysical research letters	2,019	cc-by	8,456	Citation: Plain Language Summary Plants are Earth's biggest contributor for cleaning the atmosphere of carbon dioxide and remove around one quarter of the carbon dioxide emitted by humans each year. However, this contribution cannot be measured directly and has to be inferred or modelled on the basis of related parameters. This introduces large uncertainties, which in turn undermine our ability to accurately create future climate scenarios. Recent research revealed that the trace gas carbonyl sulﬁde is taken up by plants in a very similar way as carbon dioxide and offers us an additional way of quantifying the carbon dioxide uptake by photosynthesis. Here we use joint measurements of the carbon dioxide and carbonyl sulﬁde exchange to infer plant carbon dioxide uptake, demonstrating the advantage of using multiple approaches. We apply our method at four major European ecosystems and show that previous approaches, based solely on carbon dioxide, may have underestimated the plant carbon dioxide uptake. Received 17 JAN 2019 Accepted 29 APR 2019 Accepted article online 3 MAY 2019 Published online 21 MAY 2019 Citation: Spielmann, F. M., Wohlfahrt, G., Hammerle, A., Kitz, F., Migliavacca, M., Alberti, G., et al. (2019). Gross primary productivity of four European ecosystems constrained by joint CO2 and COS ﬂux measurements. Geophysical Research Letters, 46, 5284–5293. https://doi.org/10.1029/ 2019GL082006 ©2019. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Gross Primary Productivity of Four European Ecosystems Constrained by Joint CO2 and COS Flux Measurements Gross Primary Productivity of Four European Ecosystems Constrained by Joint CO2 and COS Flux Measurements Gross Primary Productivity of Four European Ecosystems Constrained by Joint CO2 and COS Flux Measurements Key Points: F. M. Spielmann1 , G. Wohlfahrt1 , A. Hammerle1, F. Kitz1 , M. Migliavacca2 , G. Alberti3,4 , A. Ibrom5 , T. S. El‐Madany2 , K. Gerdel1 , G. Moreno6 , O. Kolle2 , T. Karl7 , A. Peressotti3, and G. Delle Vedove3 F. M. Spielmann1 , G. Wohlfahrt1 , A. Hammerle1, F. Kitz1 , M. Migliavacca2 , G. Alberti3,4 , A. Ibrom5 , T. S. El‐Madany2 , K. Gerdel1 , G. Moreno6 , O. Kolle2 , T. Karl7 , A. Peressotti3, and G. Delle Vedove3 • Traditionally gross primary productivity is inferred from ecosystem‐scale CO2 ﬂux measurements 1Department of Ecology, University of Innsbruck, Innsbruck, Austria, 2Department of Biogeochemical Integration, Max Planck Institute for Biogeochemistry, Jena, Germany, 3Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy, 4CNR‐IBIMET, Firenze, Italy, 5Department of Environmental Engineering, Technical University of Denmark, Kongens Lyngby, Denmark, 6INDEHESA‐Forest Research Group, Universidad de Extremadura, Plasencia, Spain, 7Institute of Atmospheric and Cryospheric Sciences, University of Innsbruck, Innsbruck, Austria • The proposed joint assimilation of CO2 and COS ﬂux measurements avoids the need to specify the leaf relative uptake rate of COS a priori • The additional information content of ecosystem‐scale COS ﬂux measurements increases inferred gross primary productivity estimates • The additional information content of ecosystem‐scale COS ﬂux measurements increases inferred gross primary productivity estimates Abstract Gross primary productivity (GPP), the gross uptake of carbon dioxide (CO2) by plant photosynthesis, is the primary driver of the land carbon sink, which presently removes around one quarter of the anthropogenic CO2 emissions each year. GPP, however, cannot be measured directly and the resulting uncertainty undermines our ability to project the magnitude of the future land carbon sink. Carbonyl sulﬁde (COS) has been proposed as an independent proxy for GPP as it diffuses into leaves in a fashion very similar to CO2, but in contrast to the latter is generally not emitted. Here we use concurrent ecosystem‐scale ﬂux measurements of CO2 and COS at four European biomes for a joint constraint on CO2 ﬂux partitioning. The resulting GPP estimates generally agree with classical approaches relying exclusively on CO2 ﬂuxes but indicate a systematic underestimation under low light conditions, demonstrating the importance of using multiple approaches for constraining present‐day GPP. Supporting Information: • Supporting Information S1 Correspondence to: G. Wohlfahrt, georg.wohlfahrt@uibk.ac.at Correspondence to: G. Wohlfahrt, georg.wohlfahrt@uibk.ac.at Citation: Spielmann, F. RESEARCH LETTER 10.1029/2019GL082006 Key Points: Key Points: M., Wohlfahrt, G., Hammerle, A., Kitz, F., Migliavacca, M., Alberti, G., et al. (2019). Gross primary productivity of four European ecosystems constrained by joint CO2 and COS ﬂux measurements. Geophysical Research Letters, 46, 5284–5293. https://doi.org/10.1029/ 2019GL082006 1. Introduction In contrast to CO2, whose uptake is always accompanied by release through mitochondrial respiration, the uptake of COS is a one‐way ﬂux (but see Gimeno et al., 2017), opening the opportunity to infer GPP at leaf and canopy scale as (Sandoval‐ Soto et al., 2005): In search for further constraints of GPP, the trace gas COS has recently received growing attention (Asaf et al., 2013; Berry et al., 2013; Campbell et al., 2008, 2017; Yang et al., 2018). COS, present in the atmosphere at an average mole fraction of 500 ppt, enters the plant leaf through the stomata in a similar way as CO2 where it is catalyzed to hydrogen sulﬁde (H2S) and CO2 in a one‐way reaction by the enzyme carbonic anhy- drase (CA; Notni et al., 2007; Protoschill‐Krebs & Kesselmeier, 1992). In contrast to CO2, whose uptake is always accompanied by release through mitochondrial respiration, the uptake of COS is a one‐way ﬂux (but see Gimeno et al., 2017), opening the opportunity to infer GPP at leaf and canopy scale as (Sandoval‐ Soto et al., 2005): GPP ¼ FCOS χCO2 ð Þ= χCOSLRU ð Þ (1) (1) GPP ¼ FCOS χCO2 ð Þ= χCOSLRU ð Þ GPP ¼ FCOS χCO2 ð Þ= χCOSLRU ð Þ where FCOS is the COS ﬂux (pmol m−2 s−1) and χCOS (ppt) and χCO2 (ppm) are the ambient mole fractions of COS and CO2, respectively. Equation (1) is mathematically closed by the so‐called leaf relative uptake rate (LRU) as the ratio of ﬂuxes per unit mole fraction for COS and CO2, which must be speciﬁed a priori or assessed independently. A recent literature synthesis (Whelan et al., 2018) showed LRU converging to a median of 1.7, but with a wide spread between 0.7 and 6.2 (95% conﬁdence interval of the median). Another critical assumption of applying equation (1) at the ecosystem scale is that nonleaf sources or sinks of COS must be negligible (Wohlfahrt et al., 2012). Previous studies have identiﬁed soils to contribute to the ecosystem‐scale COS exchange, either as sinks or sources of COS, even though drivers for differences in direction and magnitude of the soil COS exchange are still poorly understood (Whelan et al., 2018). 2.1. Site Description Field measurements were conducted at four different European biomes in four measurement campaigns: During spring and summer 2015 at an intensively managed temperate mountain grassland (GRA), in spring 2016 at a Mediterranean savanna ecosystem (SAV), in summer 2016 at a temperate beech forest (DBF), and in summer 2017 at an agricultural soybean ﬁeld (CRO). For further information on all sites, see Table S1 in the suppporting information (Braendholt et al., 2018; El‐Madany et al., 2018; Hörtnagl et al., 2011; Hortnagl & Wohlfahrt, 2014) and Text S1. 1. Introduction The net exchange of CO2 between an ecosystem and the atmosphere (net ecosystem exchange, NEE) consists of two major components of opposite direction, gross primary productivity (GPP), and ecosystem respiration (Reco). Of these three quantities only NEE can be directly derived at ecosystem level, whereas GPP and Reco have to be inferred from proxies or models (Wohlfahrt & Gu, 2015). For the contemporary carbon cycle, the single most important source for GPP estimates has been NEE measurements by means of the eddy covariance (EC) technique from which GPP as well as Reco are inferred in a standardized fashion by applying so‐called ﬂux partitioning (FP) models (see section 2; Beer et al., 2010; Lasslop et al., 2010; Mahecha et al., 2010; Papale et al., 2006), which exploit the fact that GPP is zero during nighttime and/or depends on solar irradiation during daytime. These FP models, however, have not escaped criticism due to acknowledged problems with some of the underlying data (e.g., potential bias of nighttime EC ﬂux measurements, Aubinet, 2008) and model structural issues (e.g., misrepresentation of sources and drivers of Reco; Heskel et al., 2013; Wehr et al., 2016; Wohlfahrt et al., 2005; Wohlfahrt & Galvagno, 2017), resulting in poorly constrained estimates of uncertainty and the potential for signiﬁcant bias of the inferred GPP and Reco estimates (Wohlfahrt & Gu, 2015). ©2019. The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 5284 SPIELMANN ET AL. SPIELMANN ET AL. Geophysical Research Letters 10.1029/2019GL082006 In search for further constraints of GPP, the trace gas COS has recently received growing attention (Asaf et al., 2013; Berry et al., 2013; Campbell et al., 2008, 2017; Yang et al., 2018). COS, present in the atmosphere at an average mole fraction of 500 ppt, enters the plant leaf through the stomata in a similar way as CO2 where it is catalyzed to hydrogen sulﬁde (H2S) and CO2 in a one‐way reaction by the enzyme carbonic anhy- drase (CA; Notni et al., 2007; Protoschill‐Krebs & Kesselmeier, 1992). 1. Introduction Previous studies that used COS to estimate ecosystem‐scale GPP relied on a constant, prescribed LRU and neglected any in situ soil contribution to the COS ﬂux (Asaf et al., 2013) or estimated LRU based on in situ branch chamber measurements of COS and CO2 (Yang et al., 2018). A third approach made use of a mechan- istic ecosystem model to quantify the relationship between COS and CO2 ﬂuxes to estimate regional GPP ﬂuxes on the base of airborne COS measurements (Hilton et al., 2017). This study seeks to address the knowledge gaps in the use of COS as a proxy for ecosystem‐scale GPP and proposes a novel approach for estimating ecosystem‐level GPP based on joint constraints from both CO2 and COS ﬂuxes. SPIELMANN ET AL. 2.3. Ecosystem Fluxes The COS and CO2 ecosystem ﬂuxes were obtained using the eddy covariance method (Aubinet et al., 1999; Baldocchi, 2014). Besides the fast retrieval of the mole fraction of these two scalars, we used available three‐ axis sonic anemometers to obtain high‐resolution data of the three wind components. The list of instruments used in this study is reported in Table S1 (Braendholt et al., 2018; El‐Madany et al., 2018; Hörtnagl et al., 2011; Hortnagl & Wohlfahrt, 2014). The raw data for scalar mole fractions as well as the sonic data were saved on the same PC in SAV and DBF, whereas we used a time synchronization software (NTP, Meinberg, NI, Germany) in GRA and CRO to synchronize two PCs (or a PC and a data logger) saving the data separately. We then used a self‐developed software to determine the time lag, introduced by the separa- tion of tube intake and the sonic anemometer and the tube length, between the mole fraction and sonic data- set (Hortnagl et al., 2010). The data were then processed using the software EdiRe (University of Edinburgh, UK) and MATLAB 2017 (MathWorks, MA, United States). We used laser drift corrected COS‐mole fraction data (see section 2.2) and linear detrending to process our data before following the procedure to correct for sensor response, tube attenuation, path averaging, and sensor separation (Gerdel et al., 2017). 2.5. Soil Models On the basis of the periodically measured soil ﬂuxes and additionally retrieved meteorological and soil data (incident shortwave radiation reaching the soil surface, soil moisture and temperature), a random forest regression model (Liaw & Wiener, 2002) was trained for each site in order to simulate the soil COS exchange at the same time scale as the ecosystem ﬂux measurements (see section 2.3). For additional information on this method see text S3 (Liaw & Wiener, 2002). 2.4. Soil Flux Chamber Measurements To quantify soil COS ﬂuxes, we installed stainless steel (grade: 316 L) rings 5 cm into the soil, which remained on site for the whole measurement campaign. The aboveground biomass eventually present within each ring was removed at least one day prior to each measurement day, if necessary (GRA, SAV). The vegetation surrounding the rings was allowed to grow and was not cut, the roots within the rings were not removed, and natural litter was left in place. During each measurement, a transparent fused silica glass chamber (Kitz et al., 2017) was placed into a water ﬁlled channel of the steel rings, while air was sucked through the chamber to the QCL. We then compared the chamber COS mole fraction with the ambient mole fraction above the chamber, using a second inlet to which we switched before the chamber measurement and after reaching stable readings inside the chamber. The COS soil ﬂux was calculated using the following equation: (2) F ¼ q C2−C1 ð Þ=A (2) where F is the COS soil ﬂux (pmol m−2 s−1), q denotes the ﬂowrate in (mol/s), C2 and C1 are the chamber and ambient mole fractions of COS in ppt, respectively, and A is the soil surface area (0.032 m2) covered by the chamber. For a more detailed description see Kitz et al. (2017). where F is the COS soil ﬂux (pmol m−2 s−1), q denotes the ﬂowrate in (mol/s), C2 and C1 are the chamber and ambient mole fractions of COS in ppt, respectively, and A is the soil surface area (0.032 m2) covered by the chamber. For a more detailed description see Kitz et al. (2017). Geophysical Research Letters 10.1029/2019GL082006 To correct for the known drift issues of the QCL (Kooijmans et al., 2016), we used a gas with known COS mole fraction to do half hourly calibrations for 1 min. The gas cylinders (working standards) used for the calibrations were either pressurized air (UN 1002), nitrogen (UN 1066), or dried ambient air, which were cross compared (when working standard cylinders were full and close to empty) to an Aculife‐treated alumi- num pressurized air cylinder obtained from the National Oceanic and Atmospheric Administration (NOAA). The latter was analyzed by the central calibration laboratory of NOAA for its COS mole fraction using gas chromatography. For additional information see Text S2 (Asaf et al., 2013; Berkelhammer et al., 2014; Campbell et al., 2017; Kooijmans et al., 2016). SPIELMANN ET AL. 2.2. Mole Fraction Measurements The COS and CO2 mole fractions were measured using a Quantum Cascade Laser (QCL) Mini Monitor (Aerodyne Research, Billerica, MA, United States) at a wave number of ~2,056 cm−1 and at a rate of 5 (SAV, DBF, and CRO) or 10 Hz (GRA). The instrument was placed in a temperature‐controlled box to mini- mize any inﬂuences of ambient temperature changes. The cooling of the QCL and its box was achieved by two Thermocubes (400, Solid State Cooling Systems, Wappinger Falls, NY, United States). We used valves (Parker‐Hannaﬁn, Cleveland, OH, United States), Teﬂon™tubing, stainless steel ﬁttings (SWAGELOK, Solon, OH, United States and FITOK, Offenbach, HE, Germany), and Teﬂon ﬁlters (Savilex, EdenPrarie, MN, United States) to ensure that only materials known not to interact with COS were used for the measurement and calibration airﬂow. At each ﬁeld site, we installed the inlet of the intake tube in close proximity to the sonic anemometer. We insulated the tube, which had a diameter of 1/4 inch in GRA and 3/8 inch in the other ﬁeld sites, and heated it to above ambient temperature to prevent condensation within the tubes. The air was sucked to the QCL at a ﬂowrate of above 7 L/min−1 using a vacuum pump (Agilent Technologies, CA, United States). SPIELMANN ET AL. 5285 Geophysical Research Letters Geophysical Research Letters 2.6. Ancillary Data Standard meteorological parameters and soil related data (e.g., soil temperature and moisture) were mea- sured at each site using state of the art sensors and provided by each site principal investigator (PI) (see Table S1; Braendholt et al., 2018; El‐Madany et al., 2018; Hörtnagl et al., 2011; Hortnagl & Wohlfahrt, 2014). SPIELMANN ET AL. 5286 Geophysical Research Letters 10.1029/2019GL082006 2.7. Flux Partitioning Models 2.7.1. FP Model Traditionally, GPP on ecosystem level is inferred by applying either a so‐called nighttime (Reichstein et al., 2005) or daytime (Lasslop et al., 2010) FP model. The nighttime FP model makes use of the assumption that the nighttime NEE represents the ecosystem respiration (Reco). Therefore, a Reco model based on a temperature‐dependent function (Lloyd & Taylor, 1994) is ﬁt against the data and used to calculate the day- time respiration. Reco ¼ rb e E0 1 TRef −T0− 1 Tair−T0 (3) (3) where Reco denotes the ecosystem respiration (μmol m−2 s−1), rb is the ecosystem base respiration at the reference temperature TRef (°C), which is set to 15 °C, Tair (°C) refers to the air temperature (°C), and E0 (°C) to the temperature sensitivity. T0 was kept constant at −46.02 °C. GPP can then be retrieved as the dif- ference between the measured NEE and the estimated daytime Reco. The daytime FP model by Lasslop et al. (2010) uses nighttime data to parameterize the temperature sensitiv- ity (E0) of Reco via equation (3) but adds a light and temperature dependent function to infer both GPP and Reco from daytime data only: NEE ¼ α β RPAR α RPAR þ β þ rb e E0 1 Tref −T0− 1 Tair−T0 (4) (4) where α denotes the canopy light utilization efﬁciency (μmol CO2/μmol photons), β the maximum CO2 uptake rate of the canopy at light saturation (μmol CO2 m−2 s−1), and RPAR the incoming photosynthetic active radiation (μmol m−2 s−1). The right‐hand side of the equation, representing ecosystem respiration, fol- lows the same notation as equation (3). where α denotes the canopy light utilization efﬁciency (μmol CO2/μmol photons), β the maximum CO2 uptake rate of the canopy at light saturation (μmol CO2 m−2 s−1), and RPAR the incoming photosynthetic active radiation (μmol m−2 s−1). The right‐hand side of the equation, representing ecosystem respiration, fol- lows the same notation as equation (3). 2.7.2. FP+ Model 2.7.2. FP+ Model We extended the FP model to include FCOS by using the GPP, resulting from the ﬁrst part on the right‐hand side of equation (4) GPP ¼ α β RPAR α RPAR þ β (5) (5) in equation (6) (rearranged equation (1)): in equation (6) (rearranged equation (1)): FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS (6) FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS (6) (6) FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS (6) FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS SPIELMANN ET AL. 3. Results and Discussion Modelled soil COS ﬂuxes ranged from an uptake of −3.57 to an emission of 9.91 pmol m−2 s−1 with a median of −0.68, 0.67, −2.60, and −0.53 pmol m−2 s−1 at GRA, SAV, DBF and CRO, respectively (Figure 1). Differences in the sign and magnitude of the soil COS exchange among sites can be explained to a large degree by the magnitude of solar radiation reaching the soil surface (see Text S3), which positively related to the soil COS emission. Sites with a sparse canopy and high amounts of direct solar radiation reaching the soil surface, like SAV, showed stronger COS emission during daytime, whereas during nighttime or at sites with a high leaf area index, uptake was the dominant process for soil COS exchange (Figure 1). Kitz et al. (2017), Whelan and Rhew (2015), and Meredith et al. (2018) suggest that daytime COS emission from soils is mainly linked to abiotic thermal or photodegradation by yet largely unknown reactions, while COS uptake is mostly governed by biological processes, notably the activity of microbial CA (Whelan et al., 2018). Even though soil COS ﬂuxes overall constituted a small fraction of ecosystem ﬂuxes during daytime (Figure 1), soils, in absolute terms, accounted for up to 10 % (SAV) of the daytime ecosystem COS ﬂux, reach- ing even higher ratios during dusk and dawn (Figure S4). On the basis of the substantial inﬂuence that soils can have on the ecosystem COS exchange during certain times, we corrected the ecosystem COS ﬂuxes for the soil contribution to retrieve the canopy COS uptake. Due to the joint control by stomatal conductance, canopy‐scale COS ﬂuxes and NEE covaried during day- time hours (Figure 2). In contrast to NEE, which turned positive in the absence of photosynthetically active radiation (PAR; i.e. net CO2 release), the light‐independent canopy uptake of COS continued at lower rates during night time (Figures 2 and S5–S12) due to incomplete stomatal closure. This ﬁnding is in agreement with other studies (Kooijmans et al., 2017, 2019; Novick et al., 2009), although we did not observe an earlier peak in COS uptake as compared to NEE (Figure 2) or GPP as Kooijmans et al. (2019) did. FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS where FCOSmodel is the modelled COS ﬂux (pmol m−2 s−1), χCOS (ppt) and χCO2 (ppm) are the measured ambient mole fractions of COS and CO2, respectively, and LRU (‐) is the leaf relative uptake rate. As COS uptake by CA is thought to be a light‐independent process, while CO2 uptake by the enzyme RUBISCO depends on solar radiation absorbed by leaf chlorophyll, LRU was, deﬁned as a light‐dependent parameter, consistent with recent experimental evidence (Kooijmans et al., 2017; Kooijmans et al., 2019; Whelan et al., 2018; Wohlfahrt et al., 2012): LRU ¼ ι e κ RPAR (7) (7) Here the parameter ι (‐) corresponds to the LRU at high light intensity, the parameter κ (μmol m−2 s−1) gov- erns the increase of LRU at low light conditions, and RPAR (μmol m−2 s−1) represents the incident PAR. In comparison to the FP model, where GPP is obtained by optimization against the measured NEE, in the FP+ model we concurrently optimize equations (4) and (6) against measured NEE and FCOS, GPP thus being derived from two independent constraints. From the 4–6 unknown model parameters (FP and FP+), we determined the temperature sensitivity para- meter of Reco (i.e., E0) using nighttime data by minimizing the root squared mean error, whereas we used DREAM (Scholz et al., 2017; Vrugt & Ter Braak, 2011), a multichain Markov Chain Monte Carlo SPIELMANN ET AL. 5287 Geophysical Research Letters 10.1029/2019GL082006 Figure 1. Carbonyl sulﬁde ﬂux distribution. Distribution plot of the measured daytime carbonyl sulﬁde (COS) ﬂuxes at ecosystem scale (colored area on the left) and the modeled daytime COS ﬂuxes from soil (brown area on the right) with a bin size of 5 pmol m−2 s−1 over the course of the campaigns for each ecosystem. Positive ﬂuxes indicate net emission, while negative ﬂuxes indicate net uptake. Figure 1. Carbonyl sulﬁde ﬂux distribution. Distribution plot of the measured daytime carbonyl sulﬁde (COS) ﬂuxes at ecosystem scale (colored area on the left) and the modeled daytime COS ﬂuxes from soil (brown area on the right) with a bin size of 5 pmol m−2 s−1 over the course of the campaigns for each ecosystem. Positive ﬂuxes indicate net emission, while negative ﬂuxes indicate net uptake. algorithm, to infer the remaining 3–5 (see Table S3) parameters based on Bayesian statistics, with daytime data. FCOSmodel ¼ GPP LRU=χCO2 ð Þ=χCOS Preliminary model runs showed that the vapor pressure deﬁcit limitation of GPP (Lasslop et al., 2010) during our ﬁeld campaigns was minor, so we excluded the parameter controlling this effect from our ﬁnal model. For additional information on the Bayesian model inversion see Text S4 (Gelman & Rubin, 1992; Schoups & Vrugt, 2010; Van Oijen et al., 2005; Vrugt & Ter Braak, 2011). SPIELMANN ET AL. 3. Results and Discussion Note that the nighttime residual uptake of COS, when GPP is zero, does not void the general approach of using COS as a proxy for GPP, as this is accounted for by the light‐dependent parameterization of LRU, which approaches inﬁnity at low light (Eq. (7)). The magnitude of the canopy COS exchange varied strongly between sites, reaching maximum mean uptake rates around 40 pmol m−2 s−1 (GRA, SAV, and CRO) and up to twice as much at DBF (Figure 2). GRA, DBF, SPIELMANN ET AL. SPIELMANN ET AL. 5288 Geophysical Research Letters 10.1029/2019GL082006 Figure 2. Mean diel carbonyl sulﬁde and carbon dioxide ﬂuxes. Mean diel variation of the net COS canopy ﬂuxes (ﬁlled circles and solid lines) and NEE (open carats and dashed lines) for (a) GRA, (b) SAV, (c) DBF, and (d) CRO over the course of the campaigns. Black xs indicate values below the limit of detection (Langford et al., 2015), which cannot be distinguished from zero ﬂuxes. Shaded areas represent ±1 standard deviation of the mean. Positive ﬂuxes indicate net emission, while negative ﬂuxes indicate net uptake. The photosynthetic active radiation is plotted as hourly means on the right y axis of each plot as a bar graph. COS = carbonyl sulﬁde; PAR = photosynthetic active radiation; NEE = net ecosystem exchange; CET = Central European Time. Figure 2. Mean diel carbonyl sulﬁde and carbon dioxide ﬂuxes. Mean diel variation of the net COS canopy ﬂuxes (ﬁlled circles and solid lines) and NEE (open carats and dashed lines) for (a) GRA, (b) SAV, (c) DBF, and (d) CRO over the course of the campaigns. Black xs indicate values below the limit of detection (Langford et al., 2015), which cannot be distinguished from zero ﬂuxes. Shaded areas represent ±1 standard deviation of the mean. Positive ﬂuxes indicate net emission, while negative ﬂuxes indicate net uptake. The photosynthetic active radiation is plotted as hourly means on the right y axis of each plot as a bar graph. COS = carbonyl sulﬁde; PAR = photosynthetic active radiation; NEE = net ecosystem exchange; CET = Central European Time. and CRO were characterized by similar maximum mean daytime net CO2 uptake rates (~20 μmol m−2 s−1), whereas SAV exhibited only half of this net uptake (~9 μmol m−2 s−1; Figure 2). 3. Results and Discussion The mean observed ecosystem COS ﬂuxes of our study lie at the upper end or above comparable ﬁeld observations of the measurements compiled in the recent review by Whelan et al. (2018). The variable canopy COS uptake to NEE ratio (Figures 2 and S13) suggests that either the LRU (equation (1)) must differ between sites, and/or that similar NEE values result from variable GPP to Reco ratios. Unaccounted ﬂuxes of COS within the ecosystems, from stems or sinks and sources of yet unknown origin are another possible but rather unlikely explanation for the differences in the COS uptake to NEE ratio. GPP resulting from the FP+ model was generally higher than the classical (FP) approach (Figure 3), the dif- ference between the sums of ﬁxed CO2 between the models over the course of the measurement campaigns amounting to GRA +5.08% ± 1.23%, SAV +6.08% ± 1.05%, DBF +4.20% ± 0.13%, CRO +1.79% ± 0.74% (the standard deviations representing the temporal variability; see Text S4 and Figures S14–S17). However, model differences were small compared to the model uncertainty calculated from the Bayesian model inver- sion (see Text S4 and Figure S18). The difference between the models is mainly attributable to a higher inferred initial quantum yield (α; equa- tion (5)) for all FP+ models (Figure 4), whereas we detected a small decrease in the maximum canopy CO2 uptake rate at light saturation (β; Figure S19). As a consequence, the absolute difference in GPP between the models for GRA, SAV and DBF increased sharply in the morning, remained relatively stable during the day, and then decreased again in the evening (Figure S20). In contrast to these sites, the FP model predicted higher GPP at higher light conditions for CRO (Figure S20), which caused the absolute model difference in GPP to decline and even reverse sign around noontime (Figure S20). A stepwise regression analysis with the absolute difference in GPP between the FP and FP+ model as dependent variable included PAR at all sites, Tair at GRA, DBF and CRO, soil temperature at GRA, and vapor pressure deﬁcit at neither site. SPIELMANN ET AL. 3. Results and Discussion As hypothesized above, the LRU at saturating light intensity, that is, parameter ι, varied strongly across the sites (Figure 4), with the optimal parameter set ranging from 0.89 (CRO) and 1.02 (GRA) for the two herbac- eous ecosystems up to 2.22 (DBF) and 2.27 (SAV) for the forest and the mixed woodland grassland site. These values and their mean (1.6) are consistent with the median (1.7) and 95 % conﬁdence interval (0.7 to 6.2) from leaf‐level studies (Whelan et al., 2018). The most productive, that is, highest GPP, ecosystem was SPIELMANN ET AL. SPIELMANN ET AL. 5289 Geophysical Research Letters 10.1029/2019GL082006 Figure 3. Comparison of model GPP output. GPP (μmol m−2 s−1) modelled on the basis of the FP (solid black lines) and the FP+ (solid colored lines) model plotted against the measured PAR (μmol m−2 s−1) for (a) GRA, (b) SAV, (c) DBF, and (d) CRO over the course of the measurement campaigns. Black shaded areas represent the 95% conﬁdence interval of the FP model, whereas the colored shading represents the corresponding 95% conﬁdence interval of the FP+ model. GPP = gross primary productivity; FP = ﬂux partitioning; PAR = photosynthetic active radiation. Figure 3. Comparison of model GPP output. GPP (μmol m−2 s−1) modelled on the basis of the FP (solid black lines) and the FP+ (solid colored lines) model plotted against the measured PAR (μmol m−2 s−1) for (a) GRA, (b) SAV, (c) DBF, and (d) CRO over the course of the measurement campaigns. Black shaded areas represent the 95% conﬁdence interval of the FP model, whereas the colored shading represents the corresponding 95% conﬁdence interval of the FP+ model. GPP = gross primary productivity; FP = ﬂux partitioning; PAR = photosynthetic active radiation. Figure 4. Comparison of model parameter output. Histogram of the probability density function of the last 2,950 runs after convergence of the DREAM algorithm of α, the canopy light utilization efﬁciency (μmol CO2/μmol photons) in the left panels for (a) GRA, (b) SAV, (c) DBF, and (d) CRO and the parameter ι, which is comparable to the LRU at high light conditions in the right panels for (e) GRA, (f) SAV, (g) DBF, and (h) CRO. The FP model is indicated by the black bars, the FP+ model by colored bars. FP = ﬂux partitioning. Figure 4. Comparison of model parameter output. 4. Conclusions During recent years COS has seen increasing use as an alternative means of inferring GPP on spatial scales from ecosystem to global (Asaf et al., 2013; Berry et al., 2013; Campbell et al., 2008; Campbell, Berry, et al., 2017; Yang et al., 2018) . The Achilles heel of these promising efforts is the need to specify the LRU a priori (Wohlfahrt et al., 2012), because its variability is not well understood, and the poorly quantiﬁed contribution of soils (Whelan et al., 2018). Our study is the ﬁrst to overcome these issues by treating the LRU as an adjus- table parameter, which is jointly optimized against both CO2 and COS ﬂux measurements, and explicitly accounts for the soil COS exchange. Although GPP inferred in this fashion agreed well with the one derived from conventional CO2 ﬂux partitioning, our FP+ model yielded a slightly higher GPP (by 4.3% ± 1.8%) on average over the course of the measurement campaigns and across all sites. Even though our study indicates a larger uptake of CO2 across multiple biomes compared to conventional CO2 ﬂux partitioning, our GPP esti- mate lies within the uncertainty of the GPP reported in Beer et al. (2010) and thus does not support recent reports of substantially higher estimates (Arneth et al., 2017; Welp et al., 2011). To take advantage of newly emerging constraints on GPP, for example, COS (Wohlfahrt et al., 2012), isotopic ﬂux partitioning (Wehr et al., 2016), and Sun‐induced ﬂuorescence (Wohlfahrt et al., 2018), should be compared and combined with traditional ﬂux partitioning to understand the differences between methods and to decrease the overall uncertainty of GPP. Acknowledgments This study was ﬁnancially supported by the Austrian National Science Fund (FWF; contracts P26931, P27176, and I03859), the Tyrolean Science Fund (contract UNI‐0404/1801), and the University of Innsbruck (Infrastructure funding by Research Area Alpine Space‐Man and Environment to G. W.; Young Academics grant to A. H.; and travel grant by Italy centre to K. G.). Financial support to F. M. S. was pro- vided through a PhD scholarship by the University of Innsbruck. 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Results and Discussion Histogram of the probability density function of the last 2,950 runs after convergence of the DREAM algorithm of α, the canopy light utilization efﬁciency (μmol CO2/μmol photons) in the left panels for (a) GRA, (b) SAV, (c) DBF, and (d) CRO and the parameter ι, which is comparable to the LRU at high light conditions in the right panels for (e) GRA, (f) SAV, (g) DBF, and (h) CRO. The FP model is indicated by the black bars, the FP+ model by colored bars. FP = ﬂux partitioning. SPIELMANN ET AL. 5290 SPIELMANN ET AL. Geophysical Research Letters 10.1029/2019GL082006 CRO, followed by similar GPP at GRA and DBF, and ﬁnally SAV (Figures 3 and S19). Not accounting for the soil COS exchange would have resulted in an overestimation of GPP by 1.3%–2.9 % for GRA and DBF and up to 5.7%–8.6 % for CRO and SAV, which cautions against neglecting the soil contribution at sites where solar radiation signiﬁcantly penetrates to the soil surface. Results were not sensitive to the chosen prior distribu- tion for the parameter ι—using a uniform prior distribution would only change the resulting ι by 0.01 (‐) in GRA to up to +0.03 (‐) in DBF (see Table S4), which decreased the GPP of DBF by 1.4 %. In contrast to our results, a recent study using isotopic ﬂux partitioning (FPiso) by Wehr et al. (2016) reported that traditional FP methods (Lasslop et al., 2010; Reichstein et al., 2005) overestimate Reco, which the authors ascribed to the Kok effect (Heskel et al., 2013), and thus in turn GPP. As the FP+ and FPiso models have a quite different theoretical basis, these conﬂicting results are difﬁcult to reconcile and most likely require joint ﬂux measure- ments of COS and the isotopologues of CO2 to be resolved. 4. Conclusions thank the “De Eccher Agricola s.r.l.” for the support during the ﬁeld campaign at CRO. 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https://openalex.org/W4377836361	https://fis.uni-bamberg.de/bitstream/uniba/58606/1/fisba58606.pdf	English	null	Visual Feedback for Maneuver-Based Driving : First Results from a Design Workshop	Schriftenreihe Logistik und Supply Chain Management	2,019	cc-by	3,190	JEL Classification: Y80 (Related Disciplines) Keywords: Automotive HMI, Design Research, User-Centered Design, Maneuver- based Driving, Head-Up Displays, Visualization 1 Introduction Automation is a key-factor in future mobility. The number of driver assistance sys- tems and information in the vehicle increases. Therefore, new challenges arise. Drivers have to operate simultaneously with assistance systems. They must under- stand how individual systems work to recognize system boundaries. A possible ap- proach to overcome these challenge, is the introduction of a global operating con- cept, which combines individual assistance systems. The driver takes a supervising role and controls the vehicle through various driving maneuvers (e.g., lane change, parking), while the vehicle performs all stabilization tasks (Franz 2014). The ma- neuver-based vehicle guidance is a complement to manual and automated driving. It ranges from simple complementation (e.g., parking assistant) to substitution (e.g., Conduct-By-Wire, see Winner & Hakuli 2006) of manual or automatic driving. In this article, we will investigate how the visual feedback for such a concept can look like. The requirements for a maneuver-based driving concept depend on the degree of automation. Under SAE level 2 and 3 (SAE 2018) it was shown that it makes sense to display current maneuvers constantly in a HUD. The reduced gaze to the instru- ment cluster and increased gaze on the road leads to a higher situational awareness (Franz 2014). In contrast to existing work, this article’s aim is to examine the prob- lem assuming SAE level 4 (complete automation of driving in critical situations). Since future display concepts in vehicles might have so-called “virtual windshields” (Haeuslschmid, Pfleging & Alt 2016), we use a HUD as foundation for our designs. In SAE level 4, there is no need to increase situational awareness as in SAE level 2 and 3, but we assume a fundamental usefulness through positive effects on ac- ceptance and trust in the system (Hoff & Bashir 2015, Walch et al. 2017). Within fully automated rides, the drivers can intervene and override the system behavior, which leads to higher acceptance. Even if the driver does not want to control the car at moment, the increased system transparency through the display of maneuvers in a HUD leads to more trust in the automation. The driver’s interaction with a global maneuver-based operating system happens in one of two basic categories: input or output. The input-interaction is the way the driver communicates with the system (“operating” from a driver’s perspective) and the output-interaction is the way the system communicates with the driver (“feed- back” from a driver’s perspective). 10.20378/irb-58606 10.20378/irb-58606 10.20378/irb-58606 1 Introduction .................................................................................................... 298 2 Method ............................................................................................................ 299 3 Results ............................................................................................................ 300 4 Discussion ....................................................................................................... 305 5 Conclusion ...................................................................................................... 306 6 Literature ........................................................................................................ 307 Abstract: Starting with the automatic gear change, the operation of a vehicle becomes more and more abstract. In the future, we could control vehicles with single, simple commands. For such a maneuver-based vehicle control system, we investigate a head-up display design in a workshop. The aims are to identify common and distinct features of various display designs through mock-ups. First results show that differ- ent sizes of GUI elements are preferred by different states. The preferred position of GUI elements in the head-up display (HUD) is the central bottom area. We found two major interface design styles: static interfaces (all elements visible) with fixed layout and dynamic interfaces (only relevant elements visible) with fixed or adap- tive layout. JEL Classification: Y80 (Related Disciplines) Keywords: Automotive HMI, Design Research, User-Centered Design, Maneuver- based Driving, Head-Up Displays, Visualization JEL Classification: Y80 (Related Disciplines) Keywords: Automotive HMI, Design Research, User-Centered Design, Maneuver- based Driving, Head-Up Displays, Visualization JEL Classification: Y80 (Related Disciplines) Henrik Detjen, Maurizio Salini, Martin Wozniak 298 1 Introduction Interaction requires an interface between the two parties involved. Interfaces for input-interaction can use touch (Franz 2014: touch- pad, Kauer et al. 2010: tablet) or touch-less (Detjen et al. 2019: speech or mid-air gesture) techniques. Commonly, the output-interaction is through the visual channel and visual techniques (Franz 2014: HUD, Kauer et al. 2010: tablet), because here, information can be communicated persistently. In this article, we focus on the out- 299 Visual Feedback for Maneuver-Based Driving put-interaction and generate first ideas for an interface from a user-centered per- spective and we present insights from a participatory design workshop. 3 Results We group our observations in size of GUI elements, HUD layout, interface style and behavior, and visualization. Overall, 352 maneuver elements in the 14 mock- ups of driving situations were used. To simplify the results, we report our following findings for all situations and participants combined. 2 Method Possible states were:  Available (blue color): Indicates that a maneuver is selectable/executable, al- so “normal” state  Unavailable (blue color, brighter): Indicates that a maneuver is not se- lectable/executable, also “inactive” or ”disabled” state Henrik Detjen, Maurizio Salini, Martin Wozniak 300 Henrik Detjen, Maurizio Salini, Martin Wozniak  Active (blue color, yellow border): Indicates that a maneuver is executed at the moment, “active” state  Active (blue color, yellow border): Indicates that a maneuver is executed at the moment, “active” state  Active (blue color, yellow border): Indicates that a maneuver is executed at the moment, “active” state The combination of state and size leads to nine sets of maneuver elements and 81 elements in total, c.f. Figure 1, Figure 2. Figure 1: Workspace with Workshop Materials – Maneuver Symbols (Left) and Explanation (Top Right), Maneuver Situation on Paper (Bottom Right) Figure 1: Workspace with Workshop Materials – Maneuver Symbols (Left) and Explanation (Top Right), Maneuver Situation on Paper (Bottom Right) After the video, participants completed a questionnaire regarding further visualiza- tion: how they would improve the maneuver state visualization, how they would integrate driving parameters (speed, distance to next car, position on lane) and if they would add some kind of animation. The answers were free text and/or sketch- es. 2 Method In the following chapter, we explain the sample, setup and procedure of our design workshop. The basic idea there was to generate mock-ups of a HUD in different maneuver situations. Therefore, we showed experts videos of maneuvers and they designed a mock-up for each video scene. The workshop took place in a laboratory of the Ruhr West University of Applied Sciences. Eight people attended. They were all male and scientific staff or students at the Ruhr West University of Applied Sciences. They had at least some expertise within design and automotive user interfaces, either through work or through com- pleted lectures in the area. Nevertheless, we introduced them into the topic of ma- neuver-based driving or refreshed their knowledge in the workshop. The setup consisted of a TV and a workspace with the workshop material (mock-up elements). On the TV screen, we played a video of a drive. The video had a playing time of about six minutes and included all driving maneuvers used in Franz (2014): turn left/right, change lane left/right, start, straight, follow lane, park, hold at stop- line, hold at side-strip and parking. At the points in the video where a maneuver began (14 times), we stopped the play- back and handed out a screenshot of the situation (DIN-A4-paper). Thus, partici- pants got a realistic impression of the maneuvers and we, therefore, a potentially improved design. The screenshot was representing a windshield or HUD. With their given maneuver elements, they designed their own mock-up of the HUD. For their mock-up, participants had a complete set of maneuver symbols in three sizes and three states for each size. Possible sizes were:  Small: 1.5cm x 1.5cm (~0.36% occlusion) Small: 1.5cm x 1.5cm ( 0.36% occlusion)  Medium: 2cm x 2cm (~0.63% occlusion)  Large: 2 25cm x 2 25cm ( 0 80% occlusion)  Medium: 2cm x 2cm (~0.63% occlusion)  Large: 2.25cm x 2.25cm (~0.80% occlusion) Occlusion means the relation of size to HUD, in this case a DIN-A4 pap Occlusion means the relation of size to HUD, in this case a DIN-A4 paper. Visual Feedback for Maneuver-Based Driving Figure 2: The Usage Frequency of Different Symbol Sizes and Their Relation to States 14 25 23 10 37 78 65 72 26 0 20 40 60 80 100 120 140 Large Medium Small Usage (n‐times) Symbol Size Active Unavailable Available Figure 2: The Usage Frequency of Different Symbol Sizes and Their Relation to States As shown in Figure 2, participants used the medium sized symbols most frequently (n = 134), followed by the small sized symbols (n = 127), while the large size was least frequent overall (n = 89). We observed that the display state has an influence on the chosen symbol size. To indicate the state “available”, participants preferred large or medium sized symbols and for the state “unavailable”, they preferred small sized symbols. 3.1 GUI Element Size We measured how often participants used a maneuver element. They were free to use one of three sizes (small, medium, large) for maneuver symbols within their GUI mock-ups. They were also free to choose the displayed state of their symbols (available, unavailable, active) for each size. 301 Visual Feedback for Maneuver-Based Driving 3.2 HUD Layout To analyse the spatial distibution of GUI elements, we divide the HUD in 9 qually sized regions: vertical (left, middle, right) x horizontal (top, middle, bottom). Figure 3 shows the distribution of elements for each region. Blue means low usage, red high usage. Figure 3: Heat-Map for Used HUD Regions Figure 3: Heat-Map for Used HUD Regions Henrik Detjen, Maurizio Salini, Martin Wozniak 302 The main observation is that the three most frequently used regions are in the sections at the bottom. Forty percent of all used symbols are in the middle-bottom region. Static UI The Static UI has mainly one fixed layout, which contains all interface elements. Each maneuver symbol is constantly visible, regardless if available or not. Their position on the HUD is permanent. So, the highlighted maneuver’s position jumps within the HUD, when a new maneuver is executed. Figure 4: Examples for the Static UI Category In Figure 4, we see mock-ups from two participants, which are prototypical for a “Static UI”. Figure 4: Examples for the Static UI Category In Figure 4, we see mock-ups from two participants, which are prototypical for a “Static UI”. In Figure 4, we see mock-ups from two participants, which are prototypical for a “Static UI”. 3.3 Interface Style and Behavior In terms of interface style and behavior, we found two major categories, in which all GUI-Designs fit in. We call these categories “Static UI” and “Dynamic UI”. They are closely related to the layout. We describe both categories in the following. Static UI Dynamic UI The Dynamic UI has basically does not contain all interface elements at the same time. It hides unavailable elements, considering they are “unneccessary” (see Discussion). While the position of the active maneuver is fixed in most cases, the position of available maneuver symbols is changed in some versions and in others they are fixed. So, the highlighted maneuver’s position stays at the same position within the HUD, when a new maneuver is executed. Further, we distinguish between a Dynamic UI with fixed layout and a Dynamic UI with a variable layout. Visual Feedback for Maneuver-Based Driving 303 Figure 5: Examples for the Dynamic UI Category Figure 5: Examples for the Dynamic UI Category In Figure 5, we see mock-ups from two participants, which are prototypical for a “Dynamic UI”. In Figure 5, we see mock-ups from two participants, which are prototypical for a “Dynamic UI”. In Figure 5, we see mock-ups from two participants, which are prototypical for a “Dynamic UI”. 3.4 Visualization To gain further insight into maneuver HUD design, we collected information about how participants would visualize maneuver states, driving parameters, GUI anima- tions. Maneuver States Maneuver States We provided a finished design of maneuver GUI elements. Participants uttered some improvements for visualization of the three maneuver states: We provided a finished design of maneuver GUI elements. Participants uttered some improvements for visualization of the three maneuver states:  Unavailable/Inactive: “just use outlines”, “increase tranparency”, “color gray”, “hide”  Available: “keep colors, known from road traffic”  Active: “not sure if necessary”, “highlight with color or border” Driving Parameters: Speed, Distance, Lane position Driving Parameters: Speed, Distance, Lane position Participants sketched a possible visualization for the driving parameters speed, distance to next car and lane position. We selected different sketches in Figure 6. Participants sketched a possible visualization for the driving parameters speed, distance to next car and lane position. We selected different sketches in Figure 6. Henrik Detjen, Maurizio Salini, Martin Wozniak 304 Figure 6: Sketches of the Driving Parameters – Speed (Top Row), Lane Position (Middle Row), Distance to Next Car (Bottom Row) Figure 6: Sketches of the Driving Parameters – Speed (Top Row), Lane Position (Middle Row), Distance to Next Car (Bottom Row) These sketches show that for distance and lane position a discrete visualization (and consequently input) is preferred, i.e. three categories of distance to the next car (“near”, “middle”, “far”). For speed, a more continuous visualization (and input), is preferred, i.e. in 1 or 5 km/h-intervals. The speed regulation contains a comparison between actual and desired speed in most cases. An arrow indicates acceleration (upwards) or slow down (downwards). Further suggestions: Maneuver Stack Figure 7 shows another mentionable sketch: A visualization of a situation in where multiple maneuvers have been uttered by the driver; a graphical stack, which shows all waiting maneuvers and hightlights the next to be executed (arrow). Figure 7: Sketch of a Maneuver Stack Figure 7: Sketch of a Maneuver Stack Animation Reguarding additional animations, participants had contrary opinions. While some said “the less the better” or “no movements”, others gave instructions on where to add animations and what to be careful with. We sorted design recommendations and issues of this group by animation target:  Active Maneuvers: “Active with a pulsing animation”  Colors/Transparency: “Change of colors for change of states”, “Color change only to gray”, “Transparency should change from inactive to available”, “Tranparency problematic for visibility”, “Colors problematic for visibility, i.e. color blindness”, “fade in/out”  Size: “Scale maneuver size to maneuver possibility”  Layout: “active maneuvers slide to the HUD edge” Visual Feedback for Maneuver-Based Driving 305 Further suggestions: Maneuver Stack 4 Discussion In our workshop, we presented predefined maneuver symbols, which proved to be understandable in previous work (Detjen et al. 2018). Another option could have been to ask the participants to draw free hand designs and therefore get more infor- mation about how to visualize symbols. We were aware of this, but focused on the layout first, and asked for improvements visualization afterwards. We planned an- other design study, where the procedure is the other way around. Another point we want to discuss are the two interface styles and their implications for user interaction. There is a trade-off between minimalistic design and learnabil- ity. The less information we present the driver the harder he remembers maneuver elements (the higher the cognitive load and frustration). When the layout is not fixed, he cannot connect a certain position of a GUI element to a certain maneuver (e.g., turn left on left side). On the other hand, this mapping of positions to elements is not necessary for an experienced user, who knows all elements and actions. This is comparable to the gear switch. A novice driver has to look at the symbol on the knob in order to find the right gear, while an experienced driver could shift gears blind. 306 Henrik Detjen, Maurizio Salini, Martin Wozniak 5 Conclusion Maneuver-based driving is a concept with high potential for future driver-vehicle interaction. In this article, we focused on the vehicle-to-driver interaction. Vehicle- to-driver interaction is important, because it improves user’s acceptance and trust in the system. We designed head-up displays / virtual windshields for maneuver-based driving in a design workshop for 14 maneuvering situations. To increase the imagination of participants, we first showed them a video of the maneuver situation which they designed. They were free to use any number of GUI elements for their mock-up. GUI elements had three sizes (small, medium, large) and three states (available, unavailable, active). For the “unavailable” state, small elements were preferred and to indicate the state “available”, participants preferred large or medium sized sym- bols. In a 9-grid layout (left/middle/right x top/middle/bottom), users placed the GUI elements frequently in the bottom row of the screen and most frequently on the middle-bottom area. We presented different visualization sketches for regulating the driving parame- ters speed, distance to next car and lane position. The sketches showed that there is a dependence on the input style. Some preferred a discrete input, some a continu- ous. Users thought critical about a possible animation of a HUD. We recommend to use animations carefully and to turn them off by default. We observed two opposite interface design approaches (static vs adaptive). For a recommendation on which UI-Style or behavior a system should implement, the space of the HUD is the limiting factor. If there is enough space, we would recommend two user interaces, split by two phases: In a first learner-phase, the “Static UI” makes most sense. Through permanent display, the user learns the position of the maneuver elements. When he is familiar with the positions, in the following expert-phase a system should use the “Dynamic UI with fixed layout” and hide the unavailable maneuver elements, because the user does not need this information any longer and the interface is cleaner. This transition between interfaces should be reversible. If there is not enough space to guarantee fixed positions for each maneuver element and maneuvers have to share position in the HUD, the “Dynamic UI with variable layout” should be the style to implement. Visual Feedback for Maneuver-Based Driving 307 6 Literature Detjen, H.; Faltous, S.; Geisler, S.; Schneegass, S. (2019): “Voice and Free-Hand Gestures for Maneuver-Based Car Control”, in preparation. Detjen, H.; Geisler, S.; Salini, M.; Wozniak, M.; Borgmann, C. (2018): “Teilauto- matisiertes Fahren via Sprachsteuerung: Erwartungen und Anforderungen”. In: Mensch und Computer 2018-Workshopband. Franz, B. (2014): “Entwicklung und Evaluation eines Interaktionskonzepts zur ma- növerbasierten Führung von Fahrzeugen”. Dissertation. TU Darmstadt. Haeuslschmid, R.; Pfleging, B.; Alt, F. (2016, May): “A design space to support the development of windshield applications for the car”. In: Proceedings of the 2016 CHI Conference on Human Factors in Computing Systems (pp. 5076– 5091). ACM. Hoff, K. A.; Bashir, M. (2015): “Trust in automation: Integrating empirical evi- dence on factors that influence trust”. In: Human Factors 57.3 (pp. 407–434). Kauer, M.; Schreiber, M.; Bruder, R. (2010, June): “How to conduct a car? A de- sign example for maneuver based driver-vehicle interaction”. In Intelligent Vehicles Symposium (IV) 2010 IEEE (pp. 1214–1221). IEEE. SAE (2018): “Levels of driving automation”, retrieved at 31.01.2019 from https://www.sae.org/news/press-room/2018/12/sae-international-releases- updated-visual-chart-for-its-%E2%80%9Clevels-of-driving- automation%E2%80%9D-standard-for-self-driving-vehicles. Walch, M.; Mühl, K.; Kraus, J.; Stoll, T.; Baumann, M.; Weber, M. (2017): “From Car-Driver-Handovers to Cooperative Interfaces: Visions for Driver-Vehicle Interaction in Automated Driving”. In Automotive User Interfaces (pp. 273– 294). Springer, Cham. Winner, H.; Hakuli, S. (2006, October): “Conduct-by-wire–following a new para- digm for driving into the future”. In: Proceedings of FISITA world automotive congress (Vol. 22, p. 27).
https://openalex.org/W3006047647	https://ojs.unud.ac.id/index.php/Manajemen/article/download/54405/33538	Indonesian	null	LEVERAGE, PROFITABILITAS, DAN KEPEMILIKAN MANAJERIAL BERPENGARUH TERHADAP NILAI PERUSAHAAN PADA PERUSAHAAN REAL ESTATE DAN PROPERTY	E-Jurnal Manajemen	2,020	cc-by	7,579	E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 DOI: https://doi.org/10.24843/EJMUNUD.2020.v09.i02.p17 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 DOI: https://doi.org/10.24843/EJMUNUD.2020.v09.i02.p17 ISSN : 2302-8912 email: adesyaw@gmail.com email: adesyaw@gmail.com ABSTRAK Nilai Perusahaan merupakan tujuan utama yang harus dicapai perusahaan karena dengan memaksimalkan nilai perusahaan berarti memaksimalkan kemakmuran pemegang saham. Investor akan mencari perusahaan yang mempunyai prospek perusahaan yang bagus sehingga menghasilkan tingkat return perusaham yang tinggi. Hal ini tentunya akan mempengaruhi tinggi rendahnya harga pasar saham suatu perusahaan, yang mencerminkan nilai perusahaan. Tujuan penelitian ini untuk mengetahui pengaruh leverage, profitabilitas, dan kepemilikan manajerial terhadap nilai perusahaan. Penelitian dilakukan pada perusahaan Real Estate dan property yang terdapat di Bursa Efek Indonesia tahun 2014- 2018. Sampel yang digunakan yaitu 15 perusahaan, dengan purposive sampling. Pengumpulan data dilakukan dengan metode observasi non partisipan melalui data laporan keuangan yang dipublikaskan di Bursa Efek Indonesia. Teknik analisis yang digunakan yaitu analisis regresi linear berganda. Dalam penelitian ini ditemukan bahwa leverage, profitabilitas dan kepemilikan manajerial berpengaruh positif terhadap nilai perusahaan. Perusahaan diharapkan dapat memperhatikan rasio leverage, profitabilitas, dan kepemilikan manajerial agar nilai perusahaan semakin meningkat. Kata kunci : Leverage, Profitabilitas, Kepemilikan Manajerial, Nilai Perusahaan Luh Putu Putri Adesia Widayanti1 I Putu Yadnya2 Luh Putu Putri Adesia Widayanti1 I Putu Yadnya2 1,2 Fakultas Ekonomi dan Bisnis Universitas Udayana (Unud), Bali, Indonesia ABSTRACT Company Value is the main goal that must be achieved by the company. Investors will look for companies that have good prospects to get high level of company return. This will certainly affect the prices of a company's stock market, which reflects the value of the company. The purpose of this study was to determine the effect of leverage, profitability, and managerial ownership on firm value. The study was conducted on Real Estate and property companies on the Indonesia Stock Exchange 2014-2018. The samples used were 15 companies, with purposive sampling. Data collected using non-participant observation through financial statement on the Indonesia Stock Exchange. Multiple linear regression was used. In this study it was found that leverage, profitability and managerial ownership had positive effect on firm value. The company is expected to pay attention to the leverage ratio, profitability, and managerial ownership so that the company's value increases. Keywords: Leverage, Profitability, Managerial Ownership, Company Value 737 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … PENDAHULUAN Tujuan tutama tperusahaan tyaitu tmemperoleh tlaba, memaksimalkan tlaba atau tkekayaan, tdan memaksimalkan tnilai tperusahaan. tPeningkatan tnilai perusahaan tyang ttinggi tmerupakan ttujuan tjangka tpanjang tyang tseharusnya dicapai tperusahaan. tNilai tperusahaan tmerupakan tpersepsi tinvestor tterhadap perusahaan tyang tsering tdikaitkan tdengan tharga tsaham. ttNilai tperusahaan yang ttinggi tmenjadi tkeingininan tpemegang tsaham tdan tpemilik tperusahaan karena tmengindikasikan ttingginya tkemakmuran tpemegang tsaham t(Rahmawati et al., 2015) Nilai tperusahaan tpada tdasarnya tdapat tdiukur tmelalui tbeberapa taspek, salah tsatunya tadalah tharga tpasar tsaham tperusahaan. tHarga tpasar tmerupakan harga tsaham tperusahaan tyang tterbentuk tantara tpembeli tdan tpenjual tdisaat terjadi ttransaksi tatau tbiasa tdisebut tnilai tpasar tperusahaan, tkarena tharga tpasar saham tdianggap tcerminan tdari tnilai taset tperusahaan tsesungguhnya. Memaksimalkan tnilai tpasar tperusahaan tsama tdengan tmemaksimalkan tharga pasar tsaham. tHarga tpasar tsaham tmerupakan tsalah tsatu tindikator tyang digunakan tuntuk tmengukur tnilai tperusahaan tyang tdiamati tmelalui tpergerakan harga tsaham tpada tperusahaan tyang tsudah tgo tpublic t(Puspitaningtyas, 2017). Harga tsaham tyang tmeningkat, takan tberdampak tpada tpeningkatan tnilai pemegang tsaham tyang tdibuktikan tmelalui ttingginya tgain tbagi tpemegang saham. tPeningkatan tnilai tperusshaan tyang ttercermin tdari ttingginya tharga saham takan tmembuat tpasar tpercaya tterhadap tkinerja tdan tprospek perusahaan dalam tmengelola tdana tinvestasinya, tsehingga treturn tyang diterima toleh pemegang tsaham takan toptimal. tMemaksimalkan tnilai perusahaan tdan meningkatkan tlaba tmerupakan ttujuan tperusahaan tyang tsaling berkaitan tuntuk meningkatkan tkesejahteraan tpara tpemegang tsaham, tsehingga tujuan ttersebut akan tmenjadi tkriteria tyang tpenting tuntuk tmenjaga kelangsungan thidup perusahaan t(Rudangga & Sudiarta, 2016) Nilai tperusahaan tyang tdicerminkan tmelalui tharga tsaham tbiasanya ditentukan toleh tdua tfaktor tutama, tyaitu tfaktor tfundamental tdan tteknikal. Biasanya tfaktor tfundamental tyang tsering tdigunakan tsebagai tdasar pengambilan tkeputusan toleh tpara tinvestor tuntuk tmenanamkan tsahamnya pada perusahaan t(Endhiarto, 2018). Faktor tfundamental tini tdigunakan tuntuk menganalisis tnilai tsuatu tperusahaan tdengan tmengolah tdata tyang tbersumber dari tlaporan tkeuangan tperusahaan t(Štangová, 2016). Faktor tfundamental tsangat tkompleks tdan tluas tcakupannya, tmeliputi faktor tfundamental tmakro tyang tberada tdi tluar tkendali tperusahaan, tterdiri dari inflasi, ttingkat tsuku tbunga, tkurs, tpertumbuhan tekonomi tdan tfaktor fundamental tmikro tyang tberada tdi tdalam tkendali tperusahaan tmeliputi trasio keuangan. tSedangkan tfaktor tteknikal tlebih tbersifat tteknis tdengan tmelihat data historis tharga tsaham tterdiri tdari tvolume tperdagangan tsaham, tnilai transaksi perdagangan tsaham, tdan tkecenderungan tnaik tturunnya tharga tsaham yang biasanya tjuga tdilakukan toleh tinvestor tdalam tmenganalisis tsaham (Rakhimsyah & Gunawan, 2014) 738 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 Industri treal testate tdan tproperty tmenjadi tsalah tsatu bahan pertimbangan investor tuntuk tberinvestasi. PENDAHULUAN tIndustri tini tdiprediksi tmengalami pertumbuhan sebagai takibat tdari tpembangunan tinfrastruktur tyang tdilakukan pemerintah selama tlima ttahun tbelakangan tini. tNamun, tnyatanya pembangunan infrastruktur tbelum tmampu tuntuk tmembuat tsektor tini ttumbuh terbukti tpada beberapa ttahun tbelakangan tini tsektor treal testate tdan tproperty masih tlesu karena tkondisi tperekonomian tIndonesia tdiketahui tmengalami perlambatan sejalan tdengan tdaya tbeli tmasyarakat tyang tmenurun tbeberapa tahun tterakhir. Daya tbeli tmasyarakat tyang tlemah ttidak tmampu tmembeli tingginya tharga properti tyang tmenyebabkan tinvestasi tpada tbeberapa ttahun terakhir tmenurun, yang tmenyebabkan tperusahaan tpengembang tsulit tmenjual aset tpropertinya. Hal tini tsejalan tdengan tharga tindeks tsaham tsektoral tReal tEsate tdan tProperty yang tmengalami tfluktuasi ttahun t2014 thingga t2018. tGambar t1. tmenyajikan indeks tharga tsaham tperusahaan treal tesate tdan tproperty ttahun t2014 thingga 2018 tsebagai tberikut: Gambar 1. Grafik Indeks Harga Saham Perusahaan Real Esate dan Property Tahun 2014 – 2018 Sumber: Data Diolah, 2019 Gambar 1. Grafik Indeks Harga Saham Perusahaan Real Esate dan Property Tahun 2014 – 2018 Sumber: Data Diolah, 2019 Gambar 1. Grafik Indeks Harga Saham Perusahaan Real Esate dan Property Tahun 2014 – 2018 Gambar t1. tmenunjukan tbahwa tindeks tharga tsaham tperusahaan tReal Esate tdan tProperty, tpada ttahun t2014 thingga t2018 tmengalami tfluktuasi. Penurunan tpertumbuhan tindeks tharga tsaham tperusahaan tReal tEsate tdan Property tpada ttahun t2015 tsebesar t490,93 tdisebabkan toleh tkenaikan tsuku bunga, tpemberlakuan trasio tkredit tterhadap tnilai taset tpinjaman, tdepresiasi rupiah tdan ttingkat tinflasi. tPuncaknya tpada ttahun t2018 tharga tsaham mengalami tpenurunan tsebesar t459,50 tyang tdisebabkan toleh tpeningkatan harga tproperti tyang tmasih ttinggi, tketidakpastian tpolitik tmenjelang tpemilu mendatang, tdan tadanya tpengawasan tdari totoritas tpajak tsehingga tmasyarakat lebih tmemilih tmenyimpan tmodalnya tpada tbank tdaripada tberinvestasi tdi sektor treal testate tdan tproperty t Selain titu, tfenomena tlain tyang tmenunjukkan tmenurunnya tnilai perusahaan tdapat tdilihat tdengan tmenurunnya tnilai trasio tPBV t(Price tBook 739 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Value) tpada tsektor treal testate tdan tproperty ttahun t2014 thingga t2018. tNilai perusahaan ttidak thanya tdapat tdigambarkan tpada tharga tsaham tsuatu perusahaan tsaja, ttetapi tdapat tdilakukan tdengan tberbagai tcara, tdan tsalah satu alat tukur tyang tdapat tdigunakan tyaitu tPrice tto tBook tValue t(PBV). PBV merupakan trasio tkeuangan tyang tmembandingkan tantara tharga saham dengan nilai tbuku tper tlembar tsaham tDibawah tini tdisajikan tgrafik trata t- trata tnilai PBV tperusahaan treal testate tdan tproperty tyang tmengalami tpenurunan ttahun 2014 thingga t2018 tsebagai tberikut Gambar 2 . PENDAHULUAN Rata-rata Nilai PBV Perusahaan Real Esate dan Property Tahun 2014 – 2018 Sumber: Data Diolah, 2019 Gambar 2 . Rata-rata Nilai PBV Perusahaan Real Esate dan Property Tahun 2014 – 2018 Sumber: Data Diolah, 2019 Berdasarkan tGambar t2. tmenyajikan tindeks tpertumbuhan trata-rata nilai perusahaan tyang tdiukur tdengan trasio tPBV tsetiap ttahunnya. tGrafik ttersebut menunjukan tbahwa tpada tperiode t2014 thingga t2018 tperusahaan tReal tEsate dan tProperty tmengalami tpenurunan tnilai tyang tsignifikan tsetiap ttahunnya. Pada ttahun t2014 tperusahaan tmemiliki tpertumbuhan tnilai tPBV tsebesar t2,36 kemudian tpada ttahun t2015 tmengalami tpenurunan tsebesar t2,04 ttpenurunan ini berlanjut thingga ttahun t2018 tsebesar t1,65. tSemakin ttinggi trasio tPBV menunjukkan tbahwa tpasar tsemakin tyakin takan tprospek tperusahaan ttersebut. Dengan tmenurunnya tnilai tPBV tmenunjukkan tbahwa tpasar tbelum tyakin terhadap tprospek tperusahaan tyang ttentunya takan tberdampak tpada menurunnya tnilai tperusahaan. y p Fenomena ttersebut tmenunjukan tbahwa tsektor treal tesate tdan tproperty mengalami tpenurunan trata-rata tPBV tmenggambarkan tmenurunnya tnilai perusahaan. tDengan tmenurunnya tnilai tperusahaan tdapat tdilihat tkinerja perusahaan tberdasarkan tlaporan tkeuangan tperusahaan tsehingga tdapat ditentukan tfaktor tyang tdapat tmempengaruhi tnilai tperusahaan. tFaktor-faktor yang mempengaruhi tnilai tperusahaan tdapat tdibedakan tmenjadi tfaktor internal dan teksternal. 740 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 Faktor tinternal tyaitu tfaktor tyang tmempengaruhi tnilai tperusahaan tdari dalam tperusahaan titu tsendiri tdengan tmenganalisis tkinerja tkeuangan tyang meliputi: tkepemilikan tmanajerial, tkepemilikan tinstitusional, tkebijakan tividen, leverage, tukuran tperusahaan, tprofitabilitas tserta tgood tcorporate tgovernance. Sedangkan tfaktor teksternal tyaitu tfaktor tyang tmempengaruhi tnilai perusahaan dari tluar tperusahaan tseperti ttingkat tsuku tbunga, timflasi tdan tnilai tkurs. Dalam tpenelitian tini tdipilih tbeberapa tfaktor tinternal tyang digunakan tsebagai variabel tkarena tsifatnya tberada tdidalam tperusahaan titu tsendiri tsehingga lebih efektif tdan tmudah tdikendalikan toleh tperusahaan t(controllable). Diantara faktor tinternal tyang tdapat tmempengaruhi tnilai tperusahaan ttersebut yang dipilih tsebagai tvariabel tdalam tpenelitian tini tyaitu tleverage, profitabilitas tdan kepemilikan tmanajerial. Perusahaan tmemiliki tsumber tpendanaan tyang tberasal tdari tdalam (intern) maupun tluar t(ekstern) tperusahaan. tPemenuhan tkebutuhan tdana tyang berasal dari tdalam tperusahaan tbersumber tdari tlaba tditahan tdan tpenyusutan. Jika pendanaan tperusahaan tyang tbersumber tdari tdalam tperusaahaan tmasih mengalami tkekurangan tmaka, tperlu tdipertimbangkan tpendanaan tyang berasal dari tluar tperusahaan tyaitu tdari tutang. tRasio tyang tmengukur tseberapa tbesar perusahaan tmenggunakan tpendanaan tyang tberasal tdari tutang tdisebut leverage. Pengelolaan tleverage tsangatlah tpenting, tsebab keputusan dalam penggunaan tutang tyang ttinggi tdapat tmeningkatkan tnilai tperusahaan dikarenakan tadanya tpengurangan tatas tpajak tpenghasilan. PENDAHULUAN tHal tini tterjadi karena tutang tyang tdimiliki toleh tperusahaan tmerupakan tbeban ttetap, tyaitu berupa tbeban tbunga. tSemakin ttinggi tutang tyang tdimiliki toleh tperusahaan, maka tbeban tbunga tyang tharus tdibayarkan tjuga trelatif tsemakin ttinggi. Perusahaan tyang tcenderung memiliki tutang tyang ttinggi takan tmendapatkan insentif tpajak tberupa tpotongan tatas tbunga tpinjaman, toleh tsebab titu perusahaan tyang tmemiliki tbeban tpajak tyang ttinggi tmelakukan tpenghematan pajak tdengan tcara tperusahaan ttersebut tmenambahkan tutang tperusahaan tyang dimilikinya Hasil tpenelitian tterdahulu tyang tdilakukan toleh tPratama & Wiksuana (2016), Mukherjee & Sen (2018) serta Farooq & Masood (2015) menyatakan bahwa tleverage tberpengaruh tpositif tterhadap tnilai tperusahaan. tPenelitian lain yang dilakukan Aggarwal & Zhao (2017) dan Ramadan (2015) menyatakan leverage tberpengaruh tnegatif tterhadap tnilai tperusahaan. tNamun, tterdapat hasil berbeda dari penelitian Sambora et al. (2014) yang menemukanbahwa leverage tidak tberpengaruh tterhadap tnilai tperusahaan. Suatu tperusahaan tharuslah tberada tdalam keadaan yang menguntungkan (profitable), tkarena ttanpa tadanya tkeuntungan takan tsulit tbagi tperusahaan untuk tmenarik tmodal tdari tluar tperusahaan tatau tinvestor (Zuhroh, 2019). Perusahaan tyang tmemiliki ttingkat tprofitabilitas tyang ttinggi takan tdiminati sahamnya toleh tinvestor, tsehingga tprofitabilitas tdapat tmempengaruhi tnilai perusahaan. Profitabilitas tmemiliki tpengaruh tyang tpositif tterhadap tnilai perusahaan. Profit tyang ttinggi takan tmemberikan tindikasi tprospek perusahaan yang baiktsehingga dapat tmemicu tinvestor tuntuk tikut tmeningkatkan 741 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … permintaan tsaham. tPermintaan tsaham tyang tmeningkat ttentunya takan menyebabkan tnilai tperusahaan tyang tmeningkat tpula (Husna & Satria, 2019) Profitabilitas tmerupakan tsalah tsatu tfaktor tyang tsecara tteoritis menentukan tnilai tsuatu tperusahaan. tPerusahaan tyang tmampu tmenghasilkan laba tyang ttinggi tdan tstabil takan tmenarik tpara tinvestor tuntuk tmenanamkan sahamnya tpada tperusahaan, karena tsecara tlangsung takan tmenguntungkan bagi investor. Kemampuantperusahaan tyang tbesar untuk tmenghasilkan tlaba juga menunjukkantmanajemen tperusahaan tyang tbaik, tsehingga tmenumbuhkan kepercayaan pada tinvestor. tKepercayaan tinvestor tini tpada takhirnya tdapat menjadi tinstrumen tyang tpaling tefektif tuntuk tmeningkatkan tharga tsaham perusahaan. Peningkatan tharga tsaham tsama tartinya tmeningkatkan tnilai perusahaan, sehingga tlebih tlanjut tdapat tmenjamin tkemakmuran tpemegang saham t(Lubis et al., 2017) Hasil ini sesuai dengan penelitian yang dilakukan oleh Tui et al. (2017), Rasyid et al. (2015), serta Lestari & Mursalim (2016) yang menyatakan bahwa profitabilitas tberpengaruh tpositif tterhadap tnilai tperusahaan. tHasil tyang berlawanan tdiperoleh tdari tpenelitian tHerawati (2014) yang menyatakan profitabilitas tberpengaruh tnegatif tterhadap tnilai tperusahaan. Faktor tlain tyang dapat tmempengaruhi tnilai tperusahaan tadalah Kepemilikan tManajerial. Kepemilikan tmanajerialtadalahtbesarnya tkepemilikan saham tyang tdimiliki toleh tpara tmanajer tpada tperusahaan. PENDAHULUAN Optimalisasi tnilai perusahan tdapat tdicapai tmelalui tpelaksanaan tfungsi tmanajemen tkeuangan, salah tsatu tkeputusan tkeuangan tyang tdiambil takan tmempengaruhi tkeputusan keuangan tlainnya tdan berdampak tpada tnilai tperusahaan t(Wijaya et al., 2017). Pada tdasarnyattujuantmanajemen tkeuangan tadalah tmemaksimalkan tnilai perusahaan, takan ttetapi tterdapat tkonflik tantara tpemegang tsaham tdengan para manajer. tHal tini tterjadi tketika tpemegang tsaham tmempercayakan pengelolaan perusahaannya tkepada tpara tmanajer. tPerbedaan tkepentingan antara tpara manajer tdan tpemegang tsaham tini tmenimbulkan tkonflik tyang biasa tdisebut agency tconflict. tKonflik tini tdisebabkan toleh tpara tmanajer cenderung mengutamakan tkepentingan tpribadi tyang ttidak tsesuai tdengan tujuan tyang ingin tdicapai toleh tpara tpemegang tsaham tyaitu tmemaksimalkan kesejahteraan pemilik. tPemegang tsaham tatau tpemilik tperusahaan takan berusaha tmengatasi konflik tini tdengan tmelakukan tpengawasan tterhadap tpara manajer, tnamun dalam tmengatasi tkonflik ttersebut takan tmenimbulkan tbiaya yang tdisebut dengan tagency tcost tatau tbiaya tkeagenan. Biaya tyang tditanggung tpemegang tsaham tuntuk tmengawasi tmanajer dikenal tdengan tagency tcost. tSalah tsatu tcara tuntuk tmemperkecil tbiaya agency cost tadalah tdengan memberikan kepemilikan tsaham tperusahaan kepada para manajer. tKepemilikan tsaham tyang tdiberikan tkepada tpara tmanajer disebut kepemilikan manajerial. Dengan tadanya tkepemilikan tmanajerial, memberikan kesempatan tbagi tpara tmanajer tuntuk tikut tterlibat tlangsung dalam kepemilikan saham,tsehingga tsecara tlangsung takan tmembuat kedudukan yang sejajar dengan para tpemegang tsaham. tHal tini tefektif bagi manajertuntuk meningkatkan tkinerja tperusahaan tdan tmeningkatkan tlaba tperusahaan tyang dapat tmeningkatkan tnilai tperusahaan t(Sujoko & Soebiataro, 2017) 742 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 Penelitian terdahulu yang dilakukan oleh Muryati & Suardikha (2014), Sholekah (2014), Basu et al. (2016) yang tmenyatakan tkepemilikan tmanajerial berpengaruh tpositif tterhadap tnilai tperusahaan. tHasil tyang tberlawanan tjuga didapat tdari tRahma (2014) menyatakan tkepemilikan tmanajerial tberpengaruh negatif tterhadap tnilai tperusahaan, tnamun tpenelitian tlain tdari tAmbarwati & Stephanus (2014), serta Georgeta (2015) menyatakan tkepemilikan tmanajerial tidak tberpengaruh tsignifikan tterhadap tnilai tperusahaan Berdasarkan tfenomena tdan tresearch tgap tyang tterdapat tdalam tlatar belakang ttersebut tmaka tyang tmenjadi trumusan tmasalah tdalam tpenelitian tini sebagai tberikut: t1) tApakah tterdapat tpengaruh tLeverage tterhadap tNilai Perusahaan? t2) tApakah tterdapat tpengaruh tProfitabilitas tterhadap tNilai Perusahaan? t3) tApakah tterdapat tpengaruh tKepemilikan tManajerial tterhadap Nilai tPerusahaan? Penelitian tini tdiharapkan tdapat tmemperkaya tbukti tempiris tsehingga menambah twawasan tatau tpengetahuan tmengenai tpengaruh tLeverage, Profitabilitas, tdan tKepemilikan tmanajerial tterhadap tNilai tPerusahaan tpada Perusahaan tReal tEstate tdan tProperty tdi tBursa tEfek tIndonesia. PENDAHULUAN tSecara tpraktis hasil tpenelitian tini tdiharapkan tdapat tdigunakan tsebagai tbahan tpertimbangan emiten tuntuk tmengevaluasi, tmemperbaiki, tdan tmeningkatkan tkinerja manajemen tdimasa tyang takan tdatang tmengenai tPengaruh Leverage, Profitabilitas, tdan tKepemilikan tManajerial tterhadap tNilai tPerusahaan tpada Perusahaan tReal tEstate tdan tProperty tdi tBursa tEfek tIndonesia. Teori tyang tdigunakan tadalah tTeori tkeagenan t(agency ttheory) tdan Signaling ttheory tadalah thubungan tatau tkontrak tantara tprincipal tdan tagent. Para tmanajer tsuatu tperusahaan tsebagai tagent tdan tpemegang tsaham tatau pemilik tperusahaan tsebagai tprincipal. tPemegang tsaham tyang tmerupakan principal tmendelegasikan tpengambilan tkeputusan tbisnis tkepada tpara tmanajer yang tmerupakan tperwakilan tatau tagen tdari tpemegang tsaham. t Permasalahan tyang tmuncul tsebagai takibattsistem tkepemilikan perusahaan tseperti tini tadalah tagen ttidak tselalu membuat keputusan-keputusan yang tbertujuan tuntuk tmemenuhitkepentingantterbaiktbagi tpemegang tsaham. Konflik tini tterjadi tkarena tpara tmanajer tsebagai tpengelola (agent) tmemiliki informasi tlebih tmengenai tperusahaan dibandingkan dengan pemilik (principal). Konflik tdapat tberkurang tketika tpihak tmanajemen tikut andil dengan tcara memiliki tproporsi tsaham tdalam tperusahaan ttersebut karena agent dan principal tmemiliki ttujuan tyang tsama, tsehingga tmanajemen tdapat sepenuhnya memperhatikan tdan tfokus tterhadap ttujuan tperusahaan tyaitu meningkatkan nilai tperusahaan. Signaling ttheory tmenjelaskan tbagaimana tseharusnya tsebuah tperusahaan memberikan tsinyal tkepada tpengguna tlaporan tkeuangan. tSinyal tini tberupa informasi tmengenai tapa tyang tsudah tdilakukan toleh tmenajemen tuntuk merealisasikan tkeinginan tpemilik. tInformasi ttersebut tselanjutnya tdikeluarkan oleh tperusahaan tsebagai tsinyal tbagi tpara tpelaku tpasar tatau tinvestor tuntuk menanamkan tmodalnya tpada tperusahaan. Leverage tdalam tSignalling ttheory tmenyatakan tbahwa tsemakin ttinggi tingkat tutang tperusahaan, tmaka tmemungkinkan tperusahaan tmampu tuntuk 743 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … membayar tutang tdari tkeuntungan tperusahaan tyang tdiperolehnya tkarena perusahaan tdianggap tmemiliki tkondisi tkeuangan tyang tbaik tyang tdapat menanggung tresiko takibat ttingginya tutang ttersebut. Tingginya tutang perusahaan tjuga takan tmeningkatkan tpertumbuhantinvestasi tkarena tmenambah sumber tpendanaan tyang tdimiliki tperusahaan. Besarnya tdana tyang tdimiliki perusahaan, sebagian besar akan digunakan dalam kegiatan reinvestasi guna pencapaian laba yang lebih tinggi dimasa mendatang. Peningkatan utang dapat memberikan sinyal positif oleh investor sebagai meningkatnya kesempatan perusahaan untuk berkembang. Hal ini akan meningkatkan harga saham yang berarti meningkatkan nilai perusahaan. Dengan tlaba tyang ttinggi takan tmembuat ttingginya ttingkat tpengembalian modal t(return) tterhadap tinvestor tsehingga tmeningkatkan tharga tsaham tyang artinya tmeningkatkan tnilai tperusahaan. Perusahaan tyang tdapat tmengelola leverage tdengan tbaik tbiasanya tdapat tmeningkatkan tkepercayaan tinvestor sehingga tdapat tmeningkatkan tnilai tperusahaan. PENDAHULUAN Hal tini tdidukung tdengan teori Modigliani tdan tMiller tmengemukakan bahwa tpenambahan tutang takan menaikkan tnilai tperusahaan.tKenaikan tutang thingga tsuatu tbatas toptimal tertentu tdipandang tsebagai tpeningkatan tkemampuan tperusahaan tdalam melunasi tkewajibannya tsehingga tdipandang tpositif toleh tpasar tdan tnilai perusahaan takan tmeningkat. tHasil tini tdidukung toleh tpenelitian tyang dilakukan toleh tAggarwal & Zhao (2017) dan Ramadan (2015) tmenyataka tbahwa leverage tberpengaruh tpositif tterhadap tnilai tperusahaan. H1: tLeverage tberpengaruh tpositif tterhadap tnilai tperusahaan Secara tteoritis tmeningkatnya tprofitabilitas tperusahaan takan meningkatkan tnilai tperusahaan. tHal ttersebut tdikarenakan tprofitabilitas merupakan tsinyal tbagi tinvestor tagar ttertarik tuntuk tberinvestasi tpada perusahaan. tTingginya tprofitabilitas tperusahaan takan tmenarik tbagi tinvestor untuk tmenanamkan tmodalnya tdengan tmelakukan tpermintaan tpembelian saham perusahaan tkarena tinvestor takan tmneilai tperusahaan tmemiliki tkinerja yang baik tsehingga tmampu tmenghasilkan tpengembalian tinvestasi t(return) yang tinggi tjuga tbagi tinvestor. Dengan tdemikian, tharga tsaham tpun takan meningkatt karena tingginya tpermintaantsaham yang tmengindikasikan meningkatnya tnilai tperusahaan. tHasil tpenelitian tini tdidukung toleh tpenelitian yang tdilakukan toleh tTui et al. (2017), Rasyid et al. (2015), serta Lestari & Mursalim (2016) yang tmenunjukkan tbahwa tprofitabilitas tberpengaruh tpositif terhadap tnilai tpeusahaan. p p H2: Profitabilitas tberpengaruh tpositif tterhadap tnilai tperusahaan Konflik tantara tpara tmanajer tdan tpemegang tsaham tatau tyang tsering disebut tdengan tmasalah tkeagenan tdapat tdiminimumkan tdengan tsuatu mekanisme tpengawasan tyang tdapat tmensejajarkan tkepentingan-kepentingan sehingga tdapat tmengurangi tbiaya tkeagenan tatau tagency tcost. tSecara teoritis, dengan tadanya tkepemilikan tmanajerial tini tdapat tmengurangi tkonflik keagenan sehingga tdapat tmeningkatkan tnilai tperusahaan tkarena tantara tpihak manajemen dengan tpemilik tatau tpemegang tsaham memiliki tujuan yang sama. Hal ini sejalan tdengan tadanya t tpenelitian tyang tdilakukan toleh tMuryati & 744 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 Suardikha (2014), Sholekah (2014), Basu et al. (2016) yang menunjukkan kepemilikan manajerial tberpengaruh tpositif tterhadap tnilai tperusahaan. H3: Kepemilikan tmanajerial tberpengaruh tpositif tterhadap tnilai tperusahaan Suardikha (2014), Sholekah (2014), Basu et al. (2016) yang menunjukkan kepemilikan manajerial tberpengaruh tpositif tterhadap tnilai tperusahaan. p j p g p p p H3: Kepemilikan tmanajerial tberpengaruh tpositif tterhadap tnilai tperusahaan Gambar 3. Kerangka Konseptual Gambar 3. Kerangka Konseptual METODE PENELITIAN Penelitian ini tergolong sebagai penelitian asosiatif. Dalam penelitian ini variabel terikat adalah Nilai Perusahaan, sedangkan variabel bebas adalah Leverage, Profitabilitas, dan Kepemilikan Manajerial. Lokasi pada penelitian ini terletak pada perusahaan real estate dan property yang terdaftar di Bursa Efek Indonesia tahun 2014-2018. Obyek pada penelitian ini adalah Nilai Perusahaan pada perusahaan real estate dan property pada tahun 2014-2018. Variabel terikat dalam penelitian ini adalah Nilai Perusahaan.Variabel bebas yang digunakan dalam penelitian ini yaitu: Leverage (X1), Profitabilitas (X2), Kepemilikan Manajerial (X3) Nilai perusahaan merupakan persepsi investor terhadap keberhasilan perusahaan secara keseluruhan yang dilihat melalui harga saham. Dalam penelitian ini nilai perusahaan diukur menggunakan rasio PBV (Price Book Value), yaitu rasio perbandingan anatara nilai kinerja pasar saham dengan nilai bukunya pada perusahaan real estate dan property periode 2014 - 2018. PBV dapat dirumuskan sebagai berikut : PBV = x 100%..........................................(1) PBV = x 100%..........................................(1) .(1) Leverage merupakan suatu rasio yang digunakan untuk mengetahui seberapa besar kemaampuan perusahaan dalam membayar seluruh kewajibannya. Dalam penelitian ini alat ukur yang digunakan untuk menghitung leverage yaitu debt to equity rasio (DER), yaitu presentase antara total utang dengan total ekuitas atau modal pada perusahaan real estate dan property tahun 2014 - 2018. Leverage d di k d b i b ik Leverage merupakan suatu rasio yang digunakan untuk mengetahui seberapa besar kemaampuan perusahaan dalam membayar seluruh kewajibannya. Dalam penelitian ini alat ukur yang digunakan untuk menghitung leverage yaitu debt to equity rasio (DER) yaitu presentase antara total utang dengan total ekuitas Leverage merupakan suatu rasio yang digunakan untuk mengetahui seberapa besar kemaampuan perusahaan dalam membayar seluruh kewajibannya. sebe apa besa e aa pua pe usa aa da a e baya se u u ewaj ba ya. Dalam penelitian ini alat ukur yang digunakan untuk menghitung leverage yaitu debt to equity rasio (DER), yaitu presentase antara total utang dengan total ekuitas atau modal pada perusahaan real estate dan property tahun 2014 - 2018. Leverage dapat diukur dengan rumus sebagai berikut: DER = / x 100%...........................................................(2) DER = / x 100%...........................................................(2) Profitabilitas merupakan rasio yang digunakan untuk menilai kemampuan perusahaan dalam mencari keuntungan atau laba dalam satu periode tertentu. Pada 745 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Kepemilikan manajerial merupakan persentase saham yang dimiliki oleh para manajer, direksi atau dewan komisaris. METODE PENELITIAN Dalam penelitian ini diproyeksikan dengan MOWN (Managerial Ownership) diukur dengan menggunakan skala rasio melalui presentase jumlah saham yang dimiliki pihak manajemen dengan jumlah saham perusahaan yang beredar pada perusahaan real estate dan property periode 2014-2018 Kepemilikan Manajerial dapat dirumuskan sebagai berikut: MOWN = / x 100%...................(4) Populasi dalam penelitian ini adalah seluruh perusahaan real estate dan property yang terdaftar di BEI tahun 2014-2018. Jumlah populasi sebanyak 48 perusahaan. Metode penentuan sampel dalam penelitian ini dilakukan dengan menggunakan purposive sampling yaitu teknik penentuan sampel dengan kriteria tertentu. Adapun kriteria sampel dalam penelitian ini adalah sebagai berikut: 1) Perusahaan real estate dan property yang terdaftar di Bursa Efek Indonesia selama 5 (lima) tahun yaitu tahun 2014-2018. 2) Perusahaan real estate dan property yang memiliki laba selama 5 (lima) tahun yaitu tahun 2014-2018. 3) Perusahaan real estate dan property yang membagikan kepemilikan saham manajemen selama 5 (lima) tahun yaitu tahun 2014-2018. Sampel yang diambil dalam penelitian ini adalah laporan keuangan tahunan yang diambil dari Perusahaan real estate dan property selama 4 (empat) tahun yaitu 2014 hingga 2018 yang terdaftar di Bursa Efek Indonesia (BEI). Terdapat sebanyak 15 perusahaan yang termasuk sampel penelitian yang sesuai dengan kriteria pada penentuan sampel tersebut. Teknik pengumpulan data dalam penelitian ini dilakukan dengan observasi non partisipan yang dilakukan dengan mengamati dan menganalisis laporan keuangan tahunan perusahaan real estate dan property tahun 2014 hingga 2018 yang diakses melalui situs resmi Bursa Efek Indonesia (BEI). Jenis data yang dipergunakan dalam penelitian ini adalah data kuantitatif, yaitu data yang dinyatakan dalam bentuk angka yang bersumber dari publikasi laporan keuangan tahunan perusahaan real estate dan property di Bursa Efek Indonesia periode 2014 - 2018. Sumber data yang digunakan dalam penelitian ini adalah data sekunder yang diperoleh melalui situs resmi BEI di http:/www.idx.co.id pada perusahaan real estate dan property selama periode 2014 hingga 2018. 746 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 HASIL DAN PEMBAHASAN Objek dalam penelitian ini adalah perusahaan real estate dan property yang terdaftar di Bursa Efek Indonesia pada tahun 2014 hingga 2018. Real estate merupakan properti yang terdiri dari tanah dan bangunan di atasnya, bersama dengan sumber daya alam, kepentingan yang dipegang di dalamnya, dan juga sebagai suatu aset nyata yang secara umum diketahui sebagai bangunan atau perumahan. Sedangkan properti merupakan kepentingan dan hak-hak yang menyangkut kepemilikan tanah, bangunan, dan perbaikan yang menyatu terhadapnya. Oleh karena itu, baik real estate maupun property merupakan dua hal yang saling berkaitan satu sama lain, sehingga dapat disimpulkan bahwa industri real estate dan property merupakan kepentingan dan hak-hak yang menyangkut kepemilikan tanah, bangunan, dan perbaikan yang menyatu terhadapnya. Bidang usaha dari sektor property dan real estate meliputi berbagai macam bentuk seperti, apartemen, mall, gedung perkantoran, hotel, maupun perumahan. Keuntungan yang diperoleh bermacam-macam, tergantung dengan tipe investasinya, bisa berupa arus kas yang stabil (recuring income), capital gain, imbal hasil suku bunga, dan dividen. g Industri property dan real estate merupakan salah satu industri yang menjanjikan untuk berkembang di Indonesia, karena potensi jumlah penduduk yang besar dengan rasio pemilikan rumah yang masih rendah. dariinvestor. Perkembangan sektor ini begitu pesat saat ini dan diperkirakan akan semakin berkembang di masa yang akan datang. Hal ini terbukti dengan semakin banyaknya sektor Real Estate dan Property yang memperluas landbank (aset berupa tanah) dan mengkspansi bisnisnya. Statistik deskriptif memberikan gambaran informasi mengenai data yang dilihat dari nilai minimum, nilai maksimum, nilai rata-rata (mean) dan standar deviasi dari variabel yang digunakan dalam penelitian ini, yaitu nilai perusahaan (PBV), leverage (DER), profitabilitas (ROE), dan kepemilikan manajerial (MOWN). Hasil deskriptif dapat dilihat pada Tabel 1. berikut: Tabel 1. Analisis Statistik Deskriptif N Minimum Maximum Mean Std. Deviation DER 75 0.07 1.97 0.7384 0.47947 ROE 75 0.004 32.29 10.9597 7.51047 MOWN 75 0.0001 32.830 2.22669 5.578961 PBV 75 0.15 8.07 1.6472 1.60897 Valid N (listwise) 75 Sumber: Data Diolah, 2019 Tabel 1. Nilai minimum variabel nilai perusahaan yang diproksikan dengan PBV dihasilkan oleh perusahaan Greenwood Sejahtera Tbk (GWSA) tahun 2015 sebesar 0,15 sedangkan nilai maksimum dihasilkan oleh perusahaan Metropolitan Kentjana Tbk (MKPI) tahun 2017 sebesar 8,07. Nilai rata-rata sebesar 1,6472 artinya rata-rata perusahaan real estate dan property memiliki nilai PBV sebesar 164,72 persen. N Kolmogorov-Smirnov Z Asymp. Sig. (2-tailed) Sumber: Data Diolah, 2019 HASIL DAN PEMBAHASAN Standar deviasi PBV sebesar 1,60897atau 160,90 persen artinya 747 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … terjadi penyimpangan nilai PBV terhadap nilai rata-rata sebesar 1,6090 atau 160,90 persen. Nilai minimum variabel Leverage yang diproksikan dengan DER dihasilkan oleh perusahaan Greenwood Sejahtera Tbk (GWSA) tahun 2016 sebesar 0,07 sedangkan nilai maksimum dihasilkan oleh perusahaan Agung Pomodoro Land Tbk (APLN) tahun 2014 sebesar 1,97. Nilai rata-rata sebesar 0,7384 artinya rata- rata perusahaan real estate dan property memiliki leverage sebesar 73,84%. Standar deviasi DER sebesar 0,47947 atau 47,95 persen artinya terjadi penyimpangan nilai DER terhadap nilai rata-rata sebesar 0,47947atau 47,947 persen Nilai minimum variabel nilai perusahaan yang diproksikan dengan ROE dihasilkan oleh perusahaan Pikko Land Development Tbk (RODA) tahun 2018 sebesar 0,004 sedangkan nilai maksimum dihasilkan oleh perusahaan Metropolitan Kentjana Tbk (MKPI) tahun 2016 sebesar 32,29. Nilai rata-rata sebesar 10,9597artinya rata-rata perusahaan real estate dan property memiliki profitabilitas sebesar 1095,97 persen. Standar deviasi ROE sebesar 7,51047atau 751,047 persen artinya terjadi penyimpangan nilai ROE terhadap nilai rata-rata sebesar 7,51047 atau 751,047 persen. Nilai minimum variabel kepemilikan manajerial yang diproksikan dengan MOWN dihasilkan oleh perusahaan Danayasa Arthatama Tbk (SCBD) sepanjang tahun 2014-2018 sebesar 0,0001 sedangkan nilai maksimum dihasilkan oleh perusahaan Pikko Land Development Tbk (RODA) tahun 2017 dan 2018 sebesar 32,830. Nilai rata-rata sebesar 2,22669artinya rata-rata perusahaan real estate dan property memiliki kepemilikan saham manajemen sebesar 222,669 persen. Standar deviasi MOWN sebesar 5,578961atau 557,8961 persen artinya terjadi penyimpangan nilai MOWN terhadap nilai rata-rata sebesar 5,578961atau 557,8961persen. p Sebelum pengujian hipotesis dilakukan, terlebih dahulu dilakukan pengujian terhadap gejala penyimpangan klasik. Uji asumsi klasik ini dilakukan untuk mengetahui kelayakan suatu model penelitian agar model regresi tidak bias. Model regresi yang baik adalah model regresi yang tidak mengandung masalah di dalam asumsi klasik. Uji normalitas dilakukan untuk menguji apakah dalam model regresi, variabel pengganggu atau residual memiliki distribusi normal. Uji ini dilakukan dengan menggunakan One Kolmogorov-Smirnov dengan melihat nilai Asymp. Sig. (2-tailed). Jika nilai Asymp. Sig. (2-tailed) lebih besar taraf signifikansi yang ditetapkan yaitu 5 persen (>0,05), maka data telah berdistribusi normal. Tabel 2. Hasil Uji Normalitas Unstandardized Residual N 75 Kolmogorov-Smirnov Z 0,802 Asymp. Sig. (2-tailed) 0,541 Sumber: Data Diolah, 2019 Tabel 2. Hasil Uji Normalitas Unstandardized Residual N 75 Kolmogorov-Smirnov Z 0,802 Asymp. Sig. (2-tailed) 0,541 Sumber: Data Diolah, 2019 N Kolmogorov-Smirnov Z Asymp. Sig. (2-tailed) Sumber: Data Diolah, 2019 748 E-Jurnal Manajemen, Vol. HASIL DAN PEMBAHASAN 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 Berdasarkan hasil analisis yang disajikan pada Tabel 2. diperoleh hasil nilai signifikan sebesar 0,541 lebih besar dari 0,05 (sig = 0,541> 0,05) yang artinya data dalam penelitian ini berdistribusi normal. Uji multikolinearitas bertujuan menguji apakah pada model regresi ditemukan adanya korelasi antar variabel bebas. Uji multikolinearitas dilakukan dengan melihat nilai variance inflation factor (VIF). Model regresi yang baik seharusnya tidak ada multikolinear. jika nilai VIF <10, maka dapat disimpulkan data bebas dari gejala multikoliearitas. Tabel 3. Hasil Uji Multikolinearitas Model Colinearity Statistic Tolerance VIF Leverage (X1) 0,974 1,027 Profitabilitas (X2) 0,945 1,058 Kepemilikan Manajerial (X3) 0,945 1,059 Sumber: Data Diolah, 2019 Tabel 3. Hasil Uji Multikolinearitas Berdasarkan hasil analisis yang disajikan Tabel 3. dapat dilihat bahwa koefisien tolerance semua variabel lebih besar dari 0,10 (tolerance = 0,974; 0,945; 0,945 > 0,10) dan nilai VIF yang lebih kecil dari 10 (VIF = 1,027; 1,058; 1,059 < 10). Hasil ini dapat disimpulkan bahwa tidak terdapat gejala multikolinear dari model regresi yang dibuat sehingga pada model regresi ditemukan korelasi antar variabel bebas. Uji heterokedastisitas untuk menguji apakah terjadi ketidaksamaan varians pada model regresi. Model regresi dianggap layak jika tidak terjadi heterokedastisitas. Untuk mengetahui gejala heteroskedastisitas salah satunya dengan melakukan Uji Glejser yaitu meregresi nilai absolut terhadap variabel independen. Apabila nilai signifikan di atas 0,05 (sig > 0,05) maka tidak terjadi heteroskedastisitas pada model regresi. Tabel 4. Hasil Uji Heteroskedastisitas Model Sig. Keterangan Leverage (X1) 0,838 Lolos Uji Profitabilitas (X2) 0,090 Lolos Uji Kepemilikan Manajerial (X3) 0,314 Lolos Uji Sumber: Data Diolah, 2019 Tabel 4. Berdasarkan hasil analisis yang disajikan pada Tabel 4. menunjukkan nilai signifikansi dari ketiga variabel lebih dari 0,05 dengan nilai signifikansi leverage (DER) sebesar 0,838, profitabilitas (ROE) sebesar 0,090 dan kepemilikan manajerial (MOWN) sebesar 0,314 artinya model regresi bebas dari gejala heteroskedastisitas. Uji autokorelasi bertujuan menguji apakah dalam model regresi linier ada korelasi antara kesalahan pengganggu pada periode t dengan kesalahan pengganggu pada periode (t -1) periode sebelumnya. Untuk menguji gejala 749 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … autokorelasi dapat menggunakan uji Durbin-Watson (DW test). HASIL DAN PEMBAHASAN Apabila kriteria yang diperoleh berupa DW test d > dU dan DW test < 4, maka model yang diteliti dapat dikatakan bebas dari autokorelasi autokorelasi dapat menggunakan uji Durbin-Watson (DW test). Apabila kriteria yang diperoleh berupa DW test d > dU dan DW test < 4, maka model yang diteliti dapat dikatakan bebas dari autokorelasi Tabel 5. Hasil Uji Autokorelasi Model R R Square Adjusted R Square Std. Error of the Estimate Durbin-Watson 1 0.702a 0.492 0.471 1.17062 2.198 Sumber: Data Diolah, 2019 Tabel 5. Dalam penelitian ini menggunakan jumlah data (n) sebanyak 75 dengan sampel variabel bebas (k’) sebanyak 3, sehingga diperoleh nilai dL sebesar 1,54 dan nilai dU sebesar 1,71, maka nilai 4-dU adalah 2,29 (4-1,71). Hasil analisis yang disajikan tabel 4.6, nilai DW sebesar 2,198 berada diantara nilai dU dan nilai dU < DW < 4-dU (1,71 < 2,198< 2,29), maka disimpulkan model regresi yang digunakan tidak ada autokorelasi. g Analisis regresi linier berganda digunakan untuk mengetahui besarnya pengarruh variabel bebas yaitu leverage, profitabilitas, dan kepemilikan manajerial terhadap variabel terikat yaitu nilai perusahaan pada perusahaan Real Estatae dan Property. Berdasarkan hasil perhitungan analisis regresi linear berganda pada Tabel 6. maka ditemukan hasil persamaan regresi berganda sebagai berikut: Y = -0,154+ 0,128X1 + 0,174X2 + 0,298X3 + e Y = -0,154+ 0,128X1 + 0,174X2 + 0,298X3 + e Tabel 6. Hasil Uji Analisis Regresi Linear Berganda Model Unstandardized Coefficients Standardized Coefficients T Sig. B Std. Error Beta 1 (Constant) -0.154 3.301 -0.047 0.963 DER 0.128 0.064 0.193 2.009 0.048 ROE 0.174 0.080 0.267 2.180 0.032 MOWN 0.298 0.120 0.297 2.473 0.016 Sumber: Data Diolah, 2019 Tabel 6. Koefisien Konstanta variabel nilai perusahaan yang diproksikan dengan Price Book Value (PBV) yaitu -0,154 dengan tanda negatif mengindikasikan konstanta berpengaruh negatif. Hal ini menunjukkan bahwa apabila leverage, profitabilitas, dan kepemilikan mnajaerial suatu perusahaan tidak menjadi perhatian penuh perusahaan atau dalam keadaan konstan (0), maka nilai perusahaan yang dihasilkan menurun. Nilai koefisien regresi (β1) variabel leverage yang diproksikan dengan Debt to Equiy Ratio (DER) sebesar 0,128. Hal ini berarti apabila DER perusahaan meningkat sebesar 1 persen, maka niai perusahaan akan meningkat sebesar 12,8 persen dan sebaliknya. Nilai koefisien regresi (β2) variabel profitabilitas yang diproksikan dengan Return On Equity (ROE) sebesar 0,174. Hal ini berarti apabila ROE perusahaan meningkat sebesar 1 persen, maka niai 750 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 E-Jurnal Manajemen, Vol. 9, No. HASIL DAN PEMBAHASAN 2, 2020 : 737-757 perusahaan akan meningkat sebesar 17,4 persen dan sebaliknya. Nilai koefisien regresi (β3) variabel kepemilikan manajerial yang diproksikan dengan Managerial Ownership (MOWN) sebesar 0,298. Hal ini berarti apabila MOWN perusahaan meningkat sebesar 1 persen, maka niai perusahaan akan meningkat sebesar 29,8 persen dan sebaliknya. p y Uji kelayakan model dilakukan dengan melakukan uji F dan koefisien determinasi. Uji F digunakan untuk mengetahui apakah variabel bebas yaitu leverage, profitabilitas, dan kepemilikan manajerial memiliki pengaruh secara simultan terhadap variable terikat yaitu nilai perusahaan. Tabel 7. Hasil Uji F Model Sum of Squares Df Mean Square F Sig. 1 Regression 94.276 3 31.425 22.932 .000b Residual 97.294 71 1.370 Total 191.570 74 Sumber: Data Diolah, 2019 Berdasarkan hasil uji F pada Tabel 7. diatas, model regresi memiliki nilai Fhitung sebesar 22,932 dengan nilai signifikansi 0,000 yang lebih kecil dari 0,05. Jumlah data (n) dalam penelitian ini adalah 75 dengan jumlah variabel sebanyak 4. Uji F dilakukan dengan melihat derajat bebas (vl = k-1); (v2 = n-k). Nilai Ftabelyang diperoleh dari derajat bebas 3;71 (v1 = 4-1; v2 = 75-4) adalah 2,733. Dengan diperolehnya nilai Fhitung lebih besar daripada nilai Ftabel (22,932> 2,733) dan nilai signifikansi lebih kecil daripada taraf nyata (0,000 < 0,05), maka dapat disimpulkan variabel bebas yaitu leverage (X1), profitabilitas (X2), dan kepemilikan manajerial (X3) berpengaruh signifikan secara simultan terhadap variabel terikat yaitu nilai perusahaan (Y). Tabel 8. Hasil Uji Koefisien Determinasi Sumber: Data Diolah, 2019 Model R R Square Adjusted R Square Std. Error of the Estimate 1 0.702a 0.492 0.471 1.17062 Tabel 8. Uji koefisien determinasi digunakan untuk mengukur seberapa jauh variabel terikat mampu dijelaskan oleh variable bebas dalam model regresi. Berdasarkan hasil analisis spss yang disajikan pada Tabel 8. diperoleh besarnya nilai Adjusted R square adalah sebesar 0,471 yang artinya sebesar 47,1 persen variasi nilai perusahaan dipengaruhi oleh leverage, profitabilitas, dan kepemilikan manajerial, sedangkan sisanya sebesar 52,9 persen dipengaruhi oleh faktor lain yang tidak dimasukkan ke dalam model penelitian. p Pada tahap ini, dilakukan pengujian dimana hasil analisis akan dinyatakan berpengaruh signifikan atau tidak sebagai pembuktian atas hipotesis yang dinyatakan dalam penelitian ini. HASIL DAN PEMBAHASAN Uji t digunakan untuk mengetahui bagaimana 751 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … pengaruh masing-masing variabel bebas yaitu leverage (DER), profitabilitas (ROE) dan kepemilikan manajerial (MOWN) secara parsial terhadap variabel terikat yaitu nilai perusahaan (PBV) Berdasarkan hasil uji regresi linear berganda, diketahui bahwa variabel leverage memiliki nilai koefisien beta positif sebesar 0,128 dengan nilai signifikansi sebesar 0,048. Nilai koefisien beta lebih dari 0 dan nilai signifikansi kurang dari 0,05 mengindikasikan bahwa leverage berpengaruh positif terhadap nilai perusahaan. Dengan demikian, H1 diterima. Berdasarkan hasil uji regresi linear berganda, diketahui bahwa variabel profitabilitas memiliki nilai koefisien beta positif sebesar 0,174 dengan nilai signifikansi sebesar 0,032. Nilai koefisien beta lebih dari 0 dan nilai signifikansi kurang 0,05 mengindikasikan bahwa profitabilitas berpengaruh positif terhadap nilai perusahaan. Dengan demikian, H2 diterima. Tabel 9. Hasil Uji T Tabel 9. Hasil Uji T Model Unstandardized Coefficients Standardized Coefficients T Sig. B Std. Error Beta 1 (Constant) -0.154 3.301 -0.047 0.963 DER 0.128 0.064 0.193 2.009 0.048 ROE 0.174 0.080 0.267 2.180 0.032 MOWN 0.298 0.120 0.297 2.473 0.016 Sumber: Data Diolah, 2019 Berdasarkan hasil uji regresi linear berganda, diketahui bahwa variabel kepemilikan manajerial memiliki nilai koefisien beta positif sebesar 0,298dengan nilai signifikansi sebesar 0,016. Nilai koefisien beta lebih dari 0 dan nilai signifikansi kurang dari 0,05 mengindikasikan bahwa kepemilikan manajerial berpengaruh positif terhadap nilai perusahaan. Dengan demikian, H3 diterima. Dari hasil uji hipotesis secara simultan (Uji F) pada Tabel 9 maka dapat disimpulkan bahwa variabel independen (leverage, profitabilitas dan kepemilikan manajerial) dalam penelitian ini secara bersama-sama berpengaruh terhadap variabel dependen yaitu nilai perusahaan. Secara parsial (Uji T) juga diperoleh hasil yang menunjukkan bahwa variabel yang memiliki pengaruh terhadap nilai perusahaan adalah ketiga variabel yaitu leverage, profitabilitas dan kepemilikan manajerial. Berdasarkan hasil penelitian menunjukkan bahwa pada penelitian ini mendukung hipotesis pertama yang menyatakan leverage berpengaruh positif terhadap nilai perusahaan pada perusahaan real estate dan property periode 2014- 2018. Arah positif tersebut memiliki arti semakin tinggi leverage maka semakin tinggi pula nilai perusahaan yang diperoleh. Perusahaan yang dapat mengelola leverage dengan baik biasanya dapat meningkatkan kepercayaan investor sehingga dapat meningkatkan nilai perusahaan. Kenaikan hutang hingga suatu batas optimal tertentu dipandang sebagai peningkatan kemampuan perusahaan dalam melunasi kewajibannya sehingga dipandang positif oleh pasar meningkatnya kesempatan perusahaan untuk 752 E-Jurnal Manajemen, Vol. 9, No. 2, 2020 : 737-757 berkembang dan menghasilkan tingkat pengembalian investasi (return) yang lebih tinggi. HASIL DAN PEMBAHASAN Bagi perusahaan hasil ini dapat digunakan untuk meningkatkan nilai perusahaan dengan menganalisis laporan keuangan salah satunya dengan menggunakan rasio leverage, profitabilitas, dan kepemilikan manajerial sehingga 753 Luh Putu Putri Adesia Widayanti, Leverage, Profitabilitas, Dan … perusahaan dapat mempertimbangkan setiap pengambilan keputusan keuangan yang diambil karena akan mempengaruhi kelangsungan hidup perusahaan di masa mendatang. Hasil ini juga bermanfaat bagi investor sebagai pertimbangan dan wawasan mengenai nilai perusahaan serta faktor-faktor yang mempengaruhi baik internal maupun eksternal. HASIL DAN PEMBAHASAN Hasil penelitian ini sejalan dengan penelitian sebelumnya yang dilakukan oleh Pratama & Wiksuana (2016), Mukherjee & Sen (2018) serta Farooq & Masood (2015) yang menunjukkan leverage berpengaruh positif terhadap nilai perusahaan. p Berdasarkan hasil penelitian menunjukkan penelitian ini mendukung hipotesis kedua yang menyatakan profitabilitas berpengaruh positif terhadap nilai perusahaan pada perusahaan real estate dan property periode 2014-2018. Arah positif tersebut menunjukkan semakin tinggi profitabilitas maka semakin tinggi pula nilai perusahaan. Hal ini terjadi karena tiingginya profitabilitas akan menjadi sinyal positif yang menarik bagi investor untuk menanamkan modalnya dengan melakukan permintaan pembelian saham pada perusahaan. Dengan tingginya profitablitas investor akan mneilai perusahaan memiliki kinerja yang baik sehingga mampu menghasilkan tingkat pengembalian investasi (return) yang tinggi bagi investor. Hasil penelitian ini sejalan dengan penelitian yang dilakukan sebelumnya oleh Tui et al. (2017), Rasyid et al. (2015), serta Lestari & Mursalim (2016) yang menunjukkan profitabilitas berpengaruh positif signifikan terhadap nilai peusahaan. Berdasarkan hasil penelitian menunjukkan penelitian ini mendukung hipotesis ketiga yang menyatakan bahwa kepemilikan manajerial berpengaruh positif terhadap nilai perusahaan pada perusahaan real estate dan property periode 2014-2018. Arah positif tersebut menunjukkan semakin tinggi kepemilikan manajerial maka semakin tinggi pula nilai perusahaan. Secara teoritis, dengan adanya kepemilikan manajerial ini dapat mengurangi konflik keagenan sehingga dapat meningkatkan nilai perusahaan karena antara pihak manajemen dengan pemilik atau pemegang saham memiliki tujuan yang sama. Dengan adanya kepemilikan saham ini biasanya efektif bagi manajer untuk meningkatkan kinerja sehingga dapat meningkatkan laba dan tingkat pengembalian investor. Hasil ini sejalan dengan penelitian sebelumnya yang dilakukan oleh Muryati & Suardikha (2014), Sholekah (2014), Basu et al. (2016) yang menunjukkan hubungan yang positif antara kepemilikan manajerial terhadap nilai perusahaan. Hasil penelitian ini memberikan tambahan informasi mengenai bagaimana pengaruh Leverage, Profitabilitas, dan Kepemilikan Manajerial terhadap Nilai Perusahaan. Dari penelitian ini terdapat bukti empiris yang diperoleh menunjukkan bahwa Leverage, Profitabilitas, dan Kepemilikan Manajerial berpengaruh positif signfikan terhadap nilai perusahaan pada perusahaanreal estate dan property. Hal ini menunjukkan bahwa tinggi rendahnya leverage, profitabilitas dan kepemilikan manajerial mempengaruhi tingkat pembelian saham pada investor sehingga menaikkan harga saham yang menjadi reflikasi dari tinggi rendahnya nilai perusahaan. Hasil ini sesuai dengan teori sinyal menyatakan bahwa informasi internal perusahaan sangat penting bagi nvestor dalam mengambil keputusan investasi pada perusahaan. Hasil penelitian ini dapat menjadi pertimbangan bagi perusahaan maupun investor. SIMPULAN Berdasarkan hasil dan pembahasan yang telah diuraikan sebelumnya, maka dapat disimpulkan Leverage berpengaruh positif terhadap nilai perusahaan. Hal ini menunjukkan bahwa semakin tinggi nilai leverage maka nilai perusahaan semakin meningkat. Profitabilitas berpengaruh positif terhadap nilai perusahaan. Hal ini menunjukkan bahwa semakin tinggi nilai profitabilitas maka nilai perusahaan semakin meningkat. Kepemilikan manajerial berpengaruh positif terhadap nilai perusahaan. Hal ini menunjukkan bahwa semakin tinggi nilai kepemilikan manajerial maka nilai perusahaan semakin meningkat. Bagi perusahaan real estate dan property diharapkan penelitian ini dapat digunkan sebagai bahan pertimbangan dalam mengambil keputusan serta membuat kebijakan dalam rangka meningkatkan nilai perusahaan dengan memerhatikan kinerja keuangan diantaranya leverage, profitabilitas dan kepemilikan manajerial. Hal yang dapat dilakukan perusahaan yaitu, meningkatkan profit dan kepemilikan saham kepada para manajer, namun perlu berhati hati dalam penggunaan leverage yang terlalu tinggi karena dapat meningkatkan risiko kebangkrutan pada perusahaan. Oleh karena itu, perusahaan diharapkan dapat dengan bijak mengelola leverage sehingga mampu meningkatkan nilai perusahaan. Bagi investor penelitian ini diharapkan menjadi pertimbangan sebelum melakukan investasi untuk memperhatikan terlebih dahulu faktor-faktor yang dapat mempengaruhi nilai perusahaan dengan menganalisis kinerja keuangan, diantaranya leverage, profiitabilitas, dan kepemilikan manajerial sehingga investor dapat memilih perusahaan yang secara optimal memberikan capital gain dan terhindar dari risiko tertentu. Bagi peneliti selanjutnya disarankan dapat melakukan penelitian dengan menambah atau mengganti faktor lain yang dapat mempengaruhi nilai perusahaan seperti: kepemilikan institusional, ukuran perusahaan, kebijakan dividen dan faktor lainnya serta menambah periode waktu penelitian dan menambah jumlah sampel penelitian dengan menambah sektor lain sehingga dapat diperoleh hasil penelitian yang lebih beragam. REFERENSI Aggarwal, R., & Zhao, X. (2017). The Leverage-Value Relationship Puzzle: An Industry Effects Resolution. 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https://openalex.org/W2344288442	https://europepmc.org/articles/pmc4880084?pdf=render	English	null	Loss of 89K Pathogenicity Island in Epidemic<i>Streptococcus suis</i>, China	Emerging infectious diseases	2,016	cc-by	1,737	Xiaolu Shi,1 Huiyan Ye,1 Jun Wang, Zhencui Li, Jingzhong Wang, Baoshan Chen, Ronghui Wen, Qinghua Hu, Youjun Feng Author affiliations: Zhejiang University School of Medicine, Hangzhou, China (X. Shi, H. Ye, J. Wang, Z. Li, Y. Feng); Shenzhen Centre for Disease Control and Prevention, Shenzhen City, China (X. Shi, J. Wang, Q. Hu); Guangxi University, Nanning City, China (H. Ye, J. Wang, B. Chen, R. Wen) DOI: http://dx.doi.org/10.32032/eid2206.152010 pp g ) Because 89K PAI is in dynamic evolution, determin ing whether it remains present in clinical strains is of interest. As previously designed (online Technical Ap pendix Table 1), a specific pair of boundary primers (1/6) was applied for PCR-based detection of the 89K PAI (Fig ure, panel A, http://wwwnc.cdc.gov/EID/article/22/6/15- 2010-F1.htm). In principle, the PCR-positive result sug gests the absence of 89K PAI, whereas the PCR-negative result indicates the presence of 89K PAI (6,8). Unlike the epidemic strain 05ZYH33 that has the 89K PAI, 9 of the 10 clinical strains examined (Stre08001, Stre08002, Stre09001, Stre09002, Stre11001, Stre11002, Stre13002, Stre13003, and Stre13004) were unexpectedly found to be PCR positive for the unique 1/6 DNA fragment with ex pected size of ≈1.5 kb (Figure, panel B). This finding indi cates that the 89K PAI is lost in these 9 clinical strains. We saw similar scenarios in the subsequent PCRs for other in ner genes/DNA fragments (943 and 944 [10]; 1/2, 3/4, and 5/6 [8]) inside of 89K PAI (Figure, panel C). Further DNA sequencing of the 1/6 PCR product showed that it match es well with the 2 boundary regions neighboring the 89K PAI, validating the loss of 89K PAI in these 9 clinical iso lates (Figure, panel D). In contrast, the strain Stre15001 behaved similarly to that of the 05ZYH33 containing the 89K PAI, in that both are PCR positive for the 4 amplicons To the Editor: Streptococcus suis serotype 2 (SS2) is a previously neglected, newly emerging human patho gen that causes occupational and opportunistic infections (1,2). Outbreaks of fatal human SS2 infections in China, featuring streptococcal toxic shock syndrome, in 2005 seri ously challenged global public health (3–5). The epidemic strain is unusual in that it contains a unique 89-kb (89K) pathogenicity island (PAI) (3,6,7). We observed the loss of genes from the 89K PAI in sporadic cases in southern China in 2007, implying the dynamic evolution of this PAI (8). Therefore, 89K PAI might be able to be used to monitor prevalent strains of S. suis in China (8). LETTERS LETTERS Stre13003, and Stre13004), and 1 in 2015 (Stre15001). Microbial and molecular assays proved that these clinical isolates were S. suis (online Technical Appendix Figures 1, 2, http://wwwnc.cdc.gov/EID/article/22/6/15-2010- Techapp1.pdf). Multiplex PCR–based molecular determi nation (16S rDNA, mrp, epf, and cps-2j) suggested that all strains except Stre13002 were SS2 (online Technical Ap pendix Figure 2) (3). To determine whether these clinical isolates derived from the same Chinese epidemic clone 05ZYH33, we sequenced an array of virulence factor–en coding genes, as well as the 16S rDNA genes. Phylogenetic trees indicated that all 10 clinical strains are classified into the same subclade as that of the strain 05ZYH33 (online Technical Appendix Figure 2).i 9. Baerlecken N, Jacobs R, Stoll M, Schmidt RE, Witte T. Recurrent, multifocal Mycobacterium avium-intercellulare infection in a patient with interferon-gamma autoantibody. Clin Infect Dis. 2009;49:e76–8. http://dx.doi.org/10.1086/605581 10. Havlir DV, Dubé MP, Sattler FR, Forthal DN, Kemper CA, Dunne MW, et al. Prophylaxis against disseminated Mycobacterium avium complex with weekly azithromycin, daily rifabutin, or both. California Collaborative Treatment Group. N Engl J Med. 1996;335:392–8. http://dx.doi.org/10.1056/ NEJM199608083350604 Address for correspondence: Florent Valour, Department of Infectious Diseases, Hospices Civils de Lyon, 103 Grande-Rue de la Croix-Rousse, 69004 Lyon, France; email: florent.valour@chu-lyon.fr Subsequent analyses by pulsed-field gel electrophore sis revealed that genotypes are diverse among these clini cal strains, which can be roughly divided into 6 groups (online Technical Appendix Figure 3). Given that the Sao surface antigen protein possesses 3 allelic variants (Sao-L [670 aa], Sao-M [580 aa], and Sao-S [489/490 aa]) (9), we thus assayed it with these clinical strains. Unexpect edly, we found 2 more new allelic variants, referred to as Sao-L1 (640 aa) and Sao-L2 (611 aa). Except for the strain BM407, which is a Chinese epidemic SS2 encoding Sao-L1, a version 30 residues shorter than Sao-L, 8 of the 10 clinical S. suis isolates consistently had the same new form of Sao protein, Sao-L2 (611 aa) (online Technical Appendix Figure 4). 1These authors contributed equally to this article. LETTERS of 1/2, 5/6, 943, and 944 but PCR negative for the 1/6 am plicon (Figure, panels B–D). The only minor difference between strains Stre15001 and 05ZYH33 lay in the 3/4 amplicon (Figure, panel C). Clearly the 3/4 DNA frag ment is present in the 89K PAI from strain 05ZYH33 but not in the counterpart of the strain Stre15001 (Figure, panel C); that is, strain Stre15001 carries a variant of 89K PAI lacking (at least part of, if not all) the 3/4 DNA frag ment. In terms of 89K PAI (and pulsed-field gel electro phoresis/Sao protein), we propose that a heterogeneous SS2 population is circulating in China. Also, we observe that the differentiation of bacterial virulence is related to the clinical strains using the infection model of Balb/c mice (online Technical Appendix Figure 5). 8. Feng Y, Shi X, Zhang H, Zhang S, Ma Y, Zheng B, et al. Recurrence of human Streptococcus suis infections in 2007: three cases of meningitis and implications that heterogeneous S. suis 2 circulates in China. Zoonoses Public Health. 2009;56:506–14. http://dx.doi.org/10.1111/j.1863-2378.2008.01225.x 9. Feng Y, Zheng F, Pan X, Sun W, Wang C, Dong Y, et al. Existence and characterization of allelic variants of Sao, a newly identified surface protein from Streptococcus suis. FEMS Microbiol Lett. 2007;275:80–8. http://dx.doi.org/10.1111/ j.1574-6968.2007.00859.x 10. Li M, Wang C, Feng Y, Pan X, Cheng G, Wang J, et al. SalK/SalR, a two-component signal transduction system, is essential for full virulence of highly invasive Streptococcus suis serotype 2. PLoS One. 2008;3:e2080. http://dx.doi.org/10.1371/journal.pone.0002080 Address for correspondence: Youjun Feng, Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China; email: fengyj@zju.edu.cn; or Qinghua Hu, Shenzhen Centre for Disease Control and Prevention, Shenzhen City, Guangdong 518055, China; email: huqinghua03@163.com In summary, the loss of 89K PAI might highlight the emergence of an epidemic SS2 population. This population appears to have genetic heterogeneity that is undergoing evolution in an adaption to some selection pressure from the environment, host restriction, or both. This work was supported by Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars (grant no. LR15H190001), the National Natural Science Foundation of China (grant no. 31570027), and the start-up package from Zhejiang University (Y.F.). Dr. Feng is a recipient of the “Young 1000 Talents” Award. Igor Mokrousov, Ekaterina Chernyaeva, Anna Vyazovaya, Viacheslav Sinkov, Viacheslav Zhuravlev, Olga Narvskaya Y.F. and Q.H. designed this project; X.S., H.Y., J.W., and L. performed experiments and analyzed the data; B.C Author affiliations: St. Petersburg Pasteur Institute, St. Petersburg, Russia (I. Mokrousov, A. Vyazovaya, O. Narvskaya); St. Petersburg State University, St. Petersburg (E. Chernyaeva); Research Institute of Phthisiopulmonology, St. Petersburg (E. Chernyaeva, V. Zhuravlev, O. Narvskaya); Scientific Center of Family Health and Reproductive Problems, Irkutsk, Russia (V. Sinkov) p p y ; R.W. contributed reagents and tools; Y.F. wrote the article. R.W. contributed reagents and tools; Y.F. wrote the article. Xiaolu Shi,1 Huiyan Ye,1 Jun Wang, Zhencui Li, Jingzhong Wang, Baoshan Chen, Ronghui Wen, Qinghua Hu, Youjun Feng We report 10 recurrent cases of human S. suis infec tions during 2008–2015 in southern China. Most of the hos pitalized patients were male workers in close contact with pigs, pork products, or both. These patients typically exhib ited clinical syndromes of meningitis, including headache, coma, vomiting, and fever. The bacterial strains acquired from humans were as follows: 2 isolates in 2008 (Stre08001 and Stre08002), 2 in 2009 (Stre09001 and Stre09002), 2 in 2011 (Stre11001 and Stre11002), 3 in 2013 (Stre13002, 1126 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 6, June 2016 References 1. Feng Y, Zhang H, Ma Y, Gao GF. Uncovering newly emerging variants of Streptococcus suis, an important zoonotic agent. Trends Microbiol. 2010;18:124–31. http://dx.doi.org/10.1016/ j.tim.2009.12.003 2. Gottschalk M, Segura M, Xu J. Streptococcus suis infections in humans: the Chinese experience and the situation in North America. Anim Health Res Rev. 2007;8:29–45. http://dx.doi.org/ 10.1017/S1466252307001247 DOI: http://dx.doi.org/10.3201/eid2206.152051 DOI: http://dx.doi.org/10.3201/eid2206.152051 To the Editor: Next-generation sequencing (NGS) technology is becoming more affordable and is increasing ly being widely used for high-resolution molecular epide miology of tuberculosis. Using an example of the emerging multidrug-resistant strain of Mycobacterium tuberculosis, we showed the value of informed understanding when in silico prediction from NGS data achieved with available bioinformatics tools is placed within the context of the ex isting genotyping framework. 3. Tang J, Wang C, Feng Y, Yang W, Song H, Chen Z, et al. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med. 2006;3:e151. Erratum in: PLoS Med. 2006;3:e377. http://dx.doi.org/10.1371/journal.pmed.0030151 4. Ye C, Zhu X, Jing H, Du H, Segura M, Zheng H, et al. Streptococcus suis sequence type 7 outbreak, Sichuan, China. Emerg Infect Dis. 2006;12:1203–8. http://dx.doi.org/10.3201/ eid1708.060232 4. Ye C, Zhu X, Jing H, Du H, Segura M, Zheng H, et al. Streptococcus suis sequence type 7 outbreak, Sichuan, China. Emerg Infect Dis. 2006;12:1203–8. http://dx.doi.org/10.3201/ eid1708.060232 5. Yu H, Jing H, Chen Z, Zheng H, Zhu X, Wang H, et al. Human Streptococcus suis outbreak, Sichuan, China. Emerg Infect Dis. 2006;12:914–20. http://dx.doi.org/10.3201/eid1206.051194 Spoligotyping is a classical method of M. tuberculo sis genotyping, and the SITVIT_WEB database contains data on 7,105 spoligotype patterns of 58,180 isolates from 153 countries (http://www.pasteur-guadeloupe.fr:8081/ SITVIT_ONLINE). Spoligotyping targets a variation of the DR/CRISPR locus, whose evolution in M. tuberculosis occurs through deletion of single or multiple spacers. By 6. Chen C, Tang J, Dong W, Wang C, Feng Y, Wang J, et al. A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates. PLoS One. 2007;2:e315. http://dx.doi.org/10.1371/journal.pone.0000315 7. Li M, Shen X, Yan J, Han H, Zheng B, Liu D, et al. GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2. Mol Microbiol. 2011;79:1670–83. http://dx.doi.org/10.1111/j.1365-2958.2011.07553.x 7. Li M, Shen X, Yan J, Han H, Zheng B, Liu D, et al. GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2. Mol Microbiol. 2011;79:1670–83. http://dx.doi.org/10.1111/j.1365-2958.2011.07553.x 1127 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 6, June 2016
https://openalex.org/W2124112856	https://madoc.bib.uni-mannheim.de/41088/1/Klotz2014_Article_PromotingWorkforceExcellenceFo.pdf	English	null	Promoting workforce excellence: formation and relevance of vocational identity for vocational educational training	Empirical research in vocational education and training	2,014	cc-by	10,861	RESEARCH Open Access * Correspondence: Viola.Klotz@wiwi.upb.de 1Chair of Business and Human Resource Education, University of Paderborn, Warburger Straße 100, D-33098, Paderborn, Germany Full list of author information is available at the end of the article © 2014 Klotz et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Promoting workforce excellence: formation and relevance of vocational identity for vocational educational training Viola Katharina Klotz1, Stephen Billett2 and Esther Winther1 Viola Katharina Klotz1, Stephen Billett2 and Esther Winther1 * Correspondence: Viola.Klotz@wiwi.upb.de 1Chair of Business and Human Resource Education, University of Paderborn, Warburger Straße 100, D-33098, Paderborn, Germany Full list of author information is available at the end of the article Abstract Vocational learning comprises more than factual knowledge and procedures; the development of a vocational identity is a key aspect and outcome of vocational education provisions and assumed to play an integral role in how students learn and perform. Despite the salience of vocational identity however, the processes that contribute to its formation are far from fully understood. It is unclear whether and which elements of vocational education and training provision shape this process and if and to what degree forms of identity really support the actual vocational performance of a vocational learner. This study seeks to provide deeper understanding of the circumstances that enable different forms of identity to develop and how they direct learning and workplace effort. Using structural equation modeling with data from 504 vocational learners and correlation analysis with data from 187 industrial apprentices, this article proposes a model to account for key influences and the impacts of vocational identity formation for the commercial sector. The results indicate that vocational identity mediates and is closely aligned to the development of vocational engagement and competence. A free career choice and the provision of maximal functional integration into operating processes at the workplace are key factors underlying identity formation. Keywords: Workplace learning; Vocational identity; Competence Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Background The purposes of vocational education and training (VET) go beyond developing the technical capacities required to perform an occupation effectively or securing students’ employment (Baethge et al. 2009; Renold 2009); they extend to the formation of students’ identity within and attachment to an occupation, as well as their integration into society through that identity (Baethge and Arends 2009; Drexel 2005). A characteristic feature of the German initial vocational training system, for example, is that those undertaking apprenticeships develop strong occupational ties and form an identity associated with that occupation, rather than loyalties to a specific company or employer (Haasler 2007; Rauner 2007). Skilled workers’ vocational identity is a crucial component of the German workforce, central to its ability to remain successful in an era of unprecedented global competition, because of its effects on vocational capacities, skill performance, and quality (Rauner 1999; Skorikov and Vondracek 2007). Without vocational identities, workers’ abilities to plan, execute, and monitor their work activities autonomously would be Page 2 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 2 of 20 far less likely. In turn, vocational identity guides workers’ practices during the course of completing complex, demanding tasks, and it offers a sustainable source of intrinsic motivation that directs and secures individual engagement with the vocation (Rauner 1999). Thus, for both institutional and personal purposes, developing strong vocational identities is an implicit goal of German vocational education; in Switzerland, it is even explicitly included in the stated learning outcomes for the vocational education system (§50 BBG 2002). Despite the salience of workplace and vocational identity however, the processes that contribute to its formation are far from fully understood. It is unclear whether and which elements of VET provision shape learners’ identities and even more unclear whether the desired effects of a strong identity in terms of an enhanced vocational performance actually materialize. To shed light on how these factors and associations contribute to vocational performance, we need an integrative approach to investigate both contextual and psychological factors. Such an inquiry would contribute to deeper understanding of the circumstances that enable different forms of identity to develop and how they affect both learning and workplace effort. To this end, we first outline the theoretical concepts vocational identity and workplace identity. We then present a background model and a conceptual model, informed by a thorough review of the literature and accounting for major hypotheses about the genesis and effects of vocational identity. Regarding the elements of VET that shape these concepts, our literature review focuses on (1) the effect of a dual versus school-based VET, (2) integration at the workplace, and (3) free career choice; for the effects and alignments of vocational identity, we focus on (4) workplace effort and (5) vocational competence as prerequisites for effectual vocational performance. We then show that associations across occupational environments, together with students’ or apprentices’ vocational performance, are mediated by fundamental motivational traits associated with vocational and workplace identities. Figure 1 presents our conceptual model graphically. As a result, we provide a clearer understanding of how vocational learners are motivated to develop such associations. We ask weather government's and employer’s expectations Figure 1 Conceptual model. Page 3 of 20 Page 3 of 20 Klotz et al. Theoretical conception of vocational identity To understand the mechanisms that lead to the formation of vocational identity, we must clarify what is formed and how to measure that formation. Next, we define the terms “vo- cation” and “identity” to provide a clearer picture of our conception of vocational identity. Billett (2011) distinguishes an “occupation,” which comprises social facts, from the per- sonal elements that individuals come to identify with and assent to as their “vocation.” Thus, we acknowledge both occupations and something else that describes people’s inter- ests or “calling.” In the second sense, vocations might be specified as those activities to which people are drawn (Estola et al. 2003) or “called” (e.g., Dror 1993), which provide lifelong fulfillment through their exercise (Hansen 1994). Separating these two usages is critical to understanding occupational identity (i.e., vocation), in that they imply distinct goals for vocational education and separate measures. The first term, occupation, refers to institutional, normative, and discourse practices associated with individual identities. Occupations offer examples, which are ordered and valued in particular ways, starting with their origins in historical and social forms (Billett 2011). Accordingly, identifiable societal expectations and factors are associated with people with an occupation, whether as an auto mechanic, nurse, accountant, or industrial clerk. In contemporary terms, the notion of vocation as occupation suggests that VET should be highly responsive to industry expectations, existing standards, and employers’ needs, often supported by government interests and economic imperatives. This influence becomes particularly salient when there are either national skill shortages or high levels of unemployment, particularly youth unemployment (Aldrich 1994; Anderson 1998; Butler 2000; Kantor 1986; Renold 2009). During these periods, vocational education is primarily a means to provide effective preparation and a smooth transition for students into the occupational practices needed in society. Governments’ typical definition of vocational education thus focuses primarily on developing the capacities needed for particular forms of work (i.e., occupations) in ways that meet societal needs (i.e., demand for skilled labor). Its purposes, forms, and key endorsements appear within and are products of society, that is, institutional facts (Searle 1995). The second term, vocations, is defined as the individual worker’s journey, calling, or personal trajectory (Dewey 1916). The essentially personal phenomenon energizes and directs a person’s individual’s intentions, activities, and interactions (Estola et al. 2003), even as they continue to be shaped by external factors (e.g., societal standing of the occupation, gendered associations). Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 of the German dual VET are realistic and how these expectations can be realized. Accordingly, we consider how students learn (Pellegrino et al. 2001; Shavelson and Ruiz-Primo 2005), how they exercise agency in participating in and learning through work, and on what bases and for what purposes they exercise this agency. Theoretical conception of vocational identity This view also accounts for the process of learning, which is a person-dependent process. From this perspective, VET functions to secure personal goals and development pathways, thus assisting students in engaging in learning that reflects their aspirations and enables them to realize their full potential. By combining these views, we propose for this study that vocations are personally directed but also socially derived practices that reflect a person’s enduring aspirations Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 4 of 20 and interests. They are usually manifested in culturally and historically derived activities that offer value for both the individual and the community (Billett 2011). Social science has different conceptualizations of identity, focusing on (1) a social identity conceptualization designating a person’s position in a social system and (2) a personal identity conceptualization denoting aspects of individual experience rooted in interactions (Côté and Levine 2002). At the level of social identity, the individual is most influenced by cultural factors and social roles, experiencing varying degrees of pressure to fit into an available identity that can be characterized mainly as a vocational habitus (e.g., Bourdieu 1990). This concept corresponds to our understanding of the term “occupation.” In contrast, at the level of personal identity, individuals find a fit between their perception of the social world and their own personality (potentials, interests, and desires, often referred to as “aspirations”). A personal identity is therefore affected by social influencing factors, but this relation is pivotally designated by individual personality and how people perceive such external influences (Mead 1934). This identity definition corresponds to our understanding of “vocations.” Accordingly, we define “vocational identity” as how people negotiate and align their personality with an occupation’s norms and practices or, more precisely, as the fit between an individual’s perception of the occupational world and his or her self-perception. We further propose, in line with Marsh and Shavelson (1985), that the analysis of a person’s identity in regard to a specific area or domain (e.g., toward a vocation) is more fruitful than a global identity conception. We acknowledge that the relative salience of identity domains may vary across cultures and socioeconomic contexts but note that several studies confirm that vocational identity development is a priority in adolescence (Kroger 1993; Skorikov and Vondracek 1998, Solomontos-Kountouri and Hurry 2008). Theoretical conception of vocational identity In the domain of vocational identity, theoretical and empirical research streams have generally confirmed that two subdomains of vocational identity exist: one referring to the actual vocation in a broad sense and one referring to a person’s workplace (also referred to as “organizational identity”; e.g., Cohen 1991; Lee et al. 2000; Wallace 1993). We refer to both conceptions for our research endeavor. We operationalized the first concept of vocational identity according to our theoretical definition: the fit between a person’s perception of the social structures of an occupational world and that person’s perception of his or her own personality. To measure this concept, we asked students about the fit between their chosen vocation and their personality (e.g. “My vocation fits me,” “My vocation is an integral part of who I am,” “I am proud of my vocation”). Workplace identity analogously refers to the perceived fit between a person’s sense of self and the workplace’s norms and practices. We accordingly asked apprentices if, for example, they feel like a part of their company, if they draw satisfaction from working for it, if they consider their company as fitting to them, and if they are proud to be a part of their company. Hypotheses: Predictors and impacts of vocational identity Our general framework (shown in Figure 2) for analyzing our theoretical conception of vocational identity is based on House’s (1977) personality and social structure perspective (PSSP) and its proposed levels of personality, interaction, and social structure. Lempert (2009) transfers the PSSP model to a theoretical model of the formation of vocational Page 5 of 20 Page 5 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Figure 2 Theoretical framework: levels and operations determining the genesis and effects of vocational identity according to Lempert (2009). Figure 2 Theoretical framework: levels and operations determining the genesis and effects of vocational identity according to Lempert (2009). learning, identifying vocational identity as a major aspect of vocational learning. The person interacts with the social structures of the occupational environment (“interaction” here refers to the person’s contact and communication with his or her surroundings, comprising several cognitive operations). This interaction is crucially influenced by the person’s personality (including cognitive structures), which then influences his or her perception and interpretation of vocational experiences, resulting in differentiated processing of experiences and ultimately reactions (impulsive) or actions (considerate). According to this model, we regard the process of identity formation as a function of both external (social) and internal (agentic) components, suggesting that both sociological and psychological perspectives are essential for a comprehensive understanding of this concept (Côté and Levine 2002; Lempert 2009). We further assume, in line with this model, that vocational identity affects a person’s actions and reactions at the workplace: it influences the person’s work results and therefore his or her vocational performance (i.e., “performance” defined, in line with Chomsky (1981), as the outward production of actual events at the workplace). Performance occurs when there is competence as well as possibility and willingness for demonstration (Achtenhagen 2004; Boekaerts 2002). To identify possible major influences and the effects of vocational identity, we began by designing a conceptual model. Next, we conducted a thorough review of the existing literature in the vocational identity domain. We then formulated five hypotheses and implemented them into one coherent and empirically testable theoretical model (Figure 3). We detail our hypotheses in the following subsections. Vocational identity is fostered better by dual training than by school-based learning Both blue- and white-collar apprentices in Germany gain work skills through a dual system that comprises both on- and off-the-job components. Off-the-job training is Page 6 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Figure 3 Theoretical model. financed by the state, with the aim of developing theoretical knowledge the worker will need in the occupation, in addition to the knowledge gained through a general education. On-the-job preparation entails participation in work activities at a firm, which includes a systematic regimen characterized by adherence to training regulations and interactions with trainers or masters who guide apprentices through the realistic components of the apprenticeship (Ashton and Green 1996; Kutscha 2000). Furthermore, apprenticeships tend to be sensitive to business cycles, such that in 2011, only 51% of students participated in the dual system, whereas 20% enrolled in regular vocational schools, and 29% prepared for regular vocational training in a so-called transitional system,a also organized by vocational schools (BMBF 2012). Because of this trend, we compared the consequences of participating in either dual or school-based training, including the implications for the formation of vocational identity. Kirpal (2006) posits that more positive outcomes derive from dual vocational preparation than from a solely school-based preparation for identity formation; we draw on this proposition and predict that continuous learning at the workplace, such as the learning gained in a dual training, positively influences identity value. We tested this hypothesis using a dummy variable indicating dual or solely school-based training. Vocational identity is enhanced by free career choice A vocation in our definition implies a calling; therefore, the ability to choose a particular occupation is critical. In line with Bühler (2007) and Heinemannet al. (2009), we suggest that free vocational choice not only is desirable for its own sake but also can shape the formation of the person’s vocational identity. We define “free career choice” as the degree to which the choice of a person for his or her preferred vocation was a voluntary one. Involuntary career choices may be the result of, for example, monetary restrictions, social pressure to choose a certain vocation, or certain forms of discrimination consti- tuting barriers for a person to choose a desired vocation (Lent et al. 2000). To assess this influence, we used the item, “My vocation was my preferred vocation of choice,” introduced by Heinemannet al. (2009, p. 10). However, we acknowledge that vocational trajectories also may arise through participation in activities that were not initially enacted or identified by workers as vocational for them but were locally and socially convenient (Billett 2011). For example, Somerville (2006) finds among elder care workers in Australia that though they did not originally intend to pursue this vocation, they became increas- ingly engaged with their work and developed a strong vocational identity over time. Workplace identity is determined by workplace integration We define “workplace integration” as the functional integration of apprentices into operat- ing working processes. Individual workplaces feature distinct practice requirements, due to their unique location, clients/customers, services offered, workplace culture, norms, history, and employees. Thus, various firms have unique ways of working and desired outcomes, even if they engage in similar occupations (Billett 2006). Apprentices’ workplace experiences, including activities and interactions, influence what they learn, mediated by social interactions with other employees. People who have been invited to participate in work activities and integrated into the work process thus may come to Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 7 of 20 Page 7 of 20 identify more with their workplace. Qualitative research affirms the importance of work participation for shaping identities and competencies (Corsten and Lempert 1992; Hartigan-Rogers et al. 2007; Henderson et al. 2007). Newton et al. (2009) find that when workplace environments welcome and support novice nurses, providing them with opportunities for active participation in the work process rather than just observational roles, they contribute richly to learning. Such experiences also should support the formation of workplace identity. Thus, whereas our first hypothesis compared the vocational identity of apprentices undergoing dual vocational versus school-based training, our second hypothesis relates solely to dual vocational apprentices, who have opportunities to experience real-life work, to predict how these experiences influence their workplace identity. To address this issue, we asked apprentices if they felt integrated into all operating processes of their back office. Workplace effort within VETs is mediated by vocational identity Several studies have examined the relationship between forms of vocational and work- place identity. Wallace (1993) cites a corrected average correlation of .45 between the two concepts, and with a meta-analysis, Lee et al. (2000) specify a similar corrected correlation of .45 between occupational commitment and organizational commitment (which we refer to as “vocational identity” and “workplace identity”) across 49 samples. However, the causality between these variables remains somewhat unclear; they may even be bidirectional. According to Peiperl and Baruch (1997), in a post–corporate career realm, vocational identity gains potency and serves as a source or predictor of workplace identity. They argue that people decide to become members of an occupation before being employed by a particular employer. Aranya et al. (1981) propose that professional affiliation is both separate from and a precedent of an affiliation with a particular workplace. Because vocational identification develops prior to workplace identity, when professionals leave a job for another employer, they should sustain their professional affiliation. This latter assertion is uncontroversial; the former is open to question. In Page 8 of 20 Page 8 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 the German VET setting, apprentices in a dual system likely develop their vocational identity while working for a company. For apprentices, after they begin working, their workplace likely shapes their vocational identity as they experience and perform work associated with the selected vocation, which also has implications for their vocational identity (Hypothesis 4). Vocational learning theory further posits that different forms of identity related to the vocation or the company increase workplace effort. We define “workplace effort” as the activities and intentional efforts that students/apprentices direct toward work performed within the training company. An increasing body of research focuses on vocational learning as a component of individual efforts to contribute to work-related activities and interactions (e.g., Hodkinson and Hodkinson 2004). However, the relation between workplace identity and workplace effort seemingly has decreased in recent decades (Baruch and Cohen 2007; Cohen 1991; Randall 1990). In the modern, individual-based labor market, companies offer weaker generators of identity or sources of personal commitment (Baruch and Cohen 2007). Therefore, we predict that vocational identity mediates the association between workplace identity and workplace effort, such that vocational identification takes the dominant role (Hypothesis 5). Workplace effort within VETs is mediated by vocational identity To test this prediction in our statistical analysis, we test vocational identity as a mediator between workplace identity and workplace effort. To operationalize our definition of workplace effort, we asked the apprentices how much effort they expend when contributing to their company’s success and how much they try to influence decisions and improve actions to ensure the quality or efficiency of their work. Vocational identity is closely related to vocational competence A successful vocational performance requires not only vocational volition but also cognitive ability. The question here is whether vocational identity is also positively aligned with vocational competence. Vocational learning theory posits that forms of identity shape the quality of learning activities. For example, Baethge and Baethge-Kinsky (1998) and Rauner (2007) depict vocational development as a coherent process of identity and competence development occurring while becoming member of a vocational community. Both studies assume bidirectionality of this relationship. Also, in our background model, vocational competence constitutes another cognitive trait on the personality level and therefore cannot be regarded as unidirectional. Accordingly, we aim to empirically investigate the strength of the interactive relation between the two concepts. We define “competence” in line with Hartig, Klieme and Leutner (2008) as a cognitive disposition that is learnable and focused on a certain field of action (domain- specific). In our research context, the field of action or domain is localized in the activities of commercial vocations. We assume that competence, as a cognitive disposition, influences the solution of domain-specific situations and tasks, so it is therefore reason- able to deduce back from the solving of authentic situations to cognitive structures through adequate item design and psychometric procedures (e.g., Wilson 2008; Shavelson 2008). Because measurement of vocational competencies includes both knowledge and action, the tasks implemented within the competence test focus on at least three competence levels, all aiming at the measurement of vocational expertise (see Greeno et al. 1984): Page 9 of 20 Page 9 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 1. Conceptual competence corresponds to factual knowledge that can be transmitted into action schemata. 2. Procedural competence subsumes the application of knowledge, that is, how to operate with facts, structures, knowledge nets, and their corresponding elements. 3. Interpretative competence focuses on an interpretation of results and on decision processes. 3. Interpretative competence focuses on an interpretation of results and on decision processes. Together, these competence levels refer to successfully completed, occupation-specific tasks. We implemented this operationalization with 36 test items to determine empirically the relation between vocational identity and vocational competence. Our data set (N = 187) included industrial apprentices who were assessed in March 2013 at four German vocational schools. Research questions The theoretical outline, according to our background model (Lempert 2009), suggests that vocational identity mediates the effects of occupational environments on vocational performance and therefore potentially provides a rich characterization of how apprentices perceive and act. With the present study, we aim to gain insight into the relevant mechanisms. The focal research questions are as follows: (1)How structurally valid and reliable are the derived measurements of our theoretically outlined definitions? (1)How structurally valid and reliable are the derived measurements of our theoretically outlined definitions? (2)How do the characteristics of vocational training, specifically, (a) dual versus school-based training, (b) integration in workplaces, and (c) a free career choice, predict vocational and workplace identity? (3)How do vocational and workplace identities mediate the relationship of the predictive variables with workplace effort? (4)Is there a detectable relation between vocational identity and vocational competence? More precisely, do apprentices who identify to a greater extent with their occupation or company also achieve higher scores in objective competence tests of their vocational expertise? Vocational identity is closely related to vocational competence Sample tasks of our instrument can be found in the Additional file 1. In summary, our literature review identified several predictors of vocational and workplace identity: (1) a dual versus school-based VET, (2) workplace integration, and (3) a free career choice, as well as two effects: (4) willingness to perform and (5) vocational competence as prerequisites for vocational performance. Our theoretical design further predicts that associations between the characteristics of occupational environments and vocational performance are mediated by the more fundamental motivational qualities of learners’ vocational and workplace identities (see Figure 3). Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Sample characteristics The data, gathered in July 2012, comprise a sample of N = 504 learners in the middle of their vocational education as industrial clerks. To compare apprentices working in a dual system against students engaged in school-based training, 290 of the participants Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 10 of 20 Page 10 of 20 were undergoing a dual and 214 a school-based VET. In total, 55% of the survey participants were women, and 47% of the students had a migrational background. However, neither gender nor migrational background correlated with the variables featured in our analysis, so we did not control for them. To research the relation between vocational identity and vocational competence, we conducted objective competence tests empirically of N = 187 industrial apprentices in March 2013 at four vocational schools (Munich, Hanover, Bielefeld, and Paderborn). 47 of the apprentices were tested after 1.5 years and 140 apprentices after 2.5 years of their vocational training. The test took 125 minutes, including the test instruction (10 minutes) and completion of the identity survey (10 minutes). Instruments The paper-and-pencil survey consisted of two sections. First, 11 items referred to socio- economic and biographical information. Second, 14 items measured the indirectly observable constructs of vocational identity, workplace identity, and workplace effort. Our scale development for these items was informed by Blau (1988), Carson and Bedeian (1994), and Heinemann and Rauner (2008). Participants were instructed to give answers that reflected their personal perceptions, using five-point Likert scales, from 1 = “I do not agree” to 5 = “I totally agree.” Thus, higher scores indicated a higher value for the related construct. The competence test comprised 36 items depicting knowledge and ability in the area of business and commerce. The item responses were scaled using a correction scheme (0 = “incorrect or no solution,” 1 = “partial solution,” and 2 = “correct solution”). The responses were then combined into one factor value for each participant. The respondents participated voluntarily during both data collections, through an agreement with their teachers and the schools’ executive boards. Method To begin our data analysis, we evaluated the measurement instrument. An exploratory factor analysis validated the survey’s factorial structure. The data seemed appropriate for factor analyses (KMO = .89; Bartlett test = .00), so we conducted an analysis with the Kaiser criterion and a parallel assessment (Horn 1965) to identify the number of latent constructs in the database. Structural equation modeling (SEM) in AMOS served to align the measurement models with the hypothesized relations of the study constructs. Missing data were addressed through full-information maximum likelihood estimation (FIML); several simulation investigations affirm that FIML outperforms most other common methods of handling missing data (Baraldi and Enders 2010; Enders and Bandalos 2001). To address the research questions, we fit baseline models of the direct effects of the four independent variables on the latent factor constructs. After establishing these direct relationships, we introduced vocational identity and workplace identity as mediators of vocational performance and tested the mediating effects. Specifically, we estimated indirect effects with delta method standard errors to confirm the mediation (MacKinnon et al. 2002; Sobel 1982). To test Hypothesis 6, we built a mere factor score for the competence test and conducted a bivariate correlation analysis with the constructs in our identity survey. Page 11 of 20 Page 11 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Measurement model As a result of an analysis with the Kaiser criterion and a parallel assessment (Horn 1965), the survey items indicated a three-factor solution. Figure 4 presents the results graphically. The empirical factor analysis of our survey instrument led to the theoretically assumed structure in which vocational identity, workplace identity, and workplace effort are empirically separate concepts. The three concepts explain 62% of the total variance and affirm strong internal consistency (Cronbach’s αs: factor 1 = .83; factor 2 = .90; factor 3 = .71). This three-dimensional structure also received confirmation in a confirmatory factor analysis in AMOS; the three-factor measurement model fit the data significantly better (.000) than a two- or one-factor model.b Table 1 presents all the questions from the measurement instrument and contains the respective factor coefficients and R-square values. The formulated items and derived instrument thus can measure vocational identity appropriately, in terms of factorial validity and reliability. All variables had low to moderate correlations (.07 to .57), suggesting little multicollinearity. Therefore, we conclude that the instrument is applicable for testing the hypotheses regarding the influences on and effects of vocational identity. Table 2 provides the correlations among the generated factors. Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Directly fitted path model The standardized path coefficients for the directly fitted model are depicted in Figure 5. Integration at the workplace was positively associated with workplace identity (r = .42), workplace effort (r = .41), and, to a lesser extent, vocational identity (r = .28). From a biographical perspective, students who decided voluntarily on their vocation were more likely to develop workplace (.34) and vocational (.52) identity and to perform effortfully (.29). In summary, the model accounts for 31% of the variance in workplace effort, 37% Figure 4 Factor extraction. Figure 4 Factor extraction. Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 Page 12 of 20 http://www.ervet-journal.com/content/6/1/6 Page 12 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Table 1 Empirical fit of items measuring vocational identity Component 1. Vocational identity 2. Workplace identity 3. Workplace effort (r2 = .59; α = .83) (r2 = .71; α = .90) (r2 = .55; α = .71) I am proud of my vocation. (Blau 1988) .81 I deeply enjoy my vocation. .81 My vocation fits me. (Carson and Bedeian 1994). .80 My vocation is an integral part of who I am. (Carson and Bedeian 1994). .73 I am highly devoted to my vocation. .69 I am proud to work for this company. .88 I deeply enjoy working in my company. .86 My company fits me. .85 My company applies to my ideas of a “good” company. .82 My company feels a little like a home to me (Heinemann and Rauner 2008). .80 I put in effort so that my work contributes to the success of my company. .78 I habitually think about how to change my work in a way to make it more efficient or of higher quality (Heinemann and Rauner 2008). .74 I consider the consequences for my company that my actions might entail. .73 I want to contribute to decisions about my work (Heinemann and Rauner 2008). .72 Table 1 Empirical fit of items measuring vocational identity in workplace identity, and 38% of the variance in vocational identity. However, the overall model fit is poor (χ2/df = 9.999, p = .000; comparative fit index [CFI] = .882; root mean square error of approximation [RMSEA] = .134). Mediated path model Modeling vocational identity as a mediator between workplace identity and workplace effort significantly improved the overall model fit (χ2/df = 1.300, p = .272; CFI = .998; RMSEA = .024). Regarding causality, we determined that the model predicting an effect Table 2 Means, standard deviations, and intercorrelations among all relevant variables 1. 2. 3. 4. 5. 6. 1. Vocational identity 1 2. Workplace identity .57* 1 3. Workplace effort .39* .34* 1 4. Workplace integration .36* .38* .36* 1 5. Free career choice .59* .45* .37* .33* 1 6. Dual versus school-based VET .14** — — — .07 1 M 3.62 3.65 3.97 3.94 3.44 .41 SD 1.05 1.02 0.87 .86 1.17 .49 p < .05. p < .01.p < .001. Table 2 Means, standard deviations, and intercorrelations among all relevant variables 1. 2. 3. 4. 5. 6. Table 2 Means, standard deviations, and intercorrelations among all relevant variables 1 2 3 4 5 6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 Page 13 of 20 http://www.ervet-journal.com/content/6/1/6 Page 13 of 20 Page 13 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Figure 5 Directly fitted path model with direct effects of indicator variables on all latent factors. Figure 5 Directly fitted path model with direct effects of indicator variables on all latent factors. of workplace identity on vocational identity fit the data significantly better than a model with the opposite effect (.000), in support of Hypothesis 4c. We present the standardized path coefficients for the mediated model in Figure 6. We excluded the significant direct effects between the independent variables and vocational engagement from the model for clarity; Table 3 presents these values. The group variable, indicating dual versus solely school-based VET, had a significant though small effect on vocational identity (r = .09). The effect of workplace identity on workplace effort was partially mediated by vocational identity (r = .10). Although the direct effect of workplace identity on vocational identity was strong (.49), its direct effect on workplace effort decreased and became quite small (r = .08) when we allowed the mediation through vocational identity. Mediated path model The effect of free career choice on workplace effort was partially mediated by vocational identity and workplace identity (r = .14); it also had a direct effect on workplace effort (r = .16), workplace identity (r = .38), and vocational identity (r = .35). However, the effect of workplace integration on vocational identity Figure 6 Mediated path model. Figure 6 Mediated path model. Figure 6 Mediated path model. Page 14 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Table 3 Standardized direct, indirect, and total effects for mediated relations Predictor, mediator Workplace effort Direct Indirect Total P: Workplace identity .08 .10 .18 M: Vocational identity .20* — — Workplace effort Direct Indirect Total P: Workplace integration .37* .08 .45* M: Workplace identity .08 — — Workplace Effort Direct Indirect Total P: Free career choice .16* .14 .30* M: Vocational identity .21* — — M: Workplace identity .08 — — Table 3 Standardized direct, indirect, and total effects for mediated relations was not significantly mediated by workplace identity (r = .08). Instead, workplace integra- tion directly increased both workplace identity (r = .46) and workplace effort (r = .37). Including the mediating variables added 8%, 12%, and 7% to the explained variance, yielding overall explanations of 39%, 50%, and 44% of the variance of vocational identity, workplace identity, and workplace effort, respectively. For the relation between vocational identity and the vocational competence of the apprentices, we found an effect of r = .25 in support of Hypothesis 6 (Table 4). Voca- tional identity further correlates with the average grade obtained at the vocational school (r = .15) and the apprentices’ self-rating of their vocational competence (r = .24). (We measured this self-rating on a six-point scale, in line with the German grading system.) Workplace identity correlates to a lesser extent with the competence tests (r = .14) and the apprentices’ self-rating of their vocational competence (r = .16). The competence tests correlate fairly well with the average grade given by the vocational school teachers (r = .40) and with the apprentices’ self-rating of their vocational competence (r = .33), suggesting high retrograde and concurrent validity of the competence measure. Hypothesis 1 In our quantitative, empirical model, we first predict that vocational identity can be fostered more effectively through continuous workplace learning experiences, which can be implemented with dual VET (Kirpal 2006). We find only a minor, albeit stable, effect in support of this prediction: Students who spend half their study time in workplace environments tend to identify slightly more strongly with their occupation than students who study only in a school setting. However, one might expect a higher group effect; theoretically, students without workplace experience cannot build a vocational identity, because they have never engaged in the occupational practice or with its practitioners. Therefore, we suggest that the issue of whether scholastic or dual apprentices identify more strongly with the occupation might be not the right question: 27% of scholastic learners stated in our context survey that they had already gathered practical experience in the business and commerce sector through pertinent internships. This finding indicates that our dummy variable of a dual versus solely school-based training does not adequately capture the amount of practical training a learner underwent. Comparing learners with practical experience (scholastic learners with prior pertinent internships or dual apprentices with continuous working experience) versus learners with no practical experience generates an effect of .19. Therefore, the amount of practical training might be a more precise predictor of vocational identity than dual versus solely school-based training. Hypothesis 2 Integrating vocational apprentices into organizational work processes strongly increases apprentices’ workplace identity and has a direct effect on their workplace effort. Posi- tive cooperation with apprentices requires their effective engagement in work groups, in support of the concept of enhanced participatory practices and enculturation (Lave and Wenger 1991). Vocational learning is a situated activity that takes place through a process of engagement in the sociocultural practices of the workplace. Chan (2011) similarly describes how bakers progress in their vocational identity, such that they first sense belonging to a particular bakery and demonstrate competence before they go on to identify as a baker. The first step is contingent on the degree to which they are accepted, recognized, and granted discretion as an apprentice in the workplace where they apprenticed. Discussion Our study of vocational identity as a personal concept, arising through interactions between the person and the mode of VET, features tests of six theoretical hypotheses Table 4 Correlation analysis of the relation between vocational identity and vocational competence 1. 2. 3. 4. 5. 1. Vocational identity 1 2. Workplace identity .53* 1 3. Self-rating of vocational competence .24*** .16* 1 4. Competence test .25*** .14* .33*** 1 5. Vocational school grades .15* .04 .48* .40* 1 Table 4 Correlation analysis of the relation between vocational identity and vocational competence Table 4 Correlation analysis of the relation between vocational identity and vocational Page 15 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 about the genesis and effects of vocational identity. We discuss the results in the fol- lowing subsections. about the genesis and effects of vocational identity. We discuss the results in the fol- lowing subsections. Hypotheses 6 Regarding Hypothesis 6, the theoretically postulated alignment between vocational identity and vocational competence is upheld. Vocational identity correlates fairly well with our developed competence tests, as well as with grades given by vocational teachers and with apprentices’ self-rated vocational competence. In summary, two conditions seem required to trigger identity and excellence. First, apprentices must be granted practical experience; during that experience, they must be welcomed and invited to participate in all operating work processes. Second, the apprentices themselves must voluntarily participate to be able to accept this invitation and decide to engage. The effects of the influencing factors on vocational performance are mediated by a personal identity conception (Allan 2005; Billett and Pavolva 2005; Etelpäpelto 2008; Fenwick 2004; Somerville and Abrahamsson 2003). Hypotheses 4 and 5 Hypotheses 4 and 5 For Hypothesis 4, we tested the causal relation between workplace and vocational identity. On the basis of theoretical considerations, we predicted an influence of workplace identity on vocational identity; this direction was empirically confirmed, such that this causal relation appears more likely among young apprentices. However, for advanced workers at later stages of their careers, or for those starting in a new company, the direction of the causal effect may switch, because they already have acquired a distinct, stable vocational identity that can shape the development of their new workplace identity when they move to a new work environment. The empirical results related to Hypothesis 5 suggest that workplace identity is not as prominent a source of workplace effort as vocational identity. Instead, much of the total influence of workplace identity on workplace effort is mediated by the more general concept of vocational identity. Hypothesis 3 We proposed and found a strong, significant influence of free career choice on both vocational identity and workplace identity. In addition, free career choice appeared to have a direct impact on workplace effort. Students voluntarily deciding on their vocational training identify more strongly with and engage more effortfully in their selected occupation. This finding is consistent with Heinemann et al.’s (2009) recognition of a positive correlation between vocational identity and free career choice (r = .27) across 23 VET programs. We thus confirm Hypothesis 3. Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 16 of 20 Page 16 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Conclusions and limitations Our results highlight that several VET characteristics exert important influences on vocational and workplace identity. We find that functional integration at the work- place (Billett 2004; Chan 2011; Hartigan-Rogers et al. 2007; Henderson et al. 2007; Lave and Wenger 1991; Newton et al. 2009), free career choice (Bühler 2007; Heinemann et al. 2009), and practical experience (Billett and Somerville 2004) foster the development of vocational identity. The mediation analyses demonstrate that learners’ vocational and workplace identities, as a form of intrinsic motivation, strengthen the relationships between VET characteristics and the apprentice’s willingness to perform, as Rauner (1999) suggests. These conclusions match Billett’s (2011) view of workplace affordances and individual bases of engagement as means to understand the duality by which learning arises through work participation. We also show that vocational identity is not only central to vocational volition but also aligned with the development of vocational competence, as Baethge and Baethge-Kinsky (1998) and Rauner (2007) suggest. We therefore conclude from our analyses that vocational identity constitutes a crucial factor for the development of an excellent workforce, in that it fosters employees’ willingness to perform in work settings and is closely related to vocational competence. We further suggest that vocational instructors have critical roles in terms of creating positive Page 17 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 vocational learning environments in schools and workplaces that can optimize learners’ perceptions of their occupations in practice, thereby shaping vocational identity, which then increases competence and willingness to perform. In line with our model results, we outline three points of intervention to strengthen a learner’s vocational identity and thereby optimize VET: (1) Grant vocational learners an opportunity to experience actual work. Vocational theory suggests that learners cannot form worthwhile, robust vocational identities without experiencing work and without actively engaging and learning in workplaces. Yet fewer firms seem willing or able to provide apprenticeship experience, which suggests the need for other options. Our results suggest that the amount of practical training is a more stable predictor of vocational identity than dual versus solely school-based training; therefore, we recommend that providing vocational internships can improve learners’ identification with their vocation and potentially overcome differences between scholastic learners and apprentices. Conclusions and limitations At a policy level, governments could cooperate with the private business sector to either train more apprentices or implement more internships as an integral part of vocational school policies. (2) Ensure maximum integration at the workplace for apprentices. Apprentices need support as they develop their sense of self as workers, through enhanced participatory practices afforded by workplaces. Our results suggest that by being allowed to participate in all operating processes, they more readily develop vocational identity and apply more workplace effort. Therefore, apprentices need opportunities for active, productive participation in work processes, rather than just taking observational roles or performing isolated, monotonous work. These practices often occur already, but this emphasis should become a central motif for securing the development of vocational identity and capacities. (3) Make vocational training consonant with individual workers’ agency. Free career choice is not only desirable for its own sake; it also encourages the development of a highly identified workforce. Our results emphasize the importance of individual interest in and consent to engage fully in the process of occupational preparation. Thus, personal and social efficiency likely can be fostered by a society’s “cultivation of power to join freely and fully in shared or common activities” (Dewey 1916, p. 85). In this respect, vocational education inhabits an important role or responsibility: to assist young people in their efforts to identify the vocation for which they are best suited and to which they are drawn. Policies and practices associated with securing occupation must consider individual agency if the goal is to achieve high engagement and effective learning outcomes. We note several shortcomings in our study approach. The measurement of vocational identity relies on self-reports to assess students’ identity concepts and work effort. This approach raises important validity concerns, in that students could offer what they perceive to be socially desirable answers. Moreover, the self-reports between the scholastic learners and dual apprentices might be biased by selection effects not captured within our data, as the scholastic system usually comprises young people who did not succeed in entering a regular training system due to a lower class rank in prior Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Page 18 of 20 schooling and/or poor grades. Endnotes aThe term “transitional system” can be misleading, because no systematic organizational structure guarantees a transition. Instead, it encompasses various training, education, and labor market schemes provided by governmental bodies to engage young people who did not enter a regular training system (Baethge et al. 2007). bOne-factor model: N = 504: χ2/df = 7.26, χ2= 559.192 and df = 77; two-factor-model: N = 504: χ2/df = 5.60, χ2= 425.741 and df = 76; three-factor-model: N = 504: χ2/df = 4.17, χ2= 308.681 and df = 74. cModel 1 (workplace identity →vocational identity): χ2/df = 1.300; Model 2 (vocational identity →workplace identity): χ2/df = 4.069. Conclusions and limitations If these variables influence the fostering of vocational identity, the effect we found might deviate from our results. Another issue resides in our objective competence test for testing Hypothesis 6 (written examination). The items are designed to capture knowledge and capability regarding the expertise of the apprentices. However, they do not capture other relevant facets such as social competence, which is a key factor in handling social interaction processes. We checked our competence tests for retrograde and concurrent validity but not for prognostic validity. Further research should investigate if the measured ability, in the sense of a disposition for expertise, also predicts the actual performance of the apprentices observed at the workplace, as captured through expert evaluations for example. Finally, with regard to the relation between vocational identity and vocational competence, we assumed a mutually re- inforcing relation, in line with Baethge and Baethge-Kinsky (1998) and Rauner (2007). However, there is no clear theoretical rationale of antecedents and consequences re- garding this relationship. Longitudinal research is needed to address this question empir- ically. Therefore, research endeavors should apply the developed identity instrument over the duration of vocational training, combined with competence tests to explicitly test causalities. Acknowledgement h l f g This article arose from the subproject “Competence-oriented assessments in VET and professional development” (Wi 3597/1-1 and Wi 3597/1-2), within the framework of the priority programme “Competence Models for Assessing Individual Learning Outcomes and Evaluating Educational Processes” (SPP 1293) of the German Research Foundation (DFG) Competing interests p g The authors declare that they have no competing interests. Authors’ contributions All authors contributed substantially to this work. VKK designed the study and analysed the data; SB contributed to the theoretical framework and helped to draft the manuscript. EW participated in the coordination of the study and helped to draft the manuscript. All authors participated in discussing the manuscript at all stages. All authors read and approved the final manuscript. Additional file Additional file 1: Sample tasks. Author details 1 1Chair of Business and Human Resource Education, University of Paderborn, Warburger Straße 100, D-33098, Paderborn, Germany. 2School of Education and Professional Studies, Griffith University, 170 Kessels Road, Nathan, QLD 4111, Australia. Page 19 of 20 Page 19 of 20 Klotz et al. Empirical Research in Vocational Education and Training 2014, 6:6 http://www.ervet-journal.com/content/6/1/6 Received: 21 November 2013 Accepted: 31 March 2014 References Achtenhagen F (2004) Prüfung von leistungsindikatoren für die berufsbildung sowie zur ausdifferenzierung beruflicher kompetenzprofile nach wissensarten. BMBF, Bonn Aldrich R (1994) Vocational education in Britain: An historical and cultural analysis. In: Heikkinen A (ed) Vocational education and culture: European prospects from history and life history. University of Tampere, Tampere, Finland Allan JK (2005) Farmers as learners: evolving identity, disposition and mastery through diverse practices. 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https://openalex.org/W4394872545	https://link.springer.com/content/pdf/10.1007/s10926-024-10182-2.pdf	English	null	Videoconference-Supervised Group Exercise Reduces Low Back Pain in Eldercare Workers: Results from the ReViEEW Randomised Controlled Trial	Journal of occupational rehabilitation	2,024	cc-by	8,540	Abstract Purpose To assess the effects of a group exercise intervention conducted by real-time videoconference on the low back pain of eldercare workers. Methods We randomly assigned 130 eldercare workers to an experimental group (EG: n = 65) or control group (CG: n = 65). Participants from both groups took part in routine prevention programs carried out in their workplace, and participants from the EG received an additional 12-week resistance-exercise intervention supervised by real-time videoconference. Assess- ments were conducted before and after the intervention, and the primary outcome was average low back pain intensity during the last 7 days, measured by the 0–10 numerical rating scale. Secondary outcomes included additional measures of low back, neck, shoulder and hand/wrist pain, as well as psycho-affective parameters, medication consumption and muscle performance. Both intention-to-treat and per-protocol analyses were applied with a group-by-time ANCOVA including baseline measurements as covariates. Results 125 participants completed post-intervention assessments (EG: n = 63, CG: n = 62). The intention-to-treat analysis showed an effect favouring the EG on average low back pain intensity (p = 0.034). Improvements in additional low back and hand/wrist pain outcomes were also observed, as well as on upper limb muscle performance (p < 0.05). The per-protocol analysis demonstrated additional benefits in depression, quality of life, hypnotic/anxiolytic medication consumption and lower limb and trunk muscle performance in participants with ≥ 50% adherence (p < 0.05).f Conclusions The intervention was effective for reducing the low back and hand/wrist pain of eldercare workers and increas- ing upper limb muscle performance. The per-protocol analysis showed additional benefits in psycho-affective parameters, medication consumption and muscle performance. Trial registration: ClinicalTrials.gov, NCT05050526. Registered 20 September 2021—Prospectively registered, https:// www.clinicaltrials.gov/study/NCT05050526 Keywords Musculoskeletal pain · Telerehabilitation · Resistance training · Occupational health · Quality of life * Ander Espin ander.espin@ehu.eus Journal of Occupational Rehabilitation https://doi.org/10.1007/s10926-024-10182-2 Journal of Occupational Rehabilitation https://doi.org/10.1007/s10926-024-10182-2 Videoconference‑Supervised Group Exercise Reduces Low Back Pain in Eldercare Workers: Results from the ReViEEW Randomised Controlled Trial Ander Espin1,2 · Jon Irazusta1,2 · Maialen Aiestaran1 · Unai Latorre Erezuma1,2 · Julia García‑García1,2 · Ismene Arrinda3 · Karmele Acedo4 · Ana Rodriguez‑Larrad1,2 Ander Espin1,2 · Jon Irazusta1,2 · Maialen Aiestaran1 · Unai Latorre Erezuma1,2 · Julia García‑García1,2 · Ismene Arrinda3 · Karmele Acedo4 · Ana Rodriguez‑Larrad1,2 Accepted: 18 February 2024 © The Author(s) 2024 Accepted: 18 February 2024 © The Author(s) 2024 1 Ageing On Research Group, Department of Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain 2 Biobizkaia Health Research Institute, Barakaldo, Spain 3 IMQ Igurco Residencias Sociosanitarias, Bilbao, Spain 4 Home Care Lab, S. Coop, Bilbao, Spain 4 Home Care Lab, S. Coop, Bilbao, Spain Study Design A parallel-assignment, two-arm, multicentre randomised controlled trial (RCT) was carried out. The study was designed so that both the assessments and the intervention could be conducted remotely via real-time videoconference. The overall study protocol is described elsewhere [20]. For participant recruitment, institutions offering eldercare ser- vices at home or in long-term facilities and located in the Basque Country (Spain) were contacted following non-prob- abilistic procedures. At each of the institutions that were interested in participating, all eldercare workers who met the selection criteria were invited to complete the baseline assessments. Following baseline measurements, partici- pants were randomly assigned (1:1 ratio) in each institution through sealed opaque envelopes to either an experimen- tal or control group by a coin-tossing sequence generation. Assessments were conducted at baseline and at the end of the intervention. Outcome assessors and researchers per- forming data analysis were blinded to group allocation. The study protocol was approved by the Ethics Commit- tee for Research Involving Human Beings of the University of the Basque Country (M10/2019/200) and prospectively registered at ClinicalTrials.gov (NCT05050526). Informed written consent was obtained from all participants before enrolling in the study. Current literature supports exercise as a tool with solid evi- dence in pain management [11]. A recent review concluded that therapeutic exercise is strongly recommended in chronic low back pain, as it has the potential to decrease pain, improve function and reduce disability [12]. There is also evidence sug- gesting that exercise could mitigate the psychological disorders associated to musculoskeletal pain [13]. Tele-rehabilitation is an increasingly used modality for delivering exercise interven- tions remotely [14]. In comparison with in-person programs, tele-exercise could be logistically and economically advanta- geous, as time and money costs associated with displacements are avoided [15]. Moreover, tele-rehabilitation is compatible with situations in which interpersonal physical distancing measures are required, such as the recent COVID-19 pandemic [16]. To date, the majority of tele-rehabilitation interventions have consisted of websites for autonomous consultation, thus lacking real-time supervision [14]. Among the few interven- tions with real-time supervision, most have followed an indi- vidual approach (e.g., individual videoconference sessions with the therapist supervising a single participant at a time) [17]. Real-time supervision and group dynamic can lead to higher participant adherence [18, 19], what could result in greater effectiveness of the intervention. Moreover, real-time supervision could be beneficial in terms of safety, as partici- pants are continuously monitored. Participants Participants had to meet all the following criteria to be eli- gible for the study: (a) formal eldercare worker on active duty, (b) ≥ 18 years of age, (c) ≥ 3 months of experience in the profession, and (d) employment contract until at least the date of study completion. Participants were excluded if (a) they were pregnant or (b) their participation was considered contraindicated according to the exercise preparticipation health screening guidelines by the American College of Sports Medicine [21]. Therefore, we conceived the ReViEEW trial (acronym for “Real-time Videoconference-based Exercise in Eldercare Workers”), which assessed, to our knowledge, the first group tele-exercise intervention with synchronous supervision in the occupational setting. The primary aim of the study was to assess the effects of the intervention on the low back pain of eldercare workers. Secondary outcomes included additional measures of musculoskeletal pain, psycho-affective param- eters, hypnotic/anxiolytic and pain medication consumption and muscle performance. Study Design However, group tele-exer- cise interventions with real-time supervision have been scarce [17] and, to our knowledge, none of them has been carried out in the workplace. Introduction Eldercare workers are qualified professionals who provide assistance with activities of daily living to dependent older people at either home or long-term facilities. These workers are of paramount importance in current and future society, as eldercare needs are increasing sharply due to the ageing of the population [1]. Eldercare work is characterized by high physically [2] and psychologically [3] demanding tasks, which can contribute to the development of musculoskeletal pain [4]. Although the presence of pain in the neck, shoulders and the upper extremity is also common, low back pain is the most (012 3456789) 3456789) Journal of Occupational Rehabilitation Methods frequent musculoskeletal disorder among eldercare work- ers [5]. Low back pain is the leading cause of disability and productivity loss worldwide [6], and it can severely affect the quality of life of the people who suffer from it [7]. Pain in the low back is often associated with mental health problems [8] and sleep [9] disturbances, which can lead to higher levels of disability, worse recovery, and greater primary healthcare utilization [10]. Musculoskeletal Pain Musculoskeletal pain outcomes referring to the last 7 days were collected separately for the low back, neck, shoulders, and hands/wrists. Average and worst intensity (0–10) were measured by the aforementioned 11-point NRS [24]. Fre- quency was defined as the number of days in pain (0–7), and interference as the number of days in which pain negatively interfered with work (0–7). Adherence and Adverse Events Additionally, participants reported the number of days in which they took pain medication during the last 7 days (0–7). In each session, the instructor collected the following infor- mation from each participant: attendance, session comple- tion, and overall perceived intensity during the session. Adherence to the intervention was defined as the percent- age of sessions in which participants performed the planned training regarding completion and intensity (i.e., 24 ses- sions completed with perceived intensity between 3 and 5 in Borg’s CR-10 scale = 100% adherence). The instructor also collected adverse events occurring during the sessions. Adverse events were divided into two types: technical (con- nection and/or operation problems with the videoconference Control Group Participants from the control group took part in the routine prevention programs carried out in their corresponding insti- tutions, which mainly consisted of regular group-based train- ing on manual and technical aid-assisted patient handling. These training activities were all held in the workplace, led by a physiotherapist, carried out annually with a duration of around 20 h, and combined theoretical classes (e.g., concepts about how to do manual and technical aid-assisted transfers Journal of Occupational Rehabilitation to dependent elderly people in biomechanically correct postures) and practical exercises (e.g., role-playing among eldercare workers to put the concepts learned into practice). system) or participant safety-related (pain, discomfort, or any other health-related problem). They were also classi- fied as minor (those slightly hindering the development of the session) or major (those preventing the development of the session). Primary Outcome The primary outcome was average pain intensity in the low back during the last 7 days, measured by an 11-point numeri- cal rating scale (NRS) ranging from 0 (complete absence of pain) to 10 (worst imaginable pain) [24]. The NRS is a valid, reliable, and widely used tool for the measurement of pain intensity, which has been proposed as the most appropriate for research purposes in comparison with other pain scales [24]. Baseline Descriptive Data In addition to the aforementioned prevention programs, participants from the experimental group took part in a 12-week exercise intervention, consisting of two sessions per week of 45 min each. Sessions were carried out in small groups of ≤ 10 participants, in the workplace but outside working hours, and remotely supervised by the instructor using real-time videoconference (Supplementary Informa- tion, Fig. SI1). Sessions started with a warm-up (5–10 min) consisting of joint mobility and aerobic activation exercises to increase heart rate. The main part of the session con- sisted of resistance exercises performed with body-weight and elastic bands (30 min). A total of 9 exercises were per- formed throughout the program (Supplementary Informa- tion, Fig. SI2). In each session, 4 sets of 6 exercises were performed. Exercises were systematically varied between sessions so that each of them was evenly performed dur- ing the whole program. In each set, exercises for the major muscle groups were alternated in a circuit format (e.g., upper limb, lower limb, trunk, upper limb, lower limb, trunk). For each exercise, three levels of progression were established: progression 1 (weeks 1–4), progression 2 (weeks 5–8) and progression 3 (weeks 9–12). Progression was achieved by modifying the exercise technique or using elastic bands of different resistance. Within each level of progression, the work:rest time ratio devoted to each exercise also increased from 30:30 to 45:15 s (Supplementary Information, Fig. SI3). Participants were monitored to reach an intensity between 3 (moderate) and 5 (strong) on the Borg’s CR-10 scale [22] and not to reach failure on any of the exercises. If an exercise caused intolerable pain, the 4-stage exercise adjustment model suggested by Jakobsen et al. was used [23]. Sessions finished with a cool-down (5–10 min) con- sisting of static stretching and breathing/relaxing exercises. Participants reported the following descriptive data at base- line: date of birth, sex, height and weight, marital status, educational level, number of children and presence of chil- dren cohabiting at home, care for dependent people outside the work environment, weekly working hours, years of expe- rience in the profession, presence of rotative and night work shifts, alcohol and tobacco consumption, compliance with World Health Organization’s physical activity guidelines, and practice of regular resistance training. Psycho‑Affective Parameters Happiness was measured by the subjective happiness scale [25]. It consists of four items in a 7-point Likert response format asking about current perceived happiness. A single composite score is obtained by averaging responses to the four items, and higher values indicate greater happiness. Anxiety and depression were measured by Goldberg’s scales [26]. They consist of two separate scales containing Journal of Occupational Rehabilitation to expected dropouts, the sample size was increased by 20%. Consequently, the required sample was 130 partici- pants (n = 65 in the experimental and n = 65 in the control groups, respectively). nine dichotomized (yes/no) response items each, ask- ing about last month’s anxious and depressive symptoms, respectively. Higher scores indicate greater anxiety/depres- sion levels. Quality of life was measured by the EuroQol-5D 0–100 health state scale [27]. It consists of a single item measuring self-perceived current health state in a scale ranging from 0 (worst imaginable) to 100 (best imaginable). Statistical Analysis Data analysis was performed with IBM SPSS Statistics 27 statistical software package (SPSS, Inc., Chicago, IL). Continuous data are expressed as means with standard deviations (SD), and categorical variables as frequency counts and percentages (%). Normality of distribution was assessed with the Shapiro–Wilk and Kolmogorov–Smirnov tests for samples < 50 and ≥ 50, respectively. Non-normally distributed variables were square-root transformed for sta- tistical analyses. Between-group baseline differences were analysed with the independent samples T and Chi-squared tests for continuous and categorical variables, respectively. Effects of the intervention were assessed with a group- by-time ANCOVA including baseline measurements as covariates, and effect size was estimated by partial eta squared (ηp 2). Values for ηp 2 of 0.01, 0.06, and 0.14 were considered small, medium and large, respectively [32]. As initially planned, the primary analysis was based on inten- tion-to-treat (ITT). A per-protocol (PP) analysis was also performed including only participants with ≥ 50% adher- ence to the intervention. Additionally, a post-hoc subgroup analysis was performed to assess the effects of the inter- vention on low back pain outcomes separately in partici- pants with (≥ 1 in average 11-point NRS) and without (< 1 in average 11-point NRS) low back pain at baseline. The level of statistical significance was set at p < 0.05. Sleep quality was assessed by the single-item sleep qual- ity scale [28]. It measures overall sleep quality during the last 7 days in a numerical scale ranging from 0 (terrible) to 10 (excellent). Additionally, participants reported the number of days of hypnotic/anxiolytic medication consumption during the last 7 days. Muscle Performance The following muscle performance tests were carried out in the same modality as the exercise sessions (i.e., with the participant located at the workplace and the assessor remotely supervising the execution of the tests via real-time videoconference). The tests were previously validated to be carried out remotely, showing they are feasible and reliable when conducted by videoconference [29]. The 5-repetition sit to stand test was used to assess lower limbs’ muscle performance. Participants had to stand up from and sit down on a chair five times as quickly as pos- sible. The time taken to complete the five repetitions is registered and reported in seconds, with shorter times indi- cating better performance. The mean of two attempts was registered. The kneeling push-up test was used to assess upper limbs’ muscle performance. Participants had to do the maximum number of push-ups possible using the knees as the pivotal point. The total number of repetitions is registered, with more repetitions indicating better performance.l Results The Shirado-Ito trunk flexor endurance test was used to assess trunk’s muscle performance. Participants had to maintain a defined trunk flexion position for as long as pos- sible. The total time was registered in seconds, with longer times indicating better performance. Participants A total of 130 participants were recruited and randomised to the experimental (n = 65) and control (n = 65) groups (Fig. 1). Participants were recruited from five long-term nursing homes (n = 11, n = 12, n = 14, n = 16, and n = 27, respectively) and one institution providing at-home elder- care services (n = 50). There were not significant differ- ences at baseline between the experimental and control groups (p > 0.05) (Table 1). Two participants from the experimental group and three participants from the control group were lost to follow-up. Study start dates differed in each of the institutions. Overall, the first participant was recruited in October 2021, and the last 12-week follow-up was in June 2023. Sample Size Calculation The sample size was calculated to detect a change in low back pain that could be relevant in terms of work absentee- ism [30]. Considering the average low back pain intensity of 5.0 (SD 2.6) in the 11-point NRS observed in a previ- ous study carried out by our research group in eldercare workers [31], and accepting an alpha error of 0.05 and a beta error of 0.20 in a bilateral contrast, 108 partici- pants were needed to detect a difference of ≥ 1 unit. Due Journal of Occupational Rehabilitation Assessed for eligibility (n=436) Enrollment Excluded (n=306) • Not meeting inclusion criteria (n=54) • Refused to participate (n=252) Randomized (n=130) Allocation Follow-up Analysis Allocated to control group (n=65) Allocated to experimental group (n=65) Received allocated intervention (n=64) Did not receive allocated intervention (n=1) Leg fracture between baseline assessment and start of intervention: 1 Lost to follow-up at 12 weeks (n=3) Refused further participation: 2 Could not be contacted: 1 Discontinued intervention (n=1) Moved country: 1 Analysed: Musculoskeletal pain and psycho-affective parameters (n=62) 5-repetition sit to stand test (n=58) Kneeling push-up test (n=52) Shirado-Ito trunk flexor endurance test (n=55) Analysed: Musculoskeletal pain and psycho-effective parameters (n=63) 5-repetition sit to stand test (n=60) Kneeling push-up test (n=59) Shirado-Ito trunk flexor endurance test (n=60) Assessed for eligibility (n=436) Excluded (n=306) • Not meeting inclusion criteria (n=54) • Refused to participate (n=252) Allocated to experimental group (n=65) Received allocated intervention (n=64) Did not receive allocated intervention (n=1) Leg fracture between baseline assessment and start of intervention: 1 Fig. Intervention Effects: ITT Analysis The group-by-time ANCOVA showed a significant effect of the intervention favouring the experimental group on aver- age low back pain intensity (p = 0.034) (Table 2). Sample Size Calculation 1 CONSORT flow diagram Table 1 Baseline characteristics of the participants Con control group, Exp experimental group, WHO World Health Organization, no differences between groups (p > 0.05) Characteristic Exp (n = 65) Con (n = 65) Age (years), mean (SD) 49 (9) 50 (9) Sex, n females (%) 61 (94) 63 (97) Height (cm), mean (SD) 163 (7) 162 (7) Weight (kg), mean (SD) 67 (11) 69 (15) Body mass index (kg/m2), mean (SD) 25.4 (4.4) 26.5 (5.3) Married, n yes (%) 35 (54) 38 (59) Children, n yes (%) 45 (69) 48 (74) Children cohabiting at home, n yes (%) 38 (58) 39 (60) Care for dependent people outside work, n yes (%) 14 (22) 12 (19) Secondary or higher education, n yes (%) 61 (94) 57 (88) Experience in profession (years), mean (SD) 16 (10) 15 (9) Working hours (hours/week), mean (SD) 33 (5) 32 (7) Rotative work shift, n yes (%) 29 (45) 35 (54) Night work shift, n yes (%) 12 (19) 13 (20) Alcohol consumption, n yes (%) 45 (69) 50 (77) Tobacco consumption, n yes (%) 24 (37) 19 (29) Meet WHO physical activity guidelines, n yes (%) 26 (40) 21 (32) Regular resistance training, n yes (%) 14 (22) 9 (14) Journal of Occupational Rehabilitation Adherence and Adverse Events day. Participant safety-related adverse events were musculo- skeletal pains that required exercise adjustment. One single physical therapist with previous experience con- ducting group therapeutic exercise programs delivered all the sessions. Mean adherence to the intervention was 67% (SD 31%). Mean number of participants in each session was 4.3 (SD 2.3). There were minor technical and participant safety-related adverse events in 31 (12%) and 24 (9%) ses- sions, respectively. Technical adverse events were mainly connection problems which slightly hindered communica- tion. The only major adverse event was a connection drop in a centre that prevented the development of the session one Table 2 Effects of the intervention: intention-to-treat (ITT) analysis Intervention Effects: PP Analysis Respecting muscle performance, there was a significant group-by-time effect favouring the experimental group in the kneeling push-up test (p = 0.040).f Forty-eight participants (74%) from the experimental group had a ≥ 50% adherence to the intervention. When including only those participants in the analysis, the group-by-time ANCOVA also showed a significant effect of the interven- tion favouring the experimental group on average low back pain intensity (p = 0.011) (Table 3).i A summary of the effect sizes of the outcomes showing statistically significant group-by-time interactions can be found in the Supplementary Information (Table SI1).i There were not significant group-by-time interactions on the remaining variables (p > 0.05). Table 3 Effects of the intervention: per-protocol (PP) analysis Intervention Effects: ITT Analysis There were also significant group-by-time interac- tions in favour of the experimental group on low back pain frequency (p = 0.010) and interference (p = 0.001), as well as on all hand/wrist pain outcomes: average Bold values are statistically significant (p < 0.05) Data are mean (SD) d b t diff i P ( 0 05) Outcome Experimental Control ANCOVA (p) ηp 2 Pre Post Pre Post Low back pain Average intensity (0–10) 3.4 (2.4) 2.4 (2.2) 3.0 (2.4) 3.1 (2.5) 0.034 0.037 Worst intensity (0–10) 4.1 (2.9) 3.1 (2.8) 3.7 (3.0) 3.8 (3.0) 0.080 0.025 Frequency (0–7) 3.0 (2.4) 1.9 (1.9) 2.7 (2.2) 2.8 (2.4) 0.010 0.054 Interference (0–7) 1.3 (2.1) 0.9 (1.6) 1.2 (1.8) 1.7 (2.0) 0.001 0.082 Neck pain Average intensity (0–10) 3.0 (2.9) 2.3 (2.6) 3.4 (3.1) 2.9 (2.6) 0.163 0.016 Worst intensity (0–10) 3.4 (3.2) 2.7 (2.8) 4.0 (3.4) 3.4 (3.0) 0.187 0.014 Frequency (0–7) 2.7 (2.5) 1.8 (2.2) 2.9 (2.6) 2.6 (2.4) 0.058 0.029 Interference (0–7) 1.5 (1.2) 1.2 (2.0) 1.5 (2.1) 1.7 (2.3) 0.137 0.018 Shoulder pain Average intensity (0–10) 2.5 (2.6) 2.0 (2.5) 3.0 (2.9) 2.4 (2.7) 0.830 < 0.001 Worst intensity (0–10) 2.7 (2.8) 2.3 (2.9) 3.7 (3.3) 2.8 (3.0) 0.943 < 0.001 Frequency (0–7) 2.3 (2.4) 1.8 (2.3) 2.9 (2.6) 2.2 (2.4) 0.788 0.001 Interference (0–7) 1.3 (2.0) 1.1 (2.0) 1.6 (2.1) 1.6 (2.2) 0.370 0.007 Hand/wrist pain Average intensity (0–10) 2.2 (2.6) 1.3 (2.1) 3.0 (3.1) 2.5 (2.9) 0.023 0.041 Worst intensity (0–10) 2.4 (2.7) 1.5 (2.3) 3.3 (3.5) 3.1 (3.4) 0.017 0.046 Frequency (0–7) 1.8 (2.2) 1.3 (1.9) 2.6 (2.8) 2.5 (2.7) 0.035 0.036 Interference (0–7) 1.2 (1.9) 0.9 (1.7) 1.9 (2.5) 1.9 (2.4) 0.049 0.031 Pain medication (0–7) 2.4 (2.4) 2.3 (2.5) 2.2 (2.6) 2.6 (2.7) 0.124 0.019 Psycho-affective parameters Happiness (1–7) 5.4 (1.0) 5.5 (1.0) 5.4 (0.8) 5.3 (0.9) 0.211 0.013 Anxiety (0–9) 4.0 (2.5) 3.4 (2.6) 4.2 (2.7) 3.7 (2.7) 0.705 0.001 Depression (0–9) 2.1 (2.0) 1.8 (1.8) 2.1 (2.1) 2.0 (1.7) 0.123 0.019 Quality of life (0–100) 75 (14) 80 (16) 70 (16) 72 (17) 0.053 0.030 Sleep quality (0–10) 6.1 (2.2) 6.5 (2.3) 6.1 (1.8) 6.5 (2.0) 0.886 < 0.001 Hypnotic/anxiolytic medication (0–7) 1.1 (2.3) 0.7 (2.1) 0.9 (2.2) 1.1 (2.4) 0.069 0.027 Muscle performance 5-repetition sit to stand test (s) 7.4 (2.3) 7.0 (2.1) 7.5 (2.0) 7.5 (2.2) 0.072 0.028 Kneeling push-up test (repetitions) 3.5 (4.9) 5.6 (6.4) 4.3 (5.5) 4.6 (6.4) 0.040 0.039 Shirado-Ito trunk flexor test (s) 47 (33) 54 (28) 52 (45) 50 (37) 0.077 0.028 Table 2 Effects of the intervention: intention-to-treat (ITT) analysis Journal of Occupational Rehabilitation intensity (p = 0.023), worst intensity (p = 0.017), frequency (p = 0.035) and interference (p = 0.049). Table 4 Effects of the intervention on low back pain outcomes: post hoc subgroup analysis of participants with (≥ 1 in average NRS) and without (< 1 in average NRS) low back pain at baseline Discussion Regarding psycho-affective parameters, the group-by- time interaction was significant for depression (p = 0.021), quality of life (p = 0.002), and hypnotic/anxiolytic medica- tion consumption (p = 0.011) favouring the experimental group in the three parameters.i The main result of this study is that the designed videocon- ference exercise intervention was effective for reducing the low back pain of eldercare workers. Improvements in hand/ wrist pain and upper limb muscle performance were also observed. In the PP analysis, additional benefits were seen on neck pain frequency, depression, quality of life, hyp- notic/anxiolytic medication, and the muscle performance of lower limbs and trunk. These findings provide evidence on an alternative and effective modality for delivering exercise to tackle musculoskeletal disorders. The few minor adverse events, together with the acceptable adherence and low drop- out rate confirm the feasibility of the intervention proposed. Concerning muscle performance, there were significant group-by-time interactions favouring the experimental group in all the performed tests: 5-repetition sit to stand (p = 0.026), kneeling push-up (p = 0.031) and Shirado-Ito trunk flexor (p = 0.030).if l There were not significant group-by-time effects on the remaining variables (p > 0.05). Intervention Effects: PP Analysis There were also significant group-by-time interactions in favour of the experimental group on low back pain worst intensity (p = 0.036), frequency (p = 0.003) and interfer- ence (p < 0.001), as well as on hand/wrist pain average Bold values are statistically significant (p < 0.05) Data are mean (SD) s seconds; no between-group differences in Pre (p > 0.05) Outcome Experimental Control ANCOVA (p) ηp 2 Pre Post Pre Post Low back pain Average intensity (0–10) 3.0 (2.5) 1.9 (2.0) 3.0 (2.4) 3.1 (2.5) 0.011 0.058 Worst intensity (0–10) 3.7 (2.9) 2.6 (2.5) 3.7 (3.0) 3.8 (3.0) 0.036 0.040 Frequency (0–7) 2.6 (2.4) 1.4 (1.7) 2.7 (2.2) 2.8 (2.4) 0.003 0.082 Interference (0–7) 0.9 (1.7) 0.6 (1.2) 1.2 (1.8) 1.7 (2.0) < 0.001 0.108 Neck pain Average intensity (0–10) 2.8 (2.9) 2.1 (2.4) 3.4 (3.1) 2.9 (2.6) 0.106 0.024 Worst intensity (0–10) 3.3 (3.2) 2.5 (2.7) 4.0 (3.4) 3.4 (3.0) 0.117 0.023 Frequency (0–7) 2.5 (2.4) 1.6 (1.9) 2.9 (2.6) 2.6 (2.4) 0.039 0.039 Interference (0–7) 1.4 (2.2) 1.0 (1.7) 1.5 (2.1) 1.7 (2.3) 0.093 0.026 Shoulder pain Average intensity (0–10) 2.4 (2.7) 1.8 (2.4) 3.0 (2.9) 2.4 (2.7) 0.475 0.005 Worst intensity (0–10) 2.6 (2.9) 2.1 (2.9) 3.7 (3.3) 2.8 (3.0) 0.700 0.001 Frequency (0–7) 2.4 (2.5) 1.5 (2.1) 2.9 (2.6) 2.2 (2.4) 0.341 0.008 Interference (0–7) 1.3 (2.2) 0.9 (1.8) 1.6 (2.1) 1.6 (2.2) 0.113 0.023 Hand/wrist pain Average intensity (0–10) 2.4 (2.6) 1.3 (2.2) 3.0 (3.1) 2.5 (2.9) 0.010 0.060 Worst intensity (0–10) 2.6 (2.8) 1.5 (2.4) 3.3 (3.5) 3.1 (3.4) 0.009 0.061 Frequency (0–7) 2.0 (2.2) 1.3 (1.9) 2.6 (2.8) 2.5 (2.7) 0.023 0.047 Interference (0–7) 1.3 (2.0) 0.9 (1.8) 1.9 (2.5) 1.9 (2.4) 0.058 0.033 Pain medication (0–7) 2.2 (2.3) 1.9 (2.2) 2.2 (2.6) 2.6 (2.7) 0.074 0.029 Psycho-affective parameters Happiness (1–7) 5.5 (0.9) 5.7 (0.9) 5.4 (0.8) 5.3 (0.9) 0.077 0.029 Anxiety (0–9) 3.7 (2.3) 2.9 (2.4) 4.2 (2.7) 3.7 (2.7) 0.274 0.011 Depression (0–9) 2.0 (1.9) 1.5 (1.8) 2.1 (2.1) 2.0 (1.7) 0.021 0.049 Quality of life (0–100) 76 (13) 83 (12) 70 (16) 72 (17) 0.002 0.084 Sleep quality (0–10) 6.3 (2.2) 6.6 (2.3) 6.1 (1.8) 6.5 (2.0) 0.946 < 0.001 Hypnotic/anxiolytic medication (0–7) 0.8 (2.0) 0.3 (1.4) 0.9 (2.2) 1.1 (2.4) 0.011 0.059 Muscle performance 5-repetition sit to stand test (s) 7.1 (1.6) 6.7 (1.3) 7.5 (2.0) 7.5 (2.2) 0.026 0.047 Kneeling push-up test (repetitions) 3.5 (5.1) 5.9 (6.6) 4.3 (5.5) 4.6 (6.4) 0.031 0.047 Shirado-Ito trunk flexor test (s) 50 (35) 58 (28) 52 (45) 50 (37) 0.030 0.046 Journal of Occupational Rehabilitation intensity (p = 0.010), worst intensity (p = 0.009) and fre- quency (p = 0.023), and neck pain frequency (p = 0.039).f Effects on Low Back Pain Among participants with low back pain at baseline, the group-by-time ANCOVA showed a significant effect of the intervention favouring the experimental group on average intensity (p = 0.036) (Table 4). There were also significant group-by-time interactions favouring the experimental group in low back pain frequency (p = 0.005) and interference (p = 0.001).if Improvements in low back pain were consistent in both the ITT and PP analyses. Moreover, the post-hoc subgroup analysis confirmed that the intervention was also effective for the treatment of participants who already had low back pain at baseline. The low number of workers without low back pain at baseline did not allow us to draw firm conclu- sions regarding the preventive capacity of the intervention for the development of low back pain. While the potential for low back pain reduction of traditional in-person exer- cise has been well established [11], this study confirms this beneficial effect could also be achieved by videoconference- supervised group exercise. We hypothesised that this posi- tive result might be due to the achieved volume and intensity of the intervention and the effective supervision of partici- pants through a remote modality. Besides, it is important to There were not significant group-by-time effects of the intervention among participants without low back pain at baseline (p > 0.05). Effects on Low Back Pain Table 4 Effects of the intervention on low back pain outcomes: post hoc subgroup analysis of participants with (≥ 1 in average NRS) and without (< 1 in average NRS) low back pain at baseline Bold values are statistically significant (p < 0.05) Data are mean (SD) No between-group differences in Pre (p > 0.05) a Experimental n = 49, Control n = 48 b Experimental n = 14, Control n = 14 Outcome Experimental Control ANCOVA (p) ηp 2 Pre Post Pre Post With low back paina Average intensity (0–10) 4.3 (1.8) 2.9 (2.2) 3.9 (2.0) 3.5 (2.4) 0.036 0.046 Worst intensity (0–10) 5.2 (2.2) 3.7 (2.7) 4.7 (2.5) 4.3 (2.8) 0.121 0.025 Frequency (0–7) 3.8 (2.0) 2.3 (1.9) 3.4 (2.0) 3.3 (2.1) 0.005 0.080 Interference (0–7) 1.6 (2.2) 1.0 (1.7) 1.5 (2.0) 1.9 (1.8) 0.001 0.116 Without low back painb Average intensity (0–10) 0.0 (0.0) 0.6 (1.0) 0.0 (0.0) 1.6 (2.5) 0.331 0.036 Worst intensity (0–10) 0.0 (0.0) 0.9 (1.4) 0.0 (0.0) 2.1 (3.0) 0.340 0.035 Frequency (0–7) 0.0 (0.0) 0.4 (0.6) 0.0 (0.0) 1.2 (2.6) 0.548 0.014 Interference (0–7) 0.0 (0.0) 0.3 (0.6) 0.0 (0.0) 1.0 (2.5) 0.646 0.008 Bold values are statistically significant (p < 0.05) Data are mean (SD) No between-group differences in Pre (p > 0.05) a Experimental n = 49, Control n = 48 bExperimental n=14, Control n=14 Outcome Experimental Control ANCOVA (p) ηp 2 Pre Post Pre Post With low back paina Average intensity (0–10) 4.3 (1.8) 2.9 (2.2) 3.9 (2.0) 3.5 (2.4) 0.036 0.046 Worst intensity (0–10) 5.2 (2.2) 3.7 (2.7) 4.7 (2.5) 4.3 (2.8) 0.121 0.025 Frequency (0–7) 3.8 (2.0) 2.3 (1.9) 3.4 (2.0) 3.3 (2.1) 0.005 0.080 Interference (0–7) 1.6 (2.2) 1.0 (1.7) 1.5 (2.0) 1.9 (1.8) 0.001 0.116 Without low back painb Average intensity (0–10) 0.0 (0.0) 0.6 (1.0) 0.0 (0.0) 1.6 (2.5) 0.331 0.036 Worst intensity (0–10) 0.0 (0.0) 0.9 (1.4) 0.0 (0.0) 2.1 (3.0) 0.340 0.035 Frequency (0–7) 0.0 (0.0) 0.4 (0.6) 0.0 (0.0) 1.2 (2.6) 0.548 0.014 Interference (0–7) 0.0 (0.0) 0.3 (0.6) 0.0 (0.0) 1.0 (2.5) 0.646 0.008 Journal of Occupational Rehabilitation (compared to 20 in our PP analysis) found no effects on depression [34] and quality of life [34, 41]. Strengths and Limitations The main strength of the present study lies in its RCT design with outcome assessor and data analyst blinding, as well as a proper sample size calculation. Study procedures and inter- vention characteristics are thoroughly described, allowing easy replicability. Moreover, the unrestrictive selection crite- ria, together with the simple exercises and few cheap materi- als used, confer the study a pragmatic nature. This allows the results be generalisable to what could happen in a real-world setting and facilitates scalability, what has been asserted as a priority in physical activity research [47]. However, it should be admitted that this could not apply to world regions in which access to the Internet and videoconference technolo- gies is not yet widespread. Regarding participant retention throughout the study, the low dropout rate suggests high acceptability and feasibility of the intervention. In this sense, Effects on Neck, Shoulder and Hand/Wrist Pain Regarding the remaining pain locations, it should be noted that the ITT analysis demonstrated improvements in all hand/wrist pain outcomes. To our knowledge, this is the first study analysing the effects of exercise on hand/wrist pain in eldercare workers. These findings are important because hand/wrist pain is highly prevalent in eldercare workers [5]. Although the hand/wrist area was not directly targeted in our exercises, it is possible that the stabilising isometric contractions of the wrist required during upper limb exercises, which have been shown to reduce wrist pain [39], were the reason for satisfactory outcomes. Concern- ing neck pain, improvements were only observed for pain frequency in the PP analysis, and no significant effects were found on shoulder pain. To our knowledge, the only RCT assessing the effects of an exercise intervention on neck- shoulder pain of eldercare workers was the one by Horneij et al., and they found no between-group differences [33]. Although exercise can be generally considered effective for the management of neck-shoulder disorders, interventions utilizing specific resistance training seem to obtain better results in comparison with other modalities such as general resistance training or general physical exercise [40]. Even though further research is needed [40], it is possible that our exercise program did not include enough specific neck- shoulder exercises. Effects on Low Back Pain The reduction in the use of hypnotic/anxiolytic medications in the interven- tion group appears to be relevant because a recent prospec- tive study with an 11-year follow-up among almost 8,000 eldercare workers found that the use of hypnotic/anxiolytic/ sedative medication increased the risk of disability pension and mortality [42]. In addition, keeping a good psychologi- cal health of eldercare workers could be important not only for ensuring their wellbeing but also for guaranteeing a high- quality care for the older people. In this regard, previous research has shown that a higher caregiver burden predicts a greater hospitalization risk of the older person [43], and burnout symptoms in nurses are related to lower quality of care [44]. note that in eldercare workers, RCTs assessing the effects of exercise on low back pain have been scarce, all limited to the face-to-face modality, and they have found conflicting results [33–36]. However, as opposed to our study, the majority of those RCTs lack an extensive description of the interven- tion, particularly regarding exercise content and criteria for progression or intensity adjustment, which makes it difficult to draw conclusions regarding the reasons explaining the dis- cordant results. Reducing low back pain in eldercare workers could have a great impact, as low back pain has shown to be a significant risk factor for increased disability [37], lowered quality of life [37] and greater risk of long-term sickness absence [38] in this population. Effects on Muscle Performance Regarding muscle performance, although the ITT analysis only showed a significant improvement in upper limb per- formance, trunk and lower limb performance also improved in the PP analysis. From a biomechanical point of view, cur- rent literature supports the idea that improvements in the structure and function of the musculoskeletal system, spe- cially muscle strength, could contribute to the pain reduction induced by exercise [45]. While improvements in muscle performance may seem obvious and expected in exercise trials, their potential impact should not be neglected. In the context of this study, a higher physical capacity could permit eldercare workers confront their daily tasks in a less strenu- ous and safer manner, therefore reducing the risk of suffering an injury or developing/increasing pain. As a consequence, a better balance between intrinsic personal resources and extrinsic job demands could be achieved, what could lead to a better health state of the workers [46]. Conclusions The group exercise intervention carried out by real- time videoconference was effective for reducing the low back pain of eldercare workers. Improvements in hand/ wrist pain and upper limb muscle performance were also observed. The results from the PP analysis suggest that a higher adherence to the intervention could lead to addi- tional benefits in psycho-affective parameters, medication consumption and muscle performance. To our knowledge, this is the first group exercise intervention conducted by videoconference in the workplace, which provides an evi- dence-based alternative modality of exercise delivery to tackle musculoskeletal disorders. i On the other hand, some limitations should be acknowl- edged. For example, despite the unrestrictive selection cri- teria, the participation rate was of only 30% (130 out of 436 participants assessed for eligibility). This could be rel- evant because the characteristics of the participants could differ from those who rejected participation and therefore the effects of the intervention might not be the same in the latter. One possible reason to explain our low participa- tion rate could be that the exercise program was carried out outside working hours, what was found to be one of the main barriers for participation in a health promotion program in healthcare workers [49]. Apart from rising par- ticipation rates, the additional benefits observed in the PP analysis make evident the necessity of finding strategies to improve adherence. With respect to the PP analysis, it should be acknowledged that the randomisation effect is lost due to excluding some participants based on their level of adherence to the intervention. Therefore, although one may tend to attribute the additional improvements found in the PP analysis to the higher adherence, it cannot be ruled out that the participants with ≥ 50% or < 50% adherence had different characteristics at baseline (e.g., motivation or expectation to improve) and that these could have inter- fered in the findings observed. Also, the inherent impos- sibility of exercise trials to blind participants could have biased the results obtained. Regarding statistical power, the secondary outcomes of the present study could be not powered enough, and new studies might be necessary to draw more reliable conclusions. Besides, the single-item sleep quality scale used in the present study has not been cross-culturally validated in Spanish yet. Although it is a numerical scale containing minimum text, it should be acknowledged that we translated it from the original Eng- lish version. Effects on Psycho‑Affective Parameters With respect to psycho-affective parameters, although the ITT analysis did not show any significant effect of the intervention, the PP analysis demonstrated improvements in depression and quality of life and a reduction in hyp- notic/anxiolytic medication use. That is, improvements in psycho-affective parameters were only observed when ana- lysing participants with ≥ 50% adherence separately. In this regard, previous RCTs in eldercare workers in which average attendance was of only 8 [34] and 12 [41] exercise sessions Journal of Occupational Rehabilitation applicable to other populations. Similarly, the great major- ity of female participants in the present study, while reflec- tive of the reality of the eldercare sector, could also limit the applicability of the findings to male eldercare workers. and based on the technical adverse events found, ensuring a high-quality connection and a good familiarisation with technology seems key for a satisfactory delivery of exercise via videoconference. Regarding outcomes, pain was studied from a broad perspective, including diverse pain character- istics and locations, as well as other pain-related variables such as medication, psycho-affective parameters and muscle performance. Despite their key contribution to pain from a biopsychosocial point of view, these variables have been understudied in low back pain research, and their inclusion in future exercise trials has been urged [48]. Finally, we only included eldercare workers. This could be important because most exercise trials in this population have merged other professionals such as nurses, physiotherapists or midwives in their samples [33–36, 41], what limits the applicability of the findings to the eldercare workers. Conclusions Finally, although including only eldercare workers can be considered a strength, it should also be recognised that due to the specific characteristics of the sample, the findings of this study could not be directly Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s10926-024-10182-2. Acknowledgements We would like to thank all the eldercare institu- tions that participated in the study: Caser Residencial Betharram, Fun- dación Aspaldiko, Grupo Servicios Sociales Integrados, IMQ Igurco Orue, IMQ Igurco Azkuna, and Grupo Colisée. Author Contributions AE, JI and ARL conceptualized the study and designed its methodology. AE coordinated the intervention and drafted the manuscript. JI and ARL managed the project, obtained funding and analysed data. MA, ULE and JGG collected data. IA and IS recruited participants, provided facilities and helped with data acquisition. All authors revised the final manuscript critically, approved its submission and are accountable for all aspects of the work. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was funded by the Basque Government (IT1538‑22 and PRE_2021_2_0056) and the University of the Basque Country (GIU20/06). 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Health-related quality of life and sick- ness absence in community nursing home employees:
https://openalex.org/W2788898308	https://www.e3s-conferences.org/10.1051/e3sconf/20183201023/pdf	English	null	Comparison of indoor air pollutants concentration in two Romanian classrooms	E3S web of conferences	2,018	cc-by	3,940	1 Introduction combustion. It is interesting to note that, the same high concentrations are found in the bedrooms leading to the conclusion that these spaces are deficient in terms of ventilation and of the assurance of necessary fresh air. Also, the average concentration values of carbon dioxide, monitored in office spaces during March-April 2012 (National Project PN 09 14 04 02, 2012) [11] were between 661 ppm and 1,018 ppm, those recorded in the period of February-March 2015, ranged between 888 and 1,668 ppm (Vasile et al., 2017) [12], and those monitored in November 2015, inside the spaces for the educational process in higher education (chemistry lab), were between 912 ppm and 990 ppm (Vasile et al., 2016) [5-9], over the permissible limit of 800 ppm [10]. Air quality of indoor environments draw considerable attention from the public as well as from the researchers [1] because relatively higher pollutant concentrations in combination with longer time spent indoors can result in higher exposures and can produce adverse health effects (Ugranli et al., 2015, Zhong et al., 2017) [2, 3]. All over the world [3], indoor monitoring of air compounds is an ongoing challenge (Ho et al., 2016) [4], numerous studies being in progress, whether it is office or dwelling buildings or schools [3, 5-9, 10]. g [ ] Educational institutions are one of the most studied indoor environments with a focus on kindergartens and primary or high schools, because of the high density population potentially vulnerable who are still growing, making them especially susceptible to the effects of pollution (Ugranli et al., 2015, Zhong et al., 2017) [2, 3]. Our previous studies relating the monitoring of indoor air from different type of spaces showed high values of the carbon dioxide concentrations in the most of cases. For example, the average values of carbon dioxide concentration monitored during three experimental campaigns in residential spaces, first in March-May 2014, ranged between 898 ppm and 1,306 ppm, the second in September-October 2014, with average values between 811 ppm and 3,111 ppm and the third in March- April 2015, with values between 1,128 ppm and 2,490 ppm (Vasile et al., 2016) [5-9]. It appears that in all three monitored residential spaces there are high concentrations of carbon dioxide, which exceeded up to three times the limit allowed in indoor air. Comparison of indoor air pollutants concentration in two Romanian classrooms Vasilica Vasile1, Alina Dima1, Elena Zorila2,3, Andrei Istrate2,3, Tiberiu Catalina2,3 1INCD URBAN-INCERC Bucharest, Laboratory PFCH Pantelimon, 266, Bucharest, Romania 2Technical University of Civil Engineering Bucharest Blvd. Pache Protopopescu 66, Bucharest, Romania 3Babes-Bolyai University, Faculty of Environmental Science and Engineering 30, Fântânele Street, Cluj Napoca Vasilica Vasile1, Alina Dima1, Elena Zorila2,3, Andrei Istrate2,3, Tiberiu Catalina2,3 1INCD URBAN-INCERC Bucharest, Laboratory PFCH Pantelimon, 266, Bucharest, Romania 2Technical University of Civil Engineering Bucharest Blvd. Pache Protopopescu 66, Bucharest, Romania 3Babes-Bolyai University, Faculty of Environmental Science and Engineering 30, Fântânele Street, Cluj Napoca Abstract. This paper investigates the air pollutions in space ventilated in two High School classrooms. The analysis consists of comparison of one classroom with hybrid ventilation system and another one stander-by classroom with natural ventilation. Several studies regarding indoor air quality during the experimental campaign have been done for VOC, CO2, CO, other pollutants, keeping monitored for humidity and temperature. The experimental demonstrated that the highest value for CO2 in stander-by classroom is 2691 ppm and in classroom with hybrid ventilation is 1897 ppm, while values for CO are 1.1 / 1.1 ppm and VOC 0.14 / 0.06 ppm, better use hybrid ventilation. * Corresponding author: tiberiu.catalina@gmail.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). ished by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attributi ivecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/20183201023 https://doi.org/10.1051/e3sconf/20183201023 E3S Web of Conferences 32, 01023 (2018) EENVIRO 2017 * Corresponding author: tiberiu.catalina@gmail.com 1 Introduction These high values of the concentrations of carbon dioxide in the space of a kitchen are justified since the cooking activities results in generation of carbon dioxide by Average concentrations of carbon dioxide between 1,720 ppm and 4,302 ppm (National Project PN 09 14 04 02, 2015) [13, 14], two to five times the tolerable limit of 800 ppm, as determined by OSHA [10], were recorded in the investigated educational sites (kindergartens, secondary and high schools) during September-October 2015, and when talking about spaces where children are working, ages ranging from 3 to 17 years, have a particularly worrying situation with a strong negative impact on the educational process, which must be an alarm signal. Carbon dioxide present in indoor air can have negative effects on the concentration and attention of children, thus affecting the learning process. It can also cause a drowsiness or headache. Due to their abundant nature, Volatile Organic Compounds (VOCs) are crucial parameters for assessing air quality both internally and externally environment. This class of compounds includes a wide range of air pollutants with a significant impact on atmospheric air and human health (Kumar et al., 2014, Lerner et al., 2012, Matsumoto et al., 2010) [15, 16, 17]. The chemical E3S Web of Conferences 32, 01023 (2018) EENVIRO 2017 https://doi.org/10.1051/e3sconf/20183201023 variety of a VOC group constitutes the cause of negative impact factor on human health, ranging from noncancerous to carcinogenic effects (Kumar et al., 2014; Lerner et al., 2012; Srivastava et al., 2005; Ramirez et al., 2012) [15, 16, 18, 19]. m3/h. During the experimental measurements the fresh air flow was set at 450 m3/h or approx. 20 m3/h/student. The extraction of the air is realized using an extraction outlet mounted in the classroom door. pp The extraction of the air is realized using an extraction outlet mounted in the classroom door. Fig. 2. Photos with the window fan + hygro-adjustable grids and the extraction outlets in the door 2 Study case The study presents the results obtained from the monitoring campaign conducted at “Mihai Viteazul” High School from Bucharest, Romania and is based on the comparative analysis of two different classrooms, one equipped with a mixed hybrid air ventilation system (I-17) and the other one does not have an air ventilation system (I-16). These two classrooms are located on the first floor of the building, consisting of ground floor and two levels, put into use in 1926. Fig. 2. Photos with the window fan + hygro-adjustable grids and the extraction outlets in the door The proposed system is functioning only if the hygroscopic air vents mounted in the windows frame don’t allow sufficient fresh flow. These air vents are Eha2 Aereco hygro-adjustable grids and six of them were installed allowing a maximum fresh air flow of 180-200 m3/h depending on the pressure difference indoor/outdoor and indoor humidity. Fig. 1. Classroom with air ventilation system and detail of the equipment’s location. 2.1. Monitoring method The volatile organic compounds, benzene, toluene and formaldehyde have been selected based on their effects on human health. Previous studies mention that the breathing benzene can cause the onset of illness, accompanied by slight irritation of the eyes and mucous membranes. If benzene is aspirated directly into the lungs it can lead to acute haemorrhagic pneumonitis. Major concerns benzene toxicity includes anaemia and acute leukaemia. Also, benzene is a known carcinogen while toluene is toxic at high concentrations (Khoder, 2006) [20]. Sources of benzene concentrations may be paints, furniture wax, lubricants and glues (Kumar et al., 2014) [15]. Formaldehyde is the simplest and most common aldehyde found in the environment and is perhaps the most important pollutant of the indoor environment due to its frequency of occurrence and the irritating potentially it has over the occupants (Occupational Health Guideline for Formaldehyde) [21], being toxic and irritating to the respiratory tract, eyes and skin and carcinogenic to humans at high concentrations (Khoder, 2006) [20]. Indoor concentrations of formaldehyde vary and are influenced by temperature, humidity, ventilation rate, building age, presence of combustion sources, type of indoor activity, etc. Fig. 1. Classroom with air ventilation system and detail of the equipment’s location. Fig. 1. Classroom with air ventilation system and detail of the equipment’s location. The two classrooms are oriented to the south and the volume of this was 299.88 m3. The interior finishes were consisted of: layered laminate parquet for flooring, water dispersion paint for wall and vinyl polychloride joinery with thermo-insulating glass for windows. The heating of spaces is normally carried out by the centralized heat supply system using steel radiators with a length of 2,000 mm. During of entire measuring period, the heating system was not active. The furniture elements are specific to classrooms and are made of MDF (medium density fibreboard) panels. Moreover, in the space equipped with the air ventilation system (I-17), are present the electronic equipment’s (computer, projector) and decorative plants. The number of occupants varied in these two classrooms, reaching a maximum value of 30 persons. All the measurements were performed in accordance with a standardized method based on the standards EN1525 and World Health Organisation -Guidelines for Indoor Air Quality: Selected Pollutants), using a monitoring protocol, which includes the specifications regarding the location area of equipment’s installation (adjacent from the ventilation system) and the setting of the chemical compounds and the monitoring interval. 2.1. Monitoring method The monitoring process of benzene and toluene was conducted using a real time detection method with GrayWolf DirectSense IQ-610 portable data-logging All the measurements were performed in accordance with a standardized method based on the standards EN1525 and World Health Organisation -Guidelines for Indoor Air Quality: Selected Pollutants), using a monitoring protocol, which includes the specifications regarding the location area of equipment’s installation (adjacent from the ventilation system) and the setting of the chemical compounds and the monitoring interval. For the first classroom (I-17) with ventilation systems, the main component of the ventilation system was the Xpelair GX9 fan with application for schools. The diameter of the ventilator fan is of 266 mm in reversible range and with enclosed external rotor motors. The system is controlled by an automatic module that allow the modification of the fresh air flow from 150 to 600 The monitoring process of benzene and toluene was conducted using a real time detection method with GrayWolf DirectSense IQ-610 portable data-logging 2 2 E3S Web of Conferences 32, 01023 (2018) EENVIRO 2017 https://doi.org/10.1051/e3sconf/20183201023 detector. In this case, the device is based on a photo- ionization detector (PID) for VOCs (Vasile et al., 2011) [22]. detector. In this case, the device is based on a photo- ionization detector (PID) for VOCs (Vasile et al., 2011) [22]. The contribution of this solution for air ventilation is also observed in the recorded values for the concentrations of the total volatile organic compounds, the mean values being lower compared to the non- ventilated space. Also, in our previous experimental campaigns in kindergartens, TCOV concentration had the highest average value of 0.52 ppm (minimum 0.23 ppm – maximum 0.85 ppm), in isobutylene units 1,193.349 μg/m3 (527.82-1,950.659 μg/m3), while in secondary or high schools, it recorded average values of 1.11 ppm (2,547.329 μg/m3), with a minimum of 0.18 ppm (413.08 μg/m3) and a maximum of 1.85 ppm (4,199.649 μg/m3) (Vasile et al., 2016c) [9]. For detection of formaldehyde present in the indoor air, we used a gas detector whose principle of operation is based on photoelectric photometry that involves the existence of a standard pill containing the test paper treated with special chemicals, an illuminating agent and a light beam. When gas is blown onto the standard pill, the paper emits illumination by chemical reaction and this causes the paper to change colour. 2.1. Monitoring method The amount of colour change is determined by the level of formaldehyde exposure and the time of exposure. In this campaign, the sampling time used for formaldehyde monitoring was 15 minutes. In every classroom, the level of formaldehyde concentration was equal (<0.01 ppm) and below the permissible limit. ( , μg ) ( , ) [ ] The mean concentration of TVOC recorded in our previous campaign in office spaces was between 0.03 ppm and 0.3 ppm (69.1-690.8 μg/m3 isobutylene units, at 24 °C and 1 atm (Vasile et al., 2015) [23, 24], in residential spaces, the values ranged between 0.16 ppm and 0.36 ppm (Vasile and Dima, 2015) [23, 24], and in a chemistry lab was recorded an average of 0.25 ppm (0.20 ppm minimum - maximum 0.35 ppm), isobutylene units 573.72 μg/m3 (minimum 458.97 μg/m3 - maximum 803.21 μg/m3) (Vasile et al., 2016a) [7]. Our results are comparable with the similar studies conducted in earlier years. Recently, Kumar et al., 2014 [15] reported the mean concentration of TVOC as 465.8 μg/m3 (145.3– 1,503.2 μg/m3) in winter and 321.8 μg/m3 (90.7–1,100.9 μg/m3) in summer for indoor air of the library of Jawaharlal Nehru University, New Delhi, a range of 100 and 538 μg/m3 for TVOC concentrations was reported in indoor air of Japanese University (Hori et al., 2012) [25]. Sarkhosh et al., 2012 [26] measured the concentrations in range from 113.4 to 486.3 ppm (227–973 μg/m3 isobutylene units) in photocopy centres and Chan et al., (2009) [27] recorded TVOC concentrations in new hotels guest rooms of the factory region which varied between 416 and 2,900 μg/m3. The volatile organic compounds have been measured continuously throughout the course hours starting with 7:41 AM, and recording the concentrations every minute. Along with the measurements of the volatile organic compounds, have also been monitored indoor environmental parameters by measuring carbon monoxide and carbon dioxide concentrations, temperature and humidity. We chose to monitor the concentration of carbon monoxide next to carbon dioxide because an accumulation of this odourless, colourless gas in indoor air can be toxic for human body due to the affinity for combination with hemoglobin and forming carboxyhemoglobin (COHb) which leads to disrupting oxygen transport. This parameter was considered an indicator of indoor air quality. In this case the source of carbon monoxide is only from outdoor air into the indoor environment. 2.1. Monitoring method This parameter was considered an indicator of indoor air quality. In this case the source of carbon monoxide is only from outdoor environment. Benzene concentration of 0.1 ppm recorded in this campaign is at the minimum level registered in kindergartens (between 0.1 ppm and 0.5 ppm) (National Project PN 09 14 04 02, phase no. 22, 2015) [13] and another secondary high schools from Bucharest (between 0.1 ppm and 0.9 ppm). Indoor air parameters monitoring was carry out by using several measuring principles, presented in table 1. Before the starting of the measurements, it was performed the calibration of the equipment. Table 1. Measuring principles for detection indoor air parameters Parameter Measuring principle Range Accuracy Carbon monoxide (CO2) Electrochemical 0÷500 ppm ±2 ppm Carbon dioxide (CO) NDIR 0÷10,000 ppm ±50 ppm Relative humidity Capacitive probe 0÷100 % ±2% ÷ ±3% Temperature Thermal resistance Pt100 -25° ÷ +70°C ±0.3°C 3 Results and interpretation Table 1. Measuring principles for detection indoor air parameters Our previous studies showed levels of benzene mean concentration of 0.3 ppm (958.4 μg/m3) in residential spaces and 0.2 ppm (638.9 μg/m3) in office spaces (Vasile et al., 2016b) [8], within PELs, and 0.13 ppm (415.30 μg/m3), with a minimum of 0.10 ppm (319.59 μg/m3) and a maximum of 0.20 ppm (638.94 μg/m3) in a chemistry lab (Vasile et al., 2016a) [7], but were higher than those reported in indoor air of Egyptian offices (4.32 ppb) (Khoder, 2006) [20], or in the library of Jawaharlal Nehru University, New Delhi (7.2-12.2 μg/m3) (Kumar et al., 2014) [15], or in indoor air of offices from NCSR “Demokritos”, Aghia Paraskevi, Athens (15.3-17.6 μg/m3) (Saraga et al., 2011) [28]. In the classroom without air ventilation system has been recorded the presence of toluene with an average value of 0.1 ppm and a maximum concentration of 0.2 ppm compared to classroom with ventilation solution, where this compound was not present in indoor air. 3 Results and interpretation 3 https://doi.org/10.1051/e3sconf/20183201023 E3S Web of Conferences 32, 01023 (2018) EENVIRO 2017 Within this article we have realized experimental campaign with the purpose to compare indoor air pollutants in case of two ventilation strategies. Two identical classrooms as shape, volume, about the same number of students, orientation and furniture was compared. 2.1. Monitoring method The difference in comparing indoor air pollutants was the using of hybrid ventilation (natural ventilation with humidity sensitive air vents added with window fan) for one classroom and only natural ventilation for other classroom. In this way, it has been demonstrated that can be obtained good air quality using hybrid ventilation. After the analysis of the measured data using professional equipment with GrayWolf Direct Sense IQ-610 portable data-logging detector we can conclude that it is necessary the introduction even more of fresh air to reduce CO2 limit at 800 ppm in both classroom, more acceptable values obtaining using hybrid ventilation. The same for VOC, CO and for other pollutants have been obtained good permissible exposure values using hybrid ventilation. In these conditions, using hybrid ventilation to achieve more suitable limits for indoor air pollutants concentration can allow more fresh airflow rates. Since the classrooms are similarly from the dimensional point of view and from the finishing products, we can consider a constant contribution of these parameters, the comparison of the recorded values based on the influence of the space ventilation solution. At the end of the monitoring program it was observed that all the specific volatile organic compounds are below of the permissible exposure limits. However, from a comparative point of view, it can be notice the presence of benzene and toluene in classroom without air ventilation system versus the classroom with ventilation solution. This concentration of the specific organic compound is also reflected in the concentration values of total volatile organic compounds. Fig. 3. Comparison between non-ventilated and ventilated classroom for TCOV, Benzene and Toluene. Acknowledgements The research article is supported by the project ID P_37_229, SMIS 103427, Contract No. 22/01.09.2016, with the title „Smart Systems for Public Safety through Control and Mitigation of Residential Radon linked with Energy Efficiency Optimization of Buildings in Romanian Major Urban Agglomerations SMART-RAD- EN” supported by the Competitiveness Operational Program 2014-2020, POC-A.1- A.1.1.4 -E- 2015 competition. References Ocupational Health Guideline for Formaldehyde – National Institute for Occupational Safty and Health, US Department of Health and Human Services [8]. V. Vasile, A. Dima, M. Ion, Monitoring of indoor air pollution in residential and office spaces, 16th International Multidisciplinary Scientific GeoConference SGEM 2016,www.sgem.org, SGEM2016 Conference Proceedings, ISBN 978-619- 7105-64-3 / ISSN 1314-2704, June 28 - July 6, 2016, Book4 Vol. 2, 467-474 pp, 2016b [22]. V. Vasile, A. 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https://openalex.org/W3182740235	https://publikationen.bibliothek.kit.edu/1000140321/133982063	English	null	Chemical composition of nanoparticles from &lt;i&gt;α&lt;/i&gt;-pinene nucleation and the influence of isoprene and relative humidity at low temperature	Atmospheric chemistry and physics	2,021	cc-by	15,199	Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. 15CERN, 1211 Geneva, Switzerland Dipartimento di Fisica, Università di Genova and INFN, 16146 Genoa, Italy 17Department of Atmospheric and Oceanic Sciences, University of Colorado Boulder, Boulder, CO 80309, USA 18 i f d li d h i i i f b k 6020 b k i 17Department of Atmospheric and Oceanic Sciences, University of Colorado Boulder, Boulder, CO 80309, USA 17Department of Atmospheric and Oceanic Sciences, University of Colorado Boulder, Boulder, CO 80309, USA p p y 18Institute for Ion and Applied Physics, University of Innsbruck, 6020 Innsbruck, Austria 19Ionicon Analytik GmbH, 6020 Innsbruck, Austria 20Forest Dynamics, Swiss Federal Institute for Forest, Snow and Landscape Research, 8903 Birm 22Atmospheric Composition Unit, Finnish Meteorological Institute, 00560 Helsinki, Finland 23 23Beijing Weather Modiﬁcation Ofﬁce, 100089 Beijing, China 24 Á 23Beijing Weather Modiﬁcation Ofﬁce, 100089 Beijing, China 24 Á 24IDL, Universidade da Beira Interior, R. Marquês de Ávila e Bolama, 6201-001 Covilhã, Portuga Chemical composition of nanoparticles from α-pinene nucleation and the inﬂuence of isoprene and relative humidity at low temperature Lucía Caudillo1, Birte Rörup2, Martin Heinritzi1, Guillaume Marie1, Mario Simon1, Andrea C. Wagner3, Tatjana Müller1,4, Manuel Granzin1, Antonio Amorim5, Farnoush Ataei6, Rima Baalbaki2, Barbara Bertozzi7, Zoé Brasseur2, Randall Chiu3, Biwu Chu2, Lubna Dada8, Jonathan Duplissy2,9, Henning Finkenzeller3, Loïc Gonzalez Carracedo10, Xu-Cheng He2, Victoria Hofbauer11, Weimeng Kong12,13, Houssni Lamkaddam8, Chuan P. Lee8, Brandon Lopez11, Naser G. A. Mahfouz11, Vladimir Makhmutov14,26, Hanna E. Manninen15, Ruby Marten8, Dario Massabò16, Roy L. Mauldin17,11, Bernhard Mentler18, Ugo Molteni8,20,21, Antti Onnela15, Joschka Pfeifer15, Maxim Philippov14, Ana A. Piedehierro22, Meredith Schervish11, Wiebke Scholz18, Benjamin Schulze12, Jiali Shen2, Dominik Stolzenburg2, Yuri Stozhkov14, Mihnea Surdu8, Christian Tauber10, Yee Jun Tham2, Ping Tian23, António Tomé24, Steffen Vogt7, Mingyi Wang11, Dongyu S. Wang8, Stefan K. Weber15, André Welti22, Wang Yonghong2, Wu Yusheng2, Marcel Zauner-Wieczorek1, Urs Baltensperger8, Imad El Haddad8, Richard C. Flagan12, Armin Hansel18,19, Kristina Höhler7, Jasper Kirkby1,15, Markku Kulmala2,9,25, Katrianne Lehtipalo2,22, Ottmar Möhler7, Harald Saathoff7, Rainer Volkamer3, Paul M. Winkler10, Neil M. Chemical composition of nanoparticles from α-pinene nucleation and the inﬂuence of isoprene and relative humidity at low temperature Donahue11, Andreas Kürten1, and Joachim Curtius1 1Institute for Atmospheric and Environmental Sciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany 2Institute for Atmospheric and Earth System Research (INAR)/Physics, Faculty of Science, 1Institute for Atmospheric and Environmental Sciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany 2Institute for Atmospheric and Earth System Research (INAR)/Physics, Faculty of Science, 3Department of Chemistry & CIRES, University of Colorado Boulder, Boulder, CO 80309-0215, USA 4 Atmospheric Chemistry Department, Max Planck Institute for Chemistry, 55128 Mainz, Germany 5CENTRA and FCUL, University of Lisbon, 1749-016 Lisbon, Portugal 7Institute of Meteorology and Climate Research, Karlsruhe Institute of Technology, 76344 E t i L ld h f G 7Institute of Meteorology and Climate Research, Karlsruhe Institute of Technology, 8Laboratory of Atmospheric Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland 9 8Laboratory of Atmospheric Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland 9 8Laboratory of Atmospheric Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland 9Helsinki Institute of Physics (HIP)/Physics, Faculty of Science, University of Helsinki, 0001 9Helsinki Institute of Physics (HIP)/Physics, Faculty of Science, University of Helsinki, 0001 10Faculty of Physics, University of Vienna, 1090 Vienna, Austria 10Faculty of Physics, University of Vienna, 1090 Vienna, Austria Center for Atmospheric Particle Studies, Carnegie Mellon University, Pittsburgh, PA 15213, USA p g y g 12Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA 13California Air Resources Board, Sacramento, CA 95814, USA hemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA 12Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA 13California Air Resources Board, Sacramento, CA 95814, USA y g g, gy, , , 13California Air Resources Board, Sacramento, CA 95814, USA 13California Air Resources Board, Sacramento, CA 95814, USA 14Lebedev Physical Institute, Russian Academy of Sciences, 119991, Moscow, Russia 15CERN, 1211 Geneva, Switzerland L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17100 25Aerosol and Haze Laboratory, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, China 26Moscow Institute of Physics and Technology, National Research University, 117303, Moscow, Russia Correspondence: Lucía Caudillo (lucia.caudillo@iau.uni-frankfurt.de) and Joachim Curtius (curtius@iau.uni-frankfurt.de) 25Aerosol and Haze Laboratory, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Received: 16 June 2021 – Discussion started: 7 July 2021 Revised: 15 October 2021 – Accepted: 17 October 2021 – Published: 25 November 2021 Received: 16 June 2021 – Discussion started: 7 July 2021 Revised: 15 October 2021 – Accepted: 17 October 2021 – Published: 25 November 2021 Abstract. Biogenic organic precursors play an important role in atmospheric new particle formation (NPF). One of the ma- jor precursor species is α-pinene, which upon oxidation can form a suite of products covering a wide range of volatilities. Highly oxygenated organic molecules (HOMs) comprise a fraction of the oxidation products formed. While it is known that HOMs contribute to secondary organic aerosol (SOA) formation, including NPF, they have not been well studied in newly formed particles due to their very low mass concen- trations. Here we present gas- and particle-phase chemical composition data from experimental studies of α-pinene oxi- dation, including in the presence of isoprene, at temperatures (−50 and −30 ◦C) and relative humidities (20 % and 60 %) relevant in the upper free troposphere. The measurements took place at the CERN Cosmics Leaving Outdoor Droplets (CLOUD) chamber. The particle chemical composition was analyzed by a thermal desorption differential mobility ana- lyzer (TD-DMA) coupled to a nitrate chemical ionization– atmospheric pressure interface–time-of-ﬂight (CI-APi-TOF) mass spectrometer. CI-APi-TOF was used for particle- and gas-phase measurements, applying the same ionization and detection scheme. Our measurements revealed the presence of C8−10 monomers and C18−20 dimers as the major com- pounds in the particles (diameter up to ∼100 nm). Particu- larly, for the system with isoprene added, C5 (C5H10O5−7) and C15 compounds (C15H24O5−10) were detected. This ob- servation is consistent with the previously observed forma- tion of such compounds in the gas phase. However, although the C5 and C15 compounds do not easily nucleate, our mea- surements indicate that they can still contribute to the particle growth at free tropospheric conditions. For the experiments reported here, most likely isoprene oxidation products en- hance the growth of particles larger than 15 nm. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Additionally, we report on the nucleation rates measured at 1.7 nm (J1.7 nm) and compared with previous studies, we found lower J1.7 nm values, very likely due to the higher α-pinene and ozone mix- ing ratios used in the present study. https://doi.org/10.5194/acp-21-17099-2021 2.1 The CLOUD chamber at CERN and the experiments Kiendler-Scharr et al. (2009) presented observations at 15 ◦C of a signiﬁcant decrease in particle number and volume concentration by the presence of isoprene in an experiment under plant-emitted VOC conditions. Subsequently, McFig- gans et al. (2019) showed that isoprene, carbon monoxide, and methane can each suppress aerosol mass and the yield from monoterpenes in mixtures of atmospheric vapors. Re- cently, a study by Heinritzi et al. (2020) revealed that the presence of isoprene in the α-pinene system suppresses new particle formation by altering the peroxy-radical termination reactions and inhibiting the formation of those molecules needed for the ﬁrst steps of cluster and particle formation (species with 19 to 20 carbon atoms). The measurements took place in the Cosmics Leaving Outdoor Droplets (CLOUD) chamber at the European Organization for Nuclear Research (CERN) during the CLOUD14 campaign (September–November 2019). The CLOUD chamber is a stainless-steel cylinder, with a vol- ume of 26.1 m3, which has been built to the highest technical standards of cleanliness (Kirkby et al., 2011; Duplissy et al., 2016). By precisely controlling several parameters, such as gas concentrations, temperature, relative humidity, ultravio- let light intensity, and internal mixing, speciﬁc atmospheric systems can be recreated in order to study the nucleation and growth processes of aerosols at atmospheric conditions. The biogenic gas concentrations, here α-pinene and isoprene, can be regulated by using individual evaporator supplies, in which dry nitrogen passes through the evaporator containing the precursors in a liquid form, at controlled temperature. In this way, the precursors are evaporated and diluted with clean air to achieve the desired concentration in the chamber. Ozone is introduced via a separate gas line. The chamber is continuously stirred by two magnetically coupled stainless- steel fans placed at the top and at the bottom of the cham- ber to provide a homogeneously mixed system (Voigtländer et al., 2012). In order to promote particle production from ions, galactic cosmic ray (GCR) conditions can be achieved by turning off the high voltage ﬁeld (30 kV m−1). The equi- librium ion-pair concentration in the chamber due to GCR is around 700 cm−3 (Kirkby et al., 2016). Despite the difﬁculties in measuring the nanoparticle chemical composition due to their very small mass, there have been several efforts for designing and improving tech- niques to face this problem. Some particle-phase studies ex- ist that report the chemical composition of newly formed nanoparticles. For instance, Kristensen et al. 2 Methods pensates for this effect by increasing the nucleation rates at lower temperatures. 2.1 The CLOUD chamber at CERN and the experiments (2017), mea- suring at −15 and +20 ◦C, showed an increased contri- bution of less oxygenated species to α-pinene SOA parti- cles formed from ozonolysis at sub-zero temperatures. Ye et al. (2019) measured the particle-phase chemical compo- sition from α-pinene oxidation between −50 and +25 ◦C with the FIGAERO inlet (Lopez-Hilﬁker et al., 2014). They found that during new particle formation from α-pinene ox- idation, gas-phase chemistry directly determines the compo- sition of the condensed phase. Highly oxygenated organic molecules are much more abundant in particles formed at higher temperatures, shifting the compounds towards higher O : C and lower volatilities. Additionally, some studies ad- dressing the chemical composition, volatility, and viscosity of organic molecules have provided important insights into their inﬂuence on the climate (Huang et al., 2018; Reid et al., 2018; Champion et al., 2019). The experiments relevant for this work were done under GCR conditions and in a ﬂow-through mode with continu- ous addition of the reactants, performed at −50 and −30 ◦C, at low and high relative humidity to simulate pure biogenic new particle formation at a range of free tropospheric condi- tions. Isoprene and α-pinene precursor gases were oxidized with O3 and ·OH (produced from O3 photolysis in the pres- ence of H2O and UV light) to induce both dark ozonoly- sis and photochemistry oxidation reactions. The α-pinene level was between 1 and 8 ppbv, the isoprene level up to 30 ppbv, and O3 approximately 100 ppbv. The ozonolysis of α-pinene was performed at −50 and −30 ◦C, while the α-pinene + isoprene experiment was performed at −30 ◦C only. The experimental overview is discussed in more detail in Sect. 3.1. Here, we present the results from gas- and particle-phase chemical composition measurements for a system where α- pinene was oxidized to simulate pure biogenic new particle formation at free tropospheric conditions in a range from −50 to −30 ◦C. The data are further compared to the mixed system of α-pinene and isoprene in order to better understand the partitioning processes. The particle chemical composi- tion was analyzed by a thermal desorption differential mobil- ity analyzer (TD-DMA) (Wagner et al., 2018), coupled to a nitrate chemical ionization–atmospheric pressure interface– time-of-ﬂight (CI-APi-TOF) mass spectrometer. This tech- nique allows for a direct comparison between the gas and particle phase as both measurements are using the identical chemical ionization source and detector. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 1 Introduction Approximately half of the global cloud condensation nuclei (CCN) are produced by nucleation (Merikanto et al., 2009; Gordon et al., 2017). In particular, biogenic emissions of volatile organic compounds (VOCs) play an important role in the formation of aerosol particles. The chemical reactions involving VOCs can lead to the formation of highly oxy- genated organic molecules (HOMs), which can be described as a class of organic compounds that are formed under at- mospherically relevant conditions by gas-phase autoxidation involving peroxy radicals (Ehn et al., 2014; Bianchi et al., 2019). These compounds possess low-saturation vapor pres- sures and are thus relevant for secondary organic aerosol (SOA) formation, including new particle formation (NPF), due to gas-to-particle partitioning. Isoprene (C5H8) has the highest global emission rate, and many studies have demonstrated the importance of isoprene in terms of SOA formation (Surratt et al., 2006, 2007, 2010; Paulot et al., 2009; Lin et al., 2012; Riva et al., 2016). α- Pinene (C10H16), while less abundant, is one of the most commonly observed and prominent contributors to biogenic SOA due to its ability to form HOMs that nucleate on their own under atmospheric conditions (Kirkby et al., 2016; Tröstl et al., 2016). The formation of SOA has been well studied in isoprene and α-pinene systems. The role of HOMs in SOA formation and NPF has also been explored in α- pinene and α-pinene with isoprene systems. However, much less is known about the particle-phase composition of HOMs in these systems and the speciﬁc controls particle formation and growth rates, including as a function of temperature and the ratio of isoprene to α-pinene. Regarding α-pinene studies, Stolzenburg et al. (2018) re- ported α-pinene dark ozonolysis experiments at +25, +5, and −25 ◦C and showed that the rapid growth of organic par- ticles is observed across these temperatures and that higher T leads to a faster autoxidation, while lower T leads to an increased partitioning due to decreased vapor pressures. Fur- thermore, Simon et al. (2020) extended the study of α-pinene gaseous oxidation products to even lower temperatures from +25 to −50 ◦C, showing that the oxygen-to-carbon ratio (O : C) and the yield for HOM formation decrease as the tem- perature decreases, whereas the reduction of volatility com- Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 17101 https://doi.org/10.5194/acp-21-17099-2021 2.3 Nitrate CI-APi-TOF mass spectrometer The gas-phase and the evaporated particulate material were measured using a nitrate chemical ionization–atmospheric pressure interface–time-of-ﬂight (CI-APi-TOF) mass spec- trometer, which has three major components: an atmospheric pressure ion–molecule reactor, where the chemical ionization takes place; an atmospheric pressure interface for transport- ing the charged ions into the mass classiﬁer; and a time-of- ﬂight mass classiﬁer, where the ions are accelerated, sepa- rated according to their mass-to-charge ratio, and detected with a microchannel plate (Jokinen et al., 2012; Kürten et al., 2014). The nitrate CI-APi-TOF mass spectrometer uses nitrate reagent ions (HNO3)n NO− 3 with n = 0–2, which are created by an ion source using a corona discharge nee- dle (Kürten et al., 2011). With this nitrate chemical ioniza- tion technique, sulfuric acid, iodic acid, dimethylamine and HOMs can be detected (Kürten et al., 2014; Simon et al., 2016; Kirkby et al., 2016; He et al., 2021). HOMs are de- tected because of the presence of functional groups such as hydroperoxy (–OOH) or hydroxy (–OH), which provide the hydrogen bonds required for clustering with the reagent ions. 2.4 Nucleation rates The particle number size distribution between ∼1 nm and 1 µm is measured using a suite of particle counters namely a particle size magniﬁer (PSM; Vanhanen et al., 2011), a con- densational particle counter (CPC 3776, TSI), a nano scan- ning mobility particle sizer (nano-SMPS 3982, TSI), and a home-built long scanning mobility particle sizer (long- SMPS). The PSM measures the size distribution between ∼1 and 3 nm as well as the total particle number concentration above a deﬁned cutoff, 1.7 nm in this study. The CPC on the other hand is used to measure the total particle number con- centration above 2.5 nm. The nano-SMPS and long-SMPS together cover the particle number size distribution between 6 nm and 1 µm. The same setup has been used in previous CLOUD experiments; see for example Lehtipalo et al. (2018) and Heinritzi et al. (2020). For the experiments that are reported in this work, a ﬁla- ment of platinum / rhodium (90 : 10) was used, and an inte- gral, non-size-selective mode of operation was chosen in or- der to maximize the mass of collected particles. For desorb- ing the sample, an electric current was applied to the ﬁlament and ramped linearly over a duration of approximately 1 min. Due to the very low experimental temperatures, cold sheath ﬂows and isolated inlet lines were installed in order to avoid drastic temperature changes between the CLOUD chamber and the instrument. Evaporation of particulate material be- fore the active heating should therefore not be substantial. The nucleation rate (Jdp), which is deﬁned as the ﬂux of particles of a certain size, is calculated using the method pro- posed by Dada et al. (2020); see Eq. (9) therein. For this study, the formation of particles with a diameter ≥1.7 nm is calculated (J1.7) using the derivative of the total concen- tration of particles measured with the PSM while accounting for size-dependent losses to the chamber wall, by coagulation or via dilution. The error on J1.7 is 30 % based on run-to-run repeatability (Dada et al., 2020). 2.2 TD-DMA The particle chemical composition was analyzed by a ther- mal desorption differential mobility analyzer (TD-DMA) coupled to a nitrate chemical ionization–atmospheric pres- sure interface–time-of-ﬂight (CI-APi-TOF) mass spectrom- eter. The TD-DMA design and characterization have been https://doi.org/10.5194/acp-21-17099-2021 Atmos. Chem. Phys., 21, 17099–17114, 2021 17102 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation described in detail by Wagner et al. (2018). This instrument allows for the direct comparison between gas- and particle- phase chemical composition as both measurements use the same ionization scheme and mass spectrometer (the detec- tion technique will be described in Sect. 2.3). Here the nitrate CI-APi-TOF mass spectrometer data for the gas and particle phase have been corrected for back- ground signals and the mass-dependent transmission efﬁ- ciency in the mass classiﬁer (Heinritzi et al., 2016). The data analysis and processing were performed using IGOR Pro 7 (WaveMetrics, Inc., USA), Tofware (Version 3.2, Aerodyne Inc., USA) and MATLAB R2019b (MathWorks, Inc., USA). The TD-DMA uses an online and semi-continuous princi- ple for the detection of the chemical composition of nanopar- ticles. The particles are sampled from the chamber and charged with an X-ray source; a speciﬁc size can be se- lected, and immediately afterwards they are electrostatically collected on a ﬁlament. Heating the ﬁlament after a deﬁned collection time evaporates the particles into a stream of clean carrier gas (N2). The particle vapor is analyzed by the ni- trate CI-APi-TOF mass spectrometer (Kürten et al., 2014). In order to estimate the instrumental background, two heat- ing proﬁles are recorded: the ﬁrst heating cycle evaporates all the particulate material collected; a second heating cycle constrains the background due to the heating of the inlet line. All reported particle-phase signals are corrected based on this background measurement. https://doi.org/10.5194/acp-21-17099-2021 3.1 Experimental overview An overview of the experiments performed at −30 and −50 ◦C at low and high relative humidity is shown in Fig. 1. The mixing ratio of ozone was stable at ∼100 ppbv for all of the experiments reported in this work (not shown). In or- der to represent pure biogenic new particle formation events, no other trace gases were added to the chamber, and the lev- els of SO2, NOx, and other trace gases were monitored to remain always below the detection limits of the respective measurement devices. By using the TD-DMA, particles were collected in every NPF system (without resolving the particle size); the shaded area in Fig. 1 refers to the period where the particle collection took place. The upper panel of Fig. 1 displays the size distribution measured by the scanning mobility particle sizer (SMPS). Four different experiments can be categorized as follows: https://doi.org/10.5194/acp-21-17099-2021 Atmos. Chem. Phys., 21, 17099–17114, 2021 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17103 Figure 1. Experimental overview for pure biogenic new particle formation. First panel: particle size distribution for four different experi- ments: α-pinene + isoprene at −30 ◦C and 20 % RH (αIP-30,20), α-pinene at −30 ◦C and 20 % RH (α-30,20), α-pinene at −50 ◦C and 20 % RH (α-50,20), and α-pinene at −50 ◦C and 60 % RH (α-50,60). The color scale represents the log 10 of the normalized particle concentration in cubic centimeters (cm−3). Second panel: particle number concentration in cm−3 measured by the PSM with a cutoff diameter of 1.7 nm and CPC 2.5 nm. Third panel: mixing ratio in parts per billion by volume (ppbv) for the biogenic precursor gases, isoprene, and α-pinene. Fourth panel: evolution of total HOM concentration in molecules per cubic centimeter (molec. cm−3), measured in the gas phase by the nitrate CI-APi-TOF mass spectrometer. The HOM total is deﬁned as the sum of C5, C10, C15, and C20 carbon classes, which are shown as well. Ozone level is not shown though remains stable over the whole period at ∼100 ppbv. The shaded areas refer to the time when the particles were collected using the TD-DMA. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17103 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17103 Figure 1. Experimental overview for pure biogenic new particle formation. 3.1 Experimental overview First panel: particle size distribution for four different experi- ments: α-pinene + isoprene at −30 ◦C and 20 % RH (αIP-30,20), α-pinene at −30 ◦C and 20 % RH (α-30,20), α-pinene at −50 ◦C and 20 % RH (α-50,20), and α-pinene at −50 ◦C and 60 % RH (α-50,60). The color scale represents the log 10 of the normalized particle concentration in cubic centimeters (cm−3). Second panel: particle number concentration in cm−3 measured by the PSM with a cutoff diameter of 1.7 nm and CPC 2.5 nm. Third panel: mixing ratio in parts per billion by volume (ppbv) for the biogenic precursor gases, isoprene, and α-pinene. Fourth panel: evolution of total HOM concentration in molecules per cubic centimeter (molec. cm−3), measured in the gas phase by the nitrate CI-APi-TOF mass spectrometer. The HOM total is deﬁned as the sum of C5, C10, C15, and C20 carbon classes, which are shown as well. Ozone level is not shown though remains stable over the whole period at ∼100 ppbv. The shaded areas refer to the time when the particles were collected using the TD-DMA. 1. α-pinene + isoprene at −30 ◦C and 20 % RH (αIP- 30,20), α-50,60 at −50 ◦C, reaching ∼2 × 105 cm−3. By comparing the experiments at −30 ◦C, α-30,20, and αIP-30,20 a lower particle concentration is observed for the system where iso- prene is present. The reduction is approximately a factor of 3; this can be attributed to the suppression of the new particle formation by isoprene oxidation. This is in line with the re- sults of Kiendler-Scharr et al. (2009), who ﬁrst reported the decrease in particle number of the nucleated particles. The effect of isoprene in terms of total HOM concentration in the gas phase and on the measured nucleation rates will be dis- cussed in more detail in Sect. 3.4.2. 2. α-pinene at −30 ◦C and 20 % RH (α-30,20), 3. α-pinene at −50 ◦C and 20 % RH (α-50,20), and 4. α-pinene at −50 ◦C and 60 % RH (α-50,60). The color scale in the upper panel of Fig. 1 indicates that the newly formed particles appear in the smallest size chan- nels of the SMPS soon after the concentration of α-pinene in the chamber is increased. 3.2 Gas- and particle-phase chemical composition Figure 2 shows the carbon distribution as an overview of the compounds detected in gas and particle phase for a sys- tem where only α-pinene was oxidized (α-30,20). C8−10 monomers (Fig. 2a) and C18−20 (Fig. 2b) dimers are ob- served in the gas as well as in the particle phase. For instance, some of the signals with the highest intensity correspond to C10H16O3−9 and C20H32O5−13; in particular C10H16O6 and C10H16O7 have an important presence in both phases. Over- all, most of the compounds that are present in the gas phase are detected as well in the particle phase, although their rel- ative contribution to the total signal can differ between the phases. The corresponding carbon distribution for the other systems can be found in Figs. S1 and S2 in the Supplement. For the experiments presented in this study, we report in Table 1 the particle growth rates (GRs) determined from the nano-scanning electrical mobility spectrometer (nSEMS) size distributions. The growth rates in 3.2–8 and 5–15 nm were calculated using the 50 % appearance time method de- scribed in Stolzenburg et al. (2018). From the calculated values in Table 1, we observe that GR3.2−8 nm for the α- pinene + isoprene system (αIP-30,20) at the ﬁrst concentra- tion stage is around 18 nm h−1 compared to ∼77 nm h−1 for the α-pinene only system (α-30,20). This is a factor of ∼4 difference, while GR5−15 nm represents a factor of 2 to 3 dif- ference between αIP-30,20 compared to α-30,20. From these values, one would conclude that isoprene does not contribute to the growth in the size range reported here. Nevertheless, by looking at the aerosol mass concentration (see Fig. S3 in the Supplement), the mass reached during the experiment αIP- 30,20 is identical in the presence and absence of isoprene at −30 ◦C and 20 % RH. Reaching the same mass with a lower number of particles for the experiment with isoprene (αIP- 30,20) compared to α-30,20 means that the growth rates at larger sizes (> 15 nm) are higher in the presence of isoprene. This is consistent with the fact that the particle size reached in the presence of isoprene is higher. Most likely, isoprene might enhance growth at larger sizes (> 15 nm) in the present study. 3.2.1 Inﬂuence of isoprene on α-pinene system at −30 ◦C and 20 % RH Figure 3 shows mass defect plots of gas and particle phase and the intensity difference between each phase at −30 ◦C. Figure 3a and d display the gas and particle composition of α-30,20, while the gas and particle composition of αIP-30,20 are shown in Fig. 3b and e, respectively. As both phases were measured with the same instrument, they can be directly inter-compared. p The intensity difference is calculated based on the normal- ized signal (each single signal divided by the total signal for each system and phase). Essentially, the normalized signal can be understood as a measure of the fraction or contribu- tion of every compound in the entire system. By looking at the intensity difference in the gas phase (Fig. 3c), it can be observed that some C5 and C15 contribute signiﬁcantly more in the system with isoprene added (αIP-30,20) that are not as pronounced in the system where only α-pinene was ox- idized (α-30,20). This observation can be attributed to the presence of isoprene in the system. As described by Hein- ritzi et al. (2020), C15 dimers are formed in the gas phase when C10 RO q 2 radicals from α-pinene ozonolysis undergo L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17104 tion is deﬁned as the sum of C5, C10, C15, and C20 carbon classes; these classes consider compounds with C2–C5, C6– C10, C11–C15, and C16–C20, respectively, and considered as a HOM such compounds with ﬁve or more oxygen atoms, as suggested in Bianchi et al. (2019). The total HOM con- centration was measured with a calibrated nitrate CI-APi- TOF mass spectrometer (Kürten et al., 2012). Additionally, a temperature-dependent sampling loss correction factor is ap- plied. From the evolution of these traces, it can be observed that C5 and C15 carbon classes have higher concentrations (approximately by a factor of 2.5) in experiment αIP-30,20 compared with α-30,20, which can be explained by the pres- ence of isoprene. However, possible fragmentation in the α- pinene ozonolysis systems also can lead to some C5 and C15 compounds produced without the presence of isoprene. terminating reactions with C5 RO q 2 radicals from the isoprene oxidation with ·OH. Additionally, C19 and C20 dimers con- tribute more in the system where only α-pinene was oxidized (α-30,20). Figure 3f shows the intensity difference in the particle phase. In contrast with what is observed in the gas phase, the particle-phase effects seem more diverse. There is an in- crease in the intensity difference for several species in the system with isoprene (αIP-30,20), such as C4−5, C13−16, and some C17−19 (see Fig. S1 in the Supplement). In par- ticular, a distinct group of C15 compounds C15H24O5−10 and C5H10O5−7 can be identiﬁed in the particle and in the gas phase. A previous study has shown that isoprene can enhance particle growth rates despite its negative effect on nucleation (Heinritzi et al., 2020). The identiﬁcation of C15 dimers in nanometer-sized particles in the present study conﬁrms this with a direct measurement. The suppressing effect of iso- prene on nucleation will further be discussed in Sect. 3.4.2. However, isoprene can still contribute to the growth of parti- cles by C5 or by C15 compounds. Additionally, these species can be an important ﬁngerprint to identify SOA formation from a mixture of biogenic vapors containing isoprene. 3.1 Experimental overview The experiments were performed such that the particles grew rapidly to reach sizes of approx- imately 100 nm, where they could potentially act as cloud condensation nuclei (CCN) or ice nucleating particles (INPs) and were used for further CCN and INP studies. The third panel of Fig. 1 shows the α-pinene and isoprene mixing ratios. For all of the systems, α-pinene was between 1 and 8 ppbv, while isoprene was only present during exper- iment αIP-30,20 up to 30 ppbv. The precursor gases were measured by using a proton transfer reaction time-of-ﬂight (PTR-TOF) mass spectrometer (Graus et al., 2010; Breiten- lechner et al., 2017), which is capable of measuring VOCs. The second panel of Fig. 1 shows the particle number concentration measured by the condensation particle counter (CPC2.5 nm) and by the particle size magniﬁer (PSM) with a cutoff diameter of 1.7 nm. A higher particle number con- centration can be observed for the experiments α-50,20 and The bottom panel of Fig. 1 shows the total HOM con- centration in the gas phase. Here, the total HOM concentra- Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 Chemical composition of nanoparticles from α-pinene nucleation Figure 2. Carbon atom distribution and oxygen atom content in gas- and particle-phase molecules for α-pinene oxidation products at −30 ◦C and 20 % RH (α-30,20). Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Carbon atom distribution C2−11 and (b) carbon atom distribution C12−20. The level of α-pinene was between 1 and 8 ppbv, and the ozone level was stable at ∼100 ppbv. The intensities are normalized by the total signal in each system and phase. Each color represents a speciﬁc number of oxygen atoms in the range of 3 to 16. Table 1. Summary of the main parameters for four pure biogenic new particle formation experiments. Experiment Isoprene α-pinene Isoprene-to- Ozone T RH HOM totala J a 1.7 Growth rate Growth rate Mass<15nm/ [ppb] [ppb] monoterpene [ppb] [◦C] [%] [molec. cm−3] [cm−3 s−1] 3.2–8 nm 5–15 nm Massb >15nm carbon ratio (R) [nm h−1] [nm h−1] [%] αIP-30,20 13.71 0.95 14.4 98.58 −30 20 1.50e8 7.29 18.0 22.8 0.29/99.7 31.38 5.12 6.1 101.56 −30 20 3.04e8 10.10 n/a 39.0 α-30,20 ∼0 3.35 n/a 102.10 −30 20 2.20e8 23.76 76.9 77.1 0.11/99.9 α-50,20 ∼0 3.04 n/a 100.55 −50 20 6.72e7 51.24 41.1 42.0 0.26/99.7 α-50,60 ∼0 7.72 n/a 110.20 −50 60 8.00e7 79.17 63.4 78.4 0.09/99.9 a Run-to-run experimental uncertainties of HOMs is ± 20 %, and for J1.7 it is ± 30 %. n/a: not applicable. b Mass fraction of particles collected on the ﬁlament during the TD-DMA collection time, the calculation of which is based on SMPS mass distributions. Table 1. Summary of the main parameters for four pure biogenic new particle formation experiments. a Run-to-run experimental uncertainties of HOMs is ± 20 %, and for J1.7 it is ± 30 %. n/a: not applicable. b Mass fraction of particles collected on the ﬁlament during the TD-DMA collection time, the calculation of which is based on SMPS mass distributions. f HOMs is ± 20 %, and for J1.7 it is ± 30 %. n/a: not applicable. b Mass fraction of particles collected on the ﬁlament during the TD-DMA collection time, the ass distributions. 60 % RH (Fig. 4b and e). In both gas and particle phase at high and low RH, we detected C8−10 monomers and C18−20 dimers. C10H16O4−7 and C20H32O5−11 are the most promi- nent signals (see Fig. S2 in the Supplement). 3.2.2 Inﬂuence of relative humidity on α-pinene system at −50 ◦C Figure 4 shows mass defect plots for the pure α-pinene exper- iments at −50 ◦C at low and high relative humidity: the gas and particle phase of α-pinene at −50 ◦C, 20 % RH (Fig. 4a and d), and the gas and particle phase of α-pinene at −50 ◦C, Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17105 Figure 2. Carbon atom distribution and oxygen atom content in gas- and particle-phase molecules for α-pinene oxidation products at −30 ◦C and 20 % RH (α-30,20). Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Carbon atom distribution C2−11 and (b) carbon atom distribution C12−20. The level of α-pinene was between 1 and 8 ppbv, and the ozone level was stable at ∼100 ppbv. The intensities are normalized by the total signal in each system and phase. Each color represents a speciﬁc number of oxygen atoms in the range of 3 to 16. 17105 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Chemical composition of nanoparticles from α-pinene nucleation low RH (α-50,20). This observation can likely be attributed to a change in the particle mass size distribution (see Fig. S3 in the Supplement), which indicates that at similar α-pinene and ozone mixing ratio, and the same temperature, the par- ticle mass concentration increases possibly due to the effect of the relative humidity in the system. In addition, a possible impact of relative humidity on particle viscosity can inﬂu- ence particle mass formed; studies by Grayson et al. (2016) and Galeazzo et al. (2021) have reported lower viscosity with higher SOA mass concentration along with RH dependence of viscosity for organic particles. The relative humidity change from 20 % to 60 % does not have a signiﬁcant inﬂuence on the gas-phase composition at temperatures of −50 ◦C (Fig. 4c), meaning that most of the gaseous compounds detected contribute practically equal to the total signal when the humidity changes over the reported range. In contrast, there are changes in the particle-phase sig- nal. The intensity difference (Fig. 4f) does not show a clear humidity effect on the particle chemical composition; how- ever, this comparison is based on the normalized signal (con- tribution of every compound to the total intensity). When looking only at the total intensity in the particle phase, we do observe an increase by a factor of ∼3 in the total signal for the system at high RH (α-50,60) compared with the system at Our ﬁndings are consistent with previous experiments. Saathoff et al. (2009) observed that humidity has a signiﬁ- cant inﬂuence on α-pinene SOA yields for lower tempera- tures. Cocker III et al. (2001) reported that the yield of SOA at higher RH for α-pinene ozonolysis (relative to the sys- tem at dry conditions) increases possibly due to the uptake Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17106 17106 17106 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Figure 3. Mass defect plots of gas and particle phase and the intensity difference between each phase. Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Gas and (d) particle phase for α-pinene oxidation products at −30 ◦C and 20 % RH (α-30,20). (b) Gas and (e) particle phase for α-pinene + isoprene oxidation products at −30 ◦C and 20 % RH (αIP-30,20). Chemical composition of nanoparticles from α-pinene nucleation The level of α-pinene was between 1 and 8 ppbv in both experiments, while isoprene was present only in experiment αIP-30,20 reaching up to 30 ppbv. Ozone levels were ∼100 ppbv in both experiments. The symbol sizes in (a), (b), (d), and (e) are the intensities normalized by the total signal in each system. The intensity difference in the gas phase (c) and in the particle phase (f) is indicated as ((αIP-30,20) −(α-30,20)) / α-30,20. The color scale represents the difference in percent. Figure 3. Mass defect plots of gas and particle phase and the intensity difference between each phase. Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Gas and (d) particle phase for α-pinene oxidation products at −30 ◦C and 20 % RH (α-30,20). (b) Gas and (e) particle phase for α-pinene + isoprene oxidation products at −30 ◦C and 20 % RH (αIP-30,20). The level of α-pinene was between 1 and 8 ppbv in both experiments, while isoprene was present only in experiment αIP-30,20 reaching up to 30 ppbv. Ozone levels were ∼100 ppbv in both experiments. The symbol sizes in (a), (b), (d), and (e) are the intensities normalized by the total signal in each system. The intensity difference in the gas phase (c) and in the particle phase (f) is indicated as ((αIP-30,20) −(α-30,20)) / α-30,20. The color scale represents the difference in percent. compounds. Therefore, it is possible that a break-up of some molecules occurs. of water. One explanation for this observation could be that the rate constant value of the α-pinene ozonolysis can be affected by the RH (Zhang et al., 2018). Nevertheless, our semi-continuous particle-phase measurements do not allow any conclusions on the magnitude of the rate constants to be drawn. Continuous particle-phase measurements under dif- ferent RH conditions are required in order to better under- stand the RH effect on the SOA formation. 3.3 Volatility distribution of particle-phase compounds Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17107 17107 L. Caudillo et al.: Chemical composition of nanoparticles from α pinene nucleation 17107 Figure 4. Mass defect plots of gas and particle phase and the intensity difference between each phase. Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Gas and (d) particle phase for α-pinene oxidation products at −50 ◦C and 20 % RH (α-50,20). (b) Gas and (e) particle phase for α-pinene oxidation products at −50 ◦C and 60 % RH (α-50,60). The level of α-pinene was between 1 and 8 ppbv, and ozone levels were ∼100 ppbv in both experiments. The symbol sizes in (a), (b), (d), and (e) are the intensities normalized by the total signal in each system. The intensity difference in the gas phase (c) and in the particle phase (f) is indicated as ((α-50,60)−(α-50,20)) / α-50,20. The color scale represents the difference in percent. Figure 4. Mass defect plots of gas and particle phase and the intensity difference between each phase. Both phases are measured with a nitrate CI-APi-TOF mass spectrometer, while the TD-DMA is coupled to it for particle-phase measurements. (a) Gas and (d) particle phase for α-pinene oxidation products at −50 ◦C and 20 % RH (α-50,20). (b) Gas and (e) particle phase for α-pinene oxidation products at −50 ◦C and 60 % RH (α-50,60). The level of α-pinene was between 1 and 8 ppbv, and ozone levels were ∼100 ppbv in both experiments. The symbol sizes in (a), (b), (d), and (e) are the intensities normalized by the total signal in each system. The intensity difference in the gas phase (c) and in the particle phase (f) is indicated as ((α-50,60)−(α-50,20)) / α-50,20. The color scale represents the difference in percent. (α-30,20 compared with αIP-30,20) or between the experi- ments at −50 ◦C (α-50,20 compared with α-50,60), which indicates that temperature is the main parameter affecting the volatility distribution for the experiments reported here. son, Table 1 gives an overview of the experimental condi- tions for the experiments α-30,20, αIP-30,20, α-50,20, and α-50,60; it further includes the HOM total concentration and derived J1.7 nm from the PSM data (see method description in Sect. 2.4). 3.4.1 New particle formation on pure α-pinene experiments Figure 6 displays pure biogenic J1.7 nm vs total HOM concen- tration at different temperatures for pure α-pinene (Simon et al., 2020) in which it can be seen that the total HOM concen- tration and their nucleation rates have a strong dependence on the temperature. As the temperature decreases, the nu- cleation rates increase strongly for a given HOM concentra- tion. In other terms, the total HOM concentration needed to reach the same nucleation rate can be up to 2 orders of mag- nitude higher for +25 ◦C compared to −50 ◦C. As described by Simon et al. (2020), this can be attributed to the reduction in volatility with decreasing temperature. In other words, at low temperatures, molecules with less oxygen content can lead to the same nucleation rate as more highly oxygenated molecules at higher temperatures. Additionally, Fig. 6 in- cludes the data points at −30 and −50 ◦C from pure α-pinene experiments reported in this study (α-30,20, α-50,20 and α- 50,60). However, it can be observed that they do not follow the trend at their corresponding temperature. 3.3 Volatility distribution of particle-phase compounds y p p We classiﬁed the volatility bins according to the regimes proposed by Donahue et al. (2012) and Schervish and Donahue (2020) and calculated the corresponding frac- tions (Table 2). Overall, the particle-phase-detected com- pounds correspond mainly to low-volatility organic com- pounds (LVOCs) and extremely low-volatility compounds (ELVOCs) by explaining more than 80 % of the signals, while ultralow-volatility organic compounds (ULVOCs) rep- resent only a small fraction (between 6 % and 17 %). With this parametrization, we are able to approximate the satu- ration mass concentration for the particle-phase compounds measured using the TD-DMA in the CLOUD chamber. For this parametrization, we assume that the elemental composi- tion is one of the main parameters to take into account. 3.3 Volatility distribution of particle-phase compounds Figure 5 shows the volatility distribution of the oxidation products in the particle phase measured by the TD-DMA for the experiments reported in this work (in linear scale Fig. S4 in the Supplement). The volatility calculation was done by using the parametrization introduced by Donahue et al. (2011) and modiﬁed by Stolzenburg et al. (2018) and Simon et al. (2020). It is expressed as the logarithm of the saturation mass concentration, log10c∗ i , in micrograms per cubic meters (µg m−3), from the number of carbon and oxy- gen atoms in the speciﬁc molecules. This approximation pa- rameterizes the volatility of a molecule based on its func- tional groups and a free parameter to distinguish between monomers and dimers. In general, for the experiments presented in this work, most of the compounds that are present in the gas phase are detected as well in the particle phase, although the rel- ative contributions to the total signal can vary depending on the phase. The more oxygenated material in the gas phase, speciﬁcally for C20 dimers with nO>13, is not observed in the particle phase. This is probably because of their very low concentrations and the difﬁculty to distinguish between real particle signal and background. We conjecture that especially at low temperatures this issue might be related to the fact that at lower temperatures, the autoxidation process to form HOMs is slower; therefore, the oxygen content and O : C de- crease (Stolzenburg et al., 2018; Ye et al., 2019; Simon et al., 2020). The low contribution of these compounds in the gas phase might be reﬂected in the particle phase. Additionally, the heating cycle that evaporates all the particulate material collected on the ﬁlament can potentially result in the ther- mal decomposition of some of the larger molecular-weight In Fig. 5 each volatility bin contains the summed inten- sity of the oxidation products measured in the particle phase, and it is normalized by the total signal. Most of the classes are distributed over the range of the volatility values that are displayed, and at lower temperatures, lower volatilities are observed (experiments α-50,20 and α-50,60). This observa- tion is due to the fact of the strong dependency between sat- uration concentration and temperature. Essentially, there are no signiﬁcant differences between the experiments at −30 ◦C Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 L. 3.4 Nucleation rates as a function of the total HOM concentration Previous CLOUD studies have reported nucleation rates (J1.7 nm) as a function of the total HOM concentration from α-pinene oxidation for different temperatures and gas mix- tures (Kirkby et al., 2016; Heinritzi et al., 2020; Simon et al., 2020). For the experiments discussed in the present study, the nucleation rates have not been reported yet. For this rea- https://doi.org/10.5194/acp-21-17099-2021 Atmos. Chem. Phys., 21, 17099–17114, 2021 17108 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Table 2. Bin volatility fractions for the different experiments. Experiment T RH ULVOCs ELVOCs LVOCs SVOCs IVOCs [◦C] [%] [%] [%] [%] [%] [%] αIP-30,20 −30 20 8.6 28.3 59.1 3.8 0.2 α-30,20 −30 20 5.9 44.1 48.1 1.9 0 α-50,20 −50 20 16.5 36.4 46.1 1.0 0 α-50,60 −50 60 13.8 50.6 34.8 0.8 0 Figure 5. TD-DMA volatility distribution of the measured ox- idation products in the particle phase for four different experi- ments: (a) α-pinene at −30 ◦C and 20 % RH (α-30,20), (b) α- pinene + isoprene at −30 ◦C and 20 % RH (αIP-30,20), (c) α- pinene at −50 ◦C and 20 % RH (α-50,20), and (d) α-pinene at −50 ◦C and 60 % RH (α-50,60). Every individual volatility bin in- cludes the sum of the intensity for the oxidation products normal- ized by the total signal in each system. Every individual volatility bin is deﬁned at 300 K, shifted, and widened according to their cor- responding temperature The color bands in the background indicate Figure 6. Pure biogenic nucleation rates of pure α-pinene at 1.7 nm diameter against total HOM concentration at different temperatures. The HOM total is deﬁned as the sum of C5, C10, C15 and C20 car- bon classes. Triangles represent galactic cosmic ray (GCR) condi- tions, and circles represent neutral conditions. Data points at −50, −25, −10, +5, and +25 ◦C are from Simon et al. (2020). The points with orange marker on the background are the contribution of this study (experiments α-30,20, α-50,20 and α-50,60). Solid and dashed lines represent power-law ﬁts to GCR and neutral con- ditions. Bars indicate 1σ run-to-run experimental uncertainty. The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. ing values are higher than previously reported (see Fig. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17108 Table 2. Bin volatility fractions for the different experiments. Table 2. Bin volatility fractions for the different experiments. αIP 30,20 30 20 8.6 α-30,20 −30 20 5.9 α-50,20 −50 20 16.5 α-50,60 −50 60 13.8 Figure 5. TD-DMA volatility distribution of the measured ox- idation products in the particle phase for four different experi- ments: (a) α-pinene at −30 ◦C and 20 % RH (α-30,20), (b) α- pinene + isoprene at −30 ◦C and 20 % RH (αIP-30,20), (c) α- pinene at −50 ◦C and 20 % RH (α-50,20), and (d) α-pinene at −50 ◦C and 60 % RH (α-50,60). Every individual volatility bin in- cludes the sum of the intensity for the oxidation products normal- ized by the total signal in each system. Every individual volatility bin is deﬁned at 300 K, shifted, and widened according to their cor- responding temperature. The color bands in the background indicate the volatility regimes as in Donahue et al. (2012) and in Schervish and Donahue (2020). The normalized intensity is dimensionless. Nevertheless, it should be noted that the particle-phase signal is given in normalized counts per second integrated over the evapo- ration time (ncps/s). Figure 6. Pure biogenic nucleation rates of pure α-pinene at 1.7 nm diameter against total HOM concentration at different temperatures. Figure 6. Pure biogenic nucleation rates of pure α-pinene at 1.7 nm diameter against total HOM concentration at different temperatures. The HOM total is deﬁned as the sum of C5, C10, C15 and C20 car- bon classes. Triangles represent galactic cosmic ray (GCR) condi- tions, and circles represent neutral conditions. Data points at −50, −25, −10, +5, and +25 ◦C are from Simon et al. (2020). The points with orange marker on the background are the contribution of this study (experiments α-30,20, α-50,20 and α-50,60). Solid and dashed lines represent power-law ﬁts to GCR and neutral con- ditions. Bars indicate 1σ run-to-run experimental uncertainty. The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. The HOM total is deﬁned as the sum of C5, C10, C15 and C20 car- bon classes. Triangles represent galactic cosmic ray (GCR) condi- tions, and circles represent neutral conditions. Data points at −50, −25, −10, +5, and +25 ◦C are from Simon et al. (2020). The points with orange marker on the background are the contribution of this study (experiments α-30,20, α-50,20 and α-50,60). 3.4 Nucleation rates as a function of the total HOM concentration S5 ical composition of nanoparticles from α-pinene nucleation ELVOCs LVOCs SVOCs IVOCs [%] [%] [%] [%] 28.3 59.1 3.8 0.2 44.1 48.1 1.9 0 36.4 46.1 1.0 0 50.6 34.8 0.8 0 Figure 6. Pure biogenic nucleation rates of pure α-pinene at 1.7 nm diameter against total HOM concentration at different temperatures. The HOM total is deﬁned as the sum of C5, C10, C15 and C20 car- bon classes. Triangles represent galactic cosmic ray (GCR) condi- tions, and circles represent neutral conditions. Data points at −50, −25, −10, +5, and +25 ◦C are from Simon et al. (2020). The points with orange marker on the background are the contribution of this study (experiments α-30,20, α-50,20 and α-50,60). Solid and dashed lines represent power-law ﬁts to GCR and neutral con- ditions. Bars indicate 1σ run-to-run experimental uncertainty. The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation different experiments L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Mass defect plots of gas phase for two different systems and the normalized difference between them. Gas phase is measured with a nitrate CI-APi-TOF mass spectrometer. (a) α-Pinene oxidation products at −10 ◦C and 90 % RH at low level of precursor gases and (b) α-pinene oxidation products at −10 ◦C and 80 % RH at high level of precursor gases. α-pinene was 0.2–0.8 ppbv and ozone ∼40– 50 ppbv in (a), and α-pinene was 2–3 ppbv and ozone ∼100 ppbv in (b). The symbol sizes and colors in (a) and (b) represent the intensities normalized by the total signal in each system. (c) The difference between the normalized signals shown in (a) and (b) is represented by the color scale. Figure 7. Mass defect plots of gas phase for two different systems and the normalized difference between them. Gas phase is measured with a nitrate CI-APi-TOF mass spectrometer. (a) α-Pinene oxidation products at −10 ◦C and 90 % RH at low level of precursor gases and (b) α-pinene oxidation products at −10 ◦C and 80 % RH at high level of precursor gases. α-pinene was 0.2–0.8 ppbv and ozone ∼40– 50 ppbv in (a), and α-pinene was 2–3 ppbv and ozone ∼100 ppbv in (b). The symbol sizes and colors in (a) and (b) represent the intensities normalized by the total signal in each system. (c) The difference between the normalized signals shown in (a) and (b) is represented by the color scale. Figure 8. Pure biogenic nucleation rates at 1.7 nm diameter against total HOM concentration at different temperatures for α-pinene and α-pinene + isoprene systems. The HOM total is deﬁned as the sum of C5, C10, C15, and C20 carbon classes. Triangles represent galac- tic cosmic ray (GCR) conditions. Data points at +5 and +25 ◦C are from Kirkby et al. (2016) and Heinritzi et al. (2020). Data points at −25 ◦C are from Simon et al. (2020). The value of isoprene-to- monoterpene carbon ratio (R) varies from 1.5 to 6.5 for Heinritzi et al. (2020), and R is equal to 14.4 and 6.1 for this study. The points with orange marker on the background indicate the contribution of this work. Solid lines represent power-law ﬁts to GCR conditions of the systems with α-pinene only, and dashed lines are the power-law ﬁts of the systems with isoprene added. Bars indicate 1σ run-to-run experimental uncertainty. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation Solid and dashed lines represent power-law ﬁts to GCR and neutral con- ditions. Bars indicate 1σ run-to-run experimental uncertainty. The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. Figure 5. TD-DMA volatility distribution of the measured ox- idation products in the particle phase for four different experi- ments: (a) α-pinene at −30 ◦C and 20 % RH (α-30,20), (b) α- pinene + isoprene at −30 ◦C and 20 % RH (αIP-30,20), (c) α- pinene at −50 ◦C and 20 % RH (α-50,20), and (d) α-pinene at −50 ◦C and 60 % RH (α-50,60). Every individual volatility bin in- cludes the sum of the intensity for the oxidation products normal- ized by the total signal in each system. Every individual volatility bin is deﬁned at 300 K, shifted, and widened according to their cor- responding temperature. The color bands in the background indicate the volatility regimes as in Donahue et al. (2012) and in Schervish and Donahue (2020). The normalized intensity is dimensionless. Nevertheless, it should be noted that the particle-phase signal is given in normalized counts per second integrated over the evapo- ration time (ncps/s). ing values are higher than previously reported (see Fig. S5 in the Supplement). In order to investigate a possible reason for this ﬁnding, we have chosen two representative experi- ments at −10 ◦C and 80 % RH to 90 % RH with different levels of α-pinene and ozone. Figure 7 shows the mass defect plots for the gas-phase chemical composition of the oxida- tion products. In one experiment (Fig. 7a) α-pinene and the ozone mixing ratio were between 0.2 and 0.8 ppbv and 40 and 50 ppbv, respectively, while for the second experiment (Fig. 7b) the mixing ratios were 2 to 3 ppbv and 100 ppbv, respectively. From Fig. 7c, it can be concluded that the for- mation of HOMs with low oxygen content is favored when For the pure α-pinene systems (α-30,20, α-50,20, and α-50,60) and complementary pure α-pinene experiments at +5 ◦C and at −10 ◦C, we have calculated the HOM yield as described in Simon et al. (2020) and found that the result- Atmos. Chem. Phys., 21, 17099–17114, 2021 https://doi.org/10.5194/acp-21-17099-2021 L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17109 Figure 7. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. the α-pinene and ozone mixing ratio are higher (relative to the system at low levels of precursor gases). An explanation for this is that the high concentration of RO2 enhances the terminating reactions before the autoxidation can lead to high oxygen content for the products. As the compounds with low oxygen content tend to have higher saturation vapor pres- sures, they do not contribute efﬁciently to new particle for- mation. For this reason, a given total HOM concentration is not unambiguously tied to a nucleation rate (even at constant temperature). The magnitude of the precursor gas mixing ra- tio (more speciﬁcally the full volatility distribution of the products and not just the simple measure of total HOM con- centration) also needs to be taken into account (see Fig. S6 in the Supplement). In summary, the lower J1.7 nm values com- pared with previous studies are very likely due to the higher α-pinene and ozone mixing ratios used in the present study. There are several compounds with low oxygen content that contribute to the total HOM concentration in the gas phase, while these do not contribute to the formation of new parti- cles. Figure 8. Pure biogenic nucleation rates at 1.7 nm diameter against total HOM concentration at different temperatures for α-pinene and α-pinene + isoprene systems. The HOM total is deﬁned as the sum of C5, C10, C15, and C20 carbon classes. Triangles represent galac- tic cosmic ray (GCR) conditions. Data points at +5 and +25 ◦C are from Kirkby et al. (2016) and Heinritzi et al. (2020). Data points at −25 ◦C are from Simon et al. (2020). The value of isoprene-to- monoterpene carbon ratio (R) varies from 1.5 to 6.5 for Heinritzi et al. (2020), and R is equal to 14.4 and 6.1 for this study. The points with orange marker on the background indicate the contribution of this work. Solid lines represent power-law ﬁts to GCR conditions of the systems with α-pinene only, and dashed lines are the power-law ﬁts of the systems with isoprene added. Bars indicate 1σ run-to-run experimental uncertainty. The overall systematic-scale uncertainty of HOMs of +78 % and −68 % is not shown. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation 17110 et al., 2016; Heinritzi et al., 2020; Simon et al., 2020). How rapidly particles are formed in a pure biogenic system de- pends strongly on the temperature and on the ion condi- tions. In general, we observe increasing nucleation rates at lower temperatures and at GCR conditions. The presence of isoprene lowers the nucleation rate (relative to the pure α- pinene system at similar conditions); this is known as iso- prene suppression of new particle formation. In this regard, there is a suppression on the new particle formation caused by adding isoprene on an α-pinene system at −30 ◦C and 20 % RH. However, it has been reported that the suppression effect is stronger when α-pinene is lower (and R is higher; see Fig. S7 in the Supplement). For instance, in a plant cham- ber experiment R = 19.5 resulted in no signiﬁcant new par- ticle formation (Kiendler-Scharr et al., 2009). Additionally, in the Michigan forest with R = 26.4, NPF events did not occur frequently (Kanawade et al., 2011). Lee et al. (2016) reported observations of NPF suppression in a rural forest in Alabama, where R = 2.0. However, one has to consider that the suppression effect at a given value of R likely decreases as temperature decreases and so does the saturation vapor pressure of the oxidation products. Lastly, the lower J1.7 values compared with previous stud- ies are very likely due to the higher α-pinene and ozone mix- ing ratios used in the present study. There are several com- pounds with low oxygen content that contribute to the total HOM concentration in the gas phase, while these do not con- tribute to the formation of new particles. For this reason, a given total HOM concentration is not unambiguously tied to a nucleation rate (even at constant temperature). The mag- nitude of the precursor gas mixing ratio, and thus the full volatility distribution, also needs to be taken into account. Data availability. Data related to this article are available upon re- quest to the corresponding authors. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/acp-21-17099-2021-supplement. Author contributions. L. Caudillo et al.: Chemical composition of nanoparticles from α-pinene nucleation LC, BR, GM, MaS, ACW, TM, MG, FA, RB, BB, ZB, RC, BC, JD, HF, LGC, XCH, VH, WK, HL, CPL, BL, NGAM, VM, HEM, RM, RLM, BM, UM, AO, JP, MP, AAP, WS, BS, JS, DS, YS, MiS, YJT, PT, AT, SV, MW, DSW, SKW, AW, WY, WY, MZW, UB, IEH, RCF, KH, JK, MK, KL, OM, RV, PMW, AK, and JC prepared the CLOUD facility and measurement instruments. LC, BR, GM, ACW, TM, MG, AA, FA, RB, BB, JD, LGC, VH, HL, CPL, NGAM, VM, RM, DM, RLM, BM, UM, WS, BS, DS, MiS, CT, YJT, AT, DSW, SKW, MZW, IEH, JK, RV, and PMW collected the data. LC, BR, MH, GM, FA, LD, RLM, UM, WS, BS, SKW, and RCF analyzed the data. LC, MH, MSim, ACW, TM, MG, LD, RLM, UM, MiS, WS, DS, UB, IEH, RCF, AH, KH, JK, MK, OM, HS, NMD, AK, and JC contributed to the scientiﬁc discussion and interpretation of the results. LC, MH, ACW, LD, UB, RCF, HS, NMD, AK, and JC contributed to the writing of the manuscript. 3.4.2 The inﬂuence of isoprene on new particle formation In order to make the present study comparable with other studies that reported a suppression effect of isoprene on bio- genic new particle formation, the values of the isoprene-to- monoterpene carbon ratio (R) are also provided in Table 1. Here and in previous studies, R is essentially the ratio be- tween isoprene and α-pinene; for experiment αIP-30,20, R is equal to 14.4 and 6.1 (for two steady-state periods in αIP- 30,20). Figure 8 shows pure biogenic nucleation rates at 1.7 nm against total HOM concentration at different temperatures for the α-pinene and α-pinene + isoprene systems (Kirkby Atmos. Chem. 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This open-access publication was funded by the Goethe University Frankfurt. This open-access publication was funded by the Goethe University Frankfurt. Review statement. This paper was edited by Kelley Barsanti and reviewed by two anonymous referees. Review statement. This paper was edited by Kelley Barsanti and reviewed by two anonymous referees. Galeazzo, T., Valorso, R., Li, Y., Camredon, M., Aumont, B., and Shiraiwa, M.: Estimation of secondary organic aerosol vis- cosity from explicit modeling of gas-phase oxidation of iso- prene and α-pinene, Atmos. Chem. Phys., 21, 10199–10213, https://doi.org/10.5194/acp-21-10199-2021, 2021. 4 Conclusions In this study, we demonstrated the suitability of a ther- mal desorption differential mobility analyzer (TD-DMA) coupled to a nitrate chemical ionization–atmospheric pres- sure interface–time-of-ﬂight (CI-APi-TOF) mass spectrome- ter for measuring HOMs in newly formed nano aerosol parti- cles. Together with the nitrate CI-APi-TOF mass spectrome- ter, this setup is capable of measuring the gas and particle phase, allowing for a direct comparison as both measure- ments use the identical chemical ionization and detector. For the pure biogenic NPF experiments performed at −50 and −30 ◦C in the CLOUD chamber at CERN, we detected in the particle phase (diameter up to ∼100 nm) compounds such as C10H16O3−9, and C20H32O5−13. Especially for the system with isoprene added, C5 (C5H10O5−7) and C15 com- pounds (C15H24O5−10) can be an important ﬁngerprint to identify secondary organic aerosol from this biogenic source. Based on the elemental composition, we calculated the sat- uration mass concentration, and according to the volatility regimes, the particle-phase compounds correspond mainly to low-volatility organic compounds (LVOCs), extremely low- volatility compounds (ELVOCs), and ultralow-volatility or- ganic compounds (ULVOCs). Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. We thank CERN for providing the CLOUD facility to perform the experiments and the CLOUD commu- nity for supporting this study. We especially would like to thank Katja Ivanova, Timo Keber, Frank Malkemper, Robert Sitals, Hanna Elina Manninen, Antti Onnela, and Robert Kristic for their contributions to the experiment. We also showed that at −30 ◦C and an isoprene-to- monoterpene carbon ratio R = 14.4 and 6.1, there is a reduc- tion of the nucleation rate (compared to the pure α-pinene system at similar conditions). In this way, isoprene sup- presses NPF at −30 ◦C. Nevertheless, this suppression effect can be stronger at higher temperatures and at high R. Financial support. 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https://openalex.org/W2316611578	http://www.mathnet.ru/php/getFT.phtml?jrnid=vsgtu&paperid=588&what=fullt&option_lang=rus	Russian	null	Об одном численно устойчивом алгоритме решения систем линейных алгебраических уравнений неполного ранга	Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ: Fiziko-matematičeskie nauki/Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ Fiziko-matematičeskie nauki	2,008	cc-by	3,104	Общероссийский математический портал А. И. Жданов, Об одном численно устойчивом алгоритме решения систем ли- нейных алгебраических уравнений неполного ранга, Вестн. Сам. гос. техн. ун-та. Сер. Физ.-мат. науки, 2008, выпуск 1(), 149–153 DOI: 10.14498/vsgtu588 DOI: 10.14498/vsgtu588 Использование Общероссийского математического портала Math-Net.Ru подразумевает, что вы прочитали и согласны с пользовательским соглашением http://www.mathnet.ru/rus/agreement Параметры загрузки: IP: 195.242.1.96 24 октября 2024 г., 08:50:38 Параметры загрузки: IP: 195.242.1.96 24 октября 2024 г., 08:50:38 Сам. гос. техн. ун-та. Сер.: Физ.-мат. науки. — 2008. — №1 (16). — С. 149–153. — ISSN 1991–8615 Вестн. Сам. гос. техн. ун-та. Сер.: Физ.-мат. науки. — 2008. — №1 (16). — С. 149–153. — ISSN 19 ОБ ОДНОМ ЧИСЛЕННО УСТОЙЧИВОМ АЛГОРИТМЕ РЕШЕНИЯ СИСТЕМ ЛИНЕЙНЫХ АЛГЕБРАИЧЕСКИХ УРАВНЕНИЙ НЕПОЛНОГО РАНГА Рассматривается новый метод решения неустойчивых задач, сводящихся к решению произвольных (возможно, с неполным рангом и несовместных) систем линейных алгебраических уравнений. Данный метод основан на пре- образовании регуляризованной нормальной системы уравнений к эквивалентной расширенной регуляризованной нормальной системе уравнений. 1. Постановка задачи. Рассмотрим произвольную (возможно неполного ранга и несовместную) систему линейных алгебраических уравнений (СЛАУ) Au = f, A ∈Rm×n, f ∈Rm. (1) Au = f, A ∈Rm×n, f ∈Rm. (1) Под «решением» СЛАУ (1) в общем случае будет пониматься нормальное псевдорешение u∗= A+f, где A+ — псевдообратная матрица. д д р р ц Пусть ˜u∗— вычисленные любым известным прямым или итерационным методом [1, 2] на ком- пьютере (в арифметике с плавающей точкой) решения u∗. Эти решения различаются по точности. Оценки погрешностей ∥˜u∗−u∗∥2 компьютерных решений ˜u∗для этих методов зависят от чисел обусловленности матриц A. Спектральное число обусловленности СЛАУ (1) очевидно определяет- ся как κ2(A) = σ1/στ, где σ1 ⩾σ2 ⩾. . . ⩾σk — сингулярные числа матрицы A, k = min(m, n), τ = rank (A) ⩽k. ( ) Если A — хорошо обусловленная матрица, т. е. её число обусловленности κ2(A) ≪1/εmach, где εmach — машинное эпсилон [4], то компьютерные решения ˜u∗, вычисленные любыми известными мето- дами [4], по точности сравнимы между собой. В противном случае, известно [1,2], что значительная часть имеющихся рекомендаций по решению плохо обусловленных (κ2(A) ≈1/εmach) и неполного ранга СЛАУ (τ < k) требует нахождения сингулярного разложения (SVD-разложения) матрицы си- стемы или действий, по существу, к этому сводящихся. Это связано с тем, что методы, основанные на SVD-разложении, являются непревзойдёнными (по точности [1]) методами решения этого класса задач. Однако методы, основанные на SVD-разложении, не позволяют эффективно решать СЛАУ большой размерности с разрежёнными матрицами, так как для их применения требуется большой объем оперативной памяти компьютера. В настоящей работе предлагается новый метод, основанный на регуляризации СЛАУ (1) с исполь- зованием эквивалентных расширенных систем. Показано, что этот метод является обратно устойчи- вым [3] и конкурентно способным (по точности) методам, основанным на SVD-разложении. Кроме того, предлагаемый метод позволяет эффективно использовать математическое обеспечение, создан- ное для решения больших разрежённых систем на высокопроизводительных параллельных вычис- лительных системах. 2. Регуляризация на основе расширенных систем. Метод регуляризации А.Н. Тихонова может использоваться для приближенного нахождения нормального псевдорешения u∗СЛАУ (1). Именно, в качестве приближения к u∗берётся решение регуляризованной нормальной системы 2. Регуляризация на основе расширенных систем. Метод регуляризации А.Н. Тихонова может использоваться для приближенного нахождения нормального псевдорешения u∗СЛАУ (1). ОБ ОДНОМ ЧИСЛЕННО УСТОЙЧИВОМ АЛГОРИТМЕ РЕШЕНИЯ СИСТЕМ ЛИНЕЙНЫХ АЛГЕБРАИЧЕСКИХ УРАВНЕНИЙ НЕПОЛНОГО РАНГА Д о к а з а т е л ь с т в о. Так как ˜Aω = ˜AT ω, то все её собственные значения λi( ˜Aω) ∈R (i = 1, 2, . . . , m + n). , , ) Если A = Om×n, тогда матрица ˜Aω — диагональная, т. е. ) Если A = Om×n, тогда матрица ˜Aω — диагональная, т. е. ˜Aω = diag (ω, . . . , ω \| {z } m , −ω, . . . , −ω \| {z } n ), ˜Aω = diag (ω, . . . , ω \| {z } m , −ω, . . . , −ω \| {z } n ), ОБ ОДНОМ ЧИСЛЕННО УСТОЙЧИВОМ АЛГОРИТМЕ РЕШЕНИЯ СИСТЕМ ЛИНЕЙНЫХ АЛГЕБРАИЧЕСКИХ УРАВНЕНИЙ НЕПОЛНОГО РАНГА Именно, в качестве приближения к u∗берётся решение регуляризованной нормальной системы (ATA + αIn)u = ATf (2) (ATA + αIn)u = ATf (2) с подходящим значением параметра регуляризации α > 0, где T – знак транспонирования, In — единичная матрица порядка n. Этот подход основан на известном факте [4], что при α →0 решение uα системы (2) стремится к нормальному псевдорешению u∗СЛАУ (1). К сожалению, в случае плохо обусловленных или неполного ранга матриц A, при достаточно малых значениях α (α ≈εmach) СЛАУ (2) оказывается очень плохо обусловленной и, как следствие, не может быть решена никаким компьютерным алгоритмом. 149 А. И. Жданов В настоящей работе регуляризованная нормальная система (2) преобразуется к эквивалентной ей расширенной системе, которая имеет существенно меньшее число обусловленности и для неё может быть получено устойчивое компьютерное решение. Регуляризованную нормальную систему уравнений (2) можно записать в виде ATr −αu = 0, ATr −αu = 0, ATr −αu = 0, где r = f −Au, или 1/2 T 1/2 где r = f Au, или α−1/2ATr −α1/2u = 0. (3) Об A f ( ) где r = f Au, или α−1/2ATr −α1/2u = 0. α−1/2ATr −α1/2u = 0. (3) или α−1/2ATr −α1/2u = 0. (3 α−1/2ATr −α1/2u = 0. где r f Au, или α−1/2ATr −α1/2u = 0. (3) α−1/2ATr −α1/2u = 0. (3) Объединяя r + Au = f и (3), получаем систему: Объединяя r + Au = f и (3), получаем систему: Объединяя r + Au = f и (3), получаем систему: r + Au = f, α−1/2Ar −α1/2u = 0 r + Au = f, α−1/2Ar −α1/2u = 0 r + Au = f, α−1/2Ar −α1/2u = 0 или или или α1/2Im A AT −α1/2In α−1/2r u = f 0 . (4) α1/2Im A AT −α1/2In α−1/2r u = f 0 . ОБ ОДНОМ ЧИСЛЕННО УСТОЙЧИВОМ АЛГОРИТМЕ РЕШЕНИЯ СИСТЕМ ЛИНЕЙНЫХ АЛГЕБРАИЧЕСКИХ УРАВНЕНИЙ НЕПОЛНОГО РАНГА (4) (4) Если обозначить y = α−1/2r и ω = α1/2, то (4) можно записать в виде Если обозначить y = α−1/2r и ω = α1/2, то (4) можно записать в виде ωIm A AT −ωIn y u = f 0 ⇐⇒ ˜Aωx = ˜f, (5) ωIm A AT −ωIn y u = f 0 ⇐⇒ ˜Aωx = ˜f, (5) де ˜Aω = ωIm A AT −ωIn , x = y u и ˜f = f 0 . (5) где ˜Aω = ωIm A AT −ωIn , x = y u и ˜f = f 0 . СЛАУ (5) является расширенной системой с квадратной матрицей ˜Aω порядка m + n. Так как α > 0, то и ω > 0. Покажем, что матрица ˜Aω – невырожденная и, следовательно, расширенная СЛАУ всегда имеет единственное решение zα = (yT α, uT α)T, совпадающее с решением регуляризованной нор- мальной системы (2) T 1 T СЛАУ (5) является расширенной системой с квадратной матрицей ˜Aω порядка m + n. Так как α > 0, то и ω > 0. Покажем, что матрица ˜Aω – невырожденная и, следовательно, расширенная СЛАУ всегда имеет единственное решение zα = (yT α, uT α)T, совпадающее с решением регуляризованной нор- мальной системы (2) (ATA I ) 1ATf uα = (ATA + αIn)−1ATf, и yα = α−1/2rα, а rα = f −Auα. Для этого сформулируем следующую теорему, которая также дает возможность оценить число обусловленности вычислительной задачи (5). и yα = α−1/2rα, а rα = f −Auα. Для этого сформулируем следующую теорему, которая также дает возможность оценить число обусловленности вычислительной задачи (5). ( ) Теорема. Если A = Om×n — нулевая m × n-матрица, то собственными значениями матрицы ˜Aω являются числа ω и −ω кратностей m и n соответственно. ˜ q Теорема. Если A = Om×n — нулевая m × n-матрица, то собственными значениями матрицы ˜Aω являются числа ω и −ω кратностей m и n соответственно. Если A ̸= Om×n, то собственными значениями матрицы ˜Aω являются числа ± q σ2 i + ω2 (i = 1, Если A ̸ Om×n, то собственными значениями матрицы Aω являются числа ± q σi + ω (i 1, 2 , . . . , τ), а также ω и −ω кратностей m −τ и n −τ соответственно, где τ = rank (A) ⩾1. откуда непосредственно следует первое утверждение теоремы. Так как dim ker A = n −τ, то −ω является собственным значением кратности n −τ матрицы ˜Aω. □ ˜ Так как матрица Aω – симметричная, то из теоремы непосредственно получаем Следствие. Если m ̸= n или m = n > τ, то максимальное сингулярное число матри σmax( ˜Aω) = q σ2 1 + ω2, σmax( ˜Aω) = q σ2 1 + ω2, а минимальное сингулярное число σmin( ˜Aω) = ω и спектральное число обусловленности матри- цы ˜Aω: а минимальное сингулярное число σmin( ˜Aω) = ω и спектральное число обусловленности матри- цы ˜Aω: цы Aω: κ2( ˜Aω) = σmax( ˜Aω) σmin( ˜Aω) = p σ2 1 + ω2 ω . Если m = n = τ, то σmax( ˜Aω) = p σ2 1 + ω2, σmin( ˜Aω) = p σ2n + ω2, где σn = στ, а κ2( ˜Aω) = s σ2 1 + ω2 σ2n + ω2 . Для регуляризованной нормальной системы (2) параметр регуляризации практически всегда дол- жен удовлетворять условию α > σk. Это условие необходимо, чтобы регуляризованное решение uα было устойчивым решением. Таким образом, на основании следствия из теоремы можно получить оценку для числа обусловленности СЛАУ (5), независящую от ранга матрицы A, κ2( ˜Aω) ⩽ p σ2 1 + ω2 ω . (8) (8) В силу трудоёмкости вычисления σ1, учитывая, что σ1 = ∥A∥2 ⩽∥A∥E, где ∥A∥E – евклидова матричная норма, вместо неравенства (8) на практике можно воспользоваться неравенством κ2( ˜Aω) ⩽ˆκ2( ˜Aω) = q ∥A∥2 E + ω2 ω . κ2( ˜Aω) ⩽ˆκ2( ˜Aω) = q ∥A∥2 E + ω2 ω . 3. Решение систем неполного ранга. Если выбрать параметр регуляризации α достаточно малым, то расширенную систему (5) можно использовать для приближенного вычисления численно устойчивых нормальных псевдорешений систем (1) неполного ранга или систем с матрицами A, имеющими неполный численный ранг [2, стр. 75]. В действительности, на практике чаще интересует ε-ранг матрицы, который для некоторого малого ε определяется по формуле rank (A, ε) = min ∥A−B∥2⩽ε rank (B). Если A ∈Rm×n — матрица из чисел с плавающей точкой A = ﬂ(A), то разумно считать A численно неполноранговой, если rank (A, ε) < min(m, n), где ε = εmach∥A∥2. откуда непосредственно следует первое утверждение теоремы. откуда непосредственно следует первое утверждение теоремы. у р Пусть A ̸= Om×n и ωIm A AT −ωIn z v = λ z v , где z ∈Rm, v ∈Rn. Тогда где z ∈R , v ∈R . Тогда ωz + Av = λz, ATz −ωv = λv. (6) Если (zT, vT)T ̸= 0 и λ ̸= ω, то, исключая в (6) z = (λ −ω)−1Av, получаем (λ −ω)−1ATAv −ωv = λv ωz + Av = λz, ATz −ωv = λv. ωz + Av = λz, ATz −ωv = λv. (6) (6) , ATz −ωv = λv. ATz −ωv = λv. Если (zT, vT)T ̸= 0 и λ ̸= ω, то, исключая в (6) z = (λ −ω)−1Av, получаем (λ −ω)−1ATAv −ωv = λv Если (zT, vT)T ̸= 0 и λ ̸= ω, то, исключая в (6) z = (λ −ω)−1Av, получаем (λ −ω)−1ATAv −ωv = λv (λ −ω)−1ATAv −ωv = λv 150 Об одном численно устойчивом алгоритме . . . или или ATAv = (λ −ω)(λ + ω)v ⇐⇒ ATAv = (λ2 −ω2)v ⇐⇒ (ATA + ω2In)v = λ2v. (7) Из (7) видно, что если v ̸= 0, тогда v – собственный вектор и λ2 соответствующее ему собственное значение матрицы ATA + ω2In, т.е. λ2 i = σ2 i + ω2 (i = 1, 2, . . . , τ). Следовательно, числа ± q σ2 i + ω2 ˜ (7) (i = 1, 2, . . . , τ) являются собственными значениями матрицы ˜Aω. T ( ) Если v = 0, а z ̸= 0, то из (7) следует, что ωz = λz и AT z = 0. Так как dim ker AT = m −τ, то ω является собственным значением кратности m −τ матрицы ˜Aω Если v = 0, а z ̸= 0, то из (7) следует, что ωz = λz и AT z = 0. Так как dim ker AT ˜ р р Если z = 0, а v ̸= 0, то из (7) следует, что −ωv = λv и Av = 0. Так как dim ker A = n −τ, то −ω является собственным значением кратности n −τ матрицы ˜Aω. □ ˜ р р Если z = 0, а v ̸= 0, то из (7) следует, что −ωv = λv и Av = 0. откуда непосредственно следует первое утверждение теоремы. ( ) ( ( ) ) Для СЛАУ (1) неполного ранга (rank (A) < k) число обусловленности регуляризованной нор- мальной системы (2) равно κ2(ATA + ω2) = λmax(ATA + ω2) λmin(ATA + ω2) = σ2 1 + ω2 ω2 , κ2(ATA + ω2) = λmax(ATA + ω2) λmin(ATA + ω2) = σ2 1 + ω2 ω2 , 151 А. И. Жданов а расширенной регуляризованной нормальной системы (РРНС) (5) — κ2( ˜Aω) = λmax( ˜Aω) λmin( ˜Aω) = p σ2 1 + ω2 ω . а расширенной регуляризованной нормальной системы (РРНС) (5) — κ2( ˜Aω) = λmax( ˜Aω) λmin( ˜Aω) = p σ2 1 + ω2 ω . а расширенной регуляризованной нормальной системы (РРНС) (5) — κ2( ˜Aω) = λmax( ˜Aω) λmin( ˜Aω) = p σ2 1 + ω2 ω . κ2( ˜Aω) = λmax( ˜Aω) λmin( ˜Aω) = p σ2 1 + ω2 ω . Таким образом, Таким образом, Таким образом, κ2(ATA + ω2) = κ2 2( ˜Aω). Таким образом, κ2(ATA + ω2) = κ2 2( ˜Aω). Если выбрать параметр ω из условий: ω2 < εmach и ω > εmach, то очевидно, что ﬂ(ATA + ω2I ) = ﬂ(ATA) κ2(ATA + ω2) = κ2 2( ˜Aω). 2( + ) 2( ω) Если выбрать параметр ω из условий: ω2 < εmach и ω > εmach, то очевидно, что ﬂ(ATA + ω2In) = ﬂ(ATA). ﬂ(ATA + ω2In) = ﬂ(ATA). ﬂ(ATA + ω2In) = ﬂ(ATA). Следовательно, при таком значении ω компьютерное решение ˜uω РРНС (5) будет достаточно точным приближением к u∗. ∗ [4, стр. 210], имеет место оценка р ∗ Как показано в [4, стр. 210], имеет место оценка р ∗ Как показано в [4, стр. 210], имеет место оценка ∥uω −u∗∥2 ⩽ ω2∥f∥2 στ(στ + ω2 ⩽ω2∥f∥2 σ3τ = ω2q, q = const. ∥uω −u∗∥2 ⩽ ω2∥f∥2 στ(στ + ω2 ⩽ω2∥f∥2 σ3τ = ω2q, q = const. Если для решения РРНС (5) применить любой обратно устойчивый алгоритм [5], например, осно- ванный на QR-разложении Хаусхолдера [2], то как показано в [3]: ∥˜xω −x∗∥2 ∥x∗∥ = O(κ2( ˜Aω)εmach), (9) (9) где ˜xω – компьютерное решение РРНС (5), x∗= (yT ∗, uT ∗)T, y∗= ω−1(f −Au∗). ( ) ( ) ( ) 4. Тестовые исследования и выводы. В качестве иллюстрации возможностей предлагаемого метода рассмотрим следующий пример. В этом примере задача решалась двумя методами: SVD- методом из пакета Matlab и предлагаемым методом на основе РРНС. откуда непосредственно следует первое утверждение теоремы. Для решения РРНС (5) (учи- тывая, что матрица ˜Aω является симметричной) был использован LDLT-метод [3], также из пакета Matlab. В РРНС (5) параметр ω = 10−15. 4. Тестовые исследования и выводы. В качестве иллюстрации возможностей предлагаемого метода рассмотрим следующий пример. В этом примере задача решалась двумя методами: SVD- методом из пакета Matlab и предлагаемым методом на основе РРНС. Для решения РРНС (5) (учи- тывая, что матрица ˜Aω является симметричной) был использован LDLT-метод [3], также из пакета Matlab. В РРНС (5) параметр ω = 10−15. ( ) р р Рассмотрим несовместную СЛАУ ( ) м несовместную СЛАУ A =    1 1 1 1 1 1 1 1 1.00000001 1 1.0000002 1   ∈R4×3, f =    −94 106 6.00000003 6.0000004   ∈R4. (10) (10) Точное псевдорешение СЛАУ (10) равно u∗= (1, 2, 3)T. Решение вычисленное SVD-методом из пакета Matlab — ˜u = (1.431, 3.668, 1.400)T, а с использованием РРНС (5) — ˜uω = (1.0000000, 1.9999999, 2 9999999)T Точное псевдорешение СЛАУ (10) равно u∗= (1, 2, 3)T. Решение вычисленное SVD-методом из пакета Matlab — ˜u = (1.431, 3.668, 1.400)T, а с использованием РРНС (5) — ˜uω = (1.0000000, 1.9999999, 2.9999999)T. ) Таким образом, предлагаемый метод на основе РРНС (при соответствующем выборе параметра ω) может даже превосходить по точности SVD-метод. При этом для СЛАУ с m ≈n РРНС превосходит по быстродействию SVD-метод. Самарский государственный аэрокосмический университет им. академика С.П. Королева, г. Самара zhdanov@smr.ru БИБЛИОГРАФИЧЕСКИЙ СПИСОК 1. Деммель, Дж. Вычислительная линейная алгебра. Теория и алгоритмы [Текст] / Дж. Деммель. — М.: Мир, 2001. — 430 с. 1. Деммель, Дж. Вычислительная линейная алгебра. Теория и алгоритмы [Текст] / Дж. Деммель. — 430 с 1. Деммель, Дж. Вычислительная линейная алгебра. Теория и алгоритмы [Текст] / Дж. Деммель. — М.: Мир, 2001. — 430 с. [ ] / 2. Голуб, Дж. Матричные вычисления [Текст] / Дж. Голуб, Ч. Ван Лоун. — М.: Мир, 1999. — 548 c 3 T f th L N N i l Li Al b [T ] / L N T f h D B SIAM Phil d l hi 1 2. Голуб, Дж. Матричные вычисления [Текст] / Дж. Голуб, Ч. Ван Лоун. М.: Мир, 1999. 548 c. 3. Trefethen, L. N. Numerical Linear Algebra [Text] / L. N. Trefethen, D. Bau. — SIAM, Philadelphia, 1997. — 373 p. у [ ] / у у 3. Trefethen, L. N. Numerical Linear Algebra [Text] / L. N. Trefethen, D. Bau. — SIAM, Philadelphia, 1997. — 373 p. б [ ] / Trefethen, L. N. Numerical Linear Algebra [Text] / L. N. Trefethen, D. Bau. SIAM, Philadelphia, 1997 Беклемишев, Д. В. Дополнительные главы линейной алгебры [Текст] / Д. В. Беклемишев. — М.: Н 336 c. [ ] / 4. Беклемишев, Д. В. Дополнительные главы линейной алгебры [Текст] / Д. В. Беклем 336 c. 5. Жданов, А. И. Введение в методы решения некорректных задач. Часть 2: учеб. пособие [Текст] / А. И. Жданов. — Самара: Изд-во Самар. гос. аэрокосм. ун-та, 2007. — 79 c. Поступила 25.09.2007 Самарский государственный аэрокосмический университет им. академика С.П. Королева, г. Самара zhdanov@smr.ru 152 Samara State Aerospace University, Samara, Russia zhdanov@smr.ru Samara State Aerospace University, Samara, Russia hd @ ABOUT ONE NUMERICAL STABLE ALGORITHM FOR SOLVING SYSTEM LINEAR ALGEBRAIC EQUATIONS OF DEFECT RANK A new method for solving unstable problems that can be reduced to arbitrary systems of linear algebraic equations (which may not be of full rank or may be inconsistent) is examined. This method is based on the reduction of regularization of normal system equations to an equivalent augmented regularization of normal system equations. Received 25.09.2007 zhdanov@smr.ru
https://openalex.org/W2127556561	https://uscholar.univie.ac.at/detail/o:243721.pdf	English	null	Noisy: Identification of problematic columns in multiple sequence alignments	Algorithms for molecular biology	2,008	cc-by	9,163	Published: 24 June 2008 Algorithms for Molecular Biology 2008, 3:7 doi:10.1186/1748-7188-3-7 This article is available from: http://www.almob.org/content/3/1/7 © 2008 Dress et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central Open Acc Software article Noisy: Identification of problematic columns in multiple sequence alignments Andreas WM Dress1,2, Christoph Flamm3, Guido Fritzsch4,5, Stefan Grünewald1,2, Matthias Kruspe5, Sonja J Prohaska3,6,7 and Peter F Stadler8,5,9,3,6 Open Access Address: 1Department of Combinatorics and Geometry (DCG), MPG/CAS Partner Institute for Computational Biology (PICB), Shanghai Institutes for Biological Sciences (SIBS), Shanghai, PR China, 2Max Planck Institute for Mathematics in the Sciences, Inselstrasse 22 -26, D 04103 Leipzig, Germany, 3Institut für Theoretische Chemie und Molekulare Strukturbiologie Universität Wien, Währingerstraße 17, A-1090 Wien, Austria, 4Institute of Biology II: Zoologie, Molekulare Evolution und Systematik der Tiere, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany, 5Interdisciplinary Center for Bioinformatics, Universität Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany, 6Santa Fe Institute, 1399 Hyde Park Rd., Santa Fe NM 87501, USA, 7Biomedical Informatics, Arizona State University, PO-Box 878809, Tempe, AZ 85287, USA, 8Bioinformatics Group, Department of Computer Science, Universität Leipzig, Härtelstraße 16-18, D-04107 Leipzig, Germany and 9RNomics Group, Fraunhofer Institut for Cell Therapy and Immunology (IZI), Perlickstraße 1, D-04103 Leipzig, Germany Email: Andreas WM Dress - andreas@picb.ac.cn; Christoph Flamm - xtof@tbi.univie.ac.at; Guido Fritzsch - gfritz@uni-leipzig.de; Stefan Grünewald - stefan@picb.ac.cn; Matthias Kruspe - matthias@bioinf.uni-leipzig.de; Sonja J Prohaska - sopr@tbi.univie.ac.at; Peter F Stadler - studla@bioinf.uni-leipzig.de * Corresponding author Received: 8 April 2008 Accepted: 24 June 2008 Received: 8 April 2008 Accepted: 24 June 2008 Published: 24 June 2008 Trees, metrics, and weighted split systems Consequently, one may try to improve the accuracy of tree reconstruction by eliminating all putative homoplastic or otherwise corrupted sites, e.g., all third-codon positions of protein-coding genes. However, since the quality of tree reconstruction decreases with decreasing sequence length, it is important not to remove too many sites from an alignment. For example, while certain first- and second- codon positions may be essentially constant (and there- fore phylogenetically useless) or hyper-variable (and hence even misleading), third-codon positions of protein- coding genes can well be informative and should not be just discarded as such [3]. There is no consensus in the lit- erature regarding the tolerance of phylogenetic methods to multiple substitutions [4,5]. Let X denote a finite set of n taxa. A split S = A\| = \|A is a bipartition of the set X of taxa, i.e., a partition of X into two disjoint, non-empty subsets A and . Two such splits A1\| and A2\| of X are called compatible if one of the four intersections A1 ∩ A2, A1 ∩ , ∩ A2 and ∩ is empty. A split system is compatible if every pair of splits is compatible. A A A A1 A2 A2 A1 A1 A2 It is a well known result that compatible split systems on X are in 1-1 correspondence with the so-called X-trees [16], i.e., finite trees T = (V, E) with vertex set V and edge set E endowed with a map from X into V whose image contains (at least) all vertices of degree less than 3. Given any alignment, it is therefore of interest to detect clearly homoplastic or otherwise corrupted sites from putative phylogenetically informative sites so that they – and no others – can be excluded or down-weighted. The complication with such an endeavor, however, is that, for- mally, homoplasy is defined relative to a given phyloge- netic tree while it is exactly a phylogenetic tree that molecular phylogenetics is attempting to derive from an alignment. Thus, care has to be taken that homoplasy detection does not implicitly presuppose a phylogenetic tree later to be derived from the same data. Abstract Motivation: Sequence-based methods for phylogenetic reconstruction from (nucleic acid) sequence data are notoriously plagued by two effects: homoplasies and alignment errors. Large evolutionary distances imply a large number of homoplastic sites. As most protein-coding genes show dramatic variations in substitution rates that are not uncorrelated across the sequence, this often leads to a patchwork pattern of (i) phylogenetically informative and (ii) effectively randomized regions. In highly variable regions, furthermore, alignment errors accumulate resulting in sometimes misleading signals in phylogenetic reconstruction. Results: We present here a method that, based on assessing the distribution of character states along a cyclic ordering of the taxa, allows the identification of phylogenetically uninformative homoplastic sites in a multiple sequence alignment. Removal of these sites appears to improve the performance of phylogenetic reconstruction algorithms as measured by various indices of "tree quality". In particular, we obtain more stable trees due to the exclusion of phylogenetically incompatible sites that most likely represent strongly randomized characters. Software: The computer program noisy implements this approach. It can be employed to improving phylogenetic reconstruction capability with quite a considerable success rate whenever (1) the average bootstrap support obtained from the original alignment is low, and (2) there are sufficiently many taxa in the data set – at least, say, 12 to 15 taxa. The software can be obtained under the GNU Public License from http://www.bioinf.uni-leipzig.de/Software/noisy/. Page 1 of 10 (page number not for citation purposes) Algorithms for Molecular Biology 2008, 3:7 http://www.almob.org/content/3/1/7 Introduction ods simply delete the most highly variable alignment columns [10,11], the S-F approach [12] presupposes well- established groups and evaluates within-group variation relative to between-groups variation. Sequence conservation in real data often varies dramati- cally along multiple sequence alignments ranging from constant sites to sequence positions that have effectively been randomized. In the context of phylogenetic recon- struction, homoplastic sites – i.e., those in which the same character appears in two distinct sequences by conver- gence (back- and parallel-mutation) rather than by com- mon ancestry – pose a well-known problem. Depending on the method, in the worst case they present a mislead- ing signal (as in the case of parsimony methods), at best they increase the noise in the data (as in most distance- based methods). In addition, alignment errors producing effectively "homoplastic sites" are known from simula- tion studies to decrease the accuracy of the reconstruction of tree topologies [1]. For real data, ref. [2] showed that alignment errors can change the result of a phylogenetic analysis significantly. In this contribution, we present a new method for deter- mining "noisy" sites in an alignment that is not a priori restricted to tree-like data. It is based on the observation that distances derived from pairwise sequence compari- sons give rise to fairly robust circular split systems [13] which, in turn, are consistent with a large number of pos- sible tree topologies [14,15]. We only use the cyclic order- ing of the taxa which some methods constructing circular split systems compute in their first step, not a recon- structed tree, to assess the degree to which an alignment site is randomized. A computer program, called noisy, implements this approach. Noise detection using circular orderings An alternative approach starts from weighted quartets. To this end, one first computes a weight for each quartet, i.e., each pair of two pairs of taxa, {{a, b} {c, d}}. This quartet weight is interpreted as the support for the hypothesis that {a, b} and {c, d} are separated by an edge in the correct phylogenetic tree. Quartet weights can be obtained in var- ious ways. In the quartet-mapping approach [20] for exam- ple, one starts with an alignment of four sequences and defines the weight of a given quartet to be the fraction of alignment sites (columns) in which a = b ≠ c = d. One may modify this score by adding 1/2 for every additional col- umn in which a = b ≠ c, d or c = d ≠ a, b holds. Quartet weights can also be derived directly from distances (although, in this case, it seems preferably to use the faster Neighbor-Net approach). A more sophisticated weighting scheme uses "expected branch lengths", i.e. the product of the posterior likelihood and the maximum likelihood branch length of the interior edge of the corresponding quartet tree. A split system is circular if the points in X (i.e., the taxa) can be arranged on a circle so that each split S ∈ is induced by a division of that circle into two arcs by delet- ing two of its (unlabeled) points. In this case, the circular ordering is said to represent the split system.   It is easy to verify that compatible split systems are circular (actually, every planar drawing of an X-tree provides such a circular ordering), and that circular split systems are weakly compatible – i.e., A1 ∩ A2 ∩ A3, A1 ∩ ∩ , ∩ A2 ∩ or ∩ ∩ A3 is empty for any three splits A1\| , A2\| , A3\| in a circular split system, cf. [13]. Any distance constructed from a weighted circular split system is called a "circular" (or Kalmanson) metric. A2 A3 A1 A3 A1 A2 A1 A2 A3 It has been observed that phylogenetic distance data are often circular or at most mildly non-circular [14,17,18]. Starting from a suitable distance measure, we can con- struct a circular split system from an alignment without significantly prejudicing later tree constructions since the circular split system still represents essentially unfiltered data. Trees, metrics, and weighted split systems More specifically, this correspondence is given by associ- ating (i) to any edge e ∈ E of such a tree T, the bipartition Se of X into those two subsets of X that are mapped into the (exactly) two distinct connected components of the graph obtained from T by deleting the edge e, (ii) and to T the collection (T) := {Se : e ∈ E} of all such splits.  Character compatibility [6] can be used to identify fast evolving sites [7,8]. Two alignment columns are compati- ble if there is a phylogenetic tree for which both columns are homoplasy-free. Fast-evolving sites are expected to be incompatible with more columns than slowly evolving ones. Consequently, sites that have more incompatibili- ties than random sites are removed from the alignment [9]. If there are conflicting signals in the data, sites sup- porting the weaker one tend to be removed. Several meth- Associating a positive weight αS to any such split S = A\| (e.g., the length of the edge e in case every edge in the tree is endowed with some predefined positive length and S = Se holds), one can define the associated metric d on X by associating, to any two taxa x, y in X, the term A Page 2 of 10 (page number not for citation purposes) http://www.almob.org/content/3/1/7 Algorithms for Molecular Biology 2008, 3:7 more, if the true phylgenetic tree T is not compatible with the pre-supposed circular order C, we can still expect that T will be compatible with a circular order C' that differs from C by only a small number of breakpoints – after all, we will compute C from the data that have evolved according to T . Hence, characters that are informative for T (and thus for C') can be expected not to "look random" when arranged according to C instead of C'. Thus, circular orders appear to offer a robust way to assess the "phyloge- netic information content" of characters (alignment col- umns) without strongly prejudicing the subsequent tree construction. Circular split systems can be obtained in various ways. The computationally most straightforward approach is the Neighbor-Net algorithm [19] that starts from a distance matrix. It computes the circular splits using an agglomerative procedure. Trees, metrics, and weighted split systems d x y x y S S S T ( , ) : ( , ) ( ) = ∈∑α δ  where one puts, for any split S = A\| ∈ (T) and all x, y ∈ X, δS (x, y) := 0 if x, y ∈ A or x, y ∈ holds, and δS (x, y) := 1 otherwise (i.e., if x and y are separated by the split S) implying that d(x, y) is the total length of the unique path from (the image of) x to (the image of) y relative to the given family of split weights . A  A ( ) ( ) α S S T ∈ where one puts, for any split S = A\| ∈ (T) and all x, y ∈ X, δS (x, y) := 0 if x, y ∈ A or x, y ∈ holds, and δS (x, y) := 1 otherwise (i.e., if x and y are separated by the split S) implying that d(x, y) is the total length of the unique path from (the image of) x to (the image of) y relative to the given family of split weights . A  A ( ) ( ) α S S T ∈ It is our goal to detect homoplasy without first determin- ing a tree; thus we have to admit more general split sys- tems. We use circular split systems which we will introduce next. Page 3 of 10 (page number not for citation purposes) Noise detection using circular orderings For each character χ • Compute the number ν(C, χ) of break points. • Compute the number ν(C, χ) of break points. • Compute N random cyclic orderings C'. For our purpose, the important property of the circular orderings computed by Neighbor-Net and Qnet is that phylogenetically more closely related taxa are preferen- tially placed closer together in the cyclic ordering. Thus, if a character χ = χi (defined by some alignment site i in a given alignment) is phylogenetically "useful", its character states will appear "clustered" along the cyclic ordering, independent of the details of the branching order in indi- vidual subtrees. In contrast, if a character is completely randomized, we will observe that character states are ran- domly arranged along the cycle. The amount of clustering can be easily quantified by the number ν = ν (C, χ) of adjacent distinct character states along the cycle C. We have ν = 0 for constant sites, and ν ≥ 2 for all non-constant sites. This number has to be compared with the numbers expected for a random distribution of character values along the cycle, given the overall distribution of the char- acter values of χ . It is in principle possible to compute this distribution. • For each cyclic ordering compute ν (C', χ). • Compute the fraction q(C, χ) of random orderings with ν(C', χ) > ν(C, χ). 3. If q(C, χ) is smaller than a given threshold, then remove the character χ. The program noisy is implemented in ISO C++ and the source code is available for download from http:// www.bioinf.uni-leipzig.de/Software/noisy/. In a first phase, a cyclic ordering of the taxa set is computed. For this purpose, noisy includes the corresponding subset of routines from the NeighborNet [19] and the QNet [21] packages. Subsequently, a reliability score q for each char- acter is calculated. The number of character-state altera- tions is counted and compared to the observed count in random shufflings. The uniform pseudo-random number generator Mersenne Twister [24] is used to generate the random shufflings. For two-state characters, a formula for the number of options to putting v ones and n - v zeros on a cycle of length n such that there are 2k ≤ min{2v, 2(n - v)} break- points (an odd number of breakpoints is impossible) is easy to derive: There are such options. Noise detection using circular orderings n k v k n v k − − ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ −− − ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ 1 1 1 1 In order to assess whether the cyclic orderings obtained using QNet and NeighborNet reduce the fraction of unin- terpretable variation, we performed the following rand- omization experiment. Given an alignment, we generated all possible cyclic orderings and computed the fraction r of sites with q > 0.8 among all variable sites in the align- ment. As shown in Fig. 1, QNet and NeighborNet nearly minimize the fraction of "noisy" alignment sites for the 10 squamate mitochondria. The program noisy exports a Postscript file, visualizing the quality of the sites of the reordered input alignment (see Fig. 2), recording their reliability score as xy-data, and containing a modified alignment for further analysis in which sites with reliabil- ity q <qcutoff are removed. Fig. 2 shows typical examples for the distribution of alignment sites with low and high reli- ability scores q. The explicit evaluation of such expressions is relatively expensive, however. Alternatively, very large tables would need to be pre-computed and stored to accomodate large numbers of sequences and/or character states. Therefore, we opted for a shuffling procedure instead: we randomly generate a cyclic ordering C' of the same charac- ter states (and their respective frequencies) as those in C and compute the fraction q = q(C, χ) of randomized sam- ples with ν(C', χ) > ν(C, χ). Hence we can interpret q as a reliability measure for the phylogenetic information con- tained in the alignment site (relative to C). Note that we obtain q = 0 for constant and singleton sites, which are phylogenetically uninformative and q 0.5 for effectively randomized sites. Sites with q << 0.5 are "worse" then ran- dom and contradict the given cyclic ordering while sup- port for the ordering is found in sites with q Ŭ 0.5. Noise detection using circular orderings The quartet {{a, b} {c, d}} is said to be realized by a cyclic ordering of X if the straight line connecting a and b and the straight line connecting c and d do not intersect in the interior of the circle. There is a circular split system repre- sented by a given cyclic ordering that contains a split that separates a and b from c and d if and only if {{a, b} {c, d}} is realized by that cyclic ordering. Hence, to ensure that as much quartet information as possible is represented, QNet [21] tries to find a cyclic ordering such that the sum of the weights of all realized quartets is maximal. Prescribing a circular order C, of course, restricts the pos- sible phylogenetic trees. Indeed, the fraction of fully resolved trees compatible with a given ordering goes to zero with the number n of leaves going to infinity. On the other hand, given any circular ordering, there are quite a few - more precisely, there are exactly - fully resolved trees that are compatible with it [15]. Further- 2 2 1 n n − − ( )! 1 1 2 4 2 n n n − − − ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ Prescribing a circular order C, of course, restricts the pos- sible phylogenetic trees. Indeed, the fraction of 2 2 1 n n − − ( )! Both, Neighbor-Net and QNet, use the same agglomera- tion process to construct a cyclic ordering. While Neigh- bor-Net tries to group those taxa close to each other that have a small distance, QNet tries to construct a cyclic ordering that maximizes the sum of the weights of the Page 3 of 10 (page number not for citation purposes) Page 3 of 10 (page number not for citation purposes) http://www.almob.org/content/3/1/7 Algorithms for Molecular Biology 2008, 3:7 1. Compute the cyclic ordering C from the input data using either Qnet or NeighborNet. quartets it realizes. Hence, both methods construct cyclic orderings with the property that groups of phylogeneti- cally closely related taxa tend to assemble along an arc. Neighbor-Net and QNet are both consistent, i.e., if the dis- tances or quartet weights correspond to a circular split sys- tem, then they find a cyclic ordering that represents that split system [22,23]. 2. For each character χ 2. The program noisy executes the following commands: Page 4 of 10 (page number not for citation purposes) Computational results As an example for the effect of removing "noisy" sites, we consider a data set of combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences of spatangoid sea urchins that was reported to have a high level of homoplasy [25]. The "raw" sequence alignments lead to phylogenetic trees that differ significantly for different methods and disagree substantially with morphology-based results. As discussed The program noisy executes the following commands: Page 4 of 10 (page number not for citation purposes) Page 4 of 10 (page number not for citation purposes) http://www.almob.org/content/3/1/7 Algorithms for Molecular Biology 2008, 3:7 Number of cyclic orderings of a set 10 complete mitochondrial genomes with a prescribed fraction of "noisy" characters, i.e., q(C, χ) ≤ 0.8) Figure 1 Number of cyclic orderings of a set 10 complete mitochondrial genomes with a prescribed fraction of "noisy" characters, i.e., q(C, χ) ≤ 0.8). The cyclic orderings computed by NeighborNet or QNet indeed essentially minimize the fraction of putative randomized alignment sites. At least in this example, QNet with quartet-mapping-derived quartet weights performs best. "ClustalW" refers to the circular ordering implicitly constructed by ClustalW from its guide tree which deter- mines the order in which sequences and profiles are combined to yield the final alignment. Fraction of noisy positions Number of circular orderings 0.64 0.66 0.68 0.70 0.72 0.74 0 5000 10000 15000 20000 NeighborNet ClustalW QNet/QM Number of cyclic orderings of a set 10 complete mitochondrial genomes with a prescribed fraction of noisy characters, i.e., q(C, χ) ≤ 0.8) Figure 1 Number of cyclic orderings of a set 10 complete mitochondrial genomes with a prescribed fraction of "noisy" characters, i.e., q(C, χ) ≤ 0.8). The cyclic orderings computed by NeighborNet or QNet indeed essentially minimize the fraction of putative randomized alignment sites. At least in this example, QNet with quartet-mapping-derived quartet weights performs best. "ClustalW" refers to the circular ordering implicitly constructed by ClustalW from its guide tree which deter- mines the order in which sequences and profiles are combined to yield the final alignment. same qualitative behavior. Table 1 shows that the fraction of effectively randomized sites varies considerably (from 26% to 37%) between different proteins even in the rela- tively benign case of mitochondrial genomes [31]. As expected, the homoplasy index is significantly reduced while the rescaled consistency index and the scaled log- likelihood values increases with increasing values of qcut- off. Computational results The other data sets show the Page 5 of 10 (page number not for citation purposes) Page 5 of 10 (page number not for citation purposes) Algorithms for Molecular Biology 2008, 3:7 http://www.almob.org/content/3/1/7 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below) Figure 2 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below). In terms of quality, the two data sets are very different. While the majority of sites in atp6 are parsimony informative and approximately one third of the sites have a reliability score above qcutoff = 0.8, this is clearly not the case for the data set by [37] where most of the sites are constant or unreliable. The black bar below the alignment indicates whether the q-value of the corresponding position is above (upper half) or below (lower half) the cutoff value. Note that only green positions have a chance to having q-value above the cutoff value. missing data constant site singleton site parsimony informative site 50 100 150 200 250 300 350 400 450 500 550 600 650 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below) Figure 2 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below). In terms of quality, the two data sets are very different. While the majority of sites in atp6 are parsimony informative and approximately one third of the sites have a reliability score above qcutoff = 0.8, this is clearly not the case for the data set by [37] where most of the sites are constant or unreliable. The black bar below the alignment indicates whether the q-value of the corresponding position is above (upper half) or below (lower half) the cutoff value. Note that only green positions have a chance to having q-value above the cutoff value. Distributio Coleopter Figure 2 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below) Figure 2 Distribution of homoplastic sites for the mitochondrial atp6 gene of squamata (2047 positions, above) and for 18S RNA of Coleoptera from an analysis of [37] (684 positions, below). In terms of quality, the two data sets are very different. While the majority of sites in atp6 are parsimony informative and approximately one third of the sites have a reliability score above qcutoff = 0.8, this is clearly not the case for the data set by [37] where most of the sites are constant or unreliable. The black bar below the alignment indicates whether the q-value of the corresponding position is above (upper half) or below (lower half) the cutoff value. Note that only green positions have a chance to having q-value above the cutoff value. An alternative measure for the stability of a phylogenetic reconstruction is the bootstrap support for trees – result- ing, in our case, from neighbor joining [33]. In some cases, the improvement can be substantial, as in the case of a Dytiscus data set provided in the supplement, where the average bootstrap support increases from 47 to 68 (neighbor-joining trees computed using PAUP 4.0b10 and 2000 bootstrap replicates [34,35]). In benign data sets, however, the changes are typically small. bootstrap support relative to the cutoff value q. Pairs of caterpillar and balanced trees with the same number of taxa were constructed such that (a) all leaves have the same evolutionary distance from the root and (b) all inter- nal edges as well as all edges leading to leaves with maxi- mal depth (maximal number of internal nodes on the path to the root) have the same "unit length". This unit length is set to 0.4 substitutions per site for the balanced trees. In the caterpillar trees the "unit length" is scaled such that the total length equals that of the balanced tree with the same number of species. Computational results missing data constant site singleton site parsimony informative site 50 100 150 200 250 300 350 400 450 500 550 600 650 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 Computational results While the tree-stability indices improve consistently indicating that the reconstructions become more stable, the absolute values of the quality indices nevertheless depend strongly on the size and quality of the input align- ments. in the original paper [25], manual removal of homoplas- tic sites improved the trees considerably. The application of noisy with cutoff qcutoff = 0.8, on the other hand, leads to consistent results for all methods including MP (Maxi- mum Parsimony) that agree with the best trees reported in [25]. In Fig. 3 we present the MP trees for the unedited and the noisy-reduced alignments. In order to assess to what extent the removal of unreliable sites from real and simulated alignments affects the com- monly used measures of tree stability, we consider the qcut- off-dependency of the most common indices for tree quality. Phylogenies were computed using maximum par- simony and neighbor joining (Kimura 2-parameter model) as implemented in PAUP 4.0b10 [26]. Scaled log- likelihood score (i.e., the log likelihood divided by the length of the alignment), homoplasy index (HI) [27], rescaled consistency index (RC) [28], and average boot- strap support (over all internal vertices) were used to assess the tree stability while topological changes were described by split distance [29]. Data sets are available for download as part of the Electronic supplement [30]. Ref. [32] suggested another way to estimate the phyloge- netic information content of an alignment. To this end, they determined the skewness-test statistics g1 of the corre- sponding tree-length distribution. We analyzed the data with the random-tree option implemented in PAUP 4.0b10 [26]. For the data matrices, we estimated 100.000 trees at random from all possible tree topologies (replace- ments allowed). The results are consistent with the tree statistics discussed above. As expected, we observe that g1 becomes more negative with increasing values of qcutoff, at least as long as one does not start to remove too many informative sites (data not shown). Fig. 4 summarizes the data for alignments of mitochon- drial protein-coding genes. Page 6 of 10 (page number not for citation purposes) Distributio Coleopter Figure 2 For each tree, we then used dawg to generate 100 independent alignments using the following parameters: alignment length 800 nt, GTR model with γ = 0.5 and ι = 0.1, and dawg's default substi- In order to study the effect of removing putative homo- plastic sites in a more systematic way, we generated artifi- cial data sets for caterpillar and balanced trees with 4 to 29 taxa using dawg [36]. Fig. 5 shows the variation of the Page 6 of 10 (page number not for citation purposes) Page 6 of 10 (page number not for citation purposes) http://www.almob.org/content/3/1/7 Algorithms for Molecular Biology 2008, 3:7 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25] Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25]. L.h.s. from original data, r.h.s. from a reduced alignment with cutoff q = 0.8. The latter tree matches the biological expectation and fits very well with those reported in [25] that were obtained from a manually reduced alignment. In particular, the noisy-reduced MP tree correctly shows Brissopsis and Allobrissus as sister groups and it correctly identifies the large monophyletic clade consisting of the Linopneustes/Metalia and Lovenia/Spatangus groups to the exclusion of Meoma and Archeopneustes. These major improve- ments are marked with a bullet. The included table compares the stability indices (HI = homoplasy index, RC = rescaled con- sistency index, RI = retention index) between the complete (unprocessed), Stockley's manually improved, and the noisy- reduced alignment. 2528 543 0.54 0.41 0.19 raw 2227 465 0.59 0.44 0.20 noisy 2076 260 0.50 0.56 0.28 RC RI HI PI−sites length Stockley Conolampas sigsbei Echinoneus cyclostomus Paraster doederleini Archeopneustes hystrix Spantagus matheyi Spantagus raschi Paramaretia multituerculata Echinocardia laevigaster Lovenia cordiformis Allobrissus agassizii Metalia spatagus Plagiobrissus grandis Linopneustes longispinus Meoma ventricosa Brissopsis atlantica Paleopneustes cristatus Brisaster fragilis Amphipneustes lorioli Abatus cavernosus Amphipneustes lorioli Abatus cavernosus Brisaster fragilis Paleopneustes cristatus Allobrissus agassizii Brissopsis atlantica Meoma ventricosa Linopneustes longispinus Plagiobrissus grandis Metalia spatagus Lovenia cordiformis Echinocardia laevigaster Paramaretia multituerculata Spantagus raschi Spantagus matheyi Archeopneustes hystrix Paraster doederleini Echinoneus cyclostomus Conolampas sigsbei MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25] Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25]. L.h.s. from original data, r.h.s. Distributio Coleopter Figure 2 In particular, the noisy-reduced MP tree correctly shows Brissopsis and Allobrissus as sister groups and it correctly identifies the large monophyletic clade consisting of the Linopneustes/Metalia and Lovenia/Spatangus groups to the exclusion of Meoma and Archeopneustes. These major improve- ments are marked with a bullet. The included table compares the stability indices (HI = homoplasy index, RC = rescaled con- sistency index, RI = retention index) between the complete (unprocessed), Stockley's manually improved, and the noisy- reduced alignment. MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25] Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25]. L.h.s. from original data, r.h.s. from a reduced alignment with cutoff q = 0.8. The latter tree matches the biological expectation and fits very well with those reported in [25] that were obtained from a manually reduced alignment. In particular, the noisy-reduced MP tree correctly shows Brissopsis and Allobrissus as sister groups and it correctly identifies the large monophyletic clade consisting of the Linopneustes/Metalia and Lovenia/Spatangus groups to the exclusion of Meoma and Archeopneustes. These major improve- ments are marked with a bullet. The included table compares the stability indices (HI = homoplasy index, RC = rescaled con- sistency index, RI = retention index) between the complete (unprocessed), Stockley's manually improved, and the noisy- reduced alignment. MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25] Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25]. L.h.s. from original data, r.h.s. from a reduced alignment with cutoff q = 0.8. The latter tree matches the biological expectation and fits very well with those reported in [25] that were obtained from a manually reduced alignment. In particular, the noisy-reduced MP tree correctly shows Brissopsis and Allobrissus as sister groups and it correctly identifies the large monophyletic clade consisting of the Linopneustes/Metalia and Lovenia/Spatangus groups to the exclusion of Meoma and Archeopneustes. These major improve- ments are marked with a bullet. The included table compares the stability indices (HI = homoplasy index, RC = rescaled con- sistency index, RI = retention index) between the complete (unprocessed), Stockley's manually improved, and the noisy- reduced alignment. number of taxa and the topology of the tree. Distributio Coleopter Figure 2 For small val- ues of qcutoff, alignment stability increases because only the most "noisy" sites are removed. (In contrast, tree sta- tution matrix for the GTR model. We observe a pro- nounced maximum of bootstrap support whose position and height, however, depends strongly on both, the Dependency of tree-quality indices on the cut-off value qcutoff for the protein-coding mitochondrial genes from all 31 currently available squamata Figure 4 Dependency of tree-quality indices on the cut-off value qcutoff for the protein-coding mitochondrial genes from all 31 currently available squamata. The stability of the trees is measured by the scaled log likelihood (ln L)/n, the homo- plasy index (HI) [27], and the rescaled consistency index (RC) [28] as computed by PAUP 4.0b10 [26]. Data sets are alignments (supplied in the electronic supplement) of individual mitochondrial protein-coding genes. They vary in size (from about 170 to 1800 nt) and randomization. 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff -30 -25 -20 -15 -10 (ln L)/n 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff 0.05 0.10 0.15 0.20 0.25 Rescaled consistency (RC) 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff 0.64 0.66 0.68 0.70 0.72 0.74 0.76 Homoplasy index (HI) ND1 ND2 ND6 ND3 COX2 ATP8 ND5 CYTB ATP6 ND4 ND4L COX1 COX3 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff -30 -25 -20 -15 -10 (ln L)/n ND1 ND2 ND6 ND3 COX2 ATP8 ND5 CYTB ATP6 ND4 ND4L COX1 COX3 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff 0.05 0.10 0.15 0.20 0.25 Rescaled consistency (RC) 0.0 0.2 0.4 0.6 0.8 1.0 qcutoff 0.64 0.66 0.68 0.70 0.72 0.74 0.76 Homoplasy index (HI) Distributio Coleopter Figure 2 from a reduced alignment with cutoff q = 0.8. The latter tree matches the biological expectation and fits very well with those reported in [25] that were obtained from a manually reduced alignment. In particular, the noisy-reduced MP tree correctly shows Brissopsis and Allobrissus as sister groups and it correctly identifies the large monophyletic clade consisting of the Linopneustes/Metalia and Lovenia/Spatangus groups to the exclusion of Meoma and Archeopneustes. These major improve- ments are marked with a bullet. The included table compares the stability indices (HI = homoplasy index, RC = rescaled con- sistency index, RI = retention index) between the complete (unprocessed), Stockley's manually improved, and the noisy- reduced alignment. 2528 543 0.54 0.41 0.19 raw 2227 465 0.59 0.44 0.20 noisy 2076 260 0.50 0.56 0.28 RC RI HI PI−sites length Stockley Conolampas sigsbei Echinoneus cyclostomus Paraster doederleini Archeopneustes hystrix Spantagus matheyi Spantagus raschi Paramaretia multituerculata Echinocardia laevigaster Lovenia cordiformis Allobrissus agassizii Metalia spatagus Plagiobrissus grandis Linopneustes longispinus Meoma ventricosa Brissopsis atlantica Paleopneustes cristatus Brisaster fragilis Amphipneustes lorioli Abatus cavernosus Amphipneustes lorioli Abatus cavernosus Brisaster fragilis Paleopneustes cristatus Allobrissus agassizii Brissopsis atlantica Meoma ventricosa Linopneustes longispinus Plagiobrissus grandis Metalia spatagus Lovenia cordiformis Echinocardia laevigaster Paramaretia multituerculata Spantagus raschi Spantagus matheyi Archeopneustes hystrix Paraster doederleini Echinoneus cyclostomus Conolampas sigsbei MP f d h f b d Fi 3 RC RI HI PI−sites length Conolampas sigsbei Echinoneus cyclostomus Paraster doederleini Archeopneustes hystrix Spantagus matheyi Spantagus raschi Paramaretia multituerculata Echinocardia laevigaste Lovenia cordiformi Allobrissus agassizii Metalia spatagus Plagiobrissus grandis Linopneustes longispinus Meoma ventricosa Brissopsis atlantica Paleopneustes cristatus Brisaster fragilis Amphipneustes lorioli Abatus cavernosus A d h d l COI [25] Amphipneustes lorioli Abatus cavernosus Brisaster fragilis Paleopneustes cristatus Allobrissus agassizii Brissopsis atlantica Meoma ventricosa Linopneustes longispinus Plagiobrissus grandis Metalia spatagus Lovenia cordiformis Echinocardia laevigaster Paramaretia multituerculata Spantagus raschi Spantagus matheyi Archeopneustes hystrix Paraster doederleini Echinoneus cyclostomus Conolampas sigsbei Allobrissus agassizii MP trees o Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25] Figure 3 MP trees of spatangoid sea urchins from combined 28S rRNA, 16S rRNA, and mitochondrial COI sequences [25]. L.h.s. from original data, r.h.s. from a reduced alignment with cutoff q = 0.8. The latter tree matches the biological expectation and fits very well with those reported in [25] that were obtained from a manually reduced alignment. Dependen available sq Figure 4 Dependency of tree-quality indices on the cut-off value qcutoff for the protein-coding mitochondrial genes from all 31 currently available squamata Figure 4 Dependency of tree-quality indices on the cut-off value qcutoff for the protein-coding mitochondrial genes from all 31 currently available squamata. The stability of the trees is measured by the scaled log likelihood (ln L)/n, the homo- plasy index (HI) [27], and the rescaled consistency index (RC) [28] as computed by PAUP 4.0b10 [26]. Data sets are alignments (supplied in the electronic supplement) of individual mitochondrial protein-coding genes. They vary in size (from about 170 to 1800 nt) and randomization. Page 7 of 10 (page number not for citation purposes) Algorithms for Molecular Biology 2008, 3:7 http://www.almob.org/content/3/1/7 Table 1: Randomized sites (at qcutoff = 0.8) in the 13 different individual protein-coding genes within the 31 currently available complete mitochondrial genomes of squamata. sngl: number of singleton positions, %rnd: percentage of randomized variable sites. Gene length sngl q ≥ 0.8 %rnd atp6 684 42 405 34.65 atp8 171 7 108 32.75 cox1 1536 88 1008 28.65 cox2 672 34 443 29.02 cox3 786 45 516 28.63 cytb 1131 74 676 33.69 nd1 942 44 589 32.80 nd2 1032 63 626 33.24 nd3 345 11 222 32.46 nd4 1371 65 831 34.65 nd4l 288 16 183 30.90 nd5 1803 103 1040 36.61 nd6 540 25 373 26.30 Table 1: Randomized sites (at qcutoff = 0.8) in the 13 different individual protein-coding genes within the 31 currently available complete mitochondrial genomes of squamata. sngl: number of singleton positions, %rnd: percentage of randomized variable sites. a good compromise between these two effects. In princi- ple, an optimal cut-off value could be estimated, provided a well-curated training set was available. For small data sets, with less than 15 taxa, we found no improvements except for rather small qcutoff values reflecting the fact that, for small data sets, there are not too many possibilities for the values of ν(C, χ) implying that noisy should be used only for at least moderately large data sets. In general, the caterpillar trees admit larger improvements in bootstrap support than the balanced ones. We remark that the balanced trees are almost correctly reconstructed while the caterpillar trees are poorly reconstructed, in par- ticular at the deep nodes (data not shown). A systematic analysis of the effects of tree shape and branch length distributions will be given elsewhere. Discussion It has been argued repeatedly that saturated – homoplas- tic – characters are detrimental to phylogeny reconstruc- tion and, thus, should be removed from multiple The relative average bootstrap support of phylogenetic trees is computed as the ratio of the average bootstrap support for the modified alignments divided by the bootstrap support obtained from the original alignment Figure 5 The relative average bootstrap support of phylogenetic trees is computed as the ratio of the average boot- strap support for the modified alignments divided by the bootstrap support obtained from the original align- ment. Values larger than 1 indicate an increase in tree robustness. The curves show a distinct maximum that depends on the number of taxa and the topology of the tree. The maximum improvement increases with the number of taxa (indicated on the right margin of both panels for the highlighted curves). For clarity, error bars obtained from 100 replicates are shown only for N = 10 and N = 25 taxa. The tree topologies, caterpillar trees on the left and balanced trees on the right, are depicted by the insets. 0.0 0.2 0.4 0.6 0.8 1.0 q cutoff 0.60 0.80 1.00 1.20 1.40 relative average bootstrap support 8 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 q cutoff 0.80 0.90 1.00 1.10 relative average bootstrap support 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 q cutoff 0.60 0.80 1.00 1.20 1.40 relative average bootstrap support 8 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 q cutoff 0.80 0.90 1.00 1.10 relative average bootstrap support 10 15 20 25 relative average bootstrap support The relativ modified a Figure 5 The relative average bootstrap support of phylogenetic trees is computed as the ratio of the average bootstrap support for the modified alignments divided by the bootstrap support obtained from the original alignment Figure 5 The relative average bootstrap support of phylogenetic trees is computed as the ratio of the average boot- strap support for the modified alignments divided by the bootstrap support obtained from the original align- ment. Values larger than 1 indicate an increase in tree robustness. The curves show a distinct maximum that depends on the number of taxa and the topology of the tree. The maximum improvement increases with the number of taxa (indicated on the right margin of both panels for the highlighted curves). Dependen available sq Figure 4 We will also discuss in that note how our algorithm can be used to deal with the alignment problems addressed in [2]. bility decreases immediately when randomly chosen alignment columns are removed; data not shown). For large values of qcutoff, tree stability starts to decrease again because noisy starts to remove too many informative sites. Empirically, we found for large data sets that qcutoff ≈ 0.8 is bility decreases immediately when randomly chosen alignment columns are removed; data not shown). For large values of qcutoff, tree stability starts to decrease again because noisy starts to remove too many informative sites. Empirically, we found for large data sets that qcutoff ≈ 0.8 is Discussion For clarity, error bars obtained from 100 replicates are shown only for N = 10 and N = 25 taxa. The tree topologies, caterpillar trees on the left and balanced trees on the right, are depicted by the insets. Page 8 of 10 (page number not for citation purposes) Algorithms for Molecular Biology 2008, 3:7 http://www.almob.org/content/3/1/7 the maximum of the gain as a function of q and to use the corresponding optimal cutoff value. sequence alignments [5]. Since homoplasy is defined rel- ative to the unknown true tree, it is not obvious, however, how to reliably identify the homoplastic characters with- out prior knowledge of that tree. In this note, we show that cyclic orderings that can be obtained robustly, e.g., from pairwise distance data, without detailed knowledge of the correct phylogenetic relationships can be employed for this task. Given a circular ordering that is consistent with the phylogeny, the variation of character states of a given site along the circle is used to determine the (puta- tive) degree of its randomization. This information can then be used to prune the sequence alignment. The com- puter program noisy that is publicly available from the authors' website implements this procedure. The analysis of several published data sets shows that removal of randomized sites consistently leads to more stable trees, irrespective of the method used for phylogeny reconstruction (neighbor joining, maximum parsimony, or maximum likelihood). While in benign data sets, the effects on consistency indices, likelihood score, or boot- strap support are typically small and we do not observe changes in the reconstructed tree topologies, the effects of removing homoplastic sites can become dramatic for poor data sets, as the example of the Cox1 genes of Dytis- cus demonstrates. More importantly, in some cases, the reconstructed tree topologies can be improved as well, see e.g. the example of the sea urchin phylogeny in Fig. 3. High rates of substitutions not equally distributed among sites in the sequences caused, e.g., by sequence constraints due to environmental pressure can produce a considera- ble amount of phylogenetic noise in the data and so- called "bad" and phylogenetically misleading alignments. Such alignments can be improved by increasing the sig- nal-to-noise ratio through exclusion of noisy sites. Align- ment modifications like concatenation of conserved blocks, known to improve phylogenetic analysis and car- ried out manually, are common practice. Acknowledgements g Partial financial support by the German DFG Bioinformatics Initiative, BIZ- 6/1-2, DFG SPP 1174 "Deep Metazoan Phylogeny", the Chinese Academy of Sciences, the German BMBF, and grants from Arizona State University is gratefully acknowledged. We also are grateful to Bill Martin for bringing [2] to our attention. (1) If the original alignment already yields trees with very high average bootstrap support, there is nothing to be gained from our method. (2) Data-sets with less than about 10 taxa are unlikely to improve. An extended abstract of this contribution was presented at the ICMSB'08 in Diliman, Feb 25–28, 2008. (3) The cutoff value of q depends on the tree topology and in particular on the number of taxa. It pays to determine Authors' contributions GF and SJP initiated this study and performed the compu- tations, SG provided a prototype of Qnet, AWMD and PFS suggested the algorithmic approach, CF and MK imple- mented noisy, and all authors closely collaborated on the interpretation of the results and the preparation of the manuscript. The analysis of artificial data sets allows us to propose a set of simple rules that allow the user to decide under which conditions it makes sense to use noisy to process multiple sequence alignments prior to using them for phylogenetic reconstruction: Competing interests Th h d l h h The authors declare that they have no competing interests. Discussion However, man- ual improvements are almost impossible for large-size alignments, and typically make it hard to reproduce the results later on. Furthermore, they are not immune to the effects of wishful thinking. On the other hand, a method such as noisy provides an essentially deterministic and unbiased solution. Our approach removes randomized sites from a pre-com- puted alignment. In contrast to manual manipulation of alignments, reducing data sets using noisy is transparent and easy to reproduce. Assuming that randomized sites are, at best, phylogenetically uninformative or, in the worst case, just misleading, we propose a new way of phy- logenetic reconstruction that is based on minimizing the number of randomized sites. Detecting homoplastic char- acters using circular orderings allows us to explore a two- stage approach: In the first step, one would construct a cir- cular ordering that minimizes the fraction of "noisy" sites (as in Fig. 1). In the second step, one would then construct the tree implied by the alignment obtained after elimina- tion of all sites that appear to be highly randomized rela- tive to that circular ordering. It is important to note that "good" alignments cannot be further improved by the reduction of alignment length. 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Algorithms for Molecular Biology 2008, 3:7 to appear Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 10 of 10 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 24. Matsumoto M: Mersenne Twister: A 623-dimensionally equid- istributed uniform pseudorandom number generator. ACM Trans Modeling Comp Simulation 1998, 8:3-30. 25. Stockley B, Smith AB, Littlewood T, Lessios HA, Mackenzie-Dodds JA: Phylogenetic relationships of spatangoid sea urchins (Echinoidea): taxon sampling density and congruence between morphological and molecular estimates. Zool Scripta 2005, 34:447-468. 26. Swofford DL: PAUP: Phylogenetic Analysis Using Parsimony ( and Other Methods) Version 4.0b10 Sunderland, MA: Sinauer Associates; 2002. 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https://openalex.org/W2339882774	https://vuir.vu.edu.au/45587/1/Extending%20gene%20ontology%20in%20the%20context%20of%20extracellular%20RNA%20and%20vesicle%20communication.pdf	English	null	Extending gene ontology in the context of extracellular RNA and vesicle communication	Journal of biomedical semantics	2,016	cc-by	7,506	Extending gene ontology in the context of extracellular RNA and vesicle communication Kei-Hoi Cheung1,2,21, Shivakumar Keerthikumar3,21, Paola Roncaglia4,22, Sai Lakshmi Subramanian5,21, Matthew E. Roth5,21, Monisha Samuel3, Sushma Anand3, Lahiru Gangoda3, Stephen Gould6,21,23, Roger Alexander7,21, David Galas7,21, Mark B. Gerstein8,9,10,21, Andrew F. Hill3,24, Robert R. Kitchen8,21, Jan Lötvall11,24, Tushar Patel12,21, Dena C. Procaccini13,21, Peter Quesenberry14,21,24, Joel Rozowsky8,10,21, Robert L. Raffai15,21, Aleksandra Shypitsyna4,22, Andrew I. Su16,21, Clotilde Théry17,24, Kasey Vickers18,21, Marca H.M. Wauben19,24, Suresh Mathivanan3,21,24, Aleksandar Milosavljevic5,21 and Louise C. Laurent20,21 This is the Published version of the following publication Cheung, K, Keerthikumar, Shivakumar, Roncaglia, Paola, Subramanian, Sai Lakshmi, Roth, Matthew E, Samuel, Monisha, Anand, Sushma, Gangoda, Lahiru, Gould, Stephen, Alexander, Roger, Galas, David, Gerstein, Mark B, Hill, Andrew F, Kitchen, Robert R, Lötvall, Jan, Patel, Tushar, Procaccini, Dena C, Quesenberry, Peter, Rozowsky, Joel, Raffai, Robert L, Shypitsyna, Aleksander, Su, Andrew L, Thery, Clotilde, Vickers, Kasey, Wauben, Marca HM, Mathivanan, Suresh, Milosavljevic, Aleksander and Laurent, Louise C (2016) Extending gene ontology in the context of extracellular RNA and vesicle communication. Journal of Biomedical Semantics, 7. ISSN 2041-1480 The publisher’s official version can be found at https://jbiomedsem.biomedcentral.com/articles/10.1186/s13326-016-0061-5 Note that access to this version may require subscription. Downloaded from VU Research Repository https://vuir.vu.edu.au/45587/ Cheung et al. Journal of Biomedical Semantics (2016) 7:19 DOI 10.1186/s13326-016-0061-5 * Correspondence: kei.cheung@yale.edu; `llaurent@ucsd.edu 1Department of Emergency Medicine, Yale Center for Medical Informatics, Yale University School of Medicine, New Haven, CT, USA 20Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, USA Full list of author information is available at the end of the article © 2016 Cheung et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated RESEARCH Open Access Background The former include ‘ectosomes’ (which refer to EVs that are produced by budding from the plasma membrane ([27, 28]) and ‘microvesicles’ (which are frequently oper- ationally defined as EVs that pellet at moderate centrifuga- tion speeds (~10,000–20,000 xg) [29]), while the latter include ‘prostasomes’ ([30]), ‘epididymosomes’ ([31]), ‘immunosomes’ ([32, 33]), ‘oncosomes’ ([34]), and ‘platelet dust’ ([23]). There are even some vesicle names that refer to observed biological activities (e.g. ‘tolerosomes’ ([35]) and ‘calcifying matrix vesicles’ ([22]). Among the many reasons cells might release exRNAs, perhaps the most intriguing possibility is that exRNAs might contribute to intercellular communication. Export of exRNAs may also be used to eliminate undesired RNAs from the originating cell. Finally, some exRNAs might be generated in a nonspecific manner, either by living cells (e.g. by a ‘bulk flow’ process) or as a conse- quence of cell death (reviewed in [6]). ExRNAs appear to be universally associated with carrier vehicles, likely due to the rapid degradation of unpro- tected RNAs in biofluids ([4, 7–10]). The biochemical properties of these carriers are likely to be the primary de- terminant of the types and specific identities of exRNAs that are secreted from cells, as well as their stability in the extracellular milieu. The first exRNA carriers identified were RNA viruses, which carry not only viral RNAs, but also varying levels of host-encoded RNAs. For example, retroviruses typically carry two host tRNAs and sub- stoichiometric levels of host mRNAs from the cell in addition to two copies of the viral RNA genome and key viral proteins ([11–16]). In fact, retrovirally infected cells produce more “empty” virus-like particles (VLPs) than in- fectious virions. These VLPs have failed to encapsulate the viral RNA genome, but can carry as much as 10 kb of host-encoded RNA ([17]). More recently, it has been dis- covered that virally uninfected cells also release RNAs into the extracellular space, and that these exRNAs are associ- ated with extracellular vesicles (EVs), lipoproteins (LPPs, most commonly HDLs ([10, 18]), LDLs ([18, 19])), and ribonucleoprotein particles (RNPs, most commonly Ago2-containing RNPs ([9, 20]). While the biogenic mechanisms underlying the release of exRNA-containing EVs, LPPs, and RNPs are still being investigated, it is clear that they are not generated by a mechanism that is com- mon to all of them. The International Society for Extracellular Vesicles (ISEV) has previously attempted to clarify the nomencla- ture in this field. Background that uninfected chick and mammalian cells release RNA- containing vesicles ([21]). However, these non-viral EVs remained largely uninvestigated for decades. EVs re- entered the literature in the late 1960’s with descriptions of calcifying matrix vesicles released by chondrocytes ([22]) and vesicular ‘dust’ released by platelets ([23]). These and other such vesicles are now commonly referred to as ‘exosomes’, a term coined by Trams et al. in 1981 ([24]) to refer to secreted vesicles that “may serve a physiologic function”, including both small vesicles of ~100 nm in diameter, and larger vesicles of ~600 nm diameter or greater. Extracellular RNAs (exRNAs) are broadly defined as RNAs that are present in the acellular portions of bio- fluids, such as body fluids (blood, cerebrospinal fluid, bile, lymph, vitreous humour, amniotic fluid, ascites, pleural, pericardial and peritoneal fluids, etc.), secretions (saliva, urine, sweat, tears, milk, seminal fluid, etc.), and cell and tissue culture supernatants. RNA sequencing analyses of exRNAs demonstrate that they represent al- most the entire range of cellular RNA species, including rRNAs, tRNAs, mRNAs, miRNAs, piRNAs, lncRNAs, and circular RNAs ([1–5]). However, the profiles of cel- lular and exRNAs are not identical, as some cellular RNAs appear to be highly enriched in the exRNA frac- tion, while others appear to be significantly underrepre- sented, and still others lie between these extremes of enrichment or exclusion. g This first definition of the term ‘exosome’ has been subsequently overlooked at least twice, first in 1987 by investigators studying the vesicular secretion of the transferrin receptor, who adopted a more restrictive def- inition of the term, conflating it with a delayed mode of vesicle secretion in which the vesicles bud at endosome membranes to create a multivesicular body (MVB), followed later by MVB fusion with the plasma mem- brane to release the vesicles into the extracellular space ([25]). In 1997, investigators studying an RNA-degrading protein complex adopted the term ‘exosome’, this time for an entirely unrelated intracellular biochemical entity ([26]). Not surprisingly, other investigators have come to different conclusions about which definition holds scien- tific precedent, resulting in variable use of the term ‘exo- some’ in different laboratories. EV-related nomenclatures are further complicated by the common use of additional terms for secreted vesicles that are variably associated with different biophysical properties or biogenesis pathways, as wells as terms based on the cell type or tissue of origin. Abstract Journal of Biomedical Semantics (2016) 7:19 Page 2 of 9 Page 2 of 9 Abstract Background: To address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research. Methods: By following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations. Results: We have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: “extracellular vesicle”, “extracellular exosome”, “apoptotic body”, and “microvesicle” were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO. Conclusions: As an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search. Keywords: Ontology, Extracellular RNA, Extracellular vesicle, Metadata, Faceted search, Atlas list of author information is available at the end of the article © 2016 Cheung et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cheung et al. Background Its primary achievements have been to (1) introduce the term ‘extracellular vesicle (EV)’ as a general term intended to encompass all secreted vesicles, and encourage its broad acceptance, and (2) encourage the use of broad-definition terms until a more compre- hensive understanding of the biogenesis and molecular compositions of different types of vesicles is developed. Given the inconsistent vesicle nomenclatures prior to these ISEV efforts, these were major advances. However, some inconsistencies still persist. For example, some Evidence for EVs and exRNA was first provided more than seventy-five years ago by Albert Claude’s observation Page 3 of 9 Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 3 of 9 data sources, it also allows DMRR data to be integrated with other data sources with metadata annotated using the same ontologies. For ontologies to be useful for bio- logical applications, it is critical that the relevant ontol- ogies contain meaningful and broadly accepted terms, as well as ensuring that the relationships between the in- cluded terms be both accurate and accepted by the per- tinent scientific community. For an ontology such as GO which is frequently used for functional enrichment analysis of genomic datasets, it is also important that terms be associated with specific gene products (coding and non-coding RNAs and proteins) in an empirically supported manner. investigators use the term ‘microvesicle’ for larger EVs (>200 nm diameter) and ‘exosome’ for smaller EVs (~30–200 nm diameter), while other investigators reject these size-based definitions and adopt a set of biogenic definitions in which ‘microvesicle’ describes vesicles that bud from the plasma membrane while ‘exosome’ de- scribes vesicles that bud into endosomes and are se- creted only later upon MVB fusion with the plasma membrane. Therefore, investigators are currently forced to make a choice between these competing definitions, the first be- ing practical but mechanistically barren, while the latter being impractical but mechanistically appealing. Unfor- tunately, there is as yet no unambiguous way to distin- guish between vesicles that bud from the plasma membrane versus those that bud at the endosome mem- brane based on either biophysical properties or molecu- lar content. This lack of standard nomenclatures creates i) a problem for individual researchers to annotate/share their data in an unambiguous manner and ii) a barrier to productive interactions with the broader research com- munity, most prominently the biomedical, genomics, and computational biology communities. Background To address such issues, we initiated our metadata standard efforts within the Metadata Working Group (MWG) of the Extracellular RNA Communication Consortium (ERCC) funded by the National Institutes of Health (NIH). As part of these efforts, MWG matched metadata terms to existing biomedical ontologies and identified the exRNA-relevant terms that were absent from major on- tologies such as the Gene Ontology (GO). pp The Gene Ontology (GO) Consortium (GOC; http:// www.geneontology.org) is a community-based bioinfor- matics effort. This Consortium develops, maintains and extends two interconnected resources: the Gene Ontol- ogy itself, and a database of GO annotations that associ- ate specific gene products with concepts in the Gene Ontology. As of March 2016, there are >42,000 GO terms for describing concepts relevant to gene product function in a species-independent manner, providing not only comprehensive coverage of biological concepts but also community-wide agreement on how those should be used to describe gene functions across all organisms. The GO is organized into three aspects [38]: these are graph structures comprised of classes for molecular functions, the biological processes these contribute to, the cellular locations where these occur (cellular compo- nents), and the relationships connecting these classes. A ‘GO annotation’ describes the association between a gene product and an ontology class, as well as references to the evidence supporting the association. In most cases, a GO annotation is a statement about gene func- tion, but because the GO cellular component aspect de- scribes a “cellular and subcellular anatomy” it can have broader applications beyond the originally specified GO annotation usage of “where a gene product is active.” Thus an association of an exRNA to a cellular component term does not necessarily imply a function per se, though a functional role may of course later be discovered. Adding gene product annotations The GOC accepts contributions of annotations from ex- ternal groups, and provides guidance in the ‘Documenta- tion’ section of its website (http://geneontology.org). The ‘Documentation’ section includes details of the recom- mended file format, called GAF (Gene Association For- mat). Annotations submitted to the GOC undergo automated checks, followed by manual review by GO curators, in this case from the Protein Function Content team at EMBL-EBI. As sources of gene product annotations in the exRNA context, ExoCarta [45] and Vesiclepedia [46] are online databases that catalogue proteins, RNAs and lipids iden- tified specifically in exosomes and other extracellular vesicle types, respectively. Recently, ExoCarta was up- dated with ISEV minimal experimental requirements feature for definition of extracellular vesicles. In collab- oration with the GOC editorial team, we have mapped the ExoCarta and Vesiclepedia gene products (coding and non-coding RNAs and proteins) onto their corre- sponding RNAcentral (http://www.rnacentral.org) and UniProt (http://www.uniprot.org/) identifiers using the newly extended GO Cellular Component terms. Use of ontologies in metadata annotation g As described in [36], the MWG of the ERCC has devel- oped the data and metadata standards for annotating exRNA profiling data for submission to the Data Man- agement and Resource Repository (DMRR) of the ERCC. Particularly, a process has been established to submit ex- periment data to DMRR along with metadata in stand- ard machine-readable formats (using Linked Data technologies). The standards cover metadata about do- nors, biosamples, experiments, studies, and analysis steps. Such metadata enable targeted selection of sam- ples of interest (e.g. specific health condition of the donor, biofluid or cell/tissue type, library preparation method, and sequencing assay) for integrative analyses. The metadata also helps organize the data for efficient interactive as well as programmatic access (e.g. REST Application Programming Interfaces (APIs)). The GOC continuously provides enhancements to the ontology and annotation database, as well as its tools and policies, ensuring that the ontology and annotations are consistent, and accurately reflect the current state of biological knowledge. In recent years, the GO has expanded the number and type of relationships used to connect terms within the ontology as well as between GO and other ontologies. A version of GO containing all relationships, including in- formation from the Uber anatomy ontology (UBERON) [39], the Chemical Entities of Biological Interest ontol- ogy (ChEBI) [40], the Plant Ontology for plant struc- ture/stage (PO) [41], the Phenotypic Quality Ontology The MWG identified existing biomedical ontologies accessible through the NCBO BioPortal [37] as a source of commonly accepted terms for annotating exRNA datasets. Such ontology-based metadata annotation does not only allow semantic retrieval/query of data in ERCC Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 4 of 9 Page 4 of 9 (PATO) [42], and the Sequence Ontology (SO) [43], is called go-plus and is available at http://geneontolo- gy.org/page/download-ontology. The GOC also makes other versions of the ontology available at this site. the GO update, the GOC editorial team routinely adds new ontology classes, or modifies existing ones, based on requests from GO curators or from community ex- perts. In the latter case, like our case here, changes to the ontology are proposed using a batch request upon direct consultation (see [44]). Defining exRNA-related terms based on community consensus Our goal was to extend the cellular component branch of GO to incorporate relevant and empirically supported terms and relationships that would be useful to and broadly accepted in the exRNA field, while both main- taining the internal consistency of GO and leveraging the knowledge previously encoded in the GO. Initially focusing on the major area of controversy (vesicles), we surveyed the literature for relevant references and sought input from domain experts, including the leader- ship of the International Society for Extracellular Vesicles (ISEV), the American Society for Exosomes and Microvesicles (ASEMV), and the ERCC. This process re- sulted in three alternative proposals, which ranged from inclusion of only the most general of terms, to inclusion of both general and moderately specific terms, to add- itional inclusion of highly specific terms (Fig. 1). As shown in Fig. 1, boxes represent GO terms, ovals repre- sent synonyms, and lines connecting boxes represent the ‘is-a’ relationship (a dashed line connecting a box and an oval corresponds to a synonymous relationship). The gray boxes and black lines represent existing terms and relationships, while the red boxes and red lines represent the proposed terms and relationships. The option-3 set of terms/relationships is a subset of the option-2 set which is a subset of option 1. We then polled the mem- bership of these societies either during their annual (ASEMV) or semi-annual (ERC Consortium) meetings or through an electronic survey (ISEV). The results of the ISEV poll are shown (Table 1), and are concordant with the consensuses reached by the other two groups: Option 2 (set B + set C) is the preferred choice. Methods As mentioned above, when we initiated our metadata ef- forts, there was a significant gap between the needs of the exRNA community and the terms and relationships present in GO. At that time, the only relevant terms in the Cellular Component branch were: ‘extracellular vesicular exosome’ (GO:0070062) and ‘prominosome’ (GO:0071914), neither of which was widely used in the exRNA community. In addition to lacking the most commonly used terms for extracellular vesicles, this version of GO lacked terms spe- cific for exRNA-containing ‘Lipoprotein Particles’ (LPPs) and ‘Ribonucleoprotein Particles’ (RNPs). Incorporating new GO terms into exRNA Atlas as a use case Incorporating new GO terms into exRNA Atlas as a use case The exRNA Atlas is a central data repository developed and maintained by the DMRR that distributes data pro- vided by the ERCC. The first public release of the exRNA Atlas, in early 2016, contained exRNA-seq pro- files of over 500 samples generated by ERCC members analyzed uniformly using standard in-house analysis pipelines and quality-controlled using standards agreed by the Consortium. The exRNA Atlas browser enables efficient searching and sub-selection of exRNA profiles for retrieval and integrative analysis. To facilitate inte- gration of the datasets into the exRNA Atlas, data and metadata standards have been developed by the Meta- data Working Group of the ERCC. The metadata stan- dards build on metadata data models we previously developed during the course of the NIH Epigenome Roadmap project, International Human Epigenome Con- sortium (IHEC) project, and in collaboration with the ENCODE3 project, and NCBI. The metadata include core objects such as biosamples, donors, experimental protocols, studies and analysis methods. The Genbor- eeKB document modeling system was used to define We then worked with the GOC team to incorporate the consensus recommendations, as well as terms rele- vant to LPPs, as supported by the literature. As part of Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 5 of 9 Fig. 1 The three different options of extending GO to include ExRNA terms and relationships Fig. 1 The three different options of extending GO to include ExRNA terms and relationships biosamples and Human Disease Ontology (DOID) [47] for disease types. To effectively identify the source from which the exRNA is extracted, the new GO terms have been used to annotate the biosamples deposited in the exRNA Atlas. syntactic and semantic models for ontology-rich multi- faceted metadata based on the lightweight JSON syntax. The latest release of the exRNA Metadata Standard as- sociates various biomedical ontologies to both the attri- bute (key) values as well as with the attributes themselves and supports dynamic validation against ontologies avail- able in NCBO Bioportal. More than twenty data elements within the metadata model are associated with ontologies, such as biofluids, disease types, anatomical locations, cell culture sources, cell lines, tissues, and other properties needed to describe biosamples and donors. Incorporating new GO terms into exRNA Atlas as a use case A number of these elements reflect the clinical focus of the ERCC, defined by ontologies such as the Systematized Nomencla- ture of Medicine - Clinical Terms (SNOMEDCT) (https:// www.nlm.nih.gov/research/umls/Snomed/snomed_main.h tml) for biofluid type as well as anatomical location of the Results and discussions ExRNA terms and relationships added to GO Based on the option-2 proposal shown in Fig. 1, the ERCC group worked with EMBL-EBI members of the GOC edi- torial team to add the proposed terms (including syno- nyms) and relationships to GO. Figure 2 shows these terms (with GO IDs) and relationships in red. Any future terms added to GO and representing types of vesicles that are part of the extracellular region will be automatically classified under GO:1903561 ‘extracellular vesicle’. Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 6 of 9 Page 6 of 9 Table 1 ISEV poll results Rank 1 2 3 4 Option 1 16 86 89 21 Option 2 162 36 8 6 Option 3 33 83 91 5 Option 4 1 7 24 180 580 ISEV members received the poll, 212 answered it. The first question was to rank the four options by order of preference. As shown in Fig. 1, the proposed options were: Option 1 = Set A + B + C; Option 2 = Set B + C; Option 3 = Set C; Option 4 = none of the above. Number of answers for each leading to understanding the molecular mechanism of extracellular vesicle biogenesis, as well as sorting and se- creting under normal and pathophysiological microenvi- ronments. While granular/specific annotations would be more informative, ontological inferencing can be applied to a given term (extracellular vesicle) to automatically retrieve its annotations and those associated with its descendant terms (e.g. extracellular exosome). GO query engines like QuickGO (http://www.ebi.ac.uk/QuickGO/) support this type of query. We anticipate that updating the gene products associated with each GO term will be an ongoing process. 580 ISEV members received the poll, 212 answered it. The first question was to rank the four options by order of preference. As shown in Fig. 1, the proposed options were: Option 1 = Set A + B + C; Option 2 = Set B + C; Option 3 = Set C; Option 4 = none of the above. Number of answers for each 580 ISEV members received the poll, 212 answered it. The first question was to rank the four options by order of preference. As shown in Fig. 1, the proposed options were: Option 1 = Set A + B + C; Option 2 = Set B + C; Option 3 = Set C; Option 4 = none of the above. Number of answers for each The use of GO terms to annotate exRNA metadata and implement faceted search within the exRNA Atlas browser At the time of writing this manuscript, 18,695 coding (mRNA and protein) and 963 non-coding (ncRNAs) gene products (including isoforms) were annotated with the GO terms: ‘extracellular vesicle’, ‘extracellular exo- some’,‘apoptotic body’ and ‘microvesicle’ identified in dif- ferent species (Table 2) from ExoCarta and Vesiclepedia. The Protein Function Content team at EMBL-EBI are currently working on importing annotations from ExoCarta and Vesiclepedia into the GOC. We believe that the gene product annotations extracted from these resources would further aid the scientific community in downstream cellular component enrichment analysis We have implemented a search interface that allows users to browse exRNA Atlas biosamples based on different facets of metadata, including the type of bio- fluid, cell culture source, disease, and exRNA source. The facets (categories of ontology concepts) provide an effective means for navigating/filtering a large set of exRNA profiles data based on different aspects of the samples. The metadata terms linked to ontologies in BioPortal have been augmented to include the newly defined exRNA terms (Fig. 3). By annotating Fig. 2 The list of exRNA-related terms, synonyms and relationships that have been added to GO Fig. 2 The list of exRNA-related terms, synonyms and relationships that have been added to GO Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 7 of 9 Table 2 Summary of the gene product annotations extracted from ExoCarta and Vesiclepedia GO-Term GO-ID Coding gene products (mRNAs or proteins) Non-coding gene products (ncRNAs) Extracellular exosome GO:0070062 10,827 953 Apoptotic body GO:0097189 168 Microvesicle GO:1903561 14,668 148 Extracellular vesicle GO:1990742 4029 Total no. of unique coding gene products Total no. of unique non-coding gene products 18,695 963 the exRNA sources using these community-agreed terms and by exposing a new facet (exRNA source) through the ExRNA browser, we have enabled more precise biologically meaningful search of the ExRNA Atlas. and for computational analysis of exRNA data. Given the rapid developments in this field, we view the current ver- sion of the relevant areas of the ontology to be a work in progress. We anticipate frequent future amendments to incorporate as well as refine additional terms, relation- ships, and associated gene products as the community de- velops a more precise and comprehensive understanding of the biogenesis, biophysical properties, molecular com- position, and functions of different types of exRNA- associated EVs, LPPs, and RNPs. The use of GO terms to annotate exRNA metadata and implement faceted search within the exRNA Atlas browser To this end, the ERCC group aims to continually interrogate the literature and to solicit input from professional societies and the wider exRNA community. The ERCC has launched the ExRNA Portal (exrna.org), a publicly accessible website and com- munity forum for dissemination of information, sharing of tools and data (primarily through the ExRNA Atlas), and aggregation of input. Future proposed amendments will be posted on the ExRNA Portal and will be subject to a public Received: 17 December 2015 Accepted: 4 April 2016 Received: 17 December 2015 Accepted: 4 April 2016 Conclusions In this manuscript, we have described an inter-community effort to make a substantial update to GO, focusing on terms and relationships pertinent to the new field of exRNA biology. We have demonstrated how these new GO terms can be used to annotate and search metadata associated with high-throughput datasets, such as RNAseq data. In addition, we anticipate that these terms will be useful as keywords for annotation and querying of the lit- erature. We have also initiated a systematic, literature- supported process for associating gene products with each exRNA-related term, such that they can be used for cellu- lar component enrichment analysis of omics datasets (e.g. RNAseq- and gene expression data). We recognize that fundamental questions regarding the composition, biogen- esis, and functions of exRNA-containing EVs, lipoproteins, and ribonucleoprotein complexes remain to be answered. Therefore, we anticipate that frequent future updates to GO will be necessary to accurately reflect continued pro- gress in this rapidly advancing field. Acknowledgements Th k g This work was supported in part by the NIH Common Fund, through the Office of Strategic Coordination and the Office of the NIH Director (AM: U54DA036134, LCL: UH2TR000906 and U01HL126494, TP: UH3TR000884, and RR: U19CA179512). Paola Roncaglia and Aleksandra Shypitsyna are funded by the Gene Ontology Consortium (P41 grant from the National Human Genome Research Institute (NHGRI), grant 5U41HG002273-14). Aleksandra Shypitsyna is also funded from the European Molecular Biology Laboratory (EMBL). We would like to give special thanks to Dena C. Procaccini for coordinating meetings and conference calls facilitating discussion and collaboration as part of the manuscript writing. We would like to thank Tony Sawford (Protein Function Development team, EMBL- EBI) for running the set of ExoCarta and Vesiclepedia annotations through the validation pipeline for inclusion in the GOA database. We would also like to thank Xandra O. Breakefield for her insights and comments. Finally we would like to acknowledge the community support offered by the following Consortiums: Extracellular RNA Communication (ERC) Consortium, Gene Ontology Consortium (GOC), and International Society for Extracellular Vesicles (ISEV), and American Society for Exosomes and Microvesicles (ASEMV). Significance of our results and other possible extensions Significance of our results and other possible extensions These substantial changes to GO reflect the current state of the exRNA field, representing the current litera- ture from the perspective of the consensus among three major professional societies in this field (ISEV, ASEMV, and ERCC). We believe that the new exRNA-relevant terms, together with their relationships to each other and with the existing GO terms, as well as the associated gene products, will be useful for annotating the increasing num- ber of manuscripts and datasets being generated by the exRNA community. We also expect that they will prove useful for literature mining, data repository integration, Fig. 3 The new GO terms have been used to annotate the biosamples deposited in the ExRNA Atlas based on the exRNA Source. The figure is a screenshot of the faceted search of biosamples in the ExRNA Atlas using the exRNA Source metadata property (facet) Fig. 3 The new GO terms have been used to annotate the biosamples deposited in the ExRNA Atlas based on the exRNA Source. The figure is a screenshot of the faceted search of biosamples in the ExRNA Atlas using the exRNA Source metadata property (facet) Cheung et al. Journal of Biomedical Semantics (2016) 7:19 Page 8 of 9 Page 8 of 9 Page 8 of 9 comment period. As described above, in the metadata as- sociated with datasets submitted to the ExRNA Atlas, we permit and request depositors to not only tag datasets with the appropriate current GO terms, but to also include more precise keywords, so that over time, frequently used keywords can be evaluated as possible new GO terms. Authors’ contributions KHC i i i d h j KHC initiated the project of extending GO in the context of exRNA. He also coordinated collaboration between ERCC and GOC. LL provided her domain expertise for guiding the consensus building process and the project as a whole. SK, MS, SA, LG and SM contributed gene product annotations from Vesiclepedia and ExoCarta. PR and AS, who are members of GOC, curated the terms and gene product annotations submitted to GO. SLS, MR and AM are involved in the development of the ExRNA Atlas, and have developed the metadata search use case. KHC, LL, SK, SM, PR, SG, SLS, MR and AM have contributed to the writing of the manuscript. KC, LL, RR, KV, SLS, MR, RA, DG, AS, RK, JR, MG, DP, TP, PQ and AM are members of ERCC. JL, AH, SM, MW, PQ, and CT are members of ISEV and handled the ISEV poll. SG is a member of ASEMV. These communities support the project. All the authors reviewed the manuscript. All authors read and approved the final manuscript. In addition, our ExRNA Atlas use case (faceted search) shows the power of exploiting multiple ontologies in a sample annotation. For example, our metadata includes a description of extracellular RNAs isolated from different types of biofluids. Ontologies such as Uberon [48], Nano- Particle Ontology [49], SNOMEDCT and Experimental Factor Ontology [50] contain concepts corresponding to different types of “Biofluid” (or “Bodily Fluid”). These con- cepts can be used alongside with the “extracellular space” concept in GO to describe extracellular RNAs that are iso- lated from different types of biofluids. Abbreviations ASEMV: American Society for Exosomes and Microvesicles; ChEBI: chemical entities of biological interest; DMRR: data management and resource repository; ERCC: Extracellular RNA Communication Consortium; EV: extracellular vesicle; ExRNA: extracellular ribonucleic acid; GAF: Gene Association Format; GO: gene ontology; GOC: Gene Ontology Consortium; HDL: high density lipoprotein; ISEV: International Society for Extracellular Vesicles; LDL: low density lipoprotein; LPP: lipoprotein particle; mRNA: messenger ribonucleic acid; lncRNA: long non-coding ribonucleic acid; miRNA: micro ribonucleic acid; MVB: multivesicular body; NCBO: National Center for Biomedical Ontology; ncRNA: non-coding RNA; PATO: phenotypic quality ontology; PR: protein ontology; piRNA: Piwi- interacting ribonucleic acid; RNP: ribonucleoprotein; rRNA: ribosomal ribonucleic acid; SO: sequence ontology; tRNA: transfer ribonucleic acid; VHDL: very high density lipoprotein; VLDL: very low density lipoprotein. Author details 1 f 1Department of Emergency Medicine, Yale Center for Medical Informatics, Yale University School of Medicine, New Haven, CT, USA. 2VA Connecticut Healthcare System, West Haven, CT, USA. 3Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia. 4European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 5Bioinformatics Research Laboratory, Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX, USA. 6Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 7Pacific Northwest Diabetes Research Institute, Seattle, WA, USA. 8Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. 9Department of Computer Science, Yale University, New Haven, CT, USA. 10Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA. 11University of Gothenburg, Gothenburg, Sweden. 12Mayo Clinic, Jacksonville, FL, USA. 13Division of Neuroscience and Behavior, National Institute on Drug Abuse (NIDA), Rockville, MD, USA. 14University Medicine Comprehensive Cancer Center, Providence, RI, USA. 15Department of Surgery, University of California San Francisco and VA Medical Center, San Francisco, CA, USA. 16Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA. 17Institut Curie, PSL Research University, INSERM U932, Paris, France. 18Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA. 19Department of Biochemistry & Cell Biology, Utrecht University, Utrecht, Netherlands. 20Department of Reproductive Medicine, University of California, San Diego, La Jolla, CA, USA. 21Extracellular RNA Communication Consortium (ERCC), , . 22Gene Ontology Consortium (GOC), , . 23American Society for Exosomes and Microvesicles (ASEMV), , . 24International Society for Extracellular Vesicles (ISEV), , . Abbreviations ASEMV A Abbreviations ASEMV: American Society for Exosomes and Microvesicles; ChEBI: chemical entities of biological interest; DMRR: data management and resource repository; ERCC: Extracellular RNA Communication Consortium; EV: extracellular vesicle; ExRNA: extracellular ribonucleic acid; GAF: Gene Association Format; GO: gene ontology; GOC: Gene Ontology Consortium; HDL: high density lipoprotein; ISEV: International Society for Extracellular Vesicles; LDL: low density lipoprotein; LPP: lipoprotein particle; mRNA: messenger ribonucleic acid; lncRNA: long non-coding ribonucleic acid; miRNA: micro ribonucleic acid; MVB: multivesicular body; NCBO: National Center for Biomedical Ontology; ncRNA: non-coding RNA; PATO: phenotypic quality ontology; PR: protein ontology; piRNA: Piwi- interacting ribonucleic acid; RNP: ribonucleoprotein; rRNA: ribosomal ribonucleic acid; SO: sequence ontology; tRNA: transfer ribonucleic acid; VHDL: very high density lipoprotein; VLDL: very low density lipoprotein. 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https://openalex.org/W2888806487	http://www.aginganddisease.org/EN/article/downloadArticleFile.do?attachType=PDF&id=147709	English	null	Progranulin Deficient Mice Develop Nephrogenic Diabetes Insipidus	Aging and disease	2,018	cc-by	9,636	[Received October 4, 2017; Revised November 26, 2017; Accepted November 27, 2017] ABSTRACT: Loss-of-function mutations of progranulin are associated with frontotemporal dementia in humans, and its deficiency in mice is a model for this disease but with normal life expectancy and mild cognitive decline on aging. The present study shows that aging progranulin deficient mice develop progressive polydipsia and polyuria under standard housing conditions starting at middle age (6-9 months). They showed high water licking behavior and doubling of the normal daily drinking volume, associated with increased daily urine output and a decrease of urine osmolality, all maintained during water restriction. Creatinine clearance, urine urea, urine albumin and glucose were normal. Hence, there were no signs of osmotic diuresis or overt renal disease, other than a concentrating defect. In line, the kidney morphology and histology revealed a 50% increase of the kidney weight, kidney enlargement, mild infiltrations of the medulla with pro-inflammatory cells, widening of tubules but no overt signs of a glomerular or tubular pathology. Plasma vasopressin levels were on average about 3-fold higher than normal levels, suggesting that the water loss resulted from unresponsiveness of the collecting tubules towards vasopressin, and indeed aquaporin-2 immunofluorescence in collecting tubules was diminished, whereas renal and hypothalamic vasopressin were increased, the latter in spite of substantial astrogliosis in the hypothalamus. The data suggest that progranulin deficiency causes nephrogenic diabetes insipidus in mice during aging. Possibly, polydipsia in affected patients - eventually interpreted as psychogenic polydipsia - may point to a similar concentrating defect. Progranulin is a multi-functional secreted neuroprotective and immune-regulatory protein. Loss-of-function mutations in humans are associated with ubiquitin positive, tau-negative frontotemporal lobar degeneration (FTLD) [1, 2], lipofuscinosis [3] and other rare neurodegenerative diseases [4]. Its deficiency in mice partly mimics the human disease, in particular the neuropsychiatric behavioral abnormalities [5], which are characteristic for FTLD patients whereas learning and Progranulin is a multi-functional secreted neuroprotective and immune-regulatory protein. Loss-of-function mutations in humans are associated with ubiquitin positive, tau-negative frontotemporal lobar degeneration (FTLD) [1, 2], lipofuscinosis [3] and other rare neurodegenerative diseases [4]. Its deficiency in mice partly mimics the human disease, in particular the neuropsychiatric behavioral abnormalities [5], which are characteristic for FTLD patients whereas learning and memory are only mildly impaired [6-8]. Progranulin is expressed in neurons of the peripheral and central nervous system, but also by activated immune cells including macrophages and microglia, but knockout models suggest that the neurodegeneration is rather the origin than sequela of the neuro-inflammation [9]. Original Article Original Article Copyright: © 2017 Hardt S et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Correspondence should be addressed to: Dr. Irmgard Tegeder, Dept. of Clinical Pharmacology, Goethe-University of Frankfurt, 60590 Frankfurt am Main, Germany. Email: tegeder@em.uni-frankfurt.de. ords: Progranulin, aging, polyuria, polydipsia, knockout mice, hypothalamus, vasopressin Progranulin Deficient Mice Develop Nephrogenic Diabetes Insipidus Stefanie Hardt1, #, Lucie Valek1, #, Jinyang Zeng-Brouwers2, Annett Wilken-Schmitz1, Liliana Schaefer2, Irmgard Tegeder1, Stefanie Hardt1, #, Lucie Valek1, #, Jinyang Zeng-Brouwers2, Annett Wilken-Schmitz1, Liliana Schaefer2, Irmgard Tegeder1, * Stefanie Hardt1, #, Lucie Valek1, #, Jinyang Zeng-Brouwers2, Annett Wilken-S Schaefer2, Irmgard Tegeder1, * 1Clinical Pharmacology, Goethe-University Hospital Frankfurt am Main, Germany 2General Pharmacology and Toxicology, Goethe-University Hospital Frankfurt am Main, Germany # These authors contributed equally to this work. [Received October 4, 2017; Revised November 26, 2017; Accepted November 27, 2017] htt //d d i /10 14336/AD 2017 1127 Volume 9, Number 5; 817-830, October 2018 http://dx.doi.org/10.14336/AD.2017.1127 Volume 9, Number 5; 817-830, October 2018 Volume 9, Number 5; 817-830, October 2018 Animals Progranulin knockout mice (Grn-/-) [34] were maintained as homozygous colony. The background is C57BL6J, so that sex and age matched C57BL6J mice (Charles River or Envigo, Germany) were used as controls. Mice were housed three to five per cage and maintained in the same room during life with constant room temperature (21 ± 1°C), standard diet and a regular light/dark schedule with light on from 7:00 A.M. to 7:00 P.M. Food and water was available ad libitum except for some experimental sessions in the IntelliCage and metabolic cage. Young mice were 9-12 weeks, aged mice 10-13 months and old mice 15-18 months. The experiments were approved by the local Ethics Committee for Animal Research (Darmstadt, Germany) and adhered to the guidelines of GV-SOLAS for animal welfare in science and the ARRIVE guidelines. We have previously observed that aged progranulin deficient mice, despite their learning deficits, make few errors in place preference learning tasks, if water was the provided award [8], suggesting a higher appetitive drive. Indeed, under standard housing conditions, they drank more water, which became apparent at 12-15 months of age. Considering its multiple functions for neuronal and peripheral-immune homeostasis, polyuria might be due to primary polydipsia like in saposin D deficient mice [26], hence reflecting FTLD-like impulsive drinking and feeding. The polydipsia may also result from neurodegeneration in hypothalamus or pituitary gland, resulting in alterations of vasopressin production, supported by a previous study showing feeding-dependent fluctuations of progranulin release in the hypothalamus [27]. Progranulin deficiency also impairs the development of sexual dimorphisms [28], suggesting that hormone- producing neurons are particularly vulnerable. Finally, the polydipsia may be caused by osmotic glucosuria or a manifestation of a nephrogenic diabetes insipidus, the latter due to unresponsiveness towards vasopressin all resulting in renal water losses. Progranulin indeed regulates insulin sensitivity [29, 30], possibly by interfering with glucose transporters of the solute carrier family [31], is involved in vesicular transport [23] and glycogen synthase kinase functions [32], which are essential mechanisms for the insertion of water channels into membranes [33]. [Received October 4, 2017; Revised November 26, 2017; Accepted November 27, 2017] It is still under debate whether progranulin released by immune cells provides a usable reservoir for neurons, either by being internalized via a transporter like sortilin [10] or via Correspondence should be addressed to: Dr. Irmgard Tegeder, Dept. of Clinical Pharmacology, Goethe-University of Frankfurt, 60590 Frankfurt am Main, Germany. Email: tegeder@em.uni-frankfurt.de. Copyright: © 2017 Hardt S et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 817 817 ISSN: 2152-5250 Hardt S., et al Progranuin & Diabetes insipidus receptor-mediated endocytosis and signaling via EGF receptors [11], Notch receptors [12] and Ephrin A2 [13], all recognized as progranulin receptors. Sortilin is likely dispensable for progranulin’s neurotrophic effects [14] but in the context of the kidney, it is noteworthy that progranulin shares sortilin as transporter with the sphingolipid activating protein, prosaposin [15, 16], a precursor of saposins, whose deficiency results in renal pathology [17] and neurodegeneration [18] due to insufficient degradation of glycosphingolipids [17, 19]. receptor-mediated endocytosis and signaling via EGF receptors [11], Notch receptors [12] and Ephrin A2 [13], all recognized as progranulin receptors. Sortilin is likely dispensable for progranulin’s neurotrophic effects [14] but in the context of the kidney, it is noteworthy that progranulin shares sortilin as transporter with the sphingolipid activating protein, prosaposin [15, 16], a precursor of saposins, whose deficiency results in renal pathology [17] and neurodegeneration [18] due to insufficient degradation of glycosphingolipids [17, 19]. To dissect out the underlying cause, which may be clinically relevant for progranulin-deficient patients, we assessed drinking and feeding behavior, metabolic functions, plasma vasopressin and other neuropeptides, morphology of the kidney and hypothalamus and aquaporins in aged progranulin deficient and control mice, and in summary, the data reveal a renal water loss without glucosuria with high vasopressin suggesting a nephrogenic diabetes insipidus. MATERIALS AND METHODS Progranulin's functions in the periphery are still somewhat enigmatic. It promotes wound healing of the skin [20], reduces joint inflammation in arthritis [21], protects the kidney against ischemia-reperfusion injury [22] and promotes tumor growth [11], all supposed to result from silencing of an activated immune system [21]. Intracellular progranulin is localized to vesicular structures, which are endosomes or autophagolysosomes and it likely promotes the degradation of protein and lipid waste via the respective pathways [23, 24]. Gene ontology enrichment analyses suggest that it contributes to the regulation of vesicular transport of proteins and metals [23]. In particular, its deficiency is associated with dysregulations of zinc transporter [5] including Slc30a9, which has been recently associated with a rare cortico- renal disease in humans [25]. Aging and Disease • Volume 9, Number 5, October 2018 Serum and plasma analyses Serum creatinine was determined with a colorimetric Microplate Assay (Oxford Biomedical Research, Biotrend, Germany), and serum urea was analyzed with an Urea Assay Kit (BioCat, Heidelberg, Germany) following the manufacturer’s instruction. Serum C- reactive protein, glucose and plasma zinc levels were analyzed by LaboKlin. Excretion of creatinine, urea, glucose and albumin Mice were placed in metabolic cages (Tecniplast) for urine collection with free access to water for 24h followed by water restriction for 2 x 12h with 1h free drinking in between. Urine was collected for 24 h in a cooling device. Body weight and drinking volume were determined daily. The 24h urine volume, urine specific weight, urine concentrations of creatinine, urea and glucose were analyzed by the veterinary laboratory LaboKlin, Bad Kissingen, Germany. Urinary albumin excretion was determined by the Bradford method and microalbumin test stripes (DIMA, Product ID M04S10-25). In addition, we used an ELISA for mouse albumin. Urinary creatinine levels were also assessed with the creatinine assay kit (Labor-Technik, Berlin, Germany) according to the manufacturer’s instruction. For analysis of vasopressin positive neurons in the hypothalamus brains were excised, postfixed in 4 % PFA (2 h, RT), cryoprotected in 20 % sucrose (overnight, 4 °C), embedded in tissue molds in cryomedium and stored at -80 °C. Cryosections of 12 µm were permeabilized with 0.1% Triton-X-100 in 1x PBS (PBST) for 25 min, then blocked for 30 min with 5% BSA (Roth) in 1x PBS. Subsequently, sections were incubated overnight with the respective primary antibody at 4 °C, and subsequent secondary antibody labelled with Alexa-488 or Cy3 (2h, RT), followed by 30 min incubation with DAPI (1 µg/µl in 1 % BSA in PBST). Primary antibodies included vasopressin (1:500, 48h, Serotec) and GFAP (1:500, Millipore). Fluorescent sections were imbedded in Fluoromount (eBioscience). Images were captured with a IntelliCage Licking behavior The Intellicage (NewBehavior AG, Zurich, Switzerland) [8, 35] consists of four operant corners, each with two water bottles, sensors, light-emitting-diodes (LEDs) and doors that control the access to the water bottles. The system fits into a large cage (20 x 55 x 38 cm, Tecniplast, 2000P). Four triangular red shelters (Tecniplast) are placed in the middle to serve as sleeping quarters and as stands to reach the food. The floor is covered with thick bedding. Mice are tagged with radio-frequency identification (RFID)-transponders, which are read with an RFID antenna integrated at corner entrance. Inside the corners, there are two holes with water bottles, which can be opened and closed by automated doors. Mice have to make nosepokes (NP) to open the doors for water access. The numbers and duration of corner visits, nosepokes, and licks are automatically recorded without the need for any handling of the mice during the recording times. Sixteen mice were housed in each cage. The IntelliCage experiments followed established protocols [8, 35, 36]. Mice were adapted to the system for 3 days with free access to every corner, with all doors Aging and Disease • Volume 9, Number 5, October 2018 818 Hardt S., et al Progranuin & Diabetes insipidus Plasma levels of vasopressin were measured by a specific enzyme immunoassays (Arg8-vasopressin EIA kit) from Enzo (Cat.No ADI-900-017, Enzo Life science GmbH, Germany) according to the instructions of the supplier. In addition, we analyzed concentration of agouti related protein (AGRP, Cat.No LS-F16813, Life Span Bioscience, USA), visinin-like protein 1 (VILIP, Cat.No CSB-EL025933MO, CusaBio Biotech, USA) and ghrelin (Cat.No EZRGRT-91K, Merck Millipore, Germany) in plasma samples using specific ELISA kits, all according to instructions of the suppliers. Vasopressin in the kidney and hypothalamus was analyzed per immune- histochemistry. open, water and food ad libitum. The free adaptation was followed by 4-days "nosepoke adaptation", during which the doors upon nosepoking at the door. Water was available for 24h. Mice were then adapted for 7 days to the "drinking-session" protocol, in which drinking was allowed between 11 and 13 a.m. and 4 and 6 p.m. Outside of these times the doors remained closed. The day-pattern was used to increase the motivation to learn. The restricted drinking times were maintained in subsequent learning tasks. Subsequently, mice returned to their home cages with free access to food and water. Morphological studies and immunohistochemistry The TSE Phenomaster offers an automated metabolic and behavioral monitoring in home cage environments. Drinking and feeding behavior were monitored with high- precision weight sensors for liquid and food dispensers, which are integrated into the lid of the cage. Mice were adapted to the drinking bottles for one week in their home cage and to the Phenomaster® cage for 3 consecutive days before starting the experiment, which consisted in 24h tap water and 24h sweetened water (20% sucrose). Drinking and feeding were recorded for 24 hours. Mice were terminally anaesthetized with isoflurane and cardially perfused with cold 1x phosphate buffered saline (PBS), pH 7.4 followed by 4% paraformaldehyde (PFA) in PBS for fixation. The kidneys were subsequently postfixed with 4% PFA in 1xPBS solution for 2h, dehydrated and embedded in paraffin. Renal sections (3.5 µm) were deparaffinized in xylene and graded ethanol and stained with haematoxylin & eosin (H&E) and periodic acid Schiff reagent (PAS). The sections were analyzed for signs of glomerulonephritis, sclerosis or tubulopathy as reported previously [37] by an investigator blinded to the genotypes. In addition, sections were processed for analysis of immune cell infiltration and aquaporin expression using immunohistology. Antigen retrieval in citrate buffer and antibody stainings followed standard procedures. Primary antibodies included rat anti-mouse F4/80 (Serotec), AQP1, AQP2 and AQP4 (Alamone labs, Jerusalem, Israel; diluted 1:100 in 1 % BSA in PBS- Triton) and vasopressin (Serotec). The numbers of neutrophils, macrophages and T cells were estimated per field (high-power field 400x, with a minimum of 10 fields counted per mouse, 3 mice per group; Soft Imaging System, Olympus, Hamburg, Germany). Aging and Disease • Volume 9, Number 5, October 2018 Statistics C) Number of lickings within 24h in the IntelliCage during free-drinking and restricted- drinking experiments. During 'free-drinking', access to the water bottles was allowed for 24h on nosepoking at the doors. During 'restricted- drinking', access to the water bottles was mostly denied except for 2x2h per day (11-12 am and 4-5 pm). D) Scatter plots showing the 24h drinking volume of aged Grn-/- and Grn+/+ mice during free drinking and water restriction for 2x12h with one-hour free drinking in between. E) Scatter plots showing the weight of food and water consumed within 24h in sex- matched young and aged Grn-/- and Grn+/+ mice in the Phenomaster cage. F) Phenomaster analysis of drinking and feeding behavior of old Grn-/- and Grn+/+ mice during presentation of tap water or sweet water with 20% sucrose (mean ± SD). The data show the food weight and drinking volume consumed within 24h. For all subpanels each scatter represents one mouse, the line is the mean and the whisker show the standard deviation (SD). Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test for each gender, organ, drinking or feeding. P< 0.05, P<0.01, * P<0.001, **** P<0.0001). RESULTS Body weight, feeding and drinking behavior Under standard housing conditions, body weight was similar in male and female young Grn+/+ and Grn-/- mice (Fig. 1A) but aged (12-15 months) female Grn-/- mice were overweight (ANOVA F(3, 62) = 10.40; P < 0.0001, posthoc adjusted P-values in the figure), which was lost again at old age (>18 months, not shown). Organ sizes and weights of the heart and spleen were similar in both genotypes irrespective of the age, but the kidneys of old Statistics Data are presented as mean ± SD unless stated otherwise and were analyzed with SPSS 23 and Graphpad Prism 6.0. Groups comprised 6-16 mice depending on the experiment, and numbers are presented in the figures and/or figure legends Data were submitted to univariate analysis of variance (ANOVA) or unpaired, 2-tailed Student's t-tests. In case of significant differences of ANOVAs, groups were compared with the respective control groups using t-tests with a correction of alpha according to Dunnett (one control group) or according to Šidák (specific control groups). Wildtype young mice were mostly used as the reference group. The alpha level was set to 0.05 for all comparisons, and adjusted P-values are reported. Figure 1. Body weight, drinking and feeding behavior of progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Scatter plots showing the body weight of young and aged male and female Grn-/- and Grn+/+ mice. B) Organ weights of aged Grn-/- and Grn+/+ mice. C) Number of lickings within 24h in the IntelliCage during free-drinking and restricted- drinking experiments. During 'free-drinking', access to the water bottles was allowed for 24h on nosepoking at the doors. During 'restricted- drinking', access to the water bottles was mostly denied except for 2x2h per day (11-12 am and 4-5 pm). D) Scatter plots showing the 24h drinking volume of aged Grn-/- and Grn+/+ mice during free drinking and water restriction for 2x12h with one-hour free drinking in between. E) Scatter plots showing the weight of food and water consumed within 24h in sex- matched young and aged Grn-/- and Grn+/+ mice in the Phenomaster cage. F) Phenomaster analysis of drinking and feeding behavior of old Grn-/- and Grn+/+ mice during presentation of tap water or sweet water with 20% sucrose (mean ± SD). The data show the food weight and drinking volume consumed within 24h. For all subpanels each scatter represents one mouse, the line is the mean and the whisker show the standard deviation (SD). Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test for each gender, organ, drinking or feeding. * P< 0.05, P<0.01, * P<0.001, **** P<0.0001). behavior of progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Scatter plots showing the body weight of young and aged male and female Grn-/- and Grn+/+ mice. B) Organ weights of aged Grn-/- and Grn+/+ mice. Aging and Disease • Volume 9, Number 5, October 2018 Analysis of vasopressin and other neuropeptides Analysis of vasopressin and other neuropeptides Aging and Disease • Volume 9, Number 5, October 2018 819 Hardt S., et al Progranuin & Diabetes insipidus Keyence (BZ-9000, Germany) microscope. For overviews, tiled images were automatically stitched. An observer who was not aware of the genotypes performed the image analyses with FIJI ImageJ for quantification of aquaporin and vasopressin immunofluorescence. The Particle Counter was employed after setting of the threshold (Isodata algorithm) and definition of size inclusion and circularity criteria. The analysis was done with sections of 4 mice per group. Results are presented as number or particles or percentage of area coverage with positive particles. RESULTS (Fig. 1A) but aged (12-15 months) female Grn-/- mice were overweight (ANOVA F(3, 62) = 10.40; P < 0.0001, posthoc adjusted P-values in the figure), which was lost again at old age (>18 months, not shown). Organ sizes and weights of the heart and spleen were similar in both genotypes irrespective of the age, but the kidneys of old Body weight, feeding and drinking behavior Body weight, feeding and drinking behavior Under standard housing conditions, body weight was similar in male and female young Grn+/+ and Grn-/- mice 820 Progranuin & Diabetes insipidus Hardt S., et al higher relative weights of the livers (ANOVA F(3, 48) = 4.906; P = 0.0047; posthoc corrected P-values in the figure). Grn-/- mice were enlarged with increased weights (2-way ANOVA for 'group X organ' F(3, 72) = 4.435; P = 0.0064; Fig. 1B), which was also obvious in tissue sections (Fig. 4). Significance was maintained when kidney weights were corrected for body weights, which also revealed Figure 2. Renal and metabolic functions of progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Scatter plots showing the 24h urine volume, urine osmolality and urine specific gravity of aged Grn-/- and Grn+/+ mice (12-16 months old). B) Concentration of glucose in 24h-urine and plasma of aged Grn-/- and Grn+/+ mice. C) Creatinine concentrations in 24h-urine and plasma, and creatinine clearance of aged Grn-/- and Grn+/+ mice. D) Concentrations of urea and albumin in 24h-urine of aged Grn-/- and Grn+/+ mice. E) Concentrations of arginine vasopressin (AVP, antidiuretic hormone) in plasma and in crude tissue extracts of the hypothalamus of aged Grn-/- and Grn+/+ mice and immunofluorescence analysis of AVP in the kidney (bottom, scale bar 50 µm). F, G) Plasma concentrations of ghrelin and agouti related protein (Agrp) of aged Grn-/- and Grn+/+ mice. For all subpanels each scatter represents one mouse, sexes were matched between groups, the line is the mean and the whisker show the standard deviation (SD). Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test. * P< 0.05, P<0.01, * P<0.001). Figure 2. Renal and metabolic functions of progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Scatter plots showing the 24h urine volume, urine osmolality and urine specific gravity of aged Grn-/- and Grn+/+ mice (12-16 months old). B) Concentration of glucose in 24h-urine and plasma of aged Grn-/- and Grn+/+ mice. C) Creatinine concentrations in 24h-urine and plasma, and creatinine clearance of aged Grn-/- and Grn+/+ mice. D) Concentrations of urea and albumin in 24h-urine of aged Grn-/- and Grn+/+ mice. E) Concentrations of arginine vasopressin (AVP, antidiuretic hormone) in plasma and in crude tissue extracts of the hypothalamus of aged Grn-/- and Grn+/+ mice and immunofluorescence analysis of AVP in the kidney (bottom, scale bar 50 µm). Aging and Disease • Volume 9, Number 5, October 2018 Body weight, feeding and drinking behavior F, G) Plasma concentrations of ghrelin and agouti related protein (Agrp) of aged Grn-/- and Grn+/+ mice. For all subpanels each scatter represents one mouse, sexes were matched between groups, the line is the mean and the whisker show the standard deviation (SD). Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test. * P< 0.05, P<0.01, * P<0.001). Aging and Disease • Volume 9, Number 5, October 2018 821 Hardt S., et al Progranuin & Diabetes insipidus Figure 3. Histomorphology of the kidney of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Immunostaining of myeloid-derived F48/80-positive immune cells (brown), with hematoxylin counterstaining of nuclei (blue). Immune cells were counted per field of view and averaged (10 fields per mouse of 3 mice per group). Numbers differed significantly between genotypes (unpaired, 2-tailed Student's t-test). Scale bars 50 µm. B) Periodic acid-Schiff (PAS) staining of polysaccharides and mucous substances. Histomorphometric scores did not differ between genotypes, except for a higher number of immune cells in Grn-/- mice. Scale bars 50 µm. C, D) Concentrations of C-reactive protein (CRP) and zinc in plasma of aged Grn-/- and Grn+/+ mice. Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test, ** P<0.0001). Figure 3. Histomorphology of the kidney of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Immunostaining of myeloid-derived F48/80-positive immune cells (brown), with hematoxylin counterstaining of nuclei (blue). Immune cells were counted per field of view and averaged (10 fields per mouse of 3 mice per group). Numbers differed significantly between genotypes (unpaired, 2-tailed Student's t-test). Scale bars 50 µm. B) Periodic acid-Schiff (PAS) staining of polysaccharides and mucous substances. Histomorphometric scores did not differ between genotypes, except for a higher number of immune cells in Grn-/- mice. Scale bars 50 µm. C, D) Concentrations of C-reactive protein (CRP) and zinc in plasma of aged Grn-/- and Grn+/+ mice. Asterisks indicate statistically significant differences between genotypes (unpaired, 2-tailed Student's t-test, ** P<0.0001). Aging and Disease • Volume 9, Number 5, October 2018 822 Hardt S., et al Progranuin & Diabetes insipidus et al Progranuin & Diabetes i Figure 4. Immunofluorescence analysis of aquaporin 1, 2 and 4 (AQP1, AQP2, AQP4) in the kidney of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Examples of AQP2 immunofluorescence at low (upper panels) and high magnifications (bottom panels and insert). Scale bars as indicated in the figure. Kidney morphology and aquaporin water channels The kidney function tests suggested that the renal water loss was caused by a defect of water reabsorption in tubules or collecting ducts. The histomorphology of the kidneys showed mildly widened tubules but no overt tubulopathy (Fig. 3A, B). The kidneys of aged Grn-/- mice showed significantly higher numbers of F4/80+ macrophages within the interstitium and perivascular regions (Fig. 3A; renal cortex P 0.039; renal medulla P = 0.0031), but H&E and PAS stainings (Fig. 3B) did not reveal signs of glomerulonephritis, tubulonephritis or tubulosclerosis. Overall, the kidney morphology agreed with the higher urine turnover [38], possibly owing to a vasopressin un-responsiveness, but without structural changes or diseases of the kidney that may result in renal water losses such as amyloidosis. Immune cell infiltration of the kidneys was not associated with higher plasma levels of the pro-inflammation markers, C-reactive protein (Fig. 3C) and euhydrated Grn-/- mice had no derangement of serum electrolytes or serum osmolality (Table 1). Urine creatinine, potassium and magnesium were reduced likely owing to secretion of diluted urine (Table 1), and levels of the trace element, zinc were reduced is plasma (Fig. 3D) confirming our previous studies [5]. We performed Phenomaster experiments (24h observation time) to quantify water and food consumption precisely. Drinking and feeding behavior was normal in young Grn-/- mice, but increased in aged Grn-/- mice as compared to the respective controls (ANOVA for 'age X genotype' for drinking F (1, 94) = 28.28; P < 0.0001, for feeding F (1, 86) = 16.45; P = 0.0001; posthoc in Fig. 1E). Polydipsia of Grn-/- was obvious at old age irrespective of tap water or sweetened water was presented, amounting to daily drinking volumes of about 7 and 18 ml/d in Grn-/- mice for tap and sweet water as compared to 3 and 10 ml/d in controls (Fig. 1E). Tap water polydipsia was accompanied with overfeeding, whereas feeding dropped to a minimum in both genotypes when sweetened water was presented (Fig. 1F). Body weight, feeding and drinking behavior The bottom panel also shows high AQP1 and AQP4 at high magnification, which did not differ between genotypes. B) Quantification of AQP2 positive particles using stitched full sections of the kidney of 4 mice per group. Each scatter is a section. Analysis of the tubule lumen as assessed by measuring the lumen area in 4 sections of 4 mice per group. Each scatter is one AQP2 positive collecting duct. The images suggest reduced AQP2 expression and widening of AQP2+ collecting ducts. Asterisks indicate significant differences between genotypes, unpaired 2-tailed Student's t-test P<0.05, *P<0.001). Figure 4. Immunofluorescence analysis of aquaporin 1, 2 and 4 (AQP1, AQP2, AQP4) in the kidney of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). A) Examples of AQP2 immunofluorescence at low (upper panels) and high magnifications (bottom panels and insert). Scale bars as indicated in the figure. The bottom panel also shows high AQP1 and AQP4 at high magnification, which did not differ between genotypes. B) Quantification of AQP2 positive particles using stitched full sections of the kidney of 4 mice per group. Each scatter is a section. Analysis of the tubule lumen as assessed by measuring the lumen area in 4 sections of 4 mice per group. Each scatter is one AQP2 positive collecting duct. The images suggest reduced AQP2 expression and widening of AQP2+ collecting ducts. Asterisks indicate significant differences between genotypes, unpaired 2-tailed Student's t-test P<0.05, ***P<0.001). Aging and Disease • Volume 9, Number 5, October 2018 823 Hardt S., et al Progranuin & Diabetes insipidus low plasma vasopressin levels. But alterations of feeding- regulating hormones, ghrelin and AgRP (Fig.2F, G), which were oppositely regulated, suggest that progranulin deficiency may cause hypothalamic hormonal dys- balances, which may account for the increase of appetite. Daily lickings in IntelliCage experiments of young and aged Grn-/- mice and respective controls showed that the number of licks was significantly increased in aged Grn-/- mice during both ‘free drinking’ and ‘restricted drinking’ as compared to controls (ANOVA for 'group' F (3, 120) = 73.28; P < 0.0001; results of posthoc t- tests presented in Fig. 1C). In contrast, young mice behaved normally. Metabolic Cage experiments showed that polydipsia of aged Grn-/- mice was associated with polyuria (Figure 1D), which was maintained during water restriction (2x12h with 1h free drinking in between). Aging and Disease • Volume 9, Number 5, October 2018 Renal and metabolic functions The increased drinking behavior was confirmed in an independent experiment in Metabolic Cages in a second set of aged Grn-/- and Grn+/+ mice. Again, the 24h-drinking volume was almost doubled and was associated with an increase of the daily urine volume and a decrease of the urine osmolality and specific urine gravity, which was maintained during water restriction suggesting a renal water loss (Fig. 2A, results of unpaired, 2-sided t-tests in the figure). Urine and plasma glucose concentrations were in the normal range (Fig. 2B), hence excluding osmotic polyuria. Plasma creatinine and creatinine clearance (Fig. 2C), urea excretion and urine albumin (Fig. 2D) were all in the normal range, suggesting an isolated concentration defect without major glomerular or tubular dysfunctions due to a renal diabetes insipidus, which is normally associated with an increase of vasopressin release. Indeed, plasma vasopressin levels in progranulin deficient mice were on average about 3-fold higher than normal levels (Fig. 2E), associated with increased vasopressin levels in the hypothalamus and in the kidney, as revealed by immunofluorescence analyses. Hence, the hypothalamic counter-regulatory loop appeared to be intact, allowing for exclusion of psychogenic polydipsia, which would suppress vasopressin production, and excluding a hypophyseal secretory defect, which would also result in Congenital nephrogenic diabetes insipidus in humans is mostly caused by loss-of-function mutations of the renal vasopressin receptor, which is required for insertion of aquaporin water channels into the outer membrane of collecting duct cells [39], or more rarely the defect is caused by mutations of aquaporin 2 [40]. To address these potential mechanisms, we performed immunofluorescence studies of aquaporin 1, 2 and 4 (AQP1, AQP2, AQP4) in the kidneys (Fig. 4). AQP1 and AQP4 immunoreactivity were similar in both genotypes. However, AQP2 immunofluorescence clearly shows the enlargement of the Grn-/- kidney and widening of the tubules associated with an apparent reduction of AQP2 immunofluorescence intensity, particularly in the inner medulla. High magnification shows that AQP2 is inserted in the apical membrane but somewhat weaker than in controls, suggesting that an AQP2 deficit in the collecting tubules may account for the water loss. Aging and Disease • Volume 9, Number 5, October 2018 824 Hardt S., et al Progranuin & Diabetes insipidus Progranuin & Diabe Figure 5. Immunofluorescence analysis of arginine vasopressin (AVP), glial fibrillary acidic protein (GFAP) of astrocytes and DAPI counterstain of nuclei in the hypothalamus of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). Renal and metabolic functions The Gensat images in the right give an overview of the localization of the paraventricular nucleus (PVN) and the nucleus supraopticus (SON), which are the major sites for vasopressin producing neurons. The upper panels show AVP, GFAP and DAPI in the PVN as overview and zoom-in images, and the lower panels show the SON. AVP immunofluorescence was more intense in Grn-/- and AVP positive neurons appear to be enlarged. GFAP staining reveals astrogliosis in Grn-/-. Scale bars 300 µm for overviews and 100 µm for zoom-in images. Figure 5. Immunofluorescence analysis of arginine vasopressin (AVP), glial fibrillary acidic protein (GFAP) of astrocytes and DAPI counterstain of nuclei in the hypothalamus of aged progranulin deficient (Grn-/-) and control mice (Grn+/+). The Gensat images in the right give an overview of the localization of the paraventricular nucleus (PVN) and the nucleus supraopticus (SON), which are the major sites for vasopressin producing neurons. The upper panels show AVP, GFAP and DAPI in the PVN as overview and zoom-in images, and the lower panels show the SON. AVP immunofluorescence was more intense in Grn-/- and AVP positive neurons appear to be enlarged. GFAP staining reveals astrogliosis in Grn-/-. Scale bars 300 µm for overviews and 100 µm for zoom-in images. Aging and Disease • Volume 9, Number 5, October 2018 825 Hardt S., et al Progranuin & Diabetes insipidus Table 1. Serum and urine electrolytes in progranulin knockout (Grn-/-) and wildtype (Grn+/+) female mice. Renal and metabolic functions Wks ml/24h g Urine (mmol/l) Serum (mmol/l) Genotype Age Drink BW Crea Ca Cl K Mg Na P Glc Urea Ca Cl K Mg Na P mosmol/kg Grn-/- 42.3 7.5 25.5 1.768 4.1 50.6 105.7 3.4 37 11.7 13.1 10 2.3 111 4.3 0.9 146 2.0 315.1 Grn-/- 42.3 9.0 26.6 1.800 3.4 55.1 96.2 3.5 42 12.5 14.5 9.6 2.3 111 4.9 1.1 145 2.0 314.1 Grn-/- 42.3 3.3 25.2 1.774 2.6 29.1 74.9 0.8 31 10.1 12.7 9.7 2.3 112 4.9 1.0 149 1.8 320.4 Grn-/- 42.3 3.8 25.4 1.298 3.2 41.9 85.4 3.0 40 12.4 14.0 8.9 2.3 108 3.8 1.0 143 2.0 308.9 Grn-/- 42.3 2.7 26.0 1.544 5.1 55.9 87.8 3.3 61 2.0 13.0 9.2 2.4 110 4.1 1.1 147 2.3 316.2 Grn-/- 46.4 8.7 24.8 0.413 0.4 17.2 28.2 0.6 16 9.2 16.3 7.3 2.3 112 4.1 0.9 146 2.0 315.6 Grn-/- 51.1 6.0 27.2 1.091 2.1 45.2 74.6 0.3 41 10.9 15.6 7.2 2.4 114 4.0 0.9 149 2.0 320.8 Grn+/+ 50.7 2.6 24.0 3.400 6.3 38.9 154.2 3.4 38 14.4 14.9 7.1 2.4 111 3.7 1.0 147 2.0 316.0 Grn+/+ 48.6 1.1 23.7 2.831 3.2 37.2 120.2 3.4 45 14.7 13.1 10.2 2.4 112 4.2 1.0 150 2.0 323.3 Grn+/+ 48.6 1.2 24.2 1.475 4.3 57.8 114.9 3.4 56 13.8 8.8 12.1 2.4 112 5.2 0.9 148 1.4 316.9 Grn+/+ 44.7 0.8 24.0 1.581 5.6 40.4 98.0 3.4 47 6.1 11.4 6.8 2.3 112 4.3 0.9 149 1.6 316.2 Grn+/+ 43.6 0.3 23.3 2.288 2.6 48.2 215.9 3.5 35 9.9 13.5 7.4 2.4 109 4.3 0.9 146 1.8 312.9 Grn+/+ 43.7 4.7 24.1 1.704 3.5 39.6 88.1 3.3 28 13.3 - 8.8 2.4 112 3.9 1.0 148 2.0 - Means Grn-/- 44.1 5.9 25.8 1.384 3.0 42.1 78.9 2.1 38.3 9.8 14.2 8.8 2.3 111.1 4.3 1.0 146.4 2.0 315.9 Grn+/+ 46.6 1.8 23.9 2.213 4.2 43.7 131.9 3.4 41.5 12.0 12.3 8.7 2.4 111.3 4.3 1.0 148.0 1.8 317.1 Grn-/-SD 3.5 2.6 0.8 0.506 1.5 14.3 25.0 1.5 13.5 3.6 1.4 1.1 0.0 1.9 0.4 0.1 2.1 0.1 4.0 Grn+/+SD 3.0 1.6 0.3 0.775 1.4 7.9 47.0 0.1 9.9 3.4 2.3 2.1 0.0 1.2 0.5 0.1 1.4 0.3 3.8 t-test P 0.196 0.007 0.0003 0.041 0.154 0.820 0.025 0.057 0.640 0.290 0.118 0.907 0.053 0.835 0.902 0.416 0.155 0.083 0.6178 Abbreviations: Wks, weeks; BW, body weight; Crea, creatinine; Ca, calcium; Cl, chloride; K, potassium; Mg, magnesium; Na, sodium; P, anorganic phosphate, Glc, glucose; SD, standard deviation; t-test 2 sided unpaired Vasopressin neurons in the hypothalamus progranulin [41]. Indeed, progranulin's plasma levels fluctuate with the feeding conditions [27], and the observed inverse dysregulations of ghrelin and agouti related protein in the knockouts point to losses of progranulin-mediated anti-orexigenic effects [27] that may explain overfeeding. High vasopressin plasma levels in combination with polyuria suggested a counter-regulatory increase of vasopression production in the hypothalamus. In addition, the hypothalamus may be target of the neurodegenerative disease caused by progranulin deficiency. Therefore, we assessed putative signs of hypothalamic gliosis, and number and distribution of vasopression-positive neurons (Fig. 5). Vasopressin (AVP; arginine vasopressin) immunofluorescence was stronger in progranulin deficient brains in the paraventricular (PVN) and supraoptical nuclei (SON), and GFAP immunoreactivity of astrocytes revealed a strong gliosis within these regions in Grn-/- mice, which is in line with the inherent neurodegenerative disease. In controls, AVP staining was finely distributed to fibers, particularly, in SON. In Grn-/ mice-, AVP-positive neurons appeared to be enlarged but rather reduced in numbers as revealed by DAPI counterstaining of the nuclei. Hence, polydipsia and polyuria could be signs of psychogenic primary polydipsia or central diabetes insipidus due to vasopressin deficiency or mal-processing [42], but both was not the case. Instead, vasopressin levels were increased, and polyuria maintained during water restriction. Except for the water loss, renal functions of progranulin deficient mice were in the normal range agreeing with previous studies [6, 34], and histo- morphometric readouts of chronic renal diseases were negative. There was also no sign of osmotic diuresis due to excretion of glucose. Hence, the results point to a resistance of collecting tubules towards vasopressin resulting in a loss of aquaporin-2 expression and adaptive hypertrophy to handle the increased rates of glomerular filtration and tubular reabsorption that occur in models of central or nephrogenic diabetes insipidus [38, 43]. Aging and Disease • Volume 9, Number 5, October 2018 DISCUSSION Vasopressin enhances water reabsorption in the renal collecting duct by vasopressin V2 receptor-mediated activation of adenylylcyclase (AC) [44], cAMP-mediated phosphorylation of aquaporin-2 (AQP2), and increased insertion of AQP2 into the apical membrane [45]. Vasopressin also determines aquaporin-2 transcription via cAMP response elements [46]. In mouse models of nephrogenic diabetes insipidus, collecting ducts are resistant towards vasopressin owing to V2 mutations [47], exaggerated phosphodiesterase-mediated AC degradation [48], interference with AC activity [49], or AQP2 We show that aged progranulin deficient mice eat and particularly, drink considerable more than controls, about twice the normal volume. Considering that progranulin knockout is a model of frontotemporal dementia, which is predominated in humans by behavioral abnormalities including compulsive eating and drinking, one may hypothesize that the observed phenotype is a manifestation of the frontotemporal neurodegeneration, possibly contributed by dysfunctions of hypothalamic neurons that show comparably high expression of 826 Progranuin & Diabetes insipidus Hardt S., et al phosphorylation, however without known interconnection with progranulin. deficiency or malfunction [43, 50, 51]. In particular, age- associated nephrogenic diabetes insipidus in some inbred mouse strains [52, 53] and WAG/Rij rats [54] are caused by an age-progressive decline of AQP2 suggesting that progranulin deficiency may lead to similar but premature AQP2 losses and hence, early manifestations of kidney aging. Considering progranulin's multi-faceted functions a number of mechanisms may contribute to the DI phenotype. The increase of vasopressin plasma levels likely reflects an intact adaptation of the hypothalamic- hypophyseal axis in response to the renal water loss and argues against a substantial degeneration of hormone producing neurons at that age. However, AVP positive neurons appear to be enlarged and are surrounded by astrogliosis suggesting that the hypothalamus is affected by progranulin deficiency. In line, the observed changes of hormones regulating appetite and feeding show that progranulin is involved in hormone homeostasis. Hypothalamic dysfunctions have been observed in some other mouse models of neurodegenerative diseases [60, 61], and primary polydipsia was found in saposin D deficient mice [26], the latter important because its precursor, prosaposin and progranulin converge on sortilin-mediated transport, interaction at the autophagolysosomal interface [16] and lysosomal functions [18, 62]. In contrast to our mice, saposin D deficient mice had reduced vasopressin levels, and water restriction resulted in a normalization of kidney morphology and functions [26], suggesting different mechanisms of the polydipsia-polyuria phenotype. DISCUSSION The observed renal immune cell infiltration in Grn-/- kidney may result from a general pro-inflammatory state in consequence of the loss of immune silencing provided by progranulin, which is expressed in immune cells of myeloid origin and suppressor T-cells and promotes the clearance of pathogens [34, 55]. Permanent immune over-activation in the absence of progranulin may not only affect the kidney, but possibly also lung, liver and brain [34] and may contribute to the observed liver enlargement, possibly a sign of liver steatosis. Hence, although the immune infiltrates do not explain the water loss they may be representatives of the overall pro- inflammatory state. Intracellular progranulin is localized to "membrane- bounded organelles" which are vesicles of altering destination, including endosomes, autophagosomes and lysosomes. Gene ontology analyses suggest that progranulin is involved in vesicle transport and vesicular transmembrane transport of ions and proteins [23]. Hence, its deficiency may impair vesicular uptake and release of ions and transport of water channels to and from the apical membrane of the collecting tubules. In line with this idea, gene regulation studies in postmortem brain of PGRN- associated FTLD-U patients show deregulations of aquaporin-1 and AQP4 among the top 100 deregulated genes (Geo data sets GDS3459, GSE13162 [56]), raising the intriguing possibility that patients with PGRN- associated frontotemporal lobar degeneration may eventually develop symptoms of polyuria. However so far, diabetes insipidus has not been described as clinical problem in these patients, possibly owing to the early onset and fatal course of the disease and/or interpretation of subtle manifestations as psychogenic polydipsia. In summary, we show that progranulin deficient mice develop a nephrogenic diabetes insipidus, which becomes manifest at about 6-9 month of age with a cumulative incidence of about 70-80%. Polyuria and polydipsia lead to a doubling of drinking volume and urine output but the dysfunction does not lead to extreme water losses, kidney damage or premature death. Nevertheless, the data show a relevant function of progranulin for the maintenance of water homeostasis and osmolality. Acknowledgements We acknowledge the financial support of the Deutsche Forschungsgemeinschaft (CRC1080, A03) to IT and SCHA 1082/6-1 to LS and the research funding program "Landesoffensive zur Entwicklung wissenschaftlich- ökonomischer Exzellenz" (LOEWE) of the State of Hessen, Research Center for Translational Medicine and Pharmacology, TMP. We thank Aihao Ding for progranulin knockout mice. p y g p y p Secreted progranulin binds to and activates a number receptors including epidermal growth factor receptor [57], Ephrin A2 [13] and Notch receptors [12]. Ephrin and Notch signaling is mainly recognized for their pro-fibrotic effects in the kidney, hence not explaining the progranulin-provided benefit, but inactivation of Notch signaling in the renal collecting duct causes nephrogenic diabetes insipidus [58] similar to that seen in our progranulin-deficient mice. 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[38] ] Naccarato R, Rizzo A, Sirigu F, Bertaglia E, Previato G, Fiaschi E (1976). Renal histologic and ultrastructural findings in psychogenic polydipsia and diabetes insipidus. Nephron, 16: 226-35. [51] [51] Ho HT, Chung SK, Law JW, Ko BC, Tam SC, Brooks HL, Knepper MA, Chung SS (2000). Aldose reductase-deficient mice develop nephrogenic diabetes insipidus. Mol Cell Biol, 20: 5840-6. [39] 39] Schliebe N, Strotmann R, Busse K, Mitschke D, Biebermann H, Schomburg L, et al. (2008). Aging and Disease • Volume 9, Number 5, October 2018 References interacts with cyclin T1 and modulates P TEFb dependent transcription. Mol Cell Biol, 23: 1688-702. [56] Chen-Plotkin AS, Geser F, Plotkin JB, Clark CM, Kwong LK, Yuan W, Grossman M, Van Deerlin VM, Trojanowski JQ, Lee VM (2008). Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration. Hum Mol Genet, 17: 1349-62. [56] Kwong LK, Yuan W, Grossman M, Van Deerlin VM, Trojanowski JQ, Lee VM (2008). 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https://openalex.org/W3176026734	https://www.frontiersin.org/articles/10.3389/fevo.2021.676961/pdf	English	null	A Disrupted Historical Fire Regime in Central British Columbia	Frontiers in ecology and evolution	2,021	cc-by	12,194	A Disrupted Historical Fire Regime in Central British Columbia Wesley Brookes, Lori D. Daniels, Kelsey Copes-Gerbitz, Jennifer N. Baron and Allan L. Carroll Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada In the 2017 and 2018, 2.55 million hectares burned across British Columbia, Canada, including unanticipated large and high-severity ﬁres in many dry forests. To transform forest and ﬁre management to achieve resilience to future megaﬁres requires improved understanding historical ﬁre frequency, severity, and spatial patterns. Our dendroecological reconstructions of 35 plots in a 161-hectare study area in a dry Douglas-ﬁr forest revealed historical ﬁres that burned at a wide range of frequencies and severities at both the plot- and study-area scales. The 23 ﬁres between 1619 and 1943 burned at intervals of 10–30 years, primarily at low- to moderate-severity that scarred trees but generated few cohorts. In contrast, current ﬁre-free intervals of 70–180 years exceed historical maximum intervals. Of the six widespread ﬁres from 1790 to 1905, the 1863 ﬁre affected 86% of plots and was moderate in severity with patches of higher severity that generated cohorts at ﬁne scales only. These results indicate the severity of ﬁres varied at ﬁne spatial scales, and offer little support for the common assertion that periodic, high-severity, stand-initiating events were a component of the mixed- severity ﬁre regime in these forest types. Many studies consider ﬁres in the late 1800s relatively severe because they generated new cohorts of trees, and thus, emphasize the importance of high-severity ﬁres in a mixed-severity ﬁre regime. In our study area, the most widespread and severe ﬁre was not a stand-initiating ﬁre. Rather, the post- 1863 cohorts persisted due disruption of the ﬁre regime in the twentieth century when land-use shifted from Indigenous ﬁre stewardship and early European settler ﬁres to ﬁre exclusion and suppression. In absence of low- to moderate-severity ﬁres, contemporary forests are dense with closed canopies that are vulnerable to high-severity ﬁre. Future management should reduce forest densities and to restore stand- and landscape-level heterogeneity and increase forest resilience. The timing and size of repeat treatments such as thinning of subcanopy trees and prescribed burning, including Indigenous ﬁre stewardship, can be guided by our reﬁned understanding of the mixed-severity ﬁre regime that was historically dominated by low- to moderate-severity ﬁres in this dry forest ecosystem ORIGINAL RESEARCH published: 28 June 2021 doi: 10.3389/fevo.2021.676961 Keywords: dendroecological reconstruction, Douglas-ﬁr, ﬁre scars, post-ﬁre cohort, frequency, severity, human impacts, mixed-severity ﬁre regime Edited by: Miguel Montoro Girona, Université du Québec en Abitibi-Témiscamingue, Canada Reviewed by: Alexandra Rouillard, Arctic University of Norway, Norway Andy Hennebelle, Université du Québec à Rimouski, Canada Correspondence: Lori D. Daniels lori.daniels@ubc.ca Specialty section: This article was submitted to Paleoecology, a section of the journal Frontiers in Ecology and Evolution Received: 06 March 2021 Accepted: 04 June 2021 Published: 28 June 2021 Citation: Brookes W, Daniels LD, Copes-Gerbitz K, Baron JN and Carroll AL (2021) A Disrupted Historical Fire Regime in Central British Columbia. Front. Ecol. Evol. 9:676961. doi: 10.3389/fevo.2021.676961 Specialty section: This article was submitted to Paleoecology, a section of the journal Frontiers in Ecology and Evolution Received: 06 March 2021 Accepted: 04 June 2021 Published: 28 June 2021 Specialty section: This article was submitted to Paleoecology, a section of the journal Frontiers in Ecology and Evolution Received: 06 March 2021 Accepted: 04 June 2021 Published: 28 June 2021 Citation: Brookes W, Daniels LD, Copes-Gerbitz K, Baron JN and Carroll AL (2021) A Disrupted Historical Fire Regime in Central British Columbia. Front. Ecol. Evol. 9:676961. doi: 10.3389/fevo.2021.676961 June 2021 \| Volume 9 \| Article 676961 1 Frontiers in Ecology and Evolution \| www.frontiersin.org Disrupted Fire Regime Brookes et al. Across BC, an average of 1,690 ﬁres burn 150,000 hectares annually (BC Government, 2018), although 92% of ﬁres are successfully suppressed before exceeding 4 ha in size (BC Wildﬁre Management Branch, 2012). Past management focused on controlling ﬁre under the premise that human lives, communities, critical infrastructure, and economically valuable resources in grasslands and forests required protection (BC Government, 2010). Despite highly successful ﬁre suppression since the 1940s, 3.4 million hectares have burned in British Columbia since 2003, costing over $3 billion (CAD) in direct ﬁre suppression and resulting in tens of thousands of evacuations (Abbott and Chapman, 2018). Combined, the 2003 and 2017 ﬁre seasons burned almost 1.5 million ha, largely in dry mixed- conifer forests with tree mortality exceeding 80% in many areas (Abbott and Chapman, 2018). Although the 2017 ﬁre season broke the British Columbian record for area burned in one ﬁre season (1.2 million ha), it was surpassed in 2018 when an additional 1.35 million hectares burned (BC Government, 2018). The threat of more frequent and severe ﬁres continues to impact the lives and property of the people living in communities surrounded by dry mixed-conifer forests (Coogan et al., 2021). INTRODUCTION Fire is a dominant disturbance in forests worldwide (Bowman et al., 2009) and interacts with multiple abiotic and biotic disturbances to drive forest composition, structure, and dynamics (Hessburg et al., 2019; Leclerc et al., 2021). In forests where ecosystem-based management aims to emulate the historical range and variability in disturbance (Landres et al., 1999; Swetnam et al., 1999; Keane et al., 2009), knowledge on historical ﬁre frequency, severity and spatial patterns guides choices on stand-level even- or uneven-aged silvicultural systems and landscape-level forest age class distributions, old-growth management areas (e.g., BC Ministries of Environment and Forests, 1995), and ﬁre suppression policies (e.g., BC Wildﬁre Management Branch, 2012). Historical baselines are also used to gauge departures from historical conditions and determine where ecological restoration or ﬁre hazard mitigation are warranted (Hessburg et al., 2005, 2016; Stephens et al., 2012, 2013; Halofsky et al., 2020). y Along the steep climatic and vegetation gradients characteristic of the montane forests of western North America, mixed-severity ﬁre regimes include ﬁres of variable frequencies and severities forming complex spatial patterns within and among events (Perry et al., 2011; Marcoux et al., 2013; Hessburg et al., 2016, 2019). In these forests, understanding the relative importance of low- versus high-severity ﬁre is critical for sustainable management and eﬀective forest restoration, but remains a point of contention (Hagmann et al., 2021). Speciﬁcally, a debate has centered on whether recent high- severity ﬁres in mixed-conifer forests are an inherent part of a mixed-severity ﬁre regimes (Klenner et al., 2008; Williams and Baker, 2012; Odion et al., 2014; Baker, 2015, 2017) or if high-severity ﬁres have exceeded the historical range of variability (Fulé et al., 2013; Stephens et al., 2014; Moritz et al., 2018). Diﬀerent management actions are associated with these two schools of thought. If the ﬁre regime is not altered, management actions are unnecessary or may have unintended negative consequences on forest productivity or critical habitat (Odion et al., 2014; Baker, 2015; DellaSala and Hanson, 2019). Where ﬁre regimes have been altered, determining the causes and degree of departure is important for taking appropriate management actions (Fulé et al., 1997; Stephens et al., 2013; Hessburg et al., 2016). There is great concern that megaﬁres such as those of 2003, 2017, and 2018 will become more common with continued climate change (Abbott and Chapman, 2018), reinforcing recommendations for transformative changes to ﬁre and forest management (North et al., 2015; Stephens et al., 2016, 2020; Daniels et al., 2020). INTRODUCTION To avoid risk of generalizing across forest types and oversimplifying treatments, ecosystem-speciﬁc reference conditions are required to develop management and restoration treatments to enhance forest resilience to ﬁre and climate change (Agee and Skinner, 2005; Keane et al., 2009; Schoennagel and Nelson, 2011; Hessburg et al., 2016). In 2017, a lightning outbreak during extreme ﬁre weather ignited multiple high-severity ﬁres that burned 33,181 ha of forests surrounding the city of Williams Lake in south central BC (Figure 1A). The combination of ignition locations, wind direction, and ﬁre suppression eﬀorts spared the forests near Knife Creek in the Alex Fraser Research Forest. Nevertheless, these ﬁres underscored the importance of reconstructing historical ﬁre regimes and their inﬂuences on forest dynamics. This research was conducted in the dry Douglas-ﬁr forests of the Research Forest to address the following questions: How frequent and severe were historical ﬁres? Did severity vary spatially within widespread ﬁres? Has the ﬁre regime changed during the twentieth century and what are the impacts on forest structure? Dendroecological analyses of ﬁre scars and increment cores sampled from a dense network of 35 plots across a 161-hectare (ha) study area allowed us to reconstruct spatio-temporal variations in ﬁre frequency, severity, and spatial patterns at a ﬁne scale. By critically assessing alternate interpretations of contemporary forest structure in the context of historical ﬁres, we elucidated disruptions to the historical ﬁre regime and provide recommendations to restore forest resilience to ﬁre. Fires of a range of severities burn in the dry mixed-conifer forests of the interior of British Columbia (BC) (Marcoux et al., 2013; Hessburg et al., 2019). Historical ignitions were by lightning and Indigenous ﬁre stewardship (Coogan et al., 2021), with low-severity surface ﬁres burning at intervals < 50 years, with high-severity stand-initiating ﬁres at intervals of 250 years (BC Ministries of Environment and Forests, 1995). Supporting the latter notion, modern ﬁre records show that large, intense ﬁres burn during extreme ﬁre weather (Klenner et al., 2008). Frontiers in Ecology and Evolution \| www.frontiersin.org Reconstructing Fire History and Forest Dynamics Following standard dendrochronological methods (Stokes and Smiley, 1996), ﬁre-scarred sections and increment cores were air-dried, mounted on wooden supports, and sanded so that individual cells and ring boundaries were clearly visible. Using high-resolution digital images (2400–4800 dpi), the ring widths of each sample were measured using the program CooRecorder (Larsson, 2011a) and crossdated against an existing regional Douglas-ﬁr chronology (Daniels, 2004) using the programs CDendro (Larsson, 2011b) and COFECHA (Holmes, 1986). By crossdating, accurate calendar years were assigned to the inner- and outer-rings of samples from living and dead trees, as well as the ﬁre scars (Brookes, 2019). Fire years were determined from the position of each scar tip within annual rings of the scarred cross-sections (Brown and Swetnam, 1994). For ﬁre scars located at the boundary between two rings, we assigned the calendar year of the completed ring, consistent with modern ﬁres in the study area that typically burn mid- to late-summer (Heyerdahl et al., 2012; BC Wildﬁre Service unpublished data). In subsequent analyses, only ﬁre years recorded by ≥2 trees to avoid scars possibly caused by disturbance agents other than ﬁre (Swetnam and Baisan, 1996). The forests near Knife Creek are dominated by interior Douglas-ﬁr [Pseudotsuga menziesii var. glauca (Beissn.) Mayr] which forms closed-canopy stands with well-developed canopy, subcanopy, and regeneration strata. Trembling aspen [Populous tremuloides Michx.] and paper birch [Betula papyrifera Marsh.] are infrequent in the canopy. Although present in the past, living, mature lodgepole pine [Pinus contorta Dougl. var. latifolia Engelm.] are absent from the canopy due to the recent mountain pine beetle [Dendroctonus ponderosae Hopkins] epidemic. Other recent biotic disturbances include defoliation by western spruce budworm [Choristoneura occidentalis Freeman] and tree mortality due to Douglas-ﬁr bark beetle [Dendroctonus pseudotsugae Hopkins] (Leclerc et al., 2021). Documentary ﬁre records from 1917 to present include two ﬁres in the Alex Fraser Research Forest, although neither burned within the boundaries of our study area (Figure 1A). A 64-ha ﬁre was recorded in 1938 and a 6-ha ﬁre was suppressed in September 2013 (BC Wildﬁre Service, 2018). Plot-level age structures were derived using the canopy and subcanopy tree ages (Brookes, 2019). To estimate the year in which each tree established, corrections were applied to the crossdated inner-ring dates (Daniels et al., 2017). Research Design and Field Sampling We reconstructed ﬁre history and forest dynamics for a subset of 35 permanent sample plots near Knife Creek in the Alex Fraser Research Forest. In 1984, 80 permanent sample plots were established along a systematic, 100 × 100 m grid that covers 161 ha (Armleder and Thomson, 1984; Koot et al., 2015). Half the plots were in forests that were partially harvested to enhance mule deer winter habitat; the other half were untreated controls. We sampled 35 plots, including 17 treated and 18 control plots (Figure 1B; Brookes, 2019). We searched a 1-ha circular plot (radius = 56.4 m) surrounding each plot center for living trees, snags, logs, or stumps with external ﬁre scars (hereafter “ﬁre-scarred trees”) (Brookes, 2019). To reduce impacts on living trees and to avoid potential bias due to partial harvesting in 1984, we preferentially sampled snags, logs and stumps. Up to 5 partial or full cross- sections were sampled per plot (Cochrane and Daniels, 2008) by selecting ﬁre-scarred trees with the most and best-preserved visible scars (Swetnam and Baisan, 1996). Even-aged cohorts provide evidence for moderate- to high- severity ﬁres (Harvey et al., 2017). In our study area, Douglas-ﬁr establishment can be prolonged because seedlings are susceptible to growing season frosts and require some protection (Klinka et al., 2000). At the plot level, an even-aged cohort was identiﬁed when ≥5 trees established within 15-years, preceded by ≥15 years with no tree establishment (after Heyerdahl et al., 2012). We tested 10- and 20-year windows but found little diﬀerence in the number of cohorts detected. To identify cohorts, we counted the number of trees that established in 15-year periods beginning with the pith date of the oldest tree in the plot and then shifted in 1-year increments until we reached the pith date of the youngest tree (Chavardès and Daniels, 2016). The year assigned to each cohort was the establishment year of the oldest tree of the cohort. Cohorts that established within 15 years of a To quantify forest composition and structure, we sampled the species and diameter at breast-height (dbh) of the 10 live canopy (emergent, dominant, and codominant crown classes) and 10 live subcanopy (intermediate and suppressed crown classes) trees (dbh ≥12.5 cm) closest to each plot center (Jonsson et al., 1992). Reconstructing Fire History and Forest Dynamics For all trees, we used an age-on-height regression developed from local Douglas- ﬁr saplings to estimate the number of years for trees to grow to coring height (Daniels, 2004). For the subset of cores that did not intercept the pith, we accounted for missed rings based on ring geometry if the core had arced rings close to the pith (Duncan, 1989). For cores that did not include arced rings but was ≥90% of the geometric radius, the length of the missing radius and average ring width were used to estimate the number of missing rings (Norton et al., 1987). Eighty-eight percent of age corrections were ≤15 years; thus, plot-level age structures were derived using 15-year classes. Study Area July is the warmest month with a mean daily temperature of 16.0◦C and mean monthly precipitation of 52.7 mm, primarily from convective storms. average annual precipitation of 450.7 mm, with approximately half falling in the growing season [1981–2000, Williams Lake Station (52◦10′59′′N 122◦03′15′′W, 940 masl), Environment Canada, 2017]. Winter months are dominated by Arctic air masses and December is the coldest month with a mean daily temperature of −7.3◦C (Environment Canada, 2017). July is the warmest month with a mean daily temperature of 16.0◦C and mean monthly precipitation of 52.7 mm, primarily from convective storms. Frontiers in Ecology and Evolution \| www.frontiersin.org Study Area The study area encompasses 161 ha near Knife Creek in the Alex Fraser Research Forest, located 24 kilometers southeast of the city of Williams Lake, BC, Canada (Figure 1). It is June 2021 \| Volume 9 \| Article 676961 2 Disrupted Fire Regime Brookes et al. n of the study area and sampled plots in the Alex Fraser Research Forest in south central British Columbia, Canada. (A) The Cariboo Wagon 97) and San Jose River lie west of the study area. Polygons in white and oranges depict historical ﬁres between 1917 and 2017. (B) Thirty-ﬁve n 161 ha of forest in the northeast corner of the Research Forest. T denotes plots in treated areas that were partially harvested in 1984; C d control plots. [Data sources (A) British Columbia Open Data Catalog and (B) Google Earth]. l territory of the T’exelcemc (Williams Lake from 761 to 879 m above sea level (masl) and the terrain is FIGURE 1 \| Location of the study area and sampled plots in the Alex Fraser Research Forest in south central British Columbia, Canada. (A) The Cariboo Wagon Road (now Highway 97) and San Jose River lie west of the study area. Polygons in white and oranges depict historical ﬁres between 1917 and 2017. (B) Thirty-ﬁve plots were sampled in 161 ha of forest in the northeast corner of the Research Forest. T denotes plots in treated areas that were partially harvested in 1984; C denotes unharvested control plots. [Data sources (A) British Columbia Open Data Catalog and (B) Google Earth]. from 761 to 879 m above sea level (masl) and the terrain is ﬂat to gently rolling. The climate is continental and strongly inﬂuenced by the rain shadow of the Coastal Mountains, yielding in the traditional territory of the T’exelcemc (Williams Lake Band), members of the Northern Secwépemc te Qelmucw (Northern Shuswap Tribal Council, 2014). Elevation varies June 2021 \| Volume 9 \| Article 676961 Frontiers in Ecology and Evolution \| www.frontiersin.org 3 Brookes et al. Disrupted Fire Regime needed, multiple cores were extracted to ensure each sample intersected or was close to the pith. average annual precipitation of 450.7 mm, with approximately half falling in the growing season [1981–2000, Williams Lake Station (52◦10′59′′N 122◦03′15′′W, 940 masl), Environment Canada, 2017]. Winter months are dominated by Arctic air masses and December is the coldest month with a mean daily temperature of −7.3◦C (Environment Canada, 2017). June 2021 \| Volume 9 \| Article 676961 Research Design and Field Sampling The distance from the center of the plot to the 10th tree in each canopy class formed the radius of a circular plot used for calculating tree densities (Jonsson et al., 1992). To assess tree ages and growth histories, an increment core was sampled c. 20–30 cm from the base of each tree and coring height was recorded. As June 2021 \| Volume 9 \| Article 676961 4 Disrupted Fire Regime Brookes et al. ﬁre year in the same plot or an adjacent plot were considered post-ﬁre cohorts (Heyerdahl et al., 2012). Mixed-severity ﬁre histories were assigned to plots with > 1 ﬁre year and ≥1 post-ﬁre cohorts. Plots with low-severity histories were plots with > 1 ﬁre scar years but no post-ﬁre cohorts. For plots not meeting these criteria, severity was unclassiﬁable. Since periods of suitable climate can facilitate tree establishment independently of disturbance, we assessed the possibility that cohorts were a consequence of favorable climate rather than ﬁre (Heyerdahl et al., 2012). Using an independent tree-ring reconstruction of June to August precipitation in Williams Lake from 1633 to 1996 (Daniels and Watson, 2003; Daniels, 2004), we calculated 15-year moving averages reported for the ﬁrst year of each 15-year window (e.g., 1901 = average values from 1901 to 1915) and compared the averaged values against the long-term mean. Positive departures indicated dry periods likely to limit establishment because a lack of summer moisture has been shown to delay seedling growth (Hermann and Lavender, 1990). Each cohort was classiﬁed as establishing during a wet or dry period. Contingency tables of the number of cohorts that established during wet versus dry periods were assessed against a random distribution using chi-squared goodness of ﬁt (α = 0.05) (Whitlock and Schluter, 2009). Trees that survived ≥1 ﬁre indicated by a ﬁre scar or post-ﬁre cohort were classiﬁed as remnant trees (Chavardès and Daniels, 2016). The severity of the most recent ﬁre at each plot was calculated as the percentage of remnant trees surviving that ﬁre relative to all trees in the plot (Chavardès and Daniels, 2016). Plots with 81−100% remnant trees indicated low-severity, 20−80% indicated moderate-severity, and < 20% indicated high- severity ﬁre (after Sherriﬀand Veblen, 2006). y Widespread ﬁres were deﬁned as those that scarred trees in ≥10 plots and ≥25% of plots with living trees present in the ﬁre year. Research Design and Field Sampling We used the distribution of ﬁre scars, post- ﬁre cohorts, and remnant trees to assess spatial variation in severity within individual widespread ﬁres (Brookes, 2019). For each ﬁre, the presence or absence of ﬁre scars, post-ﬁre cohorts, remnant trees, and potential recording trees were combined to estimate severity at the plot level. Similar to the classiﬁcation of ﬁre-severity through time, plots with ﬁre scars but no post- ﬁre cohort indicated local low-intensity surface ﬁre that scarred trees but did not initiate a post-ﬁre cohort. Plots with ﬁre scars and a corresponding post-ﬁre cohort indicated ﬁre that burned at moderate-to-high intensity, suﬃcient to provide a suitable environment for tree establishment although some trees survived. Plots with only a post-ﬁre cohort burned with suﬃciently high intensity to initiate a new cohort, leave no local ﬁre scars, and destroy evidence of previous ﬁres. Lastly, plots with remnant trees that did not scar or form cohorts indicated the plot did not burn or burned with an intensity too low to scar trees. The latter evidence was strongest at plots with existing scarred trees at the time of ﬁre, because trees that have previously scarred are more likely to scar during subsequent ﬁres. Forest Composition and Structure Forest Composition and Structure All plots comprised Douglas-ﬁr only, except one that included trembling aspen (Table 1). Canopy-dominant trees were up to 149 cm in diameter and 457 years of age. In the control plots, densities were 182 ± 84 (mean ± standard deviation) trees ha−1 in the canopy and subcanopy densities were ≤581 trees ha−1, except one plot with 3,537 trees ha−1. Densities were lower in the treated area, averaging 152 ± 85 trees ha−1 in the canopy and 224 ± 299 trees ha−1 in the subcanopy due to partial harvesting in 1984 (Leclerc et al., 2021). Determining Historical Fire Frequency and Severity y To quantify ﬁre frequency, we developed plot-level ﬁre chronologies using ﬁre-scars and post-ﬁre cohorts (Brookes, 2019). For each plot, the length of the ﬁre record, number of ﬁres, the minimum, maximum, and mean number of years between ﬁres, and the time since last ﬁre were calculated. The length of the ﬁre record at each plot was determined from the age of the oldest tree. The time since last ﬁre was the number of years between 2015 (the year of sampling) and the last ﬁre year indicated by a scar or a post-ﬁre cohort. The maximum ﬁre interval from each plot was used to determine whether the current ﬁre-free interval exceeded the historical range of variation. A visual representation of ﬁre scar and cohort occurrence for each plot was constructed using the Fire History Analysis and Exploration System (FHAES) (Brewer et al., 2016). To discern whether reconstructed ﬁre frequency diﬀered between the mule deer control and treatment units at Knife Creek, we composited the plot-level ﬁre records for the control and treatment units and used a Welch’s 2-tail t-test to compare the mean intervals between ﬁres. They did not diﬀer signiﬁcantly (p = 0.50, α = 0.05, df = 31), therefore we composited ﬁre records from all plots and calculated the study-area ﬁre frequency as the mean interval between ﬁre years. We used breakpoint analysis to test for structural changes in the cumulative number of ﬁres from 1600 to 2015. We used ordinary least squares- based CUSUM tests as an explorative tool, Bayesian information criterion to determine the optimal number of breakpoints, and built a segmented regression model following Zeileis et al. (2003) and Zeileis (2005). Breakpoint analyses were conducted in R (version 4.0.5) statistical software (R Core Team, 2021) using the package strucchange (Zeileis et al., 2002). Frontiers in Ecology and Evolution \| www.frontiersin.org Fire Occurrence and Frequency Twenty-nine of 35 plots contained ﬁre-scarred trees (Figure 2 and Table 2). Seventy-nine of 82 (96%) sections sampled from 67 ﬁre-scarred trees were successfully crossdated. Of 305 ﬁre scars, 282 scars formed in years when ≥2 trees were scarred across the study area and were included in subsequent analyses. Plot-level ﬁre chronologies ranged from 150 to 555 years and included 1-10 ﬁre years. At the 26 plots with ≥2 ﬁres, mean ﬁre intervals were The severity of ﬁres at individual plots through time was inferred using criteria from Heyerdahl et al. (2012). We classiﬁed plots with only 1 ﬁre year associated with a post-ﬁre cohort and no ﬁre scars as those recording high-severity ﬁre through time. June 2021 \| Volume 9 \| Article 676961 5 Disrupted Fire Regime Brookes et al. TABLE 1 \| Summary of forest attributes in 35 plots near Knife Creek in the Alex Fraser Research Forest, British Columbia. Fire Occurrence and Frequency Plot DBH (cm) Age (years) Density (trees ha−1) Mean (SD) Maximum Mean (SD) Maximum Canopy Subcanopy Total C01 28.9 (11.7) 46.3 116 (24) 144 158 113 271 C03 24.3 (10.6) 49.8 104 (24) 160 237 263 500 C05 30.3 (12.0) 55.0 207 (71) 373 278 345 623 C07 34.4 (16.8) 69.5 150 (45) 302 204 188 392 C09 28.5 (9.2) 43.8 122 (15) 139 108 3,537 3,645 C10 32.8 (20.5) 82.1 185 (96) 352 54 273 327 C14 30.2 (14.5) 62.5 154 (26) 228 85 120 205 C17 30.3 (19.4) 70.7 146 (21) 194 172 158 330 C18 26.6 (10.0) 44.6 134 (11) 146 353 81 434 C21 24.3 (9.8) 44.7 149 (40) 305 138 393 531 C22 32.3 (21.5) 81.9 158 (70) 340 46 581 627 C23 27.9 (8.5) 44.0 139 (5) 144 162 376 538 C24B 33.0 (29.3) 46.4 153 (31) 220 245 278 523 C27 26.1 (16.6) 90.4 153 (23) 172 237 537 774 C28 25.2 (12.4) 48.1 109 (20) 163 188 345 533 C30 27.0 (14.5) 55.0 132 (35) 212 129 111 240 C31 26.2 (10.1) 44.3 139 (13) 185 197 268 465 C33 23.6 (10.0) 43.2 144 (20) 177 300 136 436 T02 27.2 (13.4) 70.4 114 (23) 161 126 118 244 T14 36.5 (25.0) 80.8 166 (77) 356 140 241 381 T14B 32.7 (19.6) 92.6 166 (49) 347 105 289 394 T16 26.2 (17.5) 89.9 131 (9) 139 254 92 346 T16B 29.5 (14.3) 74.6 156 (41) 309 70 360 430 T19 29.4 (20.3) 86.6 124 (74) 328 71 141 212 T20 24.7 (10.8) 57.1 128 (18) 169 278 43 321 T22 35.3 (22.3) 75.9 165 (116) 457 78 140 218 T24 33.7 (21.3) 68.8 139 (66) 291 134 1,326 1,460 T24B 23.5 (9.2) 43.2 137 (7) 151 306 249 555 T27 36.4 (23.9) 109.8 133 (34) 241 180 185 365 T28 29.0 (9.8) 49.7 142 (3) 148 225 46 271 T29 29.9 (16.2) 60.6 208 (120) 391 71 229 300 T33 32.1 (20.5) 89.9 134 (50) 261 116 126 242 T35 33.5 (15.8) 65.2 215 (64) 292 278 34 312 T37 29.0 (13.4) 66.1 134 (31) 209 94 62 156 T49 27.0 (16.2) 71.8 135 (51) 289 52 130 182 Plots are stratiﬁed by control (C) or treatment (T) and listed numerically. TABLE 1 \| Summary of forest attributes in 35 plots near Knife Creek in the Alex Fraser Research Forest, British Columbia. Fire Occurrence and Frequency widespread and persistent, including scars on 41% of sampled trees in 86% of plots. 17-110 years and < 40 years in 73% of plots. Current ﬁre-free intervals of 72–184 years exceeded their corresponding historical maximum ﬁre intervals (23−110 years) at all but one plot. Tree Establishment and Cohorts (A) Horizontal lines represent individual plots, stratiﬁed by ﬁre history through time and record length (top to bottom). Dashed segments of lines indicate years prior to ﬁre-scar formation on ≥1 sampled trees; solid lines indicate presence of ≥1 scarred trees. Black triangles represent ﬁre years, gray/white triangles represent the ﬁrst year of each post-ﬁre/non-ﬁre cohort. (B) Percentage of plots recording ﬁre scars calculated as the number of plots recording ﬁre relative to the number of plots with trees present that could record ﬁre. (C) Age-structure histogram (15-year bins) for all plots combined. FIGURE 2 \| Fire record from 1600 to 2015 for 35 plots in the Alex Fraser Research Forest. (A) Horizontal lines represent individual plots, stratiﬁed by ﬁre history through time and record length (top to bottom). Dashed segments of lines indicate years prior to ﬁre-scar formation on ≥1 sampled trees; solid lines indicate presence of ≥1 scarred trees. Black triangles represent ﬁre years, gray/white triangles represent the ﬁrst year of each post-ﬁre/non-ﬁre cohort. (B) Percentage of plots recording ﬁre scars calculated as the number of plots recording ﬁre relative to the number of plots with trees present that could record ﬁre. (C) Age-structure histogram (15-year bins) for all plots combined. (p = 0.07, α = 0.05, df = 1), corroborating the inference that most cohorts established following ﬁres. Variability in Historical Widespread Fires Variability in Historical Widespread Fires The spatial distribution of ﬁre scars, combined with post-ﬁre cohorts, revealed variation in ﬁre severity within and among the 6 widespread ﬁres (Figure 4 and Table 4). As well, plots lacking ﬁre evidence were adjacent to and between plots with scars or cohorts, providing indirect evidence of spatial variation in severity. The 1790 ﬁre scarred trees in 19 plots distributed across the study area and a post-ﬁre cohort established in one plot. The 1817 ﬁre scarred trees in 13 plots, 12 of which were in the north and west of the study area. The 1840 ﬁre scarred trees in 16 plots, one of which included a post-ﬁre cohort. Cohorts established in 3 other plots. Plots with cohorts were in the north and east of the study area. This ﬁre was the last recorded in 2 plots; severity was classiﬁed as moderate in 1 plot and high in the other. Scars and cohorts in 30 plots throughout the study area provide evidence that the 1863 ﬁre was relatively severe and widespread. Tree Establishment and Cohorts Trees were scarred in 24 plots, 11 of which included post-ﬁre cohorts. Cohorts established in another 6 plots. This ﬁre was the Tree Establishment and Cohorts The composite ﬁre record for the study area indicated 23 years from 1619 to 1943 when ≥2 trees were scarred, with intervals of 7−32 years (Figure 2). We detected two signiﬁcant structural changes in the cumulative number of ﬁres over time, with breakpoints in 1757 (95% CI: 1756, 1760) and 1942 (95% CI could not be computed) (Table 3). The cumulative number of ﬁres increased from 1600 to 1757 at a rate of 0.05 ﬁres per year. The rate of ﬁre accumulation increased signiﬁcantly to 0.07 ﬁres per year from 1758 to 1942, but stagnated after 1943 when no ﬁres burned. Six widespread ﬁres burned in 1790, 1817, 1831, 1840, 1863, and 1905 (Table 4), during the period of rapid ﬁre accumulation. Evidence of the 1863 ﬁre was most We successfully estimated the ages of 656 of 700 (94%) trees that we cored; 44 trees missing inner rings due to substantive heartwood decay or with correlation coeﬃcients < 0.25 relative to the regional chronology were omitted from subsequent analyses (Table 2). Trees established from 1559 to 1962, with 76% establishing after the widespread 1863 ﬁre (Figure 3 and Table 1). Only 2% of trees recruited into the canopy since the last ﬁre in 1943. Among the 35 plots, 24 cohorts established from 1844 to 1899, 22 of which were classiﬁed as post-ﬁre cohorts and 9 included the oldest trees in their plots. Cohort establishment was signiﬁcantly associated with dry, rather than wet, periods June 2021 \| Volume 9 \| Article 676961 Frontiers in Ecology and Evolution \| www.frontiersin.org 6 Disrupted Fire Regime Brookes et al. FIGURE 2 \| Fire record from 1600 to 2015 for 35 plots in the Alex Fraser Research Forest. (A) Horizontal lines represent individual plots, stratiﬁed by ﬁre history through time and record length (top to bottom). Dashed segments of lines indicate years prior to ﬁre-scar formation on ≥1 sampled trees; solid lines indicate presence of ≥1 scarred trees. Black triangles represent ﬁre years, gray/white triangles represent the ﬁrst year of each post-ﬁre/non-ﬁre cohort. (B) Percentage of plots recording ﬁre scars calculated as the number of plots recording ﬁre relative to the number of plots with trees present that could record ﬁre. (C) Age-structure histogram (15-year bins) for all plots combined. FIGURE 2 \| Fire record from 1600 to 2015 for 35 plots in the Alex Fraser Research Forest. Frontiers in Ecology and Evolution \| www.frontiersin.org Fire Severity Through Time g Fire scars and post-ﬁre cohorts varied among plots and indicated ﬁres burned at a range of severities through time. Eighteen plots (54%) were classiﬁed as having mixed-severity ﬁres through time, 4 (11%) as high severity, 9 (26%) as low severity, and 4 (11%) were unclassiﬁable (Table 2). Eleven mixed-severity plots recorded ≥5 ﬁre-scar years and a single post-ﬁre cohort that established after the 1840 (n = 3) and 1863 (n = 8) ﬁres (Table 2). The plots with low-severity ﬁres through time recorded 2−9 ﬁres. The most recent ﬁre at 8 plots was classiﬁed as low-severity, 15 were moderate- and 6 were high-severity, based on the percentage of remnant trees. Four plots with neither a ﬁre-scar or post-ﬁre cohort could not be classiﬁed. At the study area scale, both plot- level ﬁre histories through time and severity of the most recent ﬁre indicate a mixed-severity ﬁre regime. June 2021 \| Volume 9 \| Article 676961 Frontiers in Ecology and Evolution \| www.frontiersin.org 7 Disrupted Fire Regime Brookes et al. TABLE 2 \| Fire records for 35 plots near Knife Creek in the Alex Fraser Research Forest, British Columbia. Fire Severity Through Time TABLE 2 \| Fire records for 35 plots near Knife Creek in the Alex Fraser Research Forest, British Columbia. Abbott and Chapman, 2018; BC Wildﬁre Service, 2018), low- and moderate-severity ﬁres frequently burned, dominating the historical mixed-severity ﬁre regime and driving forest dynamics in the Douglas-ﬁr forests near Knife Creek (Table 2). The composite ﬁre-scar record indicated surface ﬁres burned every 10–30 years prior to the 1940s, comparable to other last recorded in 15 plots; severity was classiﬁed as moderate in 7 plots and high in 8 plots. In contrast with 1863, the 1905 ﬁre was relatively low in severity. Trees were scarred in 13 plots, but no post-ﬁre cohorts established. All but one plot with scars were in the north and west of the study area, where young trees had established after the 1840 and 1863 ﬁres. The 1905 ﬁre was the last recorded in 11 plots; severity was classiﬁed as low in 4 plots and moderate in 7 plots. TABLE 3 \| Breakpoints in the cumulative number of ﬁres from 1600 to 2015. Period (years) Intercept Slope p-value 1600–1757 −84.03 0.05 – 1758–1942 −107.70 0.07 <2 × 10−16 1943–2015 22.00 0.00 <2 × 10−16 The period 1600–1757 is the reference period; p-values indicate whether the slope of a segment is signiﬁcantly different from that of the reference period (α = 0.05). TABLE 3 \| Breakpoints in the cumulative number of ﬁres from 1600 to 2015. Fire Severity Through Time Plot Fire evidence (n) Fire record Fire intervals (years) Fire history through time Attributes of the last ﬁre Scarred trees Scars Dated cores Cohorts Mean Range Year Time since ﬁre (years) Remnant trees (% survivors) Severity T33 3 9 18 0 1584–2015 30 9–60 Low 1905 111 67 Moderate C28 3 7 20 0 1615–2015 41 14–113 Low 1863 153 5 High C07 5 4 15 0 1635–2015 29 23–42 Low 1905 111 94 Low C03 3 8 17 0 1646–2015 27 13–42 Low 1905 111 29 Moderate T14B 2 7 20 0 1656–2015 27 9–47 Low 1863 153 65 Moderate C10 1 8 18 0 1673–2015 24 13–32 Low 1863 153 39 Moderate T22 1 2 17 0 1712–2015 111 111 Low 1840 176 22 Moderate T24 3 5 16 0 1715–2015 29 23–42 Low 1905 111 69 Moderate C21 2 7 19 0 1743–2015 25 13–32 Low 1905 111 100 Low C27 1 7 19 1 1460–2015 37 9–113 Mixed 1840 176 0 High T27 2 6 19 1 1558–2015 53 32–73 Mixed 1895 121 84 Low C31 3 11 20 1 1587–2015 19 9–73 Mixed 1863 153 5 High T14 5 5 18 1 1592–2015 29 23–42 Mixed 1905 111 94 Low T49 3 3 19 1 1597–2015 37 32–42 Mixed 1905 111 58 Moderate C24B 2 7 17 1 1621–2015 26 9–42 Mixed 1943 73 100 Low C05 5 4 19 1 1651–2015 38 23–50 Mixed 1905 111 84 Low T16B 3 9 19 1 1666–2015 18 11–32 Mixed 1863 153 26 Moderate T20 2 4 19 1 1666–2015 38 23–50 Mixed 1905 111 79 Moderate C18 1 2 19 1 1691–2015 73 73 Mixed 1863 153 0 High C22 1 7 19 1 1712–2015 18 9–26 Mixed 1863 153 21 Moderate C01 5 6 20 1 1721–2015 28 23–42 Mixed 1930 86 85 Low C23 1 7 20 1 1722–2015 19 12–32 Mixed 1863 153 0 High T37 2 6 20 1 1726–2015 23 9–42 Mixed 1905 111 75 Moderate T19 2 5 17 1 1732–2015 29 23–42 Mixed 1905 111 53 Moderate T28 1 3 19 1 1748–2015 52 23–80 Mixed 1943 73 100 Low C14 1 2 14 1 1788–2015 23 23 Mixed 1863 153 42 Moderate T16 1 2 19 1 1800–2015 32 32 Mixed 1863 153 0 High C30 0 0 20 1 1804–2015 – – High 1863 153 15 High C09 2 1 19 1 1818–2015 – – High 1863 153 0 High C17 0 0 18 1 1822–2015 – – High 1863 153 28 Moderate T24B 0 0 20 1 1865–2015 – – High 1863 153 0 High T29 0 0 20 0 1625–2015 – – Unclassiﬁed – – – – T35 0 0 19 0 1724–2015 – – Unclassiﬁed – – – – C33 1 1 19 0 1823–2015 – – Unclassiﬁed 1863 153 26 Moderate T02 0 0 20 0 1855–2015 – – Unclassiﬁed – – – – Plots are stratiﬁed by ﬁre history through time and record length as in Figure 2. Historical Mixed-Severity Fire Regime Douglas-ﬁr-dominated forests in warm and dry climates of interior British Columbia (Figure 2; Daniels, 2004; Heyerdahl et al., 2007, 2012; Marcoux et al., 2013; Greene and Daniels, 2017; Harvey et al., 2017) and, more broadly, across the western United States (Heyerdahl et al., 2008; Perry et al., 2011; Hessburg et al., 2019). p The 1790 ﬁre illustrates the challenges and limitations of inferring the severity of ﬁres in the distant past (Figure 4), although it burned during the period of rapid ﬁre accumulation, when ≥77% of plots included recorder trees (Table 3). Scars in 19 plots but a post-ﬁre cohort in only one plot suggest a low-severity ﬁre (e.g., interpretations i and ii, above). Alternately, had the 1790 ﬁre burned at higher intensity and generated post-ﬁre cohorts in multiple plots, subsequent ﬁres eliminated that evidence from contemporary age structures (e.g., interpretation iii, above). Over the 50 years from 1790 to 1840, ﬁve ﬁres burned at short intervals of 9-15 years and 66% of plots that burned in 1790 re-burned up to four times. Trees that established after 1790 would have been young, small, and lacking thick bark to resist subsequent ﬁres. In fact, the single persistent post-1790 cohort was in the only plot that remained ﬁre-free until 1840. Thus, it is plausible that other post-1790 cohorts established but were consumed by subsequent ﬁres. Conversely, such cohorts may have persisted without repeat ﬁres from 1790 to 1840. g Our ﬁne-scale spatio-temporal reconstruction of historical ﬁres detected no evidence of a stand-initiating ﬁre across the 161-ha study area over the 396-year reconstruction (Figures 2, 3). Fire scars, cohorts, and remnant trees indicated the six widespread ﬁres from 1790 to 1905 included substantive internal heterogeneity at scales of 10–100s of meters (Figure 4). Individuals and patches of trees survived even the two most widespread ﬁres in 1840 and 1863, while cohorts initiated following ﬁres in 1790, 1840, and 1863 (Table 4). Cohort establishment suggests that some patches of high-intensity ﬁre were suﬃcient to cause overstory tree mortality and improve levels of light and nutrients available to survivors and seedlings (Harvey et al., 2017). Nevertheless, these ﬁne-scale patches were embedded in an uneven-aged forest matrix maintained by frequent low- or moderate-severity ﬁres, a characteristic noted in other dry forests in south central BC (Daniels, 2004; Heyerdahl et al., 2007; Harvey et al., 2017). Historical Mixed-Severity Fire Regime Given the small size of our study area, replicating our research across a network of sites at landscape-to-regional scales will improve understanding of the frequency and relative importance of ﬁres of a range of severities. Deciphering the relative severity of the widespread 1863 ﬁre was key for understanding the contemporary forest age structure and concluding the historical ﬁre regime has been disrupted. Many studies consider ﬁres in the late 1800s relatively severe because they generated new cohorts of trees, and thus, emphasize the importance of high-severity ﬁres in a mixed- severity ﬁre regime (Daniels et al., 2017). We classiﬁed the 1863 as moderate-to-high severity since the resulting post-ﬁre cohorts account for >70% of contemporary canopy and subcanopy trees (Sherriﬀand Veblen, 2006; Chavardès and Daniels, 2016). However, emphasizing the origin versus persistence of those cohorts yields contrasting interpretations of the contemporary ﬁre regime. A focus on cohort origin supports an interpretation that the 1863 ﬁre was a periodic high-severity event in a mixed- severity regime that has not been disrupted (Odion et al., 2014). This interpretation inherently assumes a high rate of ﬁre-caused tree mortality predicated abundant post-ﬁre tree establishment, justifying the classiﬁcation of high severity. Yet, the survival of remnant trees in 29 of 35 plots is inconsistent with this assumption. Alternately, a focus on cohort persistence supports Historical Mixed-Severity Fire Regime Historical Mixed-Severity Fire Regime In stark contrast with the predominantly severe, stand- initiating megaﬁres of 2017 that burned 33,181 ha of Douglas-ﬁr forests surrounding our study area (Figure 1A; The period 1600–1757 is the reference period; p-values indicate whether the slope of a segment is signiﬁcantly different from that of the reference period (α = 0.05). June 2021 \| Volume 9 \| Article 676961 Frontiers in Ecology and Evolution \| www.frontiersin.org 8 Disrupted Fire Regime Brookes et al. TABLE 4 \| Evidence used to infer plot-level severity within six widespread ﬁres. Years Plots (n) Plots with no ﬁre evidence [n (%)] Plots with ﬁre evidence [n (%)] Plots with no subsequent ﬁre [n (%)] Scarred recorder trees absent Scarred recorder trees present Fire scar only Fire scar and post-ﬁre cohort Post-ﬁre cohort only 1790 29 7 (24) 3 (10) 18 (62) 1 (3) 0 (0) 1 (3) 1817 31 8 (26) 10 (32) 13 (42) 0 (0) 0 (0) 2 (6) 1831 33 8 (24) 12 (36) 13 (39) 0 (0) 0 (0) 2 (6) 1840 33 6 (18) 8 (24) 15 (46) 1 (3) 3 (9) 4 (12) 1863 35 3 (9) 2 (6) 13 (37) 11 (31) 6 (17) 22 (63) 1905 35 6 (17) 16 (46) 13 (37) 0 (0) 0 (0) 35 (100) Recorder trees include all living trees present at the time of ﬁre; previously scarred recorder trees are more susceptible to ﬁre than trees without scars. Fire scars indicate low-severity ﬁre; post-ﬁre cohorts indicate moderate- or high-severity ﬁre. TABLE 4 \| Evidence used to infer plot-level severity within six widespread ﬁres. (Swetnam et al., 1999; Falk et al., 2011; Daniels et al., 2017). Below, we discuss how the presence, absence, and assumptions underlying ﬁre evidence yield alternate interpretations of severity of the two widespread ﬁres in 1790 and 1863. Douglas-ﬁr-dominated forests in warm and dry climates of interior British Columbia (Figure 2; Daniels, 2004; Heyerdahl et al., 2007, 2012; Marcoux et al., 2013; Greene and Daniels, 2017; Harvey et al., 2017) and, more broadly, across the western United States (Heyerdahl et al., 2008; Perry et al., 2011; Hessburg et al., 2019). Inferring Fire Severity From Scars and Cohorts Only plots containing trees old enough to record each ﬁre are shown (n). Colors of one-hectare circular plots indicate severity: yellow plots include ﬁre scars only indicating low severity; orange plots include ﬁre scars and post-ﬁre cohorts indicating moderate severity; and, red plots include post-ﬁre cohorts only indicating high severity. White indicate plots with no evidence of ﬁre; bold boundaries indicate plots with trees that had previously been scarred and were susceptible to ﬁre damage. FIGURE 4 \| Spatial pattern of ﬁre severity for six widespread ﬁres. Only plots containing trees old enough to record each ﬁre are shown (n). Colors of one-hectare circular plots indicate severity: yellow plots include ﬁre scars only indicating low severity; orange plots include ﬁre scars and post-ﬁre cohorts indicating moderate severity; and, red plots include post-ﬁre cohorts only indicating high severity. White indicate plots with no evidence of ﬁre; bold boundaries indicate plots with trees that had previously been scarred and were susceptible to ﬁre damage. immediately west of our study area (Figure 1A). Travel corridors are generally known to have a high prevalence of Indigenous ﬁre (Lake and Christianson, 2019). Therefore, the historical ﬁre record reconstructed in this study likely reﬂects Indigenous ﬁre stewardship, including the practice of intentionally setting ﬁres to modify ﬁre regimes and increase resource availability (Turner, 1999; Turner et al., 2000; Lake and Christianson, 2019). Indigenous ﬁre stewardship in dry forest ecosystems is well documented in oral histories in BC (Simmons, 2012; Lewis et al., 2018; Xwisten Nation et al., 2018), and ﬁre was likely used to manage forests for food and medicinal plants and for hunting animals at or near Knife Creek. an interpretation that tree survival rates have been exceptionally high due to subsequent reductions in ﬁre occurrence, size, and severity (Guiterman et al., 2018). In the 70 years preceding 1863, ﬁve widespread ﬁres reburned plots multiple times (Figures 2, 4 and Table 4). In contrast, in the 150 years since 1863, small ﬁres in 1876, 1895, 1930, and 1943 scarred trees in only one or two plots and 15 plots with post-1863 cohorts have not reburned. The last widespread ﬁre in 1905 was low in severity, only scarring trees without initiating new cohorts. Excessive ﬁre-free intervals of 72–184 years indicate individual plots have missed up to six ﬁres relative to historical frequencies (Tables 2, 3). Inferring Fire Severity From Scars and Cohorts Cohorts The presence and location of ﬁre-scarred trees and post-ﬁre cohorts are used to infer the timing, spatial patterns, and severity of past ﬁres (Swetnam et al., 1999; Daniels et al., 2017). The absence of scars and cohorts can be equally informative, but yield multiple possible interpretations: (i) ﬁre did not burn, (ii) ﬁre burned at low intensity leaving no scars or cohorts, or (iii) ﬁre burned at suﬃcient intensity to leave scars or cohorts but the evidence no longer persists (Falk et al., 2007; Yocom Kent, 2014). The latter interpretations are confounded by diminishing ﬁre evidence due to subsequent disturbances, tree mortality, and wood decay through time, a widely recognized limitation of dendroecological reconstructions The presence and location of ﬁre-scarred trees and post-ﬁre cohorts are used to infer the timing, spatial patterns, and severity of past ﬁres (Swetnam et al., 1999; Daniels et al., 2017). Frontiers in Ecology and Evolution \| www.frontiersin.org June 2021 \| Volume 9 \| Article 676961 9 Disrupted Fire Regime Brookes et al. FIGURE 3 \| Plot-level ﬁre history reconstructed from ﬁre scars and post-ﬁre cohorts. Plot numbers are followed by the number of trees in the age histogram in parentheses. T denotes plots in treated areas that were partially harvested in 1984; C denotes unharvested control plots. Plots are stratiﬁed by ﬁre history through time (low, mixed, high and unclassiﬁed, from top to bottom). FIGURE 3 \| Plot-level ﬁre history reconstructed from ﬁre scars and post-ﬁre cohorts. Plot numbers are followed by the number of trees in the age histogram in parentheses. T denotes plots in treated areas that were partially harvested in 1984; C denotes unharvested control plots. Plots are stratiﬁed by ﬁre history through time (low, mixed, high and unclassiﬁed, from top to bottom). FIGURE 3 \| Plot-level ﬁre history reconstructed from ﬁre scars and post-ﬁre cohorts. Plot numbers are followed by the number of trees in the age histogram in parentheses. T denotes plots in treated areas that were partially harvested in 1984; C denotes unharvested control plots. Plots are stratiﬁed by ﬁre history through time (low, mixed, high and unclassiﬁed, from top to bottom). June 2021 \| Volume 9 \| Article 676961 Frontiers in Ecology and Evolution \| www.frontiersin.org 10 Brookes et al. Disrupted Fire Regime FIGURE 4 \| Spatial pattern of ﬁre severity for six widespread ﬁres. Inferring Fire Severity From Scars and Cohorts Absent surface ﬁres to drive tree mortality and maintain low forest densities, we conclude the persistence of the post-1863 cohorts both result from and strongly indicate disruption of the historical ﬁre regime during the twentieth century. During the Gold Rush and continuing through to the 1940s, the region surrounding our study area was rapidly colonized by Europeans as ranches and road houses were established along the Cariboo Wagon Road (currently Highway 97, the main north-south highway in BC), which is adjacent to the Knife Creek study area (Figure 1A; Day, 1998). Controlled burning continued to be used by ranchers to promote the expansion of meadows and grasslands to provide feed for livestock (Parminter, 1991; Day, 1998). In 1874, the Bush Fire Act introduced ﬁnes or imprisonment if a purposely set ﬁre resulted in damage to private or crown land and the Forest Act of 1912 provided ﬁnancial support to equip and maintain a ﬁre prevention force (Parminter, 1991). Since the 1940s, ﬁreﬁghting technology improved substantially, and both ﬁre- scar and documentary records indicate the majority of ﬁres have been successfully suppressed (Daniels, 2004; BC Wildﬁre Service unpublished data). AUTHOR CONTRIBUTIONS WB organized the data collection and analyzed the data with guidance from LDD and ALC. WB and LDD led the writing of the manuscript. All authors contributed to discussing and revising the manuscript. ACKNOWLEDGMENTS This manuscript includes excerpts from the Master of Science thesis by WB, completed at the University of British Columbia, Vancouver, BC, Canada. We respectfully acknowledge that the land on which we conducted our research is the unceded ancestral territory of the T’exelcemc (Williams Lake Band) of the Secwepemc (Shuswap) Nation. We thank the staﬀat the Alex Fraser Research Forest, K. Day, C. Koot, and S. Ewan, for facilitating this research; T. Dergousoﬀ, A. Kaufman, M-A. Leclerc, M-E. Leclerc, A. Weixelman, E. Xu, and R. Chavardès for assistance in the ﬁeld and lab; G. Preston for assistance with the maps; and B. Eskelson, J. Rhemtulla, and two reviewers for helpful comments that improved the manuscript. FUNDING This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to LDD (RGPIN-2020-06310) and ALC (RGPIN-2015-04376), and a grant from an anonymous foundation. This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to LDD (RGPIN-2020-06310) and ALC (RGPIN-2015-04376), and a grant from an anonymous foundation. Human Disruption of the Fire Regime The absence of ﬁre scars since 1943 or establishment of new post- ﬁre cohorts in the twentieth century provide strong evidence of a disrupted ﬁre regime at Knife Creek (Figure 2). All plots included young, small subcanopy trees and 19 plots included live, ﬁre-scarred trees that would have been highly susceptible to scarring had ﬁres burned (Figure 3). The lack of ﬁre is consistent with other dendroecological reconstructions, a period of cooler and wetter regional climate from 1946 to 1976 (Daniels, 2004; Heyerdahl et al., 2012; Harvey et al., 2017), and historical land use in the region (Parminter, 1991; Day, 1998). Speciﬁcally, the arrival of Europeans to this area following the Gold Rush in the 1860s severely restricted Indigenous ﬁre stewardship (Parminter, 1991; Day, 1998). This part of the T’exelcmec traditional territory includes sacred ceremonial grounds and a well-established travel route between seasonal camps along the San Jose River (oral history shared with and reported by Day, 2007), which lies Tree-ring evidence, consistent with oral histories and documentary records, indicates changes in human land-use and ﬁre suppression have disrupted the ﬁre regime at Knife Creek relative to the historical range of variation. As observed in other Frontiers in Ecology and Evolution \| www.frontiersin.org June 2021 \| Volume 9 \| Article 676961 11 Brookes et al. Disrupted Fire Regime dry mixed-conifer forests, contemporary ﬁre regimes have led to increased tree densities, especially in subcanopy strata, that have made dry forests less resilient to ﬁre (Hessburg et al., 2019). There is compelling evidence that in the absence of frequent ﬁres, a substantial degree of forest structural diversity in Knife Creek has been lost (Hessburg et al., 2005, 2016; Spies et al., 2006; Harvey et al., 2017). Stands near Knife Creek that have not been harvested have closed canopies and forest densities generally range from 200 to 775 trees ha−1, with the densest stands exceeding 3400 trees ha−1 (Table 1). Such dense canopies leave Knife Creek at increased risk of a high-severity stand-initiating ﬁre (Leclerc et al., 2021) and justify treatments to mitigate hazardous fuels and restore ecosystem structure and function (Hessburg et al., 2005, 2016; Stephens et al., 2012; Halofsky et al., 2020). low levels of ﬂammable fuels and an open canopy structure, reducing crown ﬁre potential and increasing forest resilience against future megaﬁres. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Implications for Forest Restoration and Management Our reconstruction of the historical ﬁre regime provides a framework for future management and restoration of the Knife Creek forest and other Douglas-ﬁr forests in the surrounding landscape. Since surface ﬁres have been essentially eliminated from the contemporary ﬁre regime, priority actions should aim to reduce stand densities, increase stand-level heterogeneity, and diversify forest structures across the landscape (Halofsky et al., 2011; Stephens et al., 2012, 2020; Churchill et al., 2013; Hessburg et al., 2016, 2019). Uneven-aged silviculture, thinning, and prescribed burning (Stephens et al., 2012), including the re-introduction of Indigenous ﬁre stewardship (Lake and Christianson, 2019; Long et al., 2020), can be used in combination to achieve these goals. Speciﬁcally, thinning the subcanopy emulates the common, widespread eﬀects of low-severity ﬁres and selected removal of canopy trees emulates the patchy eﬀects of moderate-to-high-severity that we reconstructed. Removing up to 76% of the relative density of the unharvested forests of Knife Creek would yield tree densities of 50–190 trees ha−1, congruent with historical densities (Hessburg et al., 2005; Leclerc et al., 2021). Recurring maintenance treatments at intervals of 10-30 years that vary in size and within-treatment severity at scales of 1-100 ha will maintain forest structures consistent with the historical mixed-severity ﬁre regime. Long-term management that emulates or includes frequent surface ﬁres will maintain Baker, W. L. (2015). Are high-severity ﬁres burning at much higher rates recently than historically in dry-forest landscapes of the western USA? PLoS One 10:e0136147. doi: 10.1371/journal.pone.0136147 Baker, W. L. (2015). 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https://openalex.org/W2802145190	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0195944&type=printable	English	null	The impact of high and low-intensity exercise in adolescents with movement impairment	PloS one	2,018	cc-by	8,939	RESEARCH ARTICLE Francesca Liu1☯, Martyn Morris1,2☯, Lisa Hicklen3‡, Hooshang Izadi4‡, Helen Dawes1,5,6,7☯ Francesca Liu1☯, Martyn Morris1,2☯, Lisa Hicklen3‡, Hooshang Izadi4‡, Helen Dawes1,5,6,7☯ 1 Department of Sport and Health Sciences, Oxford Brookes University, Oxford, Oxfordshire, United Kingdom, 2 Department of Biomolecular and Sport Sciences, Coventry University, Coventry, West Midlands, United Kingdom, 3 Department of Kinesiology, University of Texas at Arlington, Arlington, Texas, United States of America, 4 Department of Mechanical Engineering and Mathematical Sciences, Oxford Brookes University, Oxford, Oxfordshire, United Kingdom, 5 Oxford Institute of Nursing & Allied Health Research (OxINMAHR), Oxford Brookes University, Oxford, Oxfordshire, United Kingdom, 6 Department of Clinical Neurology, University of Oxford, Oxford, United Kingdom, 7 Cardiff University, Cardiff, Wales a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * francesca.liu-2013@brookes.ac.uk Abstract Five to six percent of young people have movement impairment (MI) associated with reduced exercise tolerance and physical activity levels which persist into adulthood. To bet- ter understand the exercise experience in MI, we determined the physiological and percep- tual responses during and following a bout of exercise performed at different intensities typically experienced during sport in youth with MI. Thirty-eight adolescents (11–18 years) categorised on the Bruininks-Oseretsky Test of Motor Proficiency-2 Short-Form performed a peak oxygen uptake bike test ( _VO2peak) test at visit 1 (V1). At visits 2 (V2) and 3 (V3), partic- ipants were randomly assigned to both low-intensity (LI) 30min exercise at 50% peak power output (PPO50%) and high-intensity (HI) 30s cycling at PPO100%, interspersed with 30s rest, for 30min protocol (matched for total work). Heart rate (HR) and rating of perceived exertion (RPE) for legs, breathing and overall was measured before, during and at 1, 3 and 7-min post-exercise (P1, P3, P7). There was a significant difference in _VO2peak between groups (MI:31.5±9.2 vs. NMI:40.0±9.5mlkg-1min-1, p<0.05). PPO was significantly lower in MI group (MI:157±61 vs. NMI:216±57 W)(p<0.05). HRavg during HI-cycling was reduced in MI (140±18 vs. 157±14bpm, p<0.05), but not LI (133±18 vs. 143±17bpm, p>0.05). Both groups experienced similar RPE for breathing and overall (MI:7.0±3.0 vs. NMI:6.0±2.0, p>0.05) at both intensities, but reported higher legs RPE towards the end (p<0.01). Significant differ- ences were found in HRrecovery at P1 post-HI (MI:128±25.9 vs. NMI:154±20.2, p<0.05) but not for legs RPE. Perceived fatigue appears to limit exercise in youth with MI in both high and low-intensity exercise types. Our findings suggest interventions reducing perceived fatigue during exercise may improve exercise tolerance and positively impact on engage- ment in physical activities. Introduction Motor coordination deficits and inefficient movement patterns are notable contributors to the reduced exercise capacity exhibited in individuals with movement impairment (MI). Nearly 2.6 million people in the UK [1] and an estimated 6% worldwide [2] present with movement difficulties including developmental coordination disorder (DCD) and neurodevelopmental/ neurological conditions (i.e., cerebral palsy). Children and adolescents with MI often display insufficient physical activity (PA) levels [3–6] compared to typically developing (TD) peers and correspondingly engage at lower intensities when participating in sports and play [7]. According to the World Health Organization (WHO), individuals between 5–17 years old should accumulate at least 60 min of moderate-to-vigorous physical activity (MVPA) daily and incorporate vigorous-intensity PA three days per week [8], in order to confer positive health effects [9]. Consequently, decreased levels of PA have implications for many aspects of children’s physical and cognitive development [10] and general health and well-being [3]. Adolescents with MI have also demonstrated higher rates of obesity compared to TD, and as a result may lead to an increased risk for developing metabolic syndrome [11, 12]. Of added concern is that such motor impairments and poor coordination contribute to a vicious cycle of reduced enjoyment, tolerance and participation [13, 14], which is known to persist throughout adolescence into adulthood [15]. However, understanding why these young people fail to meet recommended PA levels remains a complex phenomenon influenced by a multitude of factors [16–18]. Major factors associated with reduced PA participation in MI have been reported to relate to exercise-induced symptoms of muscle fatigue, poor physical tolerance and lower energy lev- els [14, 17]. This is further evidenced in the literature whereby young people with motor coor- dination difficulties and MI commonly exhibit lower fitness (including aerobic power, muscle strength, endurance, anaerobic power) [19, 20]. One explanation may be that children with MI experience earlier symptoms of fatigue compared to motorically proficient peers [17]. Chil- dren and adolescents with higher levels of MI have also demonstrated an inability to exercise hard enough to tax the cardiovascular system despite their ability to push themselves and stress their muscles to work anaerobically during exercise [13]. Moreover, it has been suggested that hypoactivity presented in youth with MI, is associated with lower self-perception and poor self-adequacy [14] and may be a significant determinant in predicting engagement in PA dur- ing adolescence [5]. OPEN ACCESS Citation: Liu F, Morris M, Hicklen L, Izadi H, Dawes H (2018) The impact of high and low-intensity exercise in adolescents with movement impairment. PLoS ONE 13(4): e0195944. https:// doi.org/10.1371/journal.pone.0195944 Editor: Lucas van der Woude, Rijksuniversiteit Groningen, NETHERLANDS Copyright: © 2018 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the Parkinson’s Fund (PF) Charitable Trust, awarded to FKL, and members of the CLEAR Unit. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment interest in HI exercise, there is limited information regarding the potential injury risks and adverse responses in youth performing higher intensities, specifically in children with MI. According to the literature, MI may be associated with inefficient movement patterns [4, 14, 26] contributing to poorer physical tolerance and fatigue, which can be defined as an acute impairment of exercise performance that includes both an increase in perceived effort required to produce a power output and the eventual inability to maintain power output [27]. Fatigue has been highlighted as a major factor affecting exercise performance in youth with MI [14, 17]. To date, the limiting factors surrounding exercise capacity in children with MI have been suggested to be peripherally derived (e.g., local muscle fatigue) [13] and/or due to inefficiencies in the oxygen transport system (e.g., inadequate cardiac output) [13, 26, 28]. Perceptual factors are also known to limit exercise [29] as confirmed by pain and discomfort ratings increasing with RPE when performing both progressive and interval exercise [30] in children and adults. During activity, RPE represents an integration of information concerning previous experience, whereby the self-reported changes in effort reflect the physiological and psychological pro- cesses that under certain conditions induce fatigue [31]. To this regard, the physiological and perceptual responses during either continuous or intermittent and more specifically with HI or LI activities have not been described in young people with MI. A better understanding of the exercise responses in these individuals is an important first step to help us identify how to support these children to perform at higher intensities and engage in more physical activity. Procedure This was a randomised crossover study, utilising two acute exercise exposure conditions and approved by the University Research Ethics Committee (UREC Registration No. 130773). Par- ticipants were recruited from local schools and a local Clinical Exercise and Rehabilitation Unit (CLEAR) in Oxfordshire, UK. Individuals attending the CLEAR unit include children with MI, DCD and neurological disabilities who participate in weekly gym sessions. Families indicating that they were interested in taking part were sent separate child and parent informa- tion sheets and gave their written consent prior to participating in the study. Participants attended the Movement Science Laboratory for testing on three separate occasions, with approximately seven days between each visit. Participants were asked to refrain from eating, performing exercise or drinking caffeine in the 2 h period before attending the sessions. All participants were fully familiarised with the testing protocol prior to data collection. Aims The purpose of this study is to describe exercise responses to maximal exercise and explore the extent of the physiological and perceptual responses during and following an acute cycling bout of work-matched HI and LI exercise in children and adolescents with MI compared to no-movement impairment (NMI). Introduction Aerobic moderate PA is a powerful stimulus for improving cognition [21] and is undisput- edly evidenced to improve fitness, health and wellbeing [8]. Both lower-intensity PA [22] and moderate-vigorous intensity activity approaches have equally demonstrated favorable health benefits in TD. However, based on the findings by Morris et al. [13], utilising short duration, HI exercise may provide a feasible method for targeting aerobic capacity and improving fitness parameters in MI. There is emerging evidence supporting the notion that HI exercise may serve as an effective strategy to increase muscle power and improve exercise capacity [16], allowing untrained individuals to work harder than would otherwise be possible at a steady- state intensity [16, 23]. Intermittent exercise is hypothesised to be easier for less trained indi- viduals whilst inducing similar or even superior physiological benefits on oxidative capacity and endurance performance [18]. Several studies have already highlighted the positive effects of high-intensity training (HIT) on improving measures of physical fitness and cardiometa- bolic risk factors [16, 23, 24] in TD children. In comparison to moderate intensity levels the potential advantages of HIT are the purported time-efficiency of the exercise modality and the enjoyment associated with this form of training [16, 25]. Despite the renewed popularity and 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Measures Baseline measures. Baseline measures including level of movement impairment (BOT-2 SF) and _VO2peak were used to classify individuals in the MI and NMI groups and to ascertain PPO for assignment of intensity levels (HI and LI). Assessment of movement economy or muscular efficiency ( _VO2/W), cardiac efficiency (O2 pulse), HRpeak and RER were also mea- sured from the _VO2peak test to establish baseline fitness parameters. Bruininks-Oseretsky Test of Motor Proficiency 2 Short Form (BOT-2 SF). During the first visit, the BOT-2 SF, a standardised test of motor proficiency was used to categorise level of movement impairment [32]. Four motor area composites were included in the BOT-2 SF encompassing; fine motor control, manual coordination, body coordination and strength and agility. Thirteen items were individually administered as described in the test manual [32]. Raw scores for each task were converted to a point score under each subtest and summed across to obtain a total standard score. The total standard scores were compared to normative scores and age equivalents to determine the individual’s percentile rank and to describe overall motor skill proficiency level. Based on the BOT-2 SF manual [32], individuals scoring below the 17th percentile cut-off were considered to have lower motor skill proficiency [32] and cate- gorised as MI and individuals scoring >17th percentile were indicated as having no-movement impairment (NMI). Exercise tolerance in adolescents with movement impairment medication for 12 weeks) were ineligible to take part. Furthermore, participants presenting with attentional, learning and/or mental health conditions as indicated by their parents and on the information sheets were excluded from this study due to potential confounders when examining perceptual responses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Participants Forty-three adolescents aged 11–18 years with no known neurological condition were recruited. For the inclusion criteria, participants were required to be able to walk with or with- out support for at least five meters and be able to safely take part and follow a two-step instruc- tion during testing procedures. Participants with any known contraindications to exercise participation (i.e., muscular degenerative conditions, congenital heart disease, uncontrolled exercise-induced asthma, chronic obstructive pulmonary disorder, uncontrolled epilepsy/on 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment ratio (RER) >-1.06 [36], and/or 3) subjective signs of exhaustion [37]. The PPO was deter- mined as the highest workload (W) attained at _VO2peak for the completed stage. The RER was calculated from the ratio of _VO2 to _VCO2 at each workload level throughout the exercise test. For the measurement of muscular efficiency or the relationship between the amount of oxygen utilised for a given work rate, the linear slope of the relationship between _VO2 and W ( _VO2/W) was derived. Oxygen pulse (O2), a non-invasive indicator of cardiac effi- ciency, was also calculated by dividing _VO2peak by HRpeak ( _VO2peak/HRpeak) and expressed as mL/beat. Rating of perceived exertion (RPE) was measured at the end of each stage using the Cart and Load Scale (CALER), which has previously been used to assess children’s perception of effort during exercise [38]. Exercise testing Height (m) (Holtain stadiometer), weight (kg) (Seca scales), body mass index (BMI) (kg/m2) and sexual maturation [33] were recorded prior to the exercise test at V1. For the purposes of testing individuals with a wide range of movement abilities, measurement of peak oxygen uptake ( _VO2peak) was performed with an incremental step test on a cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands). The protocol consisted of 1 min stages after an initial 2 min of unloaded cycling. Workload was progressed by 15–20 Watt (W) from unloaded cycling each minute based on the height of the participant [34]. The test was terminated when the participant reached volitional exhaustion or was unable to maintain a cadence of 60 revolu- tions per minute (rpm) despite verbal encouragement. Oxygen uptake ( _VO2), carbon dioxide produced ( _VCO2) and volume of expired air per minute ( _VE) were measured breath-by-breath using an online gas analyser (Cortex Metalyser 3B, Cortex, Leipzig, Germany). Before each testing session, the gas analysers were calibrated according to manufacturer guidelines. The gas sample line was calibrated using gases of a known concentration and flow volume was cali- brated using a 3 L syringe (Hans Rudolph). All participants wore a fitted face-mask covering the nose and mouth connected to a low resistance volume transducer (Triple V, Hoechberg, Germany). Additionally, heart rate (HR) was recorded continuously throughout the testing using short-range telemetry (Polar S810, Finland). Oxygen uptake ( _VO2) was recorded as the highest 30 s average of each stage, while _VO2peak was recorded as the highest 30 s average before the termination of the test. The criteria for obtaining a _VO2peak was considered when two of the three following criteria was achieved: 1) HR >180 beats/min [35], 2) Respiratory exchange 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Sample size Based on statistical power calculations using GPower 3.1.9.2 (Heinrich Heine University Du¨s- seldorf, Germany), a sample size of 45 participants total is required for a power of 0.95, an alpha (α) of 0.05 and an effect size (Cohen’s d) of 0.50 to detect differences between the group means. Exercise tolerance in adolescents with movement impairment session under standard temperature conditions in the laboratory (~20–22 ˚C). For the HI ses- sion, participants were asked to perform a 30 min bout of cycling consisting of pedaling for 30 s-on, then no pedaling for 30 s-off at 100% PPO (PPO100%) as determined from PPO during the maximal incremental bike test. In contrast, for the LI session participants were asked to cycle continuously for 30 min at PPO50%. Throughout the session, both HR (Polar S810, Fin- land) and RPE (CALER scale) was monitored and recorded at 5 min intervals. Participants were asked to rate their RPE for legs, breathing and overall using the CALER scale every 5 min throughout the 30 min session. Following each session, participants were informally asked to indicate enjoyment level. Cardiovascular and perceptual outcome measures. Outcomes measures of interest include cardiovascular (HR) and perceptual (RPE) responses following both HI- and LI- cycling bouts. Additional measures of interest include heart rate average (HRavg) and HR recovery (HRrecovery) at post-1, 3 and 7 min exercise (P1, P3, P7). When considering relative HR, this was represented as percentage change (%Δ) from HRpeak, which was expressed as per- centage of the maximal heart rate (HRmax) throughout the study (i.e., %HRmax). The %HRmax has been used extensively as prescription means of exercise intensity and exemplifies a close relationship with oxygen uptake [39]. Comparisons between groups (MI vs. NMI), between conditions (HI vs. LI) and across time points (average during 30 min bout and post-exercise P1, P3, P7) were made to assess the impact of exercise intensities on outcome measures as well as the interactions between group and time. Randomisation Participants were randomised using an excel program which generated a random allocation to either the HI- or LI-bout first. The researchers informed the participant which exercise inten- sity they would perform for the second session (V2) and then cross-over to the other intensity bout for the third and final session (V3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise interventions Participants were asked to complete two experimental conditions in a randomised crossover design for the exercise intervention including: a HI-cycling bout and a LI-bout of cycling (Fig 1). g The exercise was performed on a cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands) with the bike seat height adjusted to the participants’ comfort and recorded for subsequent sessions. Participants performed the exercise at the same time of day for each Fig 1. Diagram of the three-visit study protocol. BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short Form 2; HI, High- intensity; LI, Low-intensity; PPO, peak power output; P1, post-1 min; P3, post-3 min; P7, post-7 min _VO2max, Peak oxygen uptake. https://doi.org/10.1371/journal.pone.0195944.g001 Fig 1. Diagram of the three-visit study protocol. BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short Form 2; HI, High- intensity; LI, Low-intensity; PPO, peak power output; P1, post-1 min; P3, post-3 min; P7, post-7 min _VO2max, Peak oxygen uptake. https://doi.org/10.1371/journal.pone.0195944.g001 Fig 1. Diagram of the three-visit study protocol. BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short Form 2; HI, High- intensity; LI, Low-intensity; PPO, peak power output; P1, post-1 min; P3, post-3 min; P7, post-7 min _VO2max, Peak oxygen uptake. https://doi.org/10.1371/journal.pone.0195944.g001 Fig 1. Diagram of the three-visit study protocol. BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short Form 2; HI, High- intensity; LI, Low-intensity; PPO, peak power output; P1, post-1 min; P3, post-3 min; P7, post-7 min _VO2max, Peak oxygen uptake. https://doi.org/10.1371/journal.pone.0195944.g001 Fig 1. Diagram of the three-visit study protocol. BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short Form 2; HI, High- intensity; LI, Low-intensity; PPO, peak power output; P1, post-1 min; P3, post-3 min; P7, post-7 min _VO2max, Peak oxygen uptake. https://doi.org/10.1371/journal.pone.0195944.g001 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Baseline measures Seventeen of the participants considered to be MI scored below the BOT-2 SF 17th percentile (below average) and 21 were classified as having NMI (BOT-2 SF >17th percentile). Table 1 illustrates the participant characteristics from the motor proficiency assessment. There was a significant difference in the BOT-2 SF standard score between MI (36.0±2.0) and NMI (44.0±12.0) [95% CI: -14.18, -1.61; p<0.001]. Moreover, there was a significant relationship between BOT-2 SF score and _VO2peak (r = 0.62, p<0.05) in both groups and a significant differ- ence in _VO2peak between groups (MI: 31.5±9.2 vs. NMI: 40.0±9.5 mlkg-1min-1) [t(36) = -2.28, p<0.01, 95% CI: -1.08, -0.29]. The PPO (W) was significantly lower in the MI group (MI: 157.0±61.0 vs. NMI: 216.0±57.0 W) [t(33) = -3.05, p<0.01, 95% CI: -101.1, -20.12] and for the LI workload (MI: 85.0±38.0 and NMI: 121.0±29.0 W) [t(31) = -2.38, p<0.05, 95% CI: -51.4, -3.99]. With regard to assessment of movement economy, the MI group demonstrated greater inefficiencies during the maximal exercise test ( _VO2/PO) (MI: 13.3±3.0; NMI: 11.2±2.0 mL/ W) [t(21) = 2.12, p<0.05, 95% CI: -2.85, 2.18] (Table 2). However, HRmax was similar between groups (MI: 170.0±25.0 and NMI: 180.0±17.0 bpm) [t(35) = -1.31, p>0.05, 95% CI: -24.7, 5.48] and there were no significant differences in O2 pulse (MI: 19.0±0.03; NMI: 20.0±0.05 mL/beat) [t(36) = -0.34, p>0.05, 95% CI: -1.79, 1.30] (Table 2). Correspondingly, there was no difference in the perception of effort throughout the exercise test and at exercise termination. All participants reported an RPE rating of 9 or 10 at the end of the test despite the MI group demonstrating significantly lower PPO at the end of the incremental bike test as shown below in Table 2. Overall, each participant exhibited a RERmax greater than 1.06 at the end of the test, however, there was a significant difference between the groups for the RERmax value (MI: 1.20±0.20 and NMI: 1.34±0.10) [t(21) = -2.61, p = 0.008, 95% CI: -2.06, 0.88]. Results A total of 43 adolescents volunteered to participate in this study (Fig 2), with results presented for 38 (11–18 years) as five of the participants were unable to complete all visits required for the study. The participants were classified into two levels of movement impairment according to the BOT-2SF: those with MI (n = 17; 15 males, 2 females) and those who were normally coordi- nated, with NMI (n = 21; 18 males, 3 females). Baseline characteristics of participants are pre- sented in Table 1. Exercise tolerance in adolescents with movement impairment no obvious pattern should be displayed and outliers were identified. Furthermore, a Shapiro- Wilk test was performed to determine normality of residuals. Since only two repeats were assessed, an unstructured covariance structure was utilised. Data analysis All data are presented as mean ± SD. Statistical analyses were performed in SPSS for Windows v21 (SPSS Inc., Chicago, IL, USA). Normality of the data was checked by Shapiro Wilk tests. Homogeneity of variances was confirmed by Mauchley’s test of sphericity and a Greenhouse- Geisser correction was applied to the degrees of freedom if the sphericity assumption was vio- lated. Baseline exercise measurements ( _VO2peak, HRmax, RER, RPE, PPO/W) were analysed using student’s t-test. A Pearson correlation coefficient (r) was used to examine the linear rela- tionship between BOT-2 SF scores and baseline _VO2peak. Within-session exercise measure- ments (HR, RPE) were analysed using a linear mixed model (LMM) [40] for repeated measures over time by group to analyze the impact of the different exercise intensities (HI vs. LI) on outcome measures at baseline, during and post-exercise with fixed effects of group (MI vs. NMI), time (HI and LI session) and the interactions between group and time. This method prevented listwise deletion due to missing data [41]. A scatter plot of the predicted values on the x-axis and the residuals on the y-axis were plotted to visually check for linearity whereby 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment Table 1. Baseline participant characteristics (mean ± SD). N = 38 MI (n = 17) NMI (n = 21) P-value Age (years) 14.5 ± 2.0 15.5 ± 2.0 - Height (m) 1.70 ± 8.6 1.74 ± 10.6 - Weight (kg) 63.3 ± 15.6 66.4 ± 16.3 - BMI (kg/m2) 22.0 ± 0.0 22.0 ± 0.0 - Tanner 5.0 ± 0.0 5.0 ± 0.0 - BOT-2SF Raw Score 61.0 ± 6.0 71.0 ± 18.0 p < 0.05 BOT-2SF Standard Score 36.0 ± 2.0 44.0 ± 12.0 p < 0.05 BMI, body mass index; BOT-2 SF, Bruininks-Oseretsky Test of Motor Proficiency Short-Form 2; kg, kilogram; m, metre; Tanner, Tanner Scale of Sexual Maturity. p 0.05 vs. NMI at baseline. https://doi.org/10.1371/journal.pone.0195944.t001 Table 1. Baseline participant characteristics (mean ± SD). considering relative HR represented as percentage change (%Δ) from HRmax (% HRmax), sig- nificant differences were only demonstrated during an average of the 30 min cycling bout for HI (MI: 82.0±9.5 vs. NMI: 87.0±7.1%) [t(36) = -3.21, p = 0.003] (Fig 3(a) and 3(b)). In contrast, the MI group did not demonstrate a great deal of change from HI during the LI bout (MI: 78.0±19.3 vs. NMI: 79.0±23.4%) [t(36) = -0.27, p>0.05]. To further validate the findings, a paired samples t-test showed no significant difference in HRavg, however, relative change was significantly different in NMI [t(20) = 2.73, p = 0.013]. In the recovery phase, HR at post-1 min (P1) was significantly different for group following HI (MI: 128.0±24.2 vs. NMI: 145.0±22.3 bpm) and LI (MI: 121.0±23.0 vs. NMI: 131.0±23.4 bpm) [F(1,35.2) = 4.91, p<0.05]. No significant differences in HR for group were observed at Table 2. Baseline exercise intensity descriptors (mean ± SD). Avg, average; bpm, beats per minute; HRmax, heart rate maximum; HI, high intensity; LI, low intensity; L, Litre; mL, milliltre; movement impairment; NMI, no- movement impairment; O2 pulse, Oxygen pulse; PPO, peak power output; RER, respiratory exchange ratio; RPE, rating of perceived exertion; VO2peak, peak oxygen uptake; _VO2/PO, muscular efficiency; W, Watt; Wpeak watt max; %Δ HIbaseline, percentage change from baseline at HI (100%); %Δ LIbaseline, percentage change from baseline LI (100%). p 0.05 vs. NMI at same time point. https://doi.org/10.1371/journal.pone.0195944.t002 max, heart rate maximum; HI, high intensity; LI, low intensity; L, Litre; mL, milliltre; movement impairment; NMI, no- Cardiovascular and perceptual responses Mean workload during the LI-cycling bout was 85.0±38.0 W in the MI group and 121.0±29.0 W in the NMI group (p<0.05). There was a significant difference between groups [F(1,36.1) = 7.1, p = 0.012] and an effect of intensity for HRavg as demonstrated by the LMM [F(1,33.3) = 37.0, p<0.001]. Overall, HRavg during HI-cycling, which took into account an average of each cycle (i.e., 30s on and 30s off) throughout the 30 min duration, was lower in MI compared to NMI (140.0±18.0 and 157.0±14.0 bpm, p<0.05) [t(34) = -3.28, p = 0.002], but not during LI- cycling (133.0±18.0 and 143.0±17.0 bpm, p>0.05) [t(33) = -1.64, p>0.05]. This denotes that there is a significant difference with regard to how the level of intensity affects the two groups with the MI group experiencing less HRavg variability irrespective of intensity. When 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment Fig 2. Flow diagram of participant recruitment and adherence throughout study. MI, movement impairment; NMI, no-movement impairment. https://doi org/10 1371/journal pone 0195944 g002 Fig 2. Flow diagram of participant recruitment and adherence throughout study. MI, movement impairment; NMI, no-movement impairment Fig 2. Flow diagram of participant recruitment and adherence throughout study. MI, movement impairment; NMI, no-movement impairment. https://doi.org/10.1371/journal.pone.0195944.g002 https://doi.org/10.1371/journal.pone.0195944.g002 8 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 N = 38 MI (n = 17) NMI (n = 21) P-value _VO2peak (L/min) 1.90 ± 0.49 2.39 ± 0.78 p < 0.05 _VO2peak (mL/kgmin) 31.54 ± 9.2 36.0 ± 11.0 p < 0.05 HRmax (bpm) 170.0 ± 25.0 180.0 ± 17.0 - PPO (W) 157.0 ± 60.5 216.0 ± 57.0 p < 0.05 _VO2/workload (mL/W) 13.3 ± 4.0 11.2 ± 2.0 p < 0.05 O2 pulse (mL/beat) 19.0 ± 0.03 20.0 ± 0.05 - RPE overall 9.00 ± 1.0 8.00 ± 1.00 - RER 1.20 ± 0.20 1.34 ± 0.10 p < 0.05 HI workload/PO (W) 145.0 ± 65.0 216.0 ± 69.0 p < 0.05 HI HRavg (bpm) 139.0 ± 18.0 156.0 ± 13.0 p < 0.05 %Δ HIbaseline 82.0 ± 18.0 87.0 ± 14.0 - LI workload/PO (W) 80.0 ± 30.0 108.0 ± 36.0 p < 0.05 LI HRavg (bpm) 134.0 ± 18.0 143.0 ± 16.0 - %Δ LIbaseline 78.0 ± 18.0 79.0± 16.0 - Table 2. Baseline exercise intensity descriptors (mean ± SD). Avg, average; bpm, beats per minute; HRmax, heart rate maximum; HI, high intensity; LI, low intensity; L, Litre; mL, milliltre; movement impairment; NMI, no- movement impairment; O2 pulse, Oxygen pulse; PPO, peak power output; RER, respiratory exchange ratio; RPE, rating of perceived exertion; VO2peak, peak oxygen uptake; _VO2/PO, muscular efficiency; W, Watt; Wpeak watt max; %Δ HIbaseline, percentage change from baseline at HI (100%); %Δ LIbaseline, percentage change from baseline LI (100%). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 9 / 16 Exercise tolerance in adolescents with movement impairment es for high- and low-intensity bouts. Percent change from baseline heart rate maximum 7 min (P1, P3, P7) presented for high-intensity visit (HI) (a) and low-intensity visit (LI) (b). bars). Figs (c) and (e) illustrate the change in HR and ratings of perceived exertion (RPE) pre, res were recorded every 5 min throughout the cycling and following in recovery at P1, P3and ng the LI-cycling bout (dotted line). Vertical and horizontal error bars represent standard 0.05 for Intensity; §p 0.05 vs. NMI at same time point (Group x Intensity). 3 Fig 3. Cardiovascular and perceptual responses for high- and low-intensity bouts. Percent change from baseline heart rate maximum (HRmax) during cycling bout and post-1, 3 and 7 min (P1, P3, P7) presented for high-intensity visit (HI) (a) and low-intensity visit (LI) (b). MI group (Hollow bars) and NMI group (filled bars). Figs (c) and (e) illustrate the change in HR and ratings of perceived exertion (RPE) pre, during and post-HI-cycling (solid line). Measures were recorded every 5 min throughout the cycling and following in recovery at P1, P3and P7. Figs (d) and (f) represent HR and RPE during the LI-cycling bout (dotted line). Vertical and horizontal error bars represent standard deviation (SD). p0.05 vs. NMI (group); ǂp0.05 for Intensity; §p 0.05 vs. NMI at same time point (Group x Intensity). https://doi.org/10.1371/journal.pone.0195944.g003 Fig 3. Cardiovascular and perceptual responses for high- and low-intensity bouts. Percent change from baseline heart rate maximum (HRmax) during cycling bout and post-1, 3 and 7 min (P1, P3, P7) presented for high-intensity visit (HI) (a) and low-intensity visit (LI) (b). MI group (Hollow bars) and NMI group (filled bars). Figs (c) and (e) illustrate the change in HR and ratings of perceived exertion (RPE) pre, during and post-HI-cycling (solid line). Measures were recorded every 5 min throughout the cycling and following in recovery at P1, P3and P7. Figs (d) and (f) represent HR and RPE during the LI-cycling bout (dotted line). Vertical and horizontal error bars represent standard deviation (SD). p0.05 vs. NMI (group); ǂp0.05 for Intensity; §p 0.05 vs. NMI at same time point (Group x Intensity). P3 for either intensity, however, significant differences for intensity were exhibited at P7 fol- lowing HI (MI: 102.0±17.2 vs. NMI: 106.0±15.4 bpm) and LI (MI: 96.0±15.2 vs. 97.0±16.2 bpm) [F(1,26.4) = 9.80, p = 0.004] and illustrated in Fig 3(c) (HI bout) and Fig 3(d) (LI bout). Both groups experienced similar RPE for breathing and overall (MI: 7.0±3.0 vs. NMI: 6.0±2.0, p>0.05) throughout the exercise at both intensities (Fig 3(e) and 3(f)). However, sig- nificant differences were observed in RPE for legs during cycling at both intensities towards P3 for either intensity, however, significant differences for intensity were exhibited at P7 fol- lowing HI (MI: 102.0±17.2 vs. NMI: 106.0±15.4 bpm) and LI (MI: 96.0±15.2 vs. 97.0±16.2 bpm) [F(1,26.4) = 9.80, p = 0.004] and illustrated in Fig 3(c) (HI bout) and Fig 3(d) (LI bout). Both groups experienced similar RPE for breathing and overall (MI: 7.0±3.0 vs. NMI: 6.0±2.0, p>0.05) throughout the exercise at both intensities (Fig 3(e) and 3(f)). However, sig- nificant differences were observed in RPE for legs during cycling at both intensities towards Both groups experienced similar RPE for breathing and overall (MI: 7.0±3.0 vs. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Discussion Children and adolescents with MI experienced higher perceived fatigue after both high and low intensity exercise despite having similar or lower physiological HR exercise responses dur- ing and following cycling. The MI group tolerated the short duration, HI exercise and the con- tinuous lower-intensity endurance exercise similarly. Therefore, both approaches may serve as a feasible modality for improving fitness but the long-term benefits and sustainability of differ- ent exercise intensities still remains to be evaluated. We propose that perceived fatigue is limit- ing exercise for youth with MI and interventions to reduce perceived fatigue during exercise may positively impact on exercise engagement in this group. Throughout the literature, it has been consistently shown that children with movement dif- ficulties (i.e., pDCD and DCD) are disadvantaged to various degrees on exercise capacity and muscle strength. Individuals with MI perform less well on fitness parameters [15] of: aerobic power, muscle strength, endurance, anaerobic power and body composition [13, 19, 26]. Simi- lar to previous findings examining submaximal and maximal exercise test measures [13, 26], it was observed that children and adolescents with MI exhibited a lower _VO2peak and PPO com- pared to their TD peers (MI: 157.0±61.0 vs. NMI: 216.0±57.0 W). As hypothesised, partici- pants with MI had a reduced exercise capacity, demonstrating a significantly lower _VO2peak in comparison to NMI (MI: 31.5 vs. NMI: 36.0 ± 11.0 mL/kgmin). Furthermore, the _VO2peak for the MI group was below the cardiovascular fitness threshold [42] and as such, associated with an increased risk of obesity, type II diabetes and cardiovascular and metabolic conditions in adulthood [43]. Parallel to values reported by Faught et al. [26], HRmax or the term HRpeak used in this study was analogous between groups (MI: 170.0±25.0 and NMI: 180.0±17.0 bpm, p>0.05). However our findings deviated from the results in Morris et al. [13], who saw a significant mean HR difference of 12 bpm in children with higher MI (176 bpm) versus NMI (188 bpm). These differences may be due to the small sample size presented in the current study, which served as a limitation to the robustness of these findings. In addition, perceived competence to complete the task could also limit the results of the _VO2max test in children with MI. It is impor- tant to note that the MI group demonstrated a similar RPE to NMI despite significant differ- ences in _VO2peak performance and exercise capacity. NMI: 6.0±2.0, p>0.05) throughout the exercise at both intensities (Fig 3(e) and 3(f)). However, sig- nificant differences were observed in RPE for legs during cycling at both intensities towards PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 10 / 16 Exercise tolerance in adolescents with movement impairment the end of the 30 min bout (MI: 8.0±2.0; NMI: 7.0±2.0 HI and MI: 7.0±3.0; NMI: 6.0±2.0 LI) [F(1,33.0) = 9.2, p<0.01] and for group x intensity [F(1,33.0) = 4.8, p<0.05]. Moreover, there was a notable difference for RPE in the legs [F(1,28.5) = 7.6, p = 0.01], breathing [F(1,28.9) = 9.2, p<0.01] and overall [F(1,28.7) = 11.8, p<0.01](Fig 3(e)) at P1 HI (MI: 6.0±3.0 legs, 5.0±3.0 breathing, 6.0±3.0 overall vs. NMI: 5.0±2.0 legs, 5.0±2.0 breathing, 5.0±2.0 overall) and indi- cated at P1 for LI (MI: 5.0±3.0 legs, 4.0±3.0 breathing, 4.0±3.0 overall vs. NMI: 4.0±2.0 legs, 5.0±2.0 breathing, 4.0±3.0 overall) (Fig 3(f)). Interestingly, the MI group appeared to experi- ence a similar level of RPE for both intensities despite achieving lower workloads (W) to complete the exercise in comparison to the NMI group. The full set of data is available as sup- plementary material (S1 Table). PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment throughout and at test termination similar to Morris et al., [13]. All participants reported a RPE rating of 9 or 10 at the end of the exercise test. Corroborating earlier studies [13, 26], participants with MI exhibited a reduced movement economy during the incremental bike test as illustrated by their _VO2/PO relationship (MI: 13.3 ± 4.0 vs. NMI: 11.2 ± 2.0 mL/W) (Table 2). According to Wasserman et al. [45], _VO2 kinetics normally rise at a rate of approximately 8.5–11 mL/minW and are independent of sex, age, body mass or height in youth. Therefore, poorer coordination and inefficient move- ment patterns may contribute to the reduced exercise capacity in MI. Similar to the observa- tions in this study, Faught et al., [26] recognised that children with pDCD were disadvantaged from the beginning of the incremental exercise protocol at low workloads (i.e., <40 W) and worked at a greater percentage of their _VO2 compared to healthy controls throughout the test. As such, even at very low exercise intensities, children with poor motor proficiency/coordina- tion may need to utilise more energy to carry out basic movements associated with maintain- ing proper posture and posture on the cycle ergometer [26]. This further suggests that youth with MI produce inefficient movements and may exercise at a higher metabolic rate to sustain the same level of workload relative to children without. Furthermore, we suspect the MI group would require more oxygen to exercise because they do not work their cardiovascular system sufficiently as represented by the similar O2 pulse (MI: 19.0 ± 0.03 vs. 20.0 ± 0.05 mL/beat) but significantly different PPO attained. To our knowledge, this is the first study to investigate the impact of varying exercise intensi- ties and the responses during the recovery phase, particularly following HI exercise, in adoles- cents with MI. As previously alluded in Morris et al. (2013), the evidence of children with MI to push themselves maximally warrants the potential applications of performing HI exercise on improving measures of health, muscle function and movement coordination. The results in this study revealed that the NMI group was able to tax the cardiovascular system sufficiently during the HI session as indicated by the 12% difference for HRavg between the HI (157.0±14.0 bpm) and LI (143.0±17.0 bpm) cycling bouts. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Discussion Thus, the limiting factors to exercise may be more perceptually related and central in origin [28]. Both groups exceeded an average RER of 1.06 at the end of the exercise test; however, RER was notably higher in NMI (1.34 in NMI vs. 1.20 in MI), which was different to the findings in Morris et al. (2013). Substrate utilisation and fat oxidation levels during the maximal exercise test were within the range previously reported in healthy children and adolescents [44]. Although the MI group demonstrated a sig- nificantly lower PPO there was no difference in the perception of effort (RPE) reported PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 11 / 16 Exercise tolerance in adolescents with movement impairment during higher intensity exercise, the MI group reported higher levels of perceived fatigue in legs and breathing even though they performed at a lower overall exercise intensity (but still the same relatively) and achieved high RER values at _VO2peak. Therefore, as opposed to the findings observed in Morris et al. [13], the factors limiting exercise performance and perceived fatigue in MI may be more central in origin rather than metabolic or peripheral. This suggests that the lower-intensity aerobic exercise will be better tolerated in the MI group in comparison to HI. Noteworthy, all participants completed each session without any adverse events and interestingly, both MI and NMI groups anecdotally preferred the HI cycling bout to the LI bout. With this in mind, exposure to different exercise intensities may build up more self-con- fidence and self-efficacy to participate in PA [4]. Strengths of this study include objective measurement of physiological and perceptual vari- ables during and following different exercise intensities, and the examination of recovery markers in MI and NMI adolescents. Ongoing growth and maturation can confound exercise interventions unless controlled adequately with well-matched controlled groups. In this acute study, only a small sample of participants (n = 38) were measured and therefore, caution must be taken when interpreting the robustness of the outcomes. There were no statistical differ- ences found between MI and NMI groups on level of maturation or age and thus, no further analyses were undertaken to delineate potential sex differences at this time. Moreover, the wide age range of the participants and the unequal distribution of boys and girls pose limita- tions on interpreting the findings. Maturation and puberty may raise significant implications for the results and according to Tolfrey and Smallcombe (2017), may be an important factor on training effect in HIT studies. However, the authors acknowledge it is difficult to identify an independent sex effect that is not due to baseline differences in peak _VO2 or maturation [25]. New research has drawn a correlation between significant sex differences in the underly- ing pathways connecting pDCD to internalising problems, indicating more mediating path- ways through PA, BMI and global self-worth in girls, compared to boys [47]. Future research should expand on these findings and include larger sample sizes, taking into account sex, mat- uration and age in relation to physiological and perceptual exercise responses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Conversely, the MI group did not appear to demonstrate considerable difference in HR measures between HI (140.0±18.0 bpm) and LI (133.0±18.0 bpm) cycling with only a 4% difference in percentage change from HRmax between intensities. These findings potentially suggest a smaller ventilatory threshold (VT), or point during exercise at which ventilation starts to increase at a faster rate than _VO2, in adolescents with MI compared to NMI. For most individuals, this threshold lies at exercise intensities between 50% and 75% of _VO2max and is dependent on the person’s level of fitness [46]. Exercise intensity for both HI and LI workload was set relatively to each child and considering the high peak RER observed in the MI group and lower actual intensity performed, it is likely that chil- dren with MI are exercising at a lower intensity relative to their VT. A limitation of this study however, was that the exercise intensities for the HI and LI cycling bouts were not determined from the VT and instead, calculated as a percentage of PPO (PP50% and PP100%). Although dif- ficult to interpret in a minority of children, the VT is considered a useful method to determine aerobic fitness in children [46] and future studies should examine VT and _VO2max changes fol- lowing different types of training [16]. In the last decade, findings have highlighted the relationship between lower motor compe- tence and fitness performance [13, 14]; noting the influence of self-efficacy and perceived adequacy [17]. The findings in this study showed that the MI group did not experience any dif- ferences in perceived fatigue and leg muscle fatigue compared to NMI peers following either HI-or LI-exercise. These observations are similar to studies reporting that children experience less fatigue during short-burst activities and often request to repeat high-intensity exercises after their completion determined to improve their previous performance [31]. However, PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 12 / 16 Writing – review & editing: Francesca Liu, Martyn Morris, Helen Dawes. Writing – review & editing: Francesca Liu, Martyn Morris, Helen Dawes. Acknowledgments I declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation, and statement that results of the present study do not constitute endorsement by ACSM. Conclusion The findings from this study highlight the physiological, perceptual and recovery responses to different exercise intensities in children and adolescents with and without MI. The exposure to both a HI intermittent bout and a continuous LI cycling bout demonstrated a lower exercise capacity in children with MI and a higher perception of physiological symptoms while per- forming at a lower intensity generally. Furthermore, the results from the incremental exercise test alongside measures of muscle strength and fatigue before and after exercise suggest that central factors could be the limiting factor to exercise tolerance in this group. Interestingly, dif- ferences for group and intensity were observed in the recovery period, yet the pattern of recov- ery was similar between MI and NMI, which may be crucial for devising suitable exercises and activity intensities. Although the number of participants in this sample was relatively small, the results from each acute session contribute to future interventions and exercise prescriptions targeting aerobic and anaerobic fitness, strength and power and general participation in physi- cal activities. Overall, all participants successfully completed the high and moderately-low intensity cycling bouts however, more research investigating the implementation of HI and LI exercise during longer-term interventions is required to further elucidate the sustainability and tolerance of this type of activity. Whether short durations of high-intensity intermittent exercise can feasibly improve health and fitness levels and adherence to longer-term PA engagement in youth with MI still remains to be explored. 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0195944 April 26, 2018 Exercise tolerance in adolescents with movement impairment Author Contributions Conceptualization: Francesca Liu, Martyn Morris, Helen Dawes. Conceptualization: Francesca Liu, Martyn Morris, Helen Dawes. Data curation: Francesca Liu, Lisa Hicklen. Formal analysis: Francesca Liu, Hooshang Izadi, Helen Dawes. Formal analysis: Francesca Liu, Hooshang Izadi, Helen Dawes. Investigation: Francesca Liu, Martyn Morris, Lisa Hicklen. Investigation: Francesca Liu, Martyn Morris, Lisa Hicklen. Methodology: Francesca Liu, Martyn Morris, Helen Dawes. Methodology: Francesca Liu, Martyn Morris, Helen Dawes. Project administration: Francesca Liu, Lisa Hicklen. Resources: Martyn Morris, Helen Dawes. 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https://openalex.org/W2078779434	https://ccforum.biomedcentral.com/counter/pdf/10.1186/cc10973	English	null	Regional citrate anticoagulation in CVVH: a new protocol combining citrate solution with a phosphate-containing replacement fluid	Critical care	2,012	cc-by	260,843	P1 Impaired innate and adaptive immunity of accelerated-aged Klotho mice in sepsis p Methods We tested for genetic association of IL-17A SNPs with susceptibility to infection and clinical outcome of severe sepsis using two cohorts of European ancestry (St Paul’s Hospital (SPH) derivation cohort, n = 679; Vasopressin and Septic Shock Trial (VASST) validation cohort n = 517). The primary outcome variable was susceptibility to Gram-positive bacterial infection. The secondary outcome variable was 28-day mortality. p S Inoue, K Suzuki-Utsunomiya, K Suzuki-Utsunomiya, T Sato, T Chiba, K Hozumi Tokai University, Kanagawa, Japan Critical Care 2012, 16(Suppl 1):P1 (doi: 10.1186/cc10608) Introduction Sepsis is primarily a disease of the aged and 60% of sepsis occurs in patients older than 65 years, 80% of deaths due to sepsis occur in this age group. Klotho knockout mice (Klotho mice) develop a syndrome resembling human aging, and exhibit shortened life spans (8 weeks); however, details regarding the immunity of and immunological changes in Klotho mice after sepsis are still unclear. The purpose of the study is to elucidate the immunological changes that occur in Klotho mice after sepsis in order to identify therapeutic targets for sepsis that occurs in aged individuals. y y Results Of four tested tag SNPs (rs4711998, rs8193036, rs2275913, rs1974226) in the IL-17A gene, rs1974226 SNP was associated with altered susceptibility to Gram-positive bacterial infection in the derivation cohort (corrected P = 0.014). Patients who have the GG genotype of the rs1974226 SNP were more susceptible to Gram- positive bacterial infection, compared to the AG/AA genotype in the two cohorts of severe sepsis (SPH, P = 0.0036; VASST, P = 0.011) and in the subgroup having lung infection (P = 0.017). Furthermore, the G allele of the IL-17A rs1974226 SNP was associated with increased 28- day mortality in two cohorts (SPH, adjusted OR 1.44, 95% CI 1.04 to 2.02, P = 0.029; VASST, adjusted OR 1.67, 95% CI 1.17 to 2.40, P = 0.0052). Conclusion IL-17A genetic variation is associated with altered suscepti- bility to Gram-positive infection and 28-day mortality of severe sepsis. References p g Methods (1) Survival study: cecum ligation puncture (CLP) was performed to Klotho and wild-type (WT) mice and 4-day survivals were compared. (2) Cell analysis study: mice were sacrifi ced at 8 hours post CLP or sham surgery. Spleens, thymus, and serum were harvested for FACS analysis using caspase 3 as a marker for apoptosis, and blood for serum cytokine assay. MEETING ABSTRACTS MEETING ABSTRACTS P1 Impaired innate and adaptive immunity of accelerated-aged Klotho mice in sepsis S Inoue, K Suzuki-Utsunomiya, K Suzuki-Utsunomiya, T Sato, T Chiba, K Hozumi Tokai University, Kanagawa, Japan Critical Care 2012, 16(Suppl 1):P1 (doi: 10.1186/cc10608) P1 Impaired innate and adaptive immunity of accelerated-aged Klotho mice in sepsis S Inoue, K Suzuki-Utsunomiya, K Suzuki-Utsunomiya, T Sato, T Chiba, K Hozumi Tokai University, Kanagawa, Japan Critical Care 2012, 16(Suppl 1):P1 (doi: 10.1186/cc10608) P3 Prevalence of TLR4 single nucleotide polymorphisms (ASP299GLY, THR399ILE) in healthy subjects and septic patients, and association with outcome T Mohovic1, R Salomao2, E Nogueira2 1Albert Einstein Hospital, São Paulo, Brazil; 2UNIFESP, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P3 (doi: 10.1186/cc10610) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 32nd International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 20-23 March 2012 Published: 20 March 2012 to the host defense. Whether genetic variation of IL-17A is associated with altered clinical outcome of severe sepsis is unknown. P1 Impaired innate and adaptive immunity of accelerated-aged Klotho mice in sepsis S Inoue, K Suzuki-Utsunomiya, K Suzuki-Utsunomiya, T Sato, T Chiba, K Hozumi Tokai University, Kanagawa, Japan Critical Care 2012, 16(Suppl 1):P1 (doi: 10.1186/cc10608) bility to Gram References 1. Puel A, et al.: Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 2011, 332:65-68. Results (1) Klotho septic mice started to die from 8 to 12 hours after CLP, and fi nal survival of Klotho mice with CLP was signifi cantly lower than that of WT with CLP (0% vs. 100%, P <0.01). (2) Increased bacterial count in peritoneal cavity and decreased recruitment of neutrophils and macrophages to the peripheral cavity were observed in Klotho-CLP mice. Serum concentration of IL-6, TNF, and IL-10 were signifi cantly higher in Klotho-CLP mice than those in the WT-CLP mice. A dramatically increased caspase 3 positive proportion in Klotho-CLP mice was observed in both fl ow cytometric and immunohistological analysis (P <0.01). 2. Cho JS, et al.: IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest 2010, 120:1762-1773. 2. Cho JS, et al.: IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest 2010, 120:1762-1773. P1 Impaired innate and adaptive immunity of accelerated-aged Klotho mice in sepsis Bacterial colony count in peritoneal lavage was also analyzed. Prevalence of TLR4 single nucleotide polymorphisms (ASP299GLY, THR399ILE) in healthy subjects and septic patients, and association with outcome T Mohovic1, R Salomao2, E Nogueira2 1Albert Einstein Hospital, São Paulo, Brazil; 2UNIFESP, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P3 (doi: 10.1186/cc10610) T Mohovic1, R Salomao2, E Nogueira2 Conclusion Poor survival in Klotho-septic mice may be associated with impaired bacterial clearance with decreased recruitment of neutrophils/macrophages in peritoneal cavity, elevated cytokines in serum, and increased apoptosis in thymus and spleen, following to impaired innate and adaptive immunity. Introduction Our study aimed to determine the prevalence of functional SNPs (Asp299Gly, Thr399Ile) of TLR4 receptors, in healthy volunteers and septic patients in a Brazilian population and to correlate the presence of these polymorphisms in septic patients with clinical outcome.i P2 P2 IL-17A rs1974226 GG genotype is associated with increased susceptibility to Gram-positive infection and mortality of severe sepsis T Nakada, J Russell, J Boyd, K Walley University of British Columbia, Vancouver, Canada Critical Care 2012, 16(Suppl 1):P2 (doi: 10.1186/cc10609) Methods We verifi ed the presence of polymorphisms ASP299GLY, THR399 ILE by PCR-restriction fragment length polymorphism followed by digestion with enzymes NcoI for SNP 299 and HinfI for SNP399 followed by electrophoresis for identifi cation of alleles.if yi Results We observed a statistically signifi cant diff erence between the genotypes of the Thr399Ile polymorphism and respiratory dysfunction, indicating a higher frequency than wild-type genotype in subjects with respiratory dysfunction than those without this condition (P = 0.001). We also observed a statistically signifi cant diff erence between genotype groups formed by the Asp299Gly and Thr399Ile polymorphisms and respiratory dysfunction more often featuring group 299Selv/399Selv grupo299Het/399Het and less frequently in individuals with respiratory dysfunction than those without this condition (P = 0.003). Critical Care 2012, 16(Suppl 1):P2 (doi: 10.1186/cc10609) Introduction IL-17A plays a key role in host defense against microbial infection including Gram-positive bacteria. Genetic factors contribute © 2010 BioMed Central Ltd © 2012 BioMed Central Ltd © 2010 BioMed Central Ltd © 2012 BioMed Central Ltd S2 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Our study shows for the fi rst time an assessment of the prevalence of polymorphisms of TLR4 Asp299Gly and Thr399Ile con- sidering its cosegregation in healthy individuals and septic patients. References 1. O’Dwyer et al.: The occurrence of severe sepsis and septic shock are related to distinct patterns of cytokine gene expression. Shock 2006, 26:544-550. Methods We used diff erent wild-type mice strains (BALB/c, C57BL/6,129SV), and KO mice lacking diff erent leukocytes subset rag2–/–, rag2gc–/–, cd3e–/–, μ–/–, il-15–/– and Ja18–/–. We used an ex vivo model consisting of intravenous injection of LPS 20 hours prior to an in vitro stimulation of AM, peritoneal macrophages and monocytes with LPS. We pretreated the wild-type mice with anti-cytokines antibodies, and KO mice with B cells and NK cells adoptive transfer.i 1. O’Dwyer et al.: The occurrence of severe sepsis and septic shock are related to distinct patterns of cytokine gene expression. Shock 2006, 26:544-550. 2. O’Dwyer et al.: The human response to infection is associated with distinct patterns of interleukin 23 and interleukin 27 expression. Intensive Care Med 2008, 34:683-691. 2. O’Dwyer et al.: The human response to infection is associated with distinct patterns of interleukin 23 and interleukin 27 expression. Intensive Care Med 2008, 34:683-691. Results We confi rmed the absence of AM tolerance to endotoxin in all the strain of wild-type mice. Inhibiting either GM-CSF or INFγ in vivo at homeostasis led to a decrease in TNF production by AM during the in vitro stimulation by LPS, suggesting the involvement of these cytokines in the prevention of tolerance within the lungs. The fact that AM from rag2–/–, rag2gc–/–, μ–/– could be tolerated, the fact that adoptive transfer of B lymphocytes in these defi cient mice restores the wild-type response, and the presence of INFγ mRNA in the lungs at homeostasis in wild-type mice and before and after adoptive B-lymphocyte transfer Prevalence of TLR4 single nucleotide polymorphisms (ASP299GLY, THR399ILE) in healthy subjects and septic patients, and association with outcome And that septic patients who develop respiratory dysfunction have more presence and genotypes 399Selv 299Selv/399Selv and less the presence of genotype 299Het/399Het, featuring a protective eff ect of the polymorphism Thr399Ile. changes in aged sepsis is still unclear. The purpose of this study was to clarify the immunological changes in sepsis of aged patients. Conclusion Our study shows for the fi rst time an assessment of the prevalence of polymorphisms of TLR4 Asp299Gly and Thr399Ile con- sidering its cosegregation in healthy individuals and septic patients. Methods Forty-four septic patients and 48 gender-matched healthy volunteers were prospectively enrolled in the study, which included the following investigations: (1) The SOFA score and clinical outcome were compared between adult sepsis (<65 years of age) and older adult sepsis (≥65 years of age). (2) Blood samples were collected from septic and control volunteers. Separated peripheral blood mononuclear cells were stained with CD4, CD8, programmed death-1 (PD-1), CD28, and CD62L antibodies and analyzed by fl ow cytometry, and serum was used to measure cytokine concentrations by using multiplex bead assay. Values were compared among four groups: normal adult (<65 years of age), normal older adult (≥65 years of age), adult sepsis (<65 years of age), and older adult sepsis (≥65 years of age) groups. g g g y p p And that septic patients who develop respiratory dysfunction have more presence and genotypes 399Selv 299Selv/399Selv and less the presence of genotype 299Het/399Het, featuring a protective eff ect of the polymorphism Thr399Ile. p y p References 1. Lorenz E, Mira JP, Cornish KL, Arbour NC, Schwartz DA: A novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun 2000, 68:6398-6401. 1. Lorenz E, Mira JP, Cornish KL, Arbour NC, Schwartz DA: A novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun 2000, 68:6398-6401. p y 2. Janeway CA, Jr, Medzhitov R: Introduction: the role of innate immunity in the adaptive immune response. Semin Immunol 1998, 10:349-350. 2. Janeway CA, Jr, Medzhitov R: Introduction: the role of innate immunity in the adaptive immune response. Semin Immunol 1998, 10:349-350. g y g g Results (1) No diff erences in SOFA scores were observed between adult sepsis (n = 19, 39 years) and older adult sepsis (n = 25, 78 years), but 3-month survival in older adult sepsis was signifi cantly decreased compared with that in adult sepsis (36% vs. 4%, P <0.05). (2) Population of CD8+ T cells in normal older adults was signifi cantly less than that in normal adults (1.5×105 vs. 5.7×104/ml, P <0.01), and percentage of PD-1+CD8+ T cells in the older adult sepsis group was signifi cantly greater than that in the normal older adult group (40% vs. 29%, P <0.01). Population of CD4+, CD62L+CD4+, and CD28+CD4+ T cells in the older adult sepsis group was signifi cantly less than that in the normal older adult group (n = 26, 80 years) (1.8×105 vs. 5.9 ×104/ml, 1.6×105 vs. 5.4×104/ml, and 1.6×105 vs. 4.4×104/ml, respectively; P <0.01); however, these values did not diff er between the adult sepsis and normal adult (n = 22, 39 years) groups. Serum IL-12 level in older adult sepsis was increased when compared with that in the other three groups (P <0.01). Conclusion Poor prognosis in older adult sepsis may be related to both preexisting decrease of CD8+ T cells with aging and loss of CD4+ T cells with sepsis. Modelling immune responses in sepsis R Grealy1, M White2, M O’Dwyer2, P Stordeur3, DG Doherty1, R McManus1, T Ryan2 R Grealy1, M White2, M O’Dwyer2, P Stordeur3, DG Doherty1, R McManus1, T Ryan2 R Grealy1, M White2, M O’Dwyer2, P Stordeur3, DG Doherty1, R McManus1, T Ryan2 y 1Trinity College Dublin, Ireland; 2St James’s Hospital, Dublin, Ireland; 3Hopital d’Erasme, Bruxelles, Belgium y 1Trinity College Dublin, Ireland; 2St James’s Hospital, Dublin, Ireland; 3Hopital d’Erasme, Bruxelles, Belgium Critical Care 2012, 16(Suppl 1):P4 (doi: 10.1186/cc10611) Critical Care 2012, 16(Suppl 1):P4 (doi: 10.1186/cc10611) Introduction The onset and evolution of the sepsis syndrome in humans is modulated by an underlying immune suppressive state [1,2]. Signalling between immune eff ector cells plays an important part in this response. The objective of this study was to investigate peripheral blood cytokine gene expression patterns and serum protein analysis in an attempt to model immune responses in patients with sepsis of varying severity. We hypothesised that such immunologic profi ling could be of use in modelling and prediction of outcomes in sepsis in addition to the evaluation of future novel sepsis therapies. Methods A prospective observational study in a mixed medical/ surgical ICU and general wards of a large academic teaching hospital was undertaken. Eighty ICU patients with a diagnosis of severe sepsis, 50 patients with mild sepsis (bacteraemia not requiring ICU admission) and 20 healthy controls were recruited. Gene expression analysis by qPCR for INFγ, TNFα, IL-2, IL-7, IL-10, IL-23, IL-27 on peripheral blood mononuclear cells (PBMCs) and serum protein analysis for IL-6 was performed. Multivariate analysis was used to construct a model of gene expression based on cytokine copy numbers alone and in combination with serum IL-6 levels. Homeostatic pulmonary microenvironment is responsible for alveolar macrophages resistance to endotoxin tolerance F Philippart1, C Fitting2, B Misset1, J Cavaillon2 1Groupe Hospitalier Paris Saint Joseph, Paris, France; 2Institut Pasteur de Paris, Paris, France Critical Care 2012, 16(Suppl 1):P6 (doi: 10.1186/cc10613) Homeostatic pulmonary microenvironment is responsible for alveolar macrophages resistance to endotoxin tolerance F Philippart1, C Fitting2, B Misset1, J Cavaillon2 Homeostatic pulmonary microenvironment is responsible for alveolar macrophages resistance to endotoxin tolerance F Philippart1, C Fitting2, B Misset1, J Cavaillon2 pp g 1Groupe Hospitalier Paris Saint Joseph, Paris, France; 2Institut Pasteur de Paris, Paris, France Critical Care 2012, 16(Suppl 1):P6 (doi: 10 1186/cc10613) Critical Care 2012, 16(Suppl 1):P6 (doi: 10.1186/cc10613 Introduction Endotoxin tolerance (ET) is a modifi cation of immune response to a second challenge with lipopolysaccharide (LPS), which results in a decreased production of proinfl ammatory cytokines, and is considered partly responsible for the susceptibility to infectious processes in hospitalized patients [1]. We previously observed an absence of ET of alveolar macrophages (AM) to LPS in an ex vivo murine model of endotoxin tolerance [2]. We hypothesized that this singularity could be mediated by granulocyte–macrophage colony- stimulating factor (GM-CSF) (known to be predominantly produced by type II pneumocytes) and interferon-gamma (INFγ), two cytokines known to prevent the occurrence of ET [3]. The objectives were to confi rm the absence of tolerance of AM to LPS and to assess the respective roles of GM-CSF and INFγ in this phenomenon and the cellular origin of INFγ.f Results Sepsis was characterised by decreased IL-2, IL-7, IL-23, INFγ and greater TNFα, IL-10 and IL-27 gene expression levels compared to controls. Severe sepsis diff ered from mild sepsis by a decreased INFγ and increased IL-10 gene expression (P <0.0001). A composite cytokine gene expression score diff erentiated controls from mild sepsis and mild sepsis from severe sepsis (P <0.0001). A model combining these cytokine gene expression levels and serum IL-6 protein levels distinguished sepsis from severe sepsis with an ROC value of 0.89. Conclusion Accurate modelling of patient response to infection is possible using peripheral blood mononuclear cell gene expression and serum protein analysis. Molecular biological techniques provide a robust method of such profi ling. This approach may be used to evaluate novel sepsis therapies. References P7 In vivo natural killer and natural killer T-cell depletion aff ects mortality in a murine pneumococcal pneumonia sepsis model E Ch i ki1 E Di 1 SM O l2 A Pi iki3 DI D i i3 DP C 3 p gg M Georgitsi3, N Malisiovas1, P Nikolaidis1, EJ Giamarellos-Bourboulis3 1Aristotle University of Thessaloniki, Greece; 2Memorial Hospital of RI, Alpert School of Medicine of Brown University, Providence, RI, USA; 3University of Athens, Medical School, Athens, Greece Figure 2 (abstract P7). Kaplan–Meier survival curve of groups B and C. Critical Care 2012, 16(Suppl 1):P7 (doi: 10.1186/cc10614) Critical Care 2012, 16(Suppl 1):P7 (doi: 10.1186/cc10614) Conclusion Our study has shown that NK cells appear to contribute to mortality in pneumococcal pneumonia. More research is needed to explore their role in host response to bacterial infection and sepsis. Introduction Apart from macrophages and neutrophils, natural killer (NK) and natural killer T (NKT) cells have been found to play a role in the early stages of bacterial infection. In this study, we investigated the role of NK and NKT cells in host defense against Streptococcus pneumoniae, using a murine pneumococcal pneumonia sepsis model. Our hypothesis was that NK and NKT cells play an immune-regulatory role during sepsis and thus in vivo depletion of those cell populations may aff ect mortality. P8 Mobilization of hematopoietic and nonhematopoietic stem cell subpopulations in sepsis: a preliminary report. T Skirecki1, U Zielińska-Borkowska1, M Złotorowicz1, M Złotorowicz1, J Kawiak2, G Hoser1 1Medical Center of Postgraduate Education, Warsaw, Poland; 2Polish Academy of Science, Warsaw, Poland Critical Care 2012, 16(Suppl 1):P8 (doi: 10.1186/cc10615) f Methods We used four groups of C57BL/6 mice (A, B, C and D, n = 10 mice/group). Animals were infected intratracheally with 50 μl of S. pneumoniae suspension (106 cfu). Twenty-four hours prior to bacterial inoculation, Group A received 50 μl of anti-asialoGM1 rabbit polyclonal antibody (Wako Chemicals GmbH, Neuss, Germany) intravenously (i.v.) to achieve in vivo NK cell inactivation; in Group B, NKT cell depletion was performed by targeting the CD1d receptor using 2 mg/kg of the monoclonal antibody anti-CD1d, clone 1B1 (BD Pharmingen, San Diego, CA, USA) i.v.; Group C (control) received an equivalent amount of isotype antibody control (nonspecifi c Ig). Group D received sham intratracheal installation of normal saline. Animals were observed daily for 7 days and deaths were recorded. The survival analysis was plotted using the Kaplan–Meier method and diff erences in survival between groups were compared with the log-rank test. Introduction Sepsis and septic shock lead to the multiorgan damage by extensive release of infl ammatory mediators. Regenerative mechanisms include such regimens as stem cells which diff erentiate towards specifi c tissues. Ratajczak MZ, et al.: Leukemia 2010, 24:1667-1675. 5 Decreased peripheral CD4+/CD8+ lymphocytes and poor prognosis in aged sepsis g p S Inoue, K Utsunomiya-Suzuki, S Morita, T Yamagiwa, S Inokuchi Tokai University, Kanagawa, Japan g S Inoue, K Utsunomiya-Suzuki, S Morita, T Yamagiwa, S Inokuchi Tokai University, Kanagawa, Japan y g p Critical Care 2012, 16(Suppl 1):P5 (doi: 10.1186/cc10612) Introduction Aging is a signifi cant factor and is associated with a poor prognosis in sepsis; however, the mechanism of immunological Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S3 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 2 (abstract P7). Kaplan–Meier survival curve of groups B and C. in KO mice demonstrated the involvement of these cells in the wild- type phenotype.i Conclusion We confi rm the resistance of AM to endotoxin tolerance. Both GM-CSF and INFγ within the lung microenvironment at homeostasis are involved in this phenomenon. B lymphocytes play a key role in the local expression of INFγ. f g 3. Adib-Conquy M, et al.: J Biol Chem 2002, 277:27927-27934. g 3. Adib-Conquy M, et al.: J Biol Chem 2002, 277:27927-27934. g 3. Adib-Conquy M, et al.: J Biol Chem 2002, 277:27927-27934. P7 In vivo natural killer and natural killer T-cell depletion aff ects mortality in a murine pneumococcal pneumonia sepsis model E Ch i ki1 E Di 1 SM O l2 A Pi iki3 DI D i i3 DP C 3 Also, in the course of the systemic infl ammation the disruption of various regulatory axes occurs, including chemokines (VEGF, HGF) and complement proteins (C5a,C3a). Among other functions these axes maintain stem cell circulation and recruitment [1]. The aim of the study was to evaluate circulating stem cells in the peripheral blood of septic patients.i Results We found that in vivo NK cell depletion improved survival after pneumococcal pneumonia and sepsis in the group of mice that received the anti-asialoGM1 antibody when compared with animals that received nonspecifi c IgG antibody (P = 0.041) (Figure 1). Nevertheless, when NKT cell depletion was attempted, survival worsened compared to the control group; however, that diff erence did not reach statistical signifi cance (P = 0.08) (Figure 2). Methods Blood samples were obtained from fi ve patients with sepsis or septic shock on the second day after diagnosis. Blood from fi ve healthy volunteers served as control. Samples were stained with the panel of antibodies against: CD45, lineage markers (Lin), CD34, CD133, VEGFR2 and isotypic controls. Cells were analyzed by fl ow cytometry and the total cell count per milliliter was calculated. Results On the basis of cell surface phenotype the following stem cell subpopulations were distinguished: hematopoietic stem cells (HSCs) CD34+CD133+CD45+Lin–, endothelial progenitor cells (EPCs) CD34+CD133+VEGFR2+; and primitive nonhematopoietic stem cells. In the blood of septic patients we found: HSCs (5/5), median level 96/ml; EPCs (5/5), median 48/ml; and nonhematopoietic stem cells (4/5), median 48/ml. Whereas in the control group the results were as follow: HSCs (5/5), median level 644/ml; EPCs (5/5), median 70/ml; and nonhematopoietic stem cells (0/5). Two of fi ve patients died of septic shock. A trend to lower number of HSCs in nonsurvivors was observed. Conclusion Stem cells can be identifi ed phenotypically in the blood of septic patients and healthy volunteers. However, the circulating primitive nonhematopoietic stem cells could not be detected under physiological conditions. Furthermore, we suggest that stem cells analysis may have serve as prognostic tool in the future. Figure 1 (abstract P7). Kaplan–Meier survival curve of groups A and C. Acknowledgements Supported by EU Structural Funds, ‘Innovative Methods of Stem Cells Applications in Medicine’, Innovative Economy Operational Programme, POIG 01.02-00-109/09. Reference Figure 1 (abstract P7). Kaplan–Meier survival curve of groups A and C. Ratajczak MZ, et al.: Leukemia 2010, 24:1667-1675. P7 In vivo natural killer and natural killer T-cell depletion aff ects mortality in a murine pneumococcal pneumonia sepsis model E Ch i ki1 E Di 1 SM O l2 A Pi iki3 DI D i i3 DP C 3 S4 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Mean respective apoptosis of groups 0, A, B, C, D and E at 24 hours were 37.9%, 77.6%, 81.9%, 73.8%, 83.6% and 75.4%; and at 48 hours 78.5%, 79.4%, 77.7%, 78.2%, 81% and 84.9% (P <0.05 group 0 vs. others). Mean respective MFI of TREM-1 of groups 0, A, B, C, D and E at 24 hours were 2.4, 4.4, 3.4, 3, 3.2 and 3; and at 48 hours 2.7, 2.8, 2.8, 2.6, 2.8 and 2.7 (P <0.05 group 0 vs. others). Tissue cultures were sterile. Release of IL-17 was greater by splenocytes of group D (Figure 1). Conclusion Increased neutrophil apoptosis and TREM-1 expression and modulated IL-17 responses are found within burn injury. Figure 1 (abstract P10). Release of IL-17 by mice splenocytes in relation to the type of thermal injury. P9 Blunted IL-17 responses early after advent of multiple injuries M Paraschos1, M Patrani1, A Pistiki2, J Van der Meer3, M Netea3, E Giamarellos-Bourboulis2, K Mandragos1 1Korgialeneion Benakeion Hospital, Athens, Greece; 2University of Athens, Medical School, Athens, Greece; 3UMC St Radboud, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P9 (doi: 10.1186/cc10616) Figure 1 (abstract P10). Release of IL-17 by mice splenocytes in relation to the type of thermal injury. Introduction To defi ne the impact of multiple injuries without the presence of sepsis in IL-17 responses. Methods A total of 32 patients and 17 healthy volunteers were enrolled. All patients were bearing: multiple injuries necessitating ICU admission with an injury severity score more than 16; and systemic infl ammatory response syndrome. Patients with infections upon ICU admission were excluded from the study. Heparinized venous blood was sampled within the fi rst 24 hours after ICU admission. Peripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation of whole blood over Ficoll. They were incubated for 5 days in RPMI 1640 supplemented with 2 mM glutamine and 10% FBS in the presence of 10 ng/ml lipopolysaccharide (LPS) of Escherichia coli O55:B5; of 5 μg/ml phytohemmaglutin (PHA); of 5×105 cfu/ml of heat- killed Candida albicans (HKCA), of Pseudomonas aeruginosa (HKPA) or of Staphylococcus aureus (HKSA). IL-17 was measured in supernatants by an enzyme immnunoassay. P7 In vivo natural killer and natural killer T-cell depletion aff ects mortality in a murine pneumococcal pneumonia sepsis model E Ch i ki1 E Di 1 SM O l2 A Pi iki3 DI D i i3 DP C 3 Results Mean respective apoptosis of groups 0, A, B, C, D and E at 24 hours were 37.9%, 77.6%, 81.9%, 73.8%, 83.6% and 75.4%; and at 48 hours 78.5%, 79.4%, 77.7%, 78.2%, 81% and 84.9% (P <0.05 group 0 vs. others). Mean respective MFI of TREM-1 of groups 0, A, B, C, D and E at 24 hours were 2.4, 4.4, 3.4, 3, 3.2 and 3; and at 48 hours 2.7, 2.8, 2.8, 2.6, 2.8 and 2.7 (P <0.05 group 0 vs. others). Tissue cultures were sterile. Release of IL-17 was greater by splenocytes of group D (Figure 1). Conclusion Increased neutrophil apoptosis and TREM-1 expression and modulated IL-17 responses are found within burn injury. y y Results Mean APACHE II score of patients was 14. Release of IL-17 by PBMCs of patients was signifi cantly lower compared to controls, as shown in Figure 1. P values refer to comparisons between controls and patients. p Conclusion The presented fi ndings show that early upon advent of multiple injuries IL-17 responses are blunted. This may corroborate with the susceptibility of patients for superinfections. P11 Insuffi cient autophagy relates to mitochondrial dysfunction, organ failure and adverse outcome in an animal model of critical illness J Gunst, I Derese, A Aertgeerts, EJ Ververs, A Wauters, G Van den Berghe, I Vanhorebeek Katholieke Universiteit Leuven, Belgium Critical Care 2012, 16(Suppl 1):P11 (doi: 10.1186/cc10618) Insuffi cient autophagy relates to mitochondrial dysfunction, organ failure and adverse outcome in an animal model of critical illness J Gunst, I Derese, A Aertgeerts, EJ Ververs, A Wauters, G Van den Berghe, I Vanhorebeek Figure 1 (abstract P9). Release of IL-17 by PBMCs of controls and of patients. Introduction Increasing evidence implicates mitochondrial dysfunction in the pathogenesis of critical illness-induced multiple organ failure. We previously demonstrated that prevention of hyperglycemia limits mitochondrial damage in vital organs [1,2], thereby reducing morbidity and mortality [3]. We now hypothesize that inadequate activation of mitochondrial repair processes (mitochondrial clearance by autophagy, mitochondrial fusion and fi ssion, and biogenesis) may contribute to accumulation of mitochondrial damage, persistence of organ failure and adverse outcome of critical illness. Methods We addressed this hypothesis in a rabbit model of critical illness. First, we studied whether vital organ mitochondrial repair pathways are diff erentially aff ected in surviving and nonsurviving hyperglycemic animals, in relation to mitochondrial and organ function. Next, we investigated whether preventing hyperglycemia with insulin aff ects mitochondrial repair over time. We quantifi ed mRNA/protein levels of key players of these processes. Activities of respiratory chain complexes I to V were measured spectrophotometrically. Plasma transaminases and creatinine were measured as markers of liver, respectively kidney, dysfunction. P12 Modulation of mediators derived from whole blood or monocytic cells stimulated with lipopolysaccharide reduces endothelial cell activation A Schildberger, T Stoifl , D Falkenhagen, V Weber Danube University Krems, Austria Critical Care 2012, 16(Suppl 1):P12 (doi: 10.1186/cc10619) Introduction Modulation of infl ammatory mediators with specifi c or selective adsorbents may represent a promising supportive therapy for septic patients. The aims of this study were to modulate mediator concentrations from lipopolysaccharide (LPS)-stimulated whole blood or monocytic THP-1 cells with specifi c or selective adsorbents and to compare the infl uence on endothelial cell activation. Conclusion Our small sample of intensive care patients with a confi rmed A/H1N1 infection supports the scarce published data about the early immunological profi le of these patients. All our patients had a prominent lymphopenia with a most signifi cant decrease in CD4 and CD8 cells. Due to the number of patients in the season 2010/11 and the survival of all patients we could not analyse the relation of survival and the change in time of immunological profi le in this unique and probably already extinct group of patients. pl Methods Whole blood or THP-1 cells (1×106 cells per ml medium containing 10% human plasma) [1] were stimulated with 10 ng/ml LPS from Escherichia coli for 4 hours. Mediator modulation was performed with either a specifi c adsorbent for TNFα which was based on sepharose particles functionalized with anti-TNFα antibodies, or with a selective albumin-coated polystyrene divinylbenzene copolymer (PS-DVB) [2]. Human umbilical vein endothelial cell (HUVEC) activation was monitored for 15 hours by measuring secretion of IL-6 and IL-8, as well as surface expression of the adhesion molecules ICAM-1 and E-selectin. Results Conditioned media derived from whole blood (CMB) or THP- 1 cells (CMT) both contained approximately 1,300 pg/ml TNFα which is known to be an important stimulator for HUVEC [1,2]. However, CMB led to a signifi cantly higher HUVEC activation as compared to CMT, as indicated by increased secretion of IL-6 and IL-8 (IL-6: 52,000 vs. 2,000 pg/ml; IL-8: 295,000 vs. 43,000 pg/ml), as well as signifi cantly increased E-selectin surface expression (50 vs. 12 mean fl uorescence intensity for CMP and CMT, respectively). P12 P12 Modulation of mediators derived from whole blood or monocytic cells stimulated with lipopolysaccharide reduces endothelial cell activation A Schildberger, T Stoifl , D Falkenhagen, V Weber Danube University Krems, Austria Critical Care 2012, 16(Suppl 1):P12 (doi: 10.1186/cc10619) P13 P13 A/H1N1 infection: immunological parameters in ICU patients I Zykova, P Sedlák, T Zajíc, A Vitouš, F Stejskal Regional Hospital Liberec, Czech Republic Critical Care 2012, 16(Suppl 1):P13 (doi: 10.1186/cc10620) P10 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S5 Time of course CD64, a leukocyte activation marker, during extracorporeal circulation S Djebara, P Biston, F Emmanuel, A Daper, M Joris, P Cauchie, M Piagnerelli CHU Charleroi, Belgium Introduction CD64 is a high-affi nity leukocyte receptor for the Fc portion of IgG [1]. As CD64 expression on neutrophil cells (PMNs) is upregulated specifi cally after bacterial stimulation, it could be used to discriminate infl ammatory states from bacterial infections [1-3]. The objective was a comparison of the time course of CD64 expression on PMN cells between patients undergoing cardiac surgery with extracorporeal circulation (ECC) with septic patients. f g Conclusion Infl ammatory mediator modulation with specifi c or selective adsorbents reduces endothelial cell activation and thus may support the development of new therapies for sepsis. References Methods Prospective study realized in the ICU of CHU Charleroi (Belgium). Thirty-nine patients scheduled for a cardiac surgery with ECC (coronary, valvular or mixed surgery) (ECC group) and 11 patients with severe sepsis or septic shock (septic group) were included. The CD64 expression on PMNs was quantifi ed by the hematologic Cell Dyn Sapphire method (Abotte US) before T0, at ICU admission (T1) and postoperatively on day 1 (T2) and day 5 (T3) for the ECC group and on days 0, 1 and 5 for the septic group. Values are expressed as median (25th to 75th) percentiles Results Fifty patients were included among which 39 in the ECC group (nine valvular, 20 coronary artery bypass grafting and 10 mixed surgery). As expected, the infl ammatory parameters were signifi cantly increased in septic patients compared to the ECC group except on day 1. Schildberger et al.: Innate Immun 2010, 16:278-287. 2. Schildberger et al.: Blood Purif 2011, 32:286-295. 1. Schildberger et al.: Innate Immun 2010, 16:278-287. 2. Schildberger et al.: Blood Purif 2011, 32:286-295. P10 P10 Apoptosis of neutrophils, expression of TREM-1 on neutrophils and IL-17 responses in experimental burn in injury are related to the type and time of burn exposure A Alexis1, D Carrer1, A Pistiki1, K Louis1, D Droggiti1, J Van der Meer2, M Netea2, E Giamarellos-Bourboulis1 1University of Athens, Medical School, Athens, Greece; 2UMC St Radboud, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P10 (doi: 10.1186/cc10617) P10 Apoptosis of neutrophils, expression of TREM-1 on neutrophils and IL-17 responses in experimental burn in injury are related to the type and time of burn exposure p y y y Results In the liver and kidney of nonsurviving hyperglycemic rabbits, molecular markers of insuffi cient autophagy were evident, including accumulation of p62 protein (but no increase of p62 mRNA) and decreases in the autophagosome-associated protein LC3-II (microtubule-associated protein light chain 3). These changes were less prominent in surviving animals and correlated with impaired mitochondrial and organ function. In contrast, key players in mitochondrial fusion, fi ssion or biogenesis were not aff ected by survival status. Therefore, we focused on autophagy to study the impact of preventing hyperglycemia. Both after 3 and 7 days of illness, autophagy was better preserved in normoglycemic than in hyperglycemic rabbits, which correlated strongly with improved mitochondrial and organ function. Introduction To defi ne infl ammatory responses in experimental burn injury in relation with the type and time of burn exposure. j y yp p Methods Burn injury was induced in 110 C57/B6 male mice after time exposure of their back as follows: group 0, sham; group A, 60°C for 60 seconds; group B, 60°C for 45 seconds and 4°C for 45 seconds; group C, 75°C for 60 seconds; group D, 90°C for 5 seconds; and group E, 4°C for 45 seconds and 60°C for 45 seconds. Mice were sacrifi ced at 24 and 48 hours. Tissues were cultured and splenocytes were isolated and stimulated with heat-killed Staphylococcus aureus and Candida albicans for 5 days for release of IL-17. Neutrophil apoptosis and expression of TREM-1 were determined after staining for ANNEXIN-V, PI and anti- TREM-1-PE and fl ow cytometry analysis. Conclusion These fi ndings put forward insuffi cient autophagy as a potentially important contributor to mitochondrial and organ dysfunction in critical illness, and open perspectives for therapies that activate autophagy during critical illness. References Results In season 2010/11 only six patients with a confi rmed A/H1N1 infection required admission to intensive care (47% of all patients with a confi rmed A/H1N1 infection admitted to our hospital). All patients required ventilation. Median APACHE II score was 18.2. Median ICU stay was 18.5 days. Median number of ventilator days was 14. No patient died, both 28-day and 3-month mortality was 0%. Total leukocyte count was without substantial diff erences, but there was a prominent lymphopenia at the time of admission (0.05 to 0.22% of total leukocyte count) as has been described in similar studies. All lymphocyte populations were decreased but a most prominent decrease was in CD4 (T-helpers) and CD8 (T-suppressors), CD19 (B-lymphocytes) and NK cells were less decreased. Comparison of the admission sample and the second sample taken 21 days after admission: both CD4 and CD8 were most decreased at admission, immunoregulatory index had a shift to positive values in the admission sample. References 1. Vanhorebeek I, et al.: Lancet 2005, 365:53-59. 2. Vanhorebeek I, et al.: Kidney Int 2009, 76:512-520. 3. Van den Berghe G, et al.: N Engl J Med 2001, 345:1359-1367. 1. Schildberger et al.: Innate Immun 2010, 16:278-287. 2. Schildberger et al.: Blood Purif 2011, 32:286-295. i References 1. Shapovalov KG, et al.: Immunological and bacteriological monitoring of patients with pneumonia and infl uenza A/H1N1 infection. Zh Mikrobiol Epidemiol Immunobiol 2011, 1:79-82. 2. Kim JE, et al.: CD4+/CD8+ T lymphocytes imbalance in children with severe 2009 pandemic infl uenzaA/H1N1 pneumonia. Korean J Pediatr 2011, 54:207-211. P12 Modulation of mediators derived from whole blood or monocytic cells stimulated with lipopolysaccharide reduces endothelial cell activation Adsorption of infl ammatory mediators from the conditioned medium of whole blood or THP-1 cells either with the specifi c TNFα adsorbent or with the selective PS-DVB beads resulted in decreased endothelial cell activation, as shown by statistically signifi cant reduction of IL-6 and IL-8 secretion from HUVEC, as well as statistically signifi cant reduction of surface expression of the adhesion molecules ICAM-1 and E-selectin. The reduction of HUVEC activation was more pronounced when applying the selective adsorbent showing that the modulation of more than one cytokine is more eff ective than removing TNFα alone. fl fi Acknowledgements Our study was supported by a grant from Scientifi c Board of Regional Hospital Liberec. Acknowledgements Our study was supported by a grant from Scientifi c Board of Regional Hospital Liberec. References Scientifi c Board of Regional Hospital Liberec. References f Oral neutrophil quantitation in patients undergoing elective cardiopulmonary bypass Results Red blood cells preferentially metabolized pyruvate (ninefold increase) compared to heart (1.2-fold increase) or liver (–2.1-fold decrease), and were a net lactate source (2.1-fold increase). Glycolytic intermediates increased in the heart, but decreased in red blood cells, while TCA intermediates decreased in the heart and amino acids increased in the liver. Under the hypoglycemic conditions of the animal model, red blood cells were found to accumulate glycerol-3-phosphate (red cell glycerol fl ux remained normal) and 2,3BPG following C13- pyruvate injection. ATP was stable in the heart, but decreased in the liver and red blood cells. Echocardiography revealed a transient recovery of left ventricular function that correlated with shifts in red blood cell metabolism. cardiopulmonary bypass ME Wilcox1, P Perez2, C DosSantos3, M Glogauer1, E Charbonney3, A Duggal2, S Sutherland2, G Rubenfeld2 1University of Toronto, Canada; 2Sunnybrook Health Sciences Centre, Toronto, Canada; 3St Michael’s Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P15 (doi: 10.1186/cc10622) ME Wilcox1, P Perez2, C DosSantos3, M Glogauer1, E Charbonney3, A Duggal2, S Sutherland2, G Rubenfeld2 1University of Toronto, Canada; 2Sunnybrook Health Sciences Centre, Toronto, Canada; 3St Michael’s Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P15 (doi: 10.1186/cc10622) Introduction Recent research suggests that the oral cavity may provide an early opportunity to monitor the innate immune system; an oral rinse assay was found to be a reliable predictor of bone marrow engraftment and neutrophil recovery in patients undergoing bone marrow transplantation [1]. Multiorgan failure may be mediated by neutrophil extravasation and aggregation [2] in highly infl ammatory states, such as cardiopulmonary bypass (CPB). The objective of this novel pilot study was to determine whether the kinetics of oral neutrophil recovery post- CPB surgery refl ect systemic immune activation. Conclusion Metabolic investigation of diff erent septic tissues revealed shifts in metabolism between organs, suggesting that sepsis induces complex metabolic shifts in response to changing nutrient availability and cell function; moreover, enhancing red blood cell metabolism may be benefi cial to depressed organ function during the onset of endotoxemia. yl y Methods Samples [3] from four-quadrant mucosal swabs and oral cavity rinses were obtained from 41 patients undergoing on-CPB elective cardiac surgery preoperatively (t–1) and postoperatively upon arrival to the CVICU (t0), at 12 to 18 hours (t1), and on day 3 (t2). Oral neutrophil counts (/ml) were determined by hemacytometry and validated by an electronic cell counter. P13 A/H1N1 infection: immunological parameters in ICU patients I Zykova, P Sedlák, T Zajíc, A Vitouš, F Stejskal Regional Hospital Liberec, Czech Republic Critical Care 2012, 16(Suppl 1):P13 (doi: 10.1186/cc10620) Introduction The outbreak of infl uenza A/H1N1 2009 had infl uenced ICUs all over the world. In the season 2009/10 we admitted to intensive care 13 patients with A/H1N1 infection in our regional hospital. In the next season 2010/11 another outbreak of A/H1N1 infection was predicted. We decided to study the immunological profi les of these patients and its development in time. Table 1 (abstract P14) ECC Sepsis P value T0 0.8 (0.6 to 1.08) 3.24 (1.9 to 7.8) <0.001 T1 0.9 (0.6 to 1.14)† Not available T2 1.3 (0.77 to 1.8),* 4.4 (2.63 to 6.7)* <0.001 T3 1.1 (0.74 to 1.4)‡ 1.3 (0.74 to 1.4) 0.16 P <0.05 T2 vs. T3, T2 vs. T0, †T2 vs. T1, ‡T3 vs. T0. ANOVA tests. Methods We conducted a prospective study on patients admitted to our hospital with A/H1N1 infection in the season 2010/11. The diagnosis was confi rmed by RT-PCT from nasopharyngeal smear or bronchoalveolar lavage in all patients. Immunological parameters (leukocyte count, lymphocyte count, CD19, CD4, CD8, immunoregulatory index, NK cells) were analysed on admission and 3 weeks after admission. P <0.05 T2 vs. T3, *T2 vs. T0, †T2 vs. T1, ‡T3 vs. T0. ANOVA tests. S6 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 5 (for example, CRP: 0.2 (0.1 to 0.6) vs. 12.5 (5.7 to 26.9) mg/dl; WBC 6.5 (5.2 to 8.7) vs. 19.5 (12 to 20.5) 103/mm3; for respectively ECC and septic group at T0, P <0.001). The CD64 expression increased signifi cantly in both groups but index values were lower in the ECC compared to the septic group except on T3 (Table 1). changes in metabolism and the metabolic interaction between tissues and red blood cells are not well understood. The objective of this study was to assess changes in intermediary metabolism during the onset of an animal model of sepsis by determining glycolytic, TCA and PPP metabolites, amino acids and ATP levels in heart, liver and red blood cells. p g p p Conclusion ECC modifi es the infl ammatory parameters, including the expression of the CD64 on PMNs but this one presents the best specifi city to diagnose an infection. Thus, CD64 expression could be proposed as a promising marker in the early diagnosis of the infection. References Methods C57BL/6 mice (30 to 35 g) were injected intraperitoneally with lipopolysaccharide (LPS, 40 mg/kg) to induce endotoxemia. Oral neutrophil quantitation in patients undergoing elective cardiopulmonary bypass Concurrent blood samples were collected for measurement of IFNα, interleukins (IL-1β, IL-6, IL-8 and IL-10), chemokine C-C motif ligand 4 (CCL-4) and Th1 and Th2 cytokines using a 10-plex human cytokine mediator panel. Continuous variables were summarized with means (standard deviation). Preoperative and postoperative oral neutrophil counts were compared using paired t tests.i Acknowledgements Supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, Global COE Program. AMP-activated protein kinase controls liposaccharide-induced hyperpermeability P13 A/H1N1 infection: immunological parameters in ICU patients I Zykova, P Sedlák, T Zajíc, A Vitouš, F Stejskal Regional Hospital Liberec, Czech Republic Critical Care 2012, 16(Suppl 1):P13 (doi: 10.1186/cc10620) Six hours post LPS, C13-pyruvate (a key intermediate metabolite) was administered subcutaneously for fl uxome analysis of intermediate metabolites. At 20, 40 and 60 minutes, heart, liver and red blood cells were collected and stored at –80°C. Labeled metabolites were measured using capillary electrophoresis–mass spectrometry, quantifi ed by calculating the AUC/t0–60 and expressed relative to control. Heart function was monitored by echocardiography. 1. Ioan-Fascinay A, et al.: Immunity 2002, 16:391-402. 1. Ioan-Fascinay A, et al.: Immunity 2002, 16:391-402. 1. Ioan-Fascinay A, et al.: Immunity 2002, 16:391-402. 2. Nuutila J, et al.: J Immunol Methods 2007, 328:189-200. 3. Qureshi SS, et al.: Clin Exp Immunol 2001, 125:258-265. 2. Nuutila J, et al.: J Immunol Methods 2007, 328:189-200. 3. Qureshi SS, et al.: Clin Exp Immunol 2001, 125:258-265. p References 1. Cheretakis C, et al.: Bone Marrow Transplant 2005, 36:227-232. 2. Fung YL, et al.: J Crit Care 2008, 23:542-549. 3. Wright DG, et al.: Blood 1986, 67:1023-1030. y p y Methods Sepsis was induced by intraperitoneal injection of liposaccharide, 10 mg/kg (LPS). Alpha-1 AMPK knockout mice (α1KO) were compared with wild-type. Vascular permeability was characterized by Evans blue extravasation. Infl ammatory cytokine mRNA expression was determined by qPCR analysis. Left ventricular mass was assessed by echocardiography. In addition, to emphasize the benefi cial role of AMPK on heart vascular permeability, AMPK activator (acadesine) was administered to C57Bl6 mice before LPS injection. The ANOVA test with Bonferroni’s post hoc test and the log-rank test were used. P <0.05 was considered as signifi cant. AMP-activated protein kinase controls liposaccharide-induced hyperpermeability D Castanares-Zapatero1, M Overtus2, D Communi3, M Horckmans3, L Bertrand2, C Oury4, C Lecut4, P Laterre1, S De man2, C Sommereyns2, S Horman2, C Beauloye2 Results Patients were 65 (10.6) years old; 78% male; 51% had signifi cant co-morbidities (25% diabetes); 54% took a statin; APACHE II score was 22 (4.4); and multiorgan dysfunction score (MODS) was highest on hospital day 1 (6.2; 2.2). Mean delta oral neutrophil count by oral swab (between t–1 and t0) was 1.7×106 (2.0×106). A signifi cant diff erence was seen in the absolute neutrophil counts (oral swab) between t–1 (1.7×106 (1.3×106)) and t0 (3.4×106 (2.7×106); P <0.001), but not between t–1 and t1 (2.0×106 (1.7×106); P = 0.14) or t2 (6.6×105 (1.1×106); P = 0.14). Similar results were obtained by oral cavity rinse. 1Université catholique de Louvain, Cliniques universitaires Saint Luc, Brussels, Belgium; 2Université catholique de Louvain, Institut de Recherche Expérimentale et Clinique, Brussels, Belgium; 3Université libre de Bruxelles, Institut de Recherche Interdisciplinaire en Biologie humaine et moléculaire, Brussels, Belgium; 4Université de Liège, Groupe Interdisciplinaire de Génoprotéomique Appliquée, Liège, Belgium Critical Care 2012, 16(Suppl 1):P17 (doi: 10.1186/cc10624) Introduction Organ dysfunction determines the severity of sepsis and is correlated to mortality. Endothelial increased permeability contri- butes to the development of organ failure. AMP-activated protein kinase (AMPK) has been shown to modulate cytoskeleton and could mediate endothelial permeability. Our hypothesis is that AMPK controls sepsis-induced hyperpermeability in the heart and is involved in septic cardiomyopathy. y y Conclusion An oral swab assay has the potential to provide rapid, risk- free, and early data on neutrophil activation and chemotactic defects in response to CPB, obviating the need for invasive sampling. This method could provide a new perspective on the systemic infl ammatory response in surgery, traumatic injury, burns, and sepsis. References P16 Keio University, Tokyo, Japan Keio University, Tokyo, Japan y y p Critical Care 2012, 16(Suppl 1):P16 (doi: 10.1186/cc10623) i Results Increased cardiac vascular permeability was observed in the LPS group in comparison to untreated animals (2.5% vs. 16%; P <0.05). The α1KO mice exhibited an increase vascular permeability after LPS injection in comparison to wild-type mice (41.5% vs. 16%; P <0.05). Introduction The systemic infl ammatory response to bacterial infection, or sepsis, results in a hypermetabolic state; yet, systemic metabolic S7 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 tremendous fi nancial costs. Recently, the primary metabolite of sevofl urane, hexafl uoro-2-propanol (HFIP), has been found to exert immunomodulatory properties attenuating infl ammatory response to lipopolysaccharides (LPS) in vitro [1]. We investigated whether HFIP attenuates plasma and tissue infl ammatory mediator expression in a rat model of endotoxic shock. α1KO animals had a signifi cant mortality increase after LPS injection (70% vs. 10%; P <0.05). LPS markedly induced the production of proinfl ammatory cytokines (TNFα, IL-1β, IL-6) that were signifi cantly higher in the α1KO animals. More importantly, LPS treatment leads to an increased left ventricular mass in the α1KO mice within 24 hours, suggesting the onset of edema. Finally LPS-induced vascular hyperpermeability was greatly reduced after AMPK activation by acadesine (13.2% vs. 40%; P <0.05). α1KO animals had a signifi cant mortality increase after LPS injection (70% vs. 10%; P <0.05). LPS markedly induced the production of proinfl ammatory cytokines (TNFα, IL-1β, IL-6) that were signifi cantly higher in the α1KO animals. More importantly, LPS treatment leads to an increased left ventricular mass in the α1KO mice within 24 hours, suggesting the onset of edema. Finally LPS-induced vascular hyperpermeability was greatly reduced after AMPK activation by acadesine (13.2% vs. 40%; P <0.05). Methods Thirty-two male Wistar rats were anesthetized, tracheoto- mized, and mechanically ventilated. The animals were randomly assigned to one of the following groups: (I) LPS group (n = 8), which received intravenous Escherichia coli endotoxin (1 mg/kg); (II) LPS/ HFIP group (n = 8), which was treated identically to the LPS group with the additional administration of HFIP (67 μg/kg over 30 minutes) after LPS injection. Control groups received Ringer’s lactate instead of LPS. General anesthesia was maintained with propofol. P18 Reduced expression of PPAR-β/δ limits the potential benefi cial eff ects of GW0742 during septic shock in atherosclerotic swine H Bracht1, F Simon1, J Matallo1, M Gröger1, O McCook1, A Seifritz1, M Georgieff 1, E Calzia1, P Radermacher1, A Kapoor2, C Thiemermann2 1University Clinic Ulm, Germany; 2William Harvey Research Institute, London, UK Critical Care 2012, 16(Suppl 1):P18 (doi: 10.1186/cc10625) ll y Results Plasma MCP-1 protein levels assessed 6 hours after LPS injection were increased by +5,192 ng/ml compared to baseline (R2 = 0.661, P <0.001). This increase in MCP-1 protein was attenuated by –48% in the LPS/HFIP group (+2,706 ng/ml to baseline, R2 = 0.661; P = 0.004). Similar results were found in BALF, in which HFIP decreased the LPS- induced raise in MCP-1 protein concentration by –62% (diff erence of 54 ng/ml, P = 0.034). LPS-stimulated animals had a +12% higher mean arterial blood pressure after 6 hours when treated with HFIP (78 mmHg vs. 67 mmHg, R2 = 0.684, P = 0.035). No signifi cant diff erences in lactate levels were observed. HFIP attenuated base defi cit in LPS-stimulated animals by 1 mmol/l (R2 = 0.522, P = 0.034). Introduction The PPAR-β/δ agonist GW0742 was shown to attenuate cardiac dysfunction in murine septic shock [1] and renal ischemia/ reperfusion injury in diabetic rats [2]. Since these data originate from unresuscitated models, we investigated the eff ects of GW0742 during long-term, resuscitated porcine septic shock. In order to assess the role of pre-existing cardiovascular morbidity we used animals with familial hypercholesteremia (11.1 (7.4; 12.3) vs. 1.4 (1.3; 1.5) mmol/l in a healthy strain; P <0.001) and consecutive, diet-induced ubiquitous atherosclerosis resulting in coronary artery disease [3], reduced glomerular fi ltration rate (76 (60; 83) vs. 103 (79; 120) ml/minute in healthy swine; P = 0.004) and presence of chronic histological kidney injury. y Conclusion Hexafl uoro-2-propanol attenuated LPS-induced infl amma- tory mediator secretion, the decrease in mean arterial blood pressure, and base defi cit. These results suggest that hexafl uoro-2-propanol may partly inhibit infl ammatory response, hypotension and the development of metabolic acidosis during endotoxic shock. Reference Methods Anesthetized and instrumented animals randomly received vehicle (n = 9) or GW0742 (n = 10; 0.03 mg/kg) at 6, 12, 18 hours after induction of fecal peritonitis [4]. Hydroxyethyl starch and noradrenaline were infused to maintain normotensive, hyperdynamic hemodynamics. P20f Eff ects of noradrenaline and lipopolysaccharide exposure on mitochondrial respiration in alveolar macrophages M Gröger, M Widman, J Matallo, P Radermacher, M Georgieff University of Ulm, Germany Critical Care 2012, 16(Suppl 1):P20 (doi: 10.1186/cc10627) y Conclusion Even early post-treatment with the PPAR-β/δ agonist GW0742 did not benefi cially infl uence acute kidney injury during long- term, resuscitated fecal peritonitis-induced septic shock in swine with pre-existing impairment of kidney function and histological damage. The lacking benefi cial eff ect of GW0742 was most likely due to the reduced expression of the PPAR-β/δ receptor. Introduction Mitochondrial respiratory capacity of immune cells seems to be impaired in septic patients [1]. On the other hand, the eff ects of catecholamines on mitochondrial function are still controversial [2] and may confound the genuine mitochondrial response to the septic event. In order to test if catecholamine therapy may infl uence the impairment of mitochondrial function in immune cells during sepsis, we measured mitochondrial respiration in cultured murine alveolar macrophages (AMJ2-C11) after 24 hours of incubation with noradrenaline and lipopolysaccharide (LPS). Acknowledgements Supported by the Else-Kröner-Fresenius-Stiftung. References 1. Kapoor et al.: Am J Respir Crit Care Med 2010, 182:1506-1515. 2. Collino et al.: Free Radic Biol Med 2011, 50:345-353. 3. Thim D: Med Bull 2010, 57:B4161. 4. Simon F, et al.: Crit Care 2009, 13:R113. Methods Three states of mitochondrial respiratory activity were quantifi ed in terms of O2-fl ux (JO2) in intact cells at 37°C by means of an O2K (Oroboros® Instruments Corp., Innsbruck, Austria) according to a previously published protocol [3] yielding routine respiration (R) as the standard respiratory level of the cells without any intervention, proton leak compensation (L) after blocking ATP synthesis by 2.5 μM oligomycine, and maximum capacity of the electron transport system (E) after uncoupling by 1 μM FCCP. The cells were studied after fi ve diff erent exposure conditions: control (C), 15 μmol/ml noradrenaline (high NoA), 5 nmol/ml noradrenaline (medium NoA), LPS, and LPS + high NoA. All data are presented in pmol/(smillion cells) as medians and 25 to 75% quartiles. Statistical signifi cance was tested by means of the Kruskal–Wallis one-way ANOVA followed by Dunn’s method. P16 All animals received additional 30 ml/kg Ringer’s lactate after injection of LPS over a time period of 1 hour. Arterial blood gases were measured every hour. Animals were euthanized 6 hours after endotoxin injection. The concentrations of monocyte chemoattractant protein-1, key player in the recruitment of monocytes during endotoxemia, was analyzed in bronchoalveolar lavage fl uid and in plasma. Linear regression was used to evaluate infl uence of HFIP on infl ammatory mediator expression. Conclusion AMPK importantly regulates cardiac vascular permeability and could control the sepsis-induced cardiomyopathy. AMPK could represent a new pharmacological target of sepsis. 1. Gustot T: Curr Opin Crit Care 2011, 17:153-159. 1. Gustot T: Curr Opin Crit Care 2011, 17:153-159. P18 Creatinine clearance was measured from 0 to 12 hours and from 12 to 24 hours of sepsis, respectively. Data are median (quartiles).f 1. Urner et al.: Am J Respir Cell Mol Biol 2011, 45:617-624. 1. Urner et al.: Am J Respir Cell Mol Biol 2011, 45:617-624. Results GW0742 did not aff ect the noradrenaline infusion rate required to achieve target hemodynamics (0.57 (0.30; 3.83) vs. 0.56 (0.41; 0.91) μg/kg/minute; P = 0.775) nor the fall in creatinine clearance (GW0742: from 129 (114; 140) to 78 (55; 95) ml/minute, P = 0.002; vehicle: from 130 (91; 142) to 41(31; 84) ml/minute, P = 0.004; P = 0.967 and P = 0.191 between groups). Immune histochemistry analysis of kidney biopsies in sham-operated swine showed markedly reduced tissue expression of the PPAR-β/δ receptor in atherosclerotic swine (281 (277; 404) vs. 57 (53; 77)×103 densitometric units in healthy swine; P = 0.008). P19f P19 Eff ects of hexafl uoro-2-propanol on infl ammatory and hemodynamic responses in a rat model of endotoxic shock M Urner, IK Herrmann, M Hasler, C Booy, B Beck-Schimmer University Hospital Zurich, Institute of Anesthesiology, Zurich, Switzerland Critical Care 2012, 16(Suppl 1):P19 (doi: 10.1186/cc10626) P21f P21 Eff ects of the anti-diabetic imeglimin in hyperglycemic mice with septic shock F Wagner1, J Vogt1, U Wachter1, S Weber1, B Stahl1, M Groeger1, O McCook1, M Georgieff 1, P Fouqueray2, T Kuhn2, E Calzia1, P Radermacher1, E Fontaine3, K Wagner1 1University Medical School Ulm, Anesthesia, Ulm, Germany, 2Poxel, Lyon, France, 3Université Joseph Fourier, LBFA, Grenoble, France Critical Care 2012, 16(Suppl 1):P21 (doi: 10.1186/cc10628) References References 1. Wu R, et al.: Mol Med 2009, 15:28-33. 2. Ertmer C, et al.: Br J Anaesth 2007, 99:830-836. 3. Guignant C, et al.: Intensive Care Med 2009, 35:1859-1867. 4. Mazzocchi G, et al.: Life Sci 2000, 66:1445-1450. 5. Hyvelin JM, et al.: J Card Surg 2002, 17:328-335. 6. Wagner F, et al.: Shock 2011, 35:396-402. 7. Wagner F, et al.: J Trauma 2011. [Epub ahead of print] References 1. Wu R, et al.: Mol Med 2009, 15:28-33. 2. Ertmer C, et al.: Br J Anaesth 2007, 99:830-836. 3. Guignant C, et al.: Intensive Care Med 2009, 35:1859-1867. 4. Mazzocchi G, et al.: Life Sci 2000, 66:1445-1450. 5. Hyvelin JM, et al.: J Card Surg 2002, 17:328-335. 6. Wagner F, et al.: Shock 2011, 35:396-402. 7. Wagner F, et al.: J Trauma 2011. [Epub ahead of print] 1. Wu R, et al.: Mol Med 2009, 15:28-33. q Results Imeglimin decreased blood glucose levels (165 (153; 180) vs. 192 (184; 221) mg/dl, P = 0.007) by increasing whole body glucose oxidation (55 (52; 57) vs. 51 (49; 55)% of infused isotope, P = 0.085), which coincided with partial restoration of gluconeogenesis (0.38 (0.34; 0.41) vs. 0.31 (0.27; 0.33) mg/g/hour, P = 0.032), liver mitochondrial activity (oxidative phosphorylation (136 (134; 160) vs. 116 (97; 122) pmol O2/ second/mg tissue, P = 0.003); maximal oxidative capacity (166 (154; 174) vs. 147 (130; 159) pmol O2/second/mg tissue, P = 0.064). Imeglimin increased liver HO-1, reduced liver Bax expression and attenuated NF- κB activation (all P <0.001). P22 Adrenomedullin blockade improves catecholamine responsiveness and kidney function in resuscitated murine septic shock K Wagner1, U Wachter1, J Vogt1, S Weber1, M Groeger1, O McCook1, M Georgieff 1, A Bergmann2, H Luettgen2, E Calzia1, P Radermacher1, F Wagner1 1University Medical School Ulm, Germany; 2AdrenoMed AG, Henningsdorf, Germany Critical Care 2012, 16(Suppl 1):P22 (doi: 10.1186/cc10629) Adrenomedullin blockade improves catecholamine responsiveness and kidney function in resuscitated murine septic shock K Wagner1, U Wachter1, J Vogt1, S Weber1, M Groeger1, O McCook1, M Georgieff 1, A Bergmann2, H Luettgen2, E Calzia1, P Radermacher1, F Wagner1 1University Medical School Ulm, Germany; 2AdrenoMed AG, Henningsdorf, Germany Critical Care 2012, 16(Suppl 1):P22 (doi: 10.1186/cc10629) Conclusion High but not moderate doses of noradrenaline reduced mitochondrial respiration in alveolar macrophages in vitro. Surprisingly, LPS increased routine respiration regardless of simultaneous noradrenaline exposure. Introduction The eff ects of adrenomedullin in circulatory shock states are controversially discussed: while its exogenous supplementation improved organ function and survival [1] in experimental models due to maintenance of hyperdynamic hemodynamics [2] in otherwise hypo dynamic conditions, high blood levels were associated with increased mortality in patients with septic shock [3], most likely as a result of excessive vasodilatation [4] and/or impaired systolic heart function [5]. P21 Eff ects of the anti-diabetic imeglimin in hyperglycemic mice with septic shock P21 Eff ects of the anti-diabetic imeglimin in hyperglycemic mice with septic shock F Wagner1, J Vogt1, U Wachter1, S Weber1, B Stahl1, M Groeger1, O McCook1, M Georgieff 1, P Fouqueray2, T Kuhn2, E Calzia1, P Radermacher1, E Fontaine3, K Wagner1 1University Medical School Ulm, Anesthesia, Ulm, Germany, 2Poxel, Lyon, France, 3Université Joseph Fourier, LBFA, Grenoble, France Critical Care 2012, 16(Suppl 1):P21 (doi: 10.1186/cc10628) i Results Adrenomedullin antagonism decreased the noradrenaline requirements needed to achieve target hemodynamics (0.009 (0.009; 0.012) vs. 0.02 (0.015; 0.044) μg/g/hour, P <0.001), increased total diuresis (2.6 (2.3; 3.9) vs. 0.6 (0.5; 2.7) ml, P = 0.028) resulting in improved fl uid balance (0.18 (0.14; 0.2) vs. 0.26 (0.19; 0.27), P = 0.011) and kidney function (creatinine levels at the end of the experiment: 1.3 (1.2; 1.5) vs. 2.0 (1.5; 2.9) μg/ml, P = 0.006; creatinine clearance: 400 (316; 509) vs. 197 (110; 301) μl/minute, P = 0.006). Introduction Shock-related hyperglycemia impairs mitochondrial function and integrity [1], ultimately leading to apoptosis and organ failure [1,2]. Imeglimin is a new anti-diabetic drug with anti- hyperglycemic and anti-apoptotic properties [3]. Therefore we investigated its eff ects in hyperglycemic mice with septic shock. Conclusion In resuscitated murine septic shock, early modulation of excess adrenomedullin activity via antibody HAM1101 improves cardiovascular catecholamine responsiveness, ultimately associated with attenuation of acute kidney injury. f Methods Immediately after cecal ligation and puncture, mice randomly received s.c. vehicle (n = 9) or imeglimin (n = 10; 100 μg/g). Fifteen hours later animals were anesthetized, mechanically ventilated and instrumented for a consecutive 6-hour observation period. After a second imeglimin bolus, colloid fl uid resuscitation and continuous i.v. noradrenaline were titrated to maintain normotensive and hyperdynamic hemodynamics. Then 2 mg/g/hour glucose was infused to induce hyperglycemia. Glucose oxidation and gluconeogenesis were derived from blood 13C6-glucose and mixed expiratory 13CO2/12CO2 isotope enrichment during continuous isotope infusion. Liver mito- chondrial activity was assessed using high-resolution respirometry [4,5], Bax, HO-1 and NF-κB expression by immunoblotting and EMSA. All data are median (quartiles). Acknowledgements Supported by an unrestricted grant from AdrenoMed AG. Eff ects of hexafl uoro-2-propanol on infl ammatory and hemodynamic responses in a rat model of endotoxic shoc l k h University Hospital Zurich, Institute of Anesthesiology, Zurich, Switzerland Critical Care 2012, 16(Suppl 1):P19 (doi: 10.1186/cc10626) Introduction Sepsis with multiple organ failure remains a leading cause of hospital morbidity and mortality on ICUs imparting S8 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 References 1. Japiassú AM, et al.: Crit Care Med 2011, 39:1056-1063.l 1. Japiassú AM, et al.: Crit Care Med 2011, 39:1056-1063.l 2. Porta F, et al.: Infl ammation 2009, 32:315-321. l 3. Renner K, et al.: Biochim Biophys Acta 2003, 1642:115-123. 3. Renner K, et al.: Biochim Biophys Acta 2003, 1642:115-123. Methods Immediately after cecal ligation and puncture to induce peritonitis, mice randomly received vehicle (n = 11) or the adreno- medullin antibody HAM1101 (n = 9; 2 μg/g to achieve antibody concentrations >4 ng/ml). Fifteen hours later animals were anesthetized, mechanically ventilated and instrumented for a consecutive 6-hour observation period. Colloid fl uid resuscitation and continuous i.v. noradrenaline were titrated to maintain normotensive (mean blood pressure >60 mmHg) and hyperdynamic hemodynamics. Creatinine blood levels and clearance were assessed as surrogate for glomerular fi ltration [6,7]. All data are median (quartiles). P22 Results After exposure with high but not with medium NoA we observed a statistically signifi cant decrease in maximum mitochondrial respiratory capacity (E-state, C 133 (118; 148) vs. high NoA 111 (106; 113), and medium NoA 129 (123; 140), P <0.05 C vs. high NoA). Both LPS and LPS + high NoA did not aff ect E-state respiration (LPS: 152 (136; 179), and LPS + NoA 129 (125; 137)), but increased routine (R) respiration when compared to control (C 45 (40; 55) vs. LPS 66 (51; 72) and LPS + NoA 65 (55; 68), P <0.05; high NoA 41 (37; 47), and medium NoA 52 (51; 57), NS). P24 Soluble usokinase plasminogen activator receptor as a useful biomarker to defi ne advent of sepsis in patients with multiple injuries Role of serum biomarkers in the diagnosis of infection in patients undergoing extracorporeal membrane oxygenation M Pieri1, T Greco1, AM Scandroglio1, M De Bonis1, G Maj1, L Fumagalli2, A Zangrillo1, F Pappalardo1 1Istituto Scientifi co San Raff aele, Milan, Italy; 2Istituto Scientifi co San Raff aele Turro, Milan, Italy Critical Care 2012, 16(Suppl 1):P26 (doi: 10.1186/cc10633) Introduction Although rates and causal organisms of infections occur ring in patients on extracorporeal membrane oxygenation (ECMO) have already been described [1], diagnosis of infection itself is challenging in clinical practice. In addition, a signifi cant heterogeneity in infection surveillance practice patterns among ELSO centers has recently been reported [2]. The aim of the study was to analyze the role of C-reactive protein (CRP) and procalcitonin (PCT) in the diagnosis of bacterial and fungal infection in critically ill patients requiring ECMO, and to assess the diff erence between venovenous (VV) and venoarterial (VA) ECMO setting. Introduction Soluble usokinase plasminogen activator receptor (suPAR) has been considered a useful biomarker to defi ne prognosis in patients with sepsis [1]. The present study aimed to defi ne the kinetics of suPAR during the physical course of patients with multiple injuries. Introduction Soluble usokinase plasminogen activator receptor (suPAR) has been considered a useful biomarker to defi ne prognosis in patients with sepsis [1]. The present study aimed to defi ne the kinetics of suPAR during the physical course of patients with multiple injuries. Methods A total of 62 patients were enrolled. All patients were bearing: multiple injuries necessitating ICU admission with an injury severity score (ISS) more than 8; and systemic infl ammatory response syndrome. Patients with infections upon ICU admission were excluded from the study. Peripheral venous blood was sampled within the fi rst 24 hours after ICU admission. Blood sampling was repeated within the fi rst 24 hours upon advent of sepsis. suPAR was measured in serum by an enzyme immnunoassay. Methods A total of 62 patients were enrolled. All patients were bearing: multiple injuries necessitating ICU admission with an injury severity score (ISS) more than 8; and systemic infl ammatory response syndrome. Patients with infections upon ICU admission were excluded from the study. Peripheral venous blood was sampled within the fi rst 24 hours after ICU admission. Blood sampling was repeated within the fi rst 24 hours upon advent of sepsis. suPAR was measured in serum by an enzyme immnunoassay. P25 comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Role of mannose-binding lectin on pneumococcal infections J Solé Violán1, I García-Laorden1, F Rodríguez de Castro1, A Payeras2, J Ferrer Agüero1, M Briones3, L Borderías4, J Aspa5, J Blanquer3, O Rajas5, M García-Bello1, J Noda1, J Rello6, C Rodríguez Gallego1 1Hospital Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital Son Llatzer, Palma de Mallorca, Spain; 3Hospital Clínico y Universitario, Valencia, Spain; 4Hospital San Jorge, Huesca, Spain; 5Hospital de La Princesa, Madrid, Spain; 6Hospital Universitario Vall d´Hebró, Barcelona, Spain Critical Care 2012, 16(Suppl 1):P25 (doi: 10.1186/cc10632) Role of mannose-binding lectin on pneumococcal infections J Solé Violán1, I García-Laorden1, F Rodríguez de Castro1, A Payeras2, J Ferrer Agüero1, M Briones3, L Borderías4, J Aspa5, J Blanquer3, O Rajas5, M García-Bello1, J Noda1, J Rello6, C Rodríguez Gallego1 1Hospital Dr Negrín, Las Palmas de Gran Canaria, Spain; 2Hospital Son Llatzer, Palma de Mallorca, Spain; 3Hospital Clínico y Universitario, Valencia, Spain; 4Hospital San Jorge, Huesca, Spain; 5Hospital de La Princesa, Madrid, Spain; 6Hospital Universitario Vall d´Hebró, Barcelona, Spain Critical Care 2012, 16(Suppl 1):P25 (doi: 10.1186/cc10632) Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the ICU. The median age was 64 years old (interquartile range, 48.7 to 71); male: 60%. The beginning of severe sepsis took place in the emergency area in 46% of cases. The main sources of infection were respiratory tract 38% and intra- abdomen 45%; 70.7% had medical pathology. The 28-day mortality was 22.7%. The profi le of death patients were men (64.7%, n = 22), with signifi cantly higher average age (63 vs. 57 years; P = 0.049), as well as clinical severity scores, APACHE II (29.8 vs. 24.1; P <0.001) and SOFA (12.1 vs. 8.9; P <0.001) and major dysfunction organs (4.6 vs. 3.6; P <0,001); we observed signifi cantly major consumption of PC (55.2 vs. 70.1, P = 0.011). Lower levels of PC were found in surgery septic shock patients, neurological focus or catheter-related infection and Gram- negative pathogens from blood cultures. The ROC analysis showed superior risk prediction of SOFA score for 28-day mortality, AUC 0.81 (95% CI: 0.73 to 0.88, sensitivity: 73.5%; specifi city: 76.7%, P = 0.001), that improves by combining with PC, AUC 0.83 (95% CI: 0.75 to 0.90, sensitivity: 77%; specifi city: 83%, P = 0.001). P25 Introduction The role of mannose-binding lectin (MBL) defi ciency (MBL2 XA/O + O/O genotypes) in host defences remains controversial. The surfactant proteins (SP)-A1, SP-A2 and SP-D, and other collectins whose genes are located near MBL2, are part of the fi rst-line lung defence against infection. We analyzed the role of MBL on susceptibility to pneumococcal infection and the existence of linkage disequilibrium (LD) among the four genes. g g Methods We studied 348 patients with pneumococcal community- acquired pneumonia (P-CAP) and 1,591 controls. A meta-analysis of MBL2 genotypes in susceptibility to P-CAP and to invasive pneumo- coccal disease (IPD) was also performed. The extent of LD of MBL2 with SFTPA1, SFTPA2 and SFTPD was analyzed. i Conclusion This cohort study showed an improvement in the survival in septic patients under a lower consumption of PC. Low levels of PC are associated with more severity in Sepsis, dysfunction organ and poor outcome. Results MBL2 genotypes did not associate with either P-CAP or bacteraemic P-CAP in the case–control study. The MBL-defi cient O/O genotype was signifi cantly associated with higher risk of IPD in a meta- analysis, whereas the other MBL-defi cient genotype (XA/O) showed a trend towards a protective role. We evidenced the existence of LD between MBL2 and SPs genes. References 1. Brunkhorst F, et al.: Protein C concentrations correlate with organ dysfunction and predict outcome independent of the presence of sepsis. Anesthesiology 2007, 107:15-23. g dysfunction and predict outcome independent of the presence of sepsis. Anesthesiology 2007, 107:15-23. 2. Yan SB, et al.: Low levels of protein C are associated with poor outcome in severe sepsis. Chest 2001, 120:915-922. 2. Yan SB, et al.: Low levels of protein C are associated with poor outcome in severe sepsis. Chest 2001, 120:915-922. g Conclusion The data do not support a role of MBL defi ciency on susceptibility to P-CAP or to IPD. LD among MBL2 and SP genes must be considered in studies on the role of MBL in infectious diseases. 1. Savva A, et al.: J Infect 2011, 63:344-350. P24 P24 P24 Soluble usokinase plasminogen activator receptor as a useful biomarker to defi ne advent of sepsis in patients with multiple injuries M Patrani1, M Paraschos1, M Georgitsi2, E Giamarellos-Bourboulis2, K Mandragos1 1Korgialeneion Benakeion Hospital, Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2012, 16(Suppl 1):P24 (doi: 10.1186/cc10631) P24 Soluble usokinase plasminogen activator receptor as a useful biomarker to defi ne advent of sepsis in patients with multiple injuries M Patrani1, M Paraschos1, M Georgitsi2, E Giamarellos-Bourboulis2, K Mandragos1 1Korgialeneion Benakeion Hospital, Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2012, 16(Suppl 1):P24 (doi: 10.1186/cc10631) P24 Soluble usokinase plasminogen activator receptor as a useful biomarker to defi ne advent of sepsis in patients with multiple injuries M Patrani1, M Paraschos1, M Georgitsi2, E Giamarellos-Bourboulis2, K Mandragos1 1Korgialeneion Benakeion Hospital, Athens, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2012, 16(Suppl 1):P24 (doi: 10.1186/cc10631) P24 Soluble usokinase plasminogen activator receptor as a useful biomarker to defi ne advent of sepsis in patients with multiple injuries Methods A case–control study on 27 patients. We analyzed serum values of PCT and CRP according to the presence of infection. Results Forty-eight percent of patients had infection. Gram-negative bacteria were the predominant pathogens (54%), and Candida was the most frequent isolated microorganism overall (15%). PCT had an AUC of 0.681 (P = 0.0062), for the diagnosis of infection in patients on VA ECMO, but failed to discriminate infection in the VV ECMO group (P = 0.14). The AUC of CRP was 0.707 (P ≤0.001) in all ECMO patients. In patients receiving VA ECMO, PCT had good accuracy with 1.89 ng/ml as the cut-off (SE = 87.8%, SP = 50%) and CRP as well with 97.70 mg/l as the cut-off (SE = 85.3%, SP = 41.6%). PCT and CRP tests in parallel had SE = 87.2%, and SP = 25.9%. Four variables were identifi ed as statistically signifi cant predictors of infection: PCT and CRP tests in parallel (OR = 1.184; P = 0.0008), age (OR = 0.980; P ≤0.001), presence of infection before ECMO implantation (OR = 1.782; P ≤0.001), and the duration of ECMO support (OR = 1.056; P ≤0.001).l y y Results Mean ISS of patients was 14.6. Median suPAR upon ICU admission was 3.74 ng/ml (range: 1.57 to 16.77 ng/ml). No correlation was found between ISS and suPAR. Sepsis was presented in 27 patients. Median suPAR upon sepsis diagnosis was 7.05 ng/ml (range: 2.18 to 32.51 ng/ml) (P <0.0001 compared with ICU admission). This change corresponded to median increase of 57.81%.i p Conclusion The presented fi ndings show that measurement of serum suPAR may help diagnosis of sepsis presenting in patients with multiple injuries. P23 Activated protein C, severe sepsis and 28-day mortality M De La Torre-Prados, A García-de la Torre, M Nieto-González, I Lucena-González, R Escobar-Conesa, A García-Alcántara, A Enguix-Armada Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2012, 16(Suppl 1):P23 (doi: 10.1186/cc10630) Activated protein C, severe sepsis and 28-day mortality M De La Torre-Prados, A García-de la Torre, M Nieto-González, I Lucena-González, R Escobar-Conesa, A García-Alcántara, A Enguix-Armada Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2012, 16(Suppl 1):P23 (doi: 10.1186/cc10630) Conclusion Imeglimin improved whole body glucose utilization and gluconeogenesis, a well-established marker of liver metabolic capacity [4,5], and attenuated organ injury, at least in part due to inhibition of the mitochondrial apoptosis pathway. Introduction Protein C (PC) defi ciency is prevalent in severe sepsis, studies showing that more than 80% of patients with severe sepsis have a baseline PC level below the lower limit of normal [1,2]. The aim of the study was to relate the anticoagulation activity evaluated by PC, with clinical parameters and 28-day mortality. Acknowledgements In memoriam of Xavier Leverve who initiated this project; supported by an unrestricted grant from Poxel. References Acknowledgements In memoriam of Xavier Leverve who initiated this project; supported by an unrestricted grant from Poxel. References 1. Vanhorebeek I, et al.: Crit Care Med 2009, 37:1355-1364. Methods A cohort study of 150 patients >18 years with severe sepsis according to the Surviving Sepsis Campaign, in an ICU of a university hospital. Demographic, clinical parameters and coagulation markers during the fi rst 24 hours were studied. PC activity was analysed using a haemostasis laboratory analyser (BCS® XP; Siemens). Descriptive and 2. Devos P, et al.: Curr Opin Clin Nutr Metab Care 2006, 9:131-139. 3. Fouqueray, et al.: J Diabetes Metab 2011, 2:4. 4. Albuszies G, et al.: Intensive Care Med 2007, 33:1094-1101. 5. Baumgart K, et al.: Crit Care Med 2010, 38:588-595. S9 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P27 Correlation of VAP diagnosis with parameters of critically ill patients in a general ICU Introduction We aimed to describe various parameters of critically ill patients who developed VAP and correlate them with its outcome. Introduction We aimed to describe various parameters of critically ill patients who developed VAP and correlate them with its outcome. Introduction We aimed to describe various parameters of critically ill patients who developed VAP and correlate them with its outcome. Methods Twenty-three VAP cases out of 338 ICU patients were studied retrospectively. Data regarding age, sex, etiology, scores (APACHE II, SOFA, CPIS), CRP, miniBAL cultures, comorbidities, antibiotic exposure, duration of mechanical ventilation, length of ICU and total stay, VAP and patient outcome were recorded. Chi-square and Mann–Whitney U tests were used for statistical analyses. y Results VAP incidence was 23/338 (6.8%). Fourteen of 23(60.9%) were males, and 9/23(39.1%) were surgical patients. Their age was 63.5 ± 16.6 years. APACHE II was 20.5 ± 6.7, initial SOFA was 8.8 ± 3.7, SOFA at VAP was 9.4 ± 3.1, CPIS 2 days before VAP was 4.6 ± 2, CPIS the day before VAP was 6 ± 1.2, and CPIS at VAP was 7.6 ± 1.3. Length of stay was 25.5 ± 13.1 days, ICU stay was 24.8 ± 13.4 days, and duration of mechanical ventilation was 22.5 ± 12.1 days. Previous antibiotic exposure included: linezolid 10/23 (43.5%), vancomycin 2/23 (8.7%), antipseudomonadic penicillins 14/23 (60.9%), β-lactams ± β-lactamase inhibitor 7/23 (30.4%), quinolones 14/23 (60.9%), aminoglycosides 6/23 (26.1%), antifungals 4/23 (17.4%), carbapenems 1/23 (4.3%), tigecycline 3/23 (13%), and colistin 8/23 (34.8%). Antibiotic therapy after the positive miniBAL was modifi ed according to antibiograms. The isolated microorganisms in miniBAL were A. baumannii 10/23 (43.5%), P. aeruginosa 5/23 (21.7%), K. pneumoniae 4/23 (17.4%), Candida spp. 2/23 (8.7%), and other 4/23 (17.4%); one infection was polymicrobial. In 20/23 cases (87%) VAP was of late onset (>4 days) (9.7 ± 6.8 days). VAP was improved in 17/23 cases (73.9%), but 15/23 patients (65.2%) died. High overall mortality may be attributed to grave condition. Most patients were admitted to the ICU hours after they were admitted to the hospital. Increased SOFA scores during admission (12 ± 2 vs. 7.7 ± 3.4, P = 0.009) and on the day of VAP diagnosis (11.5 ± 2.1 vs. 8.6 ± 3, P = 0.016) were associated with VAP deterioration. Increased CPIS on the last 2 days before VAP was also associated with worse VAP outcomes (6.2 ± 1.7 vs. j Reference Reference Conclusion Both traditional and emerging infl ammatory biomarkers can help in the diagnosis of infection in patients receiving ECMO. Indeed, we demonstrated for the fi rst time that PCT is a reliable infection marker in patients undergoing VA ECMO. We suggest routine S10 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 concomitant PCT and CRP assay with defi nite cut-off values as a new test to identify infection in patients undergoing VA ECMO. References 12th day. C-reactive protein (CRP) and PCT were measured daily. We compared infected and noninfected patients. concomitant PCT and CRP assay with defi nite cut-off values as a new test to identify infection in patients undergoing VA ECMO. References 12th day. C-reactive protein (CRP) and PCT were measured daily. We compared infected and noninfected patients. p p Results A total of 50 patients were included during a 12-month period (age 70.5 ± 9.4 years, 50% male). The 21 patients (42%) that subsequently developed infection (16 surgical wound infections) had age, Charlson comorbidity score, primary diagnosis, surgical procedure, intestinal preparation and antibiotic prophylaxis similar to those who had an uneventful recovery. Infection was less frequent in men (28% vs. 72%, P = 0.042). Moreover PCT and CRP before surgery were equally low in patients with or without postoperative infection (0.10 ± 0.06 vs. 0.07 ± 0.04 ng/ml; 1.81 ± 2.83 vs. 0.72 ± 1.12 mg/dl, respectively). After surgery, both PCT and CRP increased markedly: PCT increased around 10× the basal level and peaked at 24 to 48 hours; CRP increased more than 15× and peaked at 48 hours. Infection was diagnosed a median of 7 days after surgery. The CRP time-course from the day of surgery onwards was signifi cantly diff erent in infected and noninfected patients (P = 0.001). In opposition, the PCT time-course was almost parallel in both groups (P = 0.866). To assess the diagnostic performance of each biomarker, we performed multiple comparisons between infected and noninfected patients between day 5 and day 9. The CRP concentration was signifi cantly diff erent (P < 0.01, Bonferroni correction) on days 6, 7 and 8. The area under the ROC curve of CRP of days 6, 7 and 8 were 0.74, 0.73 and 0.75, respectively. Procalcitonin as a predictive marker for PCR test and blood culture results in suspected invasive candidemia A Cortegiani, SM Raineri, F Montalto, MT Strano, A Giarratano University Hospital Policlinico P. Giaccone, Palermo, Italy Critical Care 2012, 16(Suppl 1):P29 (doi: 10.1186/cc10636) A Cortegiani, SM Raineri, F Montalto, MT Strano, A Giarratano University Hospital Policlinico P. Giaccone, Palermo, Italy Critical Care 2012, 16(Suppl 1):P29 (doi: 10.1186/cc10636) Introduction Procalcitonin (PTC) seems to have potential to predict the result of blood culture (BC) supporting the diagnosis of invasive candidemia. Although blood culture is still the gold standard, PCR assays are able to quickly and reliably detect fungi in blood in suspected invasive candidemia. Our aim is to verify the potential of PTC values to predict the result of PCR assay in suspected invasive candidemia. y Methods We retrospectively analyzed 78 patients with suspected invasive candidemia from whom we obtained PCT value, BC and PCR assay. All tests have been obtained on the day in which patients reached a Candida score ≥4. We calculated PTC mean values according to BC and PCR results and compared data using the Mann–Whitney U test. We performed the ROC analysis to test the diagnostic performance of PTC with regards to BC and PCR result. Conclusion Our fi ndings support the prognostic value of SOFA score. CPIS values of 6, although not diagnostic, may need increased alertness on behalf of the clinician. R f Results PCR tests and BC were both negative in 48 patients and the PTC mean value in this group was 21.5 ng/ml while 19 patients were PCR-positive and BC-positive with a PTC mean value of 2.07 ng/ml. The diff erence between these PCT mean values was signifi cant (P = 0.0001). In eight cases BC were negative whereas PCR tests were positive with the PCT mean level in this group being 1.82 ng/ml. No patient resulted PCR-negative and BC-positive. According to PCR results only, there was a signifi cant diff erence between PTC mean values in positives and negatives (P = 0.0001). The ROC analysis showed that the best PTC cut- off value for prediction of BC result was 4.57 with AUC of 0.91 (CI 0.83 to 0.96, sensitivity 99%, specifi city 80.39%). Concerning the PCR result, the calculated cut-off was 4.31 with AUC of 0.96 (CI 0.948 to 1, sensitivity 96.6%, specifi city 97.9%; positive predictive value 94.51%; negative predictive value 97.83%). References 1. Vincent JL, et al.: Intensive Care Med 1996, 22:707-710. 2. P27 Correlation of VAP diagnosis with parameters of critically ill patients in a general ICU 4.1 ± 1.8 and 6.8 ± 1.2 vs. 5.7 ± 1.1, P = 0.03 and P = 0.03, respectively).i Conclusion After a major elective surgical insult both CRP and PCT serum levels increased independently of the presence of infection. The CRP time-course showed to be useful in the early detection of an infectious complication whereas PCT was unhelpful. Procalcitonin as a predictive marker for PCR test and blood culture results in suspected invasive candidemia A Cortegiani, SM Raineri, F Montalto, MT Strano, A Giarratano University Hospital Policlinico P. Giaccone, Palermo, Italy Critical Care 2012, 16(Suppl 1):P29 (doi: 10.1186/cc10636) Procalcitonin as a predictive marker for PCR test and blood culture results in suspected invasive candidemia Papazian L, et al.: Am J Respir Crit Care Med 1995, 152:1982-1991. 1. Vincent JL, et al.: Intensive Care Med 1996, 22:707-710. 2. Papazian L, et al.: Am J Respir Crit Care Med 1995, 152:1982-1991. P27 P27 Correlation of VAP diagnosis with parameters of critically ill patients in a general ICU DK Matthaiou, A Ioannidis, G Gounti, D Lathyris, A Vathis, S Vasiliagkou, K Kontopoulou, K Mandraveli, E Antoniadou ‘G. Gennimatas’ General Hospital, Thessaloniki, Greece Critical Care 2012, 16(Suppl 1):P27 (doi: 10.1186/cc10634) j Reference A CRP concentration >5.0 mg/dl at day 6 was predictive of infection with a sensitivity of 85% and a specifi city of 62% (positive likelihood ratio 2.2, negative likelihood ratio 0.2). 1. Bizzarro MJ, et al.: Pediatr Crit Care Med 2011, 12:277-281. 2. Kao LS, et al.: ASAIO J 2011, 57:231-238. P30 P30 Would procalcitonin measurement aid antimicrobial stewardship in a UK district general hospital mixed adult critical care population? J Clayton, J White, L Wilson, M Leonard, J Cuniff e Wirral University Teaching Hospital NHS Foundation Trust, Wirral, UK Critical Care 2012, 16(Suppl 1):P30 (doi: 10.1186/cc10637) Introduction We sought to establish what impact knowledge of procalcitonin (PCT) levels could have on antimicrobial prescribing and stewardship within our 18-bed mixed critical care unit. Assicot and colleagues demonstrated that PCT levels are raised during sepsis and can correlate with the severity [1]. The PCT level peaks after 6 to 12 hours and has a half-life of approximately 25 to 36 hours in critically ill patients [2], declining with adequate treatment. A recent multicentre trial demonstrated reduced duration of antibiotic therapy by using PCT-guided treatment strategy; however, only 10% of the cohort was surgical patients and therefore this fi nding cannot be extrapolated to a general critical care population [3]. Conclusion The late-onset candidemia are more likely to be associated with death than earlier episodes. Unresolved organ failure as assessed through SOFA score despite eff ective antifungal treatment was associated with death, while PCT failed to predict the outcome. References Methods The question was posed: would knowledge of PCT levels have altered real-time clinical management of patients on established antimicrobial therapy? Over a 2-month period patients were treated in a conventional manner based on clinical fi ndings and standard investigations. Plasma samples from days 0 (respective to antimicrobial therapy) 1, 3, 5 and 7 were analysed for PCT. Nonparametric statistical analysis of PCT levels was available for a retrospective multidisciplinary team review of case notes. This was performed within the context of a local service review and the chair of the local ethics committee gave approval for analysis of plasma samples and case-note reviewi 1. Charles PE, et al.: Intensive Care Med 2006, 32:1577-1583. 2. Charles PE, et al.: Intensive Care Med 2009, 35:2146-2150. 3. Martini A, et al.: J Infect 2010, 60:425-430. Usefulness of daily monitoring of procalcitonin and C-reactive protein in the early diagnosis of infection after elective colonic surgery g y J Rebanda, P Povoa p , , , g Critical Care 2012, 16(Suppl 1):P28 (doi: 10.1186/cc10635) Introduction The diagnosis of infectious complications after elective colonic surgery is frequently misleading, delaying its resolution. Recently several biomarkers, namely procalcitonin (PCT), have been described as more specifi c in infection diagnosis. Conclusion According to our data, PTC seems to be characterized by a remarkable diagnostic performance and predictive value for both BC and PCR assay in suspected invasive candidemia. PCT could be considered as the fi rst step of the diagnostic process for suspected invasive candidemia in order to spare as much time as possible before starting a pre-emptive antifungal therapy. This may lead to less useless therapies in negative patients and quicker and more reliable start of i Methods We conducted a prospective observational study segregating patients submitted to elective colonic surgery. Patients were assessed before surgery, and then from the day of surgery until discharge or the Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S11 follow-up data including PCT measurement were collected. The SOFA score was calculated daily during the fi rst week of antifungal treatment. Survivors at discharge from the ICU were compared to nonsurvivors by univariate followed by a Cox regression analysis. follow-up data including PCT measurement were collected. The SOFA score was calculated daily during the fi rst week of antifungal treatment. Survivors at discharge from the ICU were compared to nonsurvivors by univariate followed by a Cox regression analysis. treatment in positive patients while waiting for the BC and antibiogram results. R f Assessment of the usefulness of presepsin (soluble CD14 subtype) in septic patients Assessment of the usefulness of presepsin (soluble CD14 subtype) in septic patients T Nishida1, H Ishikura1, A Murai1, Y Irie1, R Yuge1, T Kamitani1, S Endo2 1Fukuoka University Hospital, Fukuoka City, Japan; 2Iwate Medical University, Iwate, Japan Critical Care 2012, 16(Suppl 1):P32 (doi: 10.1186/cc10639) Results Twenty-seven patients were identifi ed. Antimicrobial cessation was deemed possible in seven of these cases at day 5. Nonescalation of treatment was supported in six further cases. In one case treatment had been escalated and PCT supported this decision. This would have resulted in 19 fewer days of antibiotic therapy. T Nishida1, H Ishikura1, A Murai1, Y Irie1, R Yuge1, T Kamitani1, S Endo2 1Fukuoka University Hospital, Fukuoka City, Japan; 2Iwate Medical University Iwate, Japan T Nishida1, H Ishikura1, A Murai1, Y Irie1, R Yuge1, T Kamitani1, S Endo2 1Fukuoka University Hospital, Fukuoka City, Japan; 2Iwate Medical University, Iwate, Japan Critical Care 2012, 16(Suppl 1):P32 (doi: 10.1186/cc10639) Critical Care 2012, 16(Suppl 1):P32 (doi: 10.1186/cc10639) Introduction Sepsis is a life-threatening condition that is characterized by a whole-body infl ammatory state. The early diagnosis and treatments of sepsis will improve the outcome of the patients. The aims of this study were to investigate the most useful biomarkers which are serum levels of soluble CD14 subtype (sCD14-ST) named presepsin, procalcitonin (PCT), IL-6, and C-reactive protein (CRP) as markers for early diagnosis of sepsis. Conclusion Our experience suggests the availability of the PCT response between days 0 and 5 would have been a useful adjunct in monitoring treatment of sepsis on our unit and would have facilitated timely de-escalation and hence exposure to antimicrobial therapy. We hypothesise such a reduction could help to prevent antimicrobial resistance, lead to decreased pharmacy and consumable costs and reduce the incidence of adverse antimicrobial-related events. Reference 1. Charles PE, Castro C, Ruiz-Santana S, et al.: Serum procalcitonin levels in critically ill patients colonized with Candida spp: new clues for the early recognition of invasive candidiasis? Intensive Care Med 2009, 35:2146-2150. y g y Results Fifty patients were included among whom 28 (56%) died in the ICU. Candida albicans was the most common isolated yeast (58%), regardless of the outcome. Nonsurvivors were elder and had a greater SAPS II score value on admission than survivors (55.8 ± 21.7 vs. 42.5 ± 14.9 points, P = 0.01). The time elapsed between the ICU admission and the onset of invasive candidiasis was signifi cantly longer in the nonsurvivors than in the survivors (8.3 ± 12.8 vs. 1.2 ± 2.8 days, P = 0.01). At the onset of candidemia, the nonsurvivors were more severely ill as assessed through SOFA score calculation (10.4 ± 4.4 vs. 7.8 ± 3.9 points, P = 0.04). Antifungal treatment was given within the fi rst 24 hours following the onset of candidemia in 60% of the whole patients and was always appropriate, regardless of the survival. During therapy, the SOFA score remained greater in the nonsurvivors than in the survivors. In contrast, PCT failed to diff erentiate the survivors from the nonsurvivors the day antifungals were started (8.7 ± 13.1 vs. 4.5 ± 4.1 ng/ml, P = 0.21), as well as the following days. The SAPS II, the SOFA score and the time elapsed between ICU admission and candidemia onset were the sole independent predictors of death in our study population. References Methods A single-center, prospective, observational study. Patients who had one or more systemic infl ammatory response syndrome (SIRS) criteria were included in this study. The blood samples for measuring the markers were collected and the severity of sepsis was evaluated at the time of admission and every other day for a week. Eighty-four patients were enrolled for this prospective study from June 2010 to June 2011. 1. Assicot M, et al.: Lancet 1993, 341:515-518. 2. Meisner M, et al.: Intensive Care Med 2000, 26:1193-1200. 3. Bouadma L, et al.: Lancet. 2010, 375:463-474. P33 Circulating cell-free DNA levels measured by a novel simple fl uorescent assay are predictive for outcome of severe sepsis A Douvdevani, A Avriel, M Paryente Wiessman, V Novack, Y Almog Soroka University Medical Center and Ben-Gurion University of the Negev, Beer-Sheva, Israel Critical Care 2012, 16(Suppl 1):P33 (doi: 10.1186/cc10640) Results We enrolled 49 subjects. There was no signifi cant correlation between EA levels and endotoxin concentration measured by LAL assay. There were no signifi cant diff erence in the EA levels of the Gram- negative infection patients and the others. The diagnostic value of EA levels was investigated using ROC curve analysis. For the diagnosis of sepsis, area under the curve of EA levels, PCT, presepsin, IL-6 and CRP were calculated as 0.76, 0.83, 0.89, 0.88 and 0.72, respectively. Both the EA levels and ICU mortalities of the patients who met the criteria for severe sepsis were signifi cantly higher than those of the patients who did not have sepsis (0.44 ± 0.21 vs. 0.22 ± 0.17, P = 0.0004; EA levels, 33% vs. 5%, P = 0.022; ICU mortalities). There was a positive relationship between EA levels and thrombomodulin (r = 0.30, P = 0.049), EA levels and lactate (r = 0.31, P = 0.028), and EA levels and SOFA score (r = 0.34, P = 0.02). There was a negative relationship between EA levels and platelet counts (r = –0.34, P = 0.018), EA levels and antithrombin (r = – 0.41, P = 0.004), and EA levels and protein C (r = –0.38, P = 0.010). Conclusion EA levels in the patients on ICU admission correlated with disease severity. Moreover, we strongly suggested that EAA may have the potential to assess organ dysfunction with sepsis, especially coagulopathy. Introduction Circulating cell-free DNA (CFD) was found to be a predic- tor of outcome in severe sepsis and septic shock [1]. The standard CFD assays are work-intensive and not practical for routine clinical laboratory use. We have recently developed a new simple, fast and reliable assay for CFD measurement. The aim was to evaluate the association between admission levels of CFD and severe sepsis outcome in patients hospitalized in intensive care utilizing the new assay. y Methods Seventy-six patients diagnosed with severe sepsis hospitalized in the ICU were enrolled in the study. Prognostic value of serum galactomannan in mixed ICU patients: a retrospective observational study Prognostic value of serum galactomannan in mixed ICU patients: a retrospective observational study S Teering, A Verreth, W Verlinden, J Jacobs, S Pilate, M Peetermans, A Verrijcken, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, M Van De Vyvere, M Malbrain ZNA Stuivenberg, Antwerp, Belgium Critical Care 2012, 16(Suppl 1):P35 (doi: 10.1186/cc10642) Conclusion By using a simple fl uorometric assay, we were able to measure CFD levels in severe septic patients. CFD levels were found to be an independent predictors for 28-day mortality. We believe that CFD is an objective, reliable and integrative prognostic marker that will allow fast evaluation of intensive care patients and predicting mortality. References Introduction Little is known about galactomannan (GM) testing in mixed ICU patients that are often not neutropenic. The aim of this study was to look for the incidence and outcome of invasive aspergillosis (IA) in critically ill patients, to validate previous reported GM thresholds and to evaluate the prognostic value of GM. g Methods A retrospective study of 474 GM samples in 160 patients from 1 January 2003 to 1 February 2004. GM tests were ordered because of clinical suspicion of IA or on a regular basis in immune compromised patients. The number of samples per patient was 3 ± 2.6. Similarly to the EORTC criteria we defi ned ‘proven IA’ as those patients with positive tissue specimen, ‘probable IA’ as those with positive cultures, and ‘possible IA’ as those treated with antifungals (high clinical index of suspicion). The number of positive samples (GM >0.5 ng/ml) was 230 (48.5%). Patient characteristics: M/F ratio 1/1, age 64.5 ± 15.9, SAPS 45.5 ± 16.8, APACHE II 19.3 ± 8, SOFA 5.8 ± 3.5, mean days on ventilation 12.9 ± 8.7, mean CRP 10.4 ± 11.2 mg/dl. 1. Saukkonen K, et al.: Clin Chem 2008, 54:1000-1007. 2. Goldshtein H, et al.: Ann Clin Biochem 2009, 46:488-494. 1. Saukkonen K, et al.: Clin Chem 2008, 54:1000-1007. 1. Saukkonen K, et al.: Clin Chem 2008, 54:1000-1007. 2. Goldshtein H, et al.: Ann Clin Biochem 2009, 46:488-494. , , 2. Goldshtein H, et al.: Ann Clin Biochem 2009, 46:488-494. Procalcitonin has a poor prognosis value in critically ill patients with candidemia Procalcitonin has a poor prognosis value in critically ill patients with candidemia PE Charles, R Bruyère, H Roche, JP Quenot, S Prin, A Pavon, F Dalle University Hospital, Dijon, France Critical Care 2012, 16(Suppl 1):P31 (doi: 10.1186/cc10638) Results Eighteen were SIRS and 42 were sepsis at the time of registration. In the receiver operating characteristics (ROC) analysis, the area under the curve (AUC) to distinguish sepsis was the highest for presepsin (0.92) followed by IL-6 (0.89), PCT (0.88), and CRP (0.83). The ROC analysis showed that at a cut-off value 647 pg/ml, presepsin may be able to discriminate between patients with and without sepsis with a sensitivity and a specifi city of 92.9% and 83.3% respectively with 95% confi dence intervals of 0.929 (0.805 to 0.985). And the presepsin values were signifi cantly higher in the patients with the more severe septic condition (for example, sepsis, severe sepsis, septic shock). In addition, a signifi cant correlation was found between the SOFA scores and the presepsin values (r2 = 0.258; P <0.01). But there was only weak correlation between APACHE II scores and the presepsin values (r2 = 0.053). PE Charles, R Bruyère, H Roche, JP Quenot, S Prin, A Pavon, F Dalle University Hospital, Dijon, France Critical Care 2012, 16(Suppl 1):P31 (doi: 10.1186/cc10638) Introduction Candidemia is an infrequent but serious infection in the critically ill patients. Although eff ective antifungal drugs are available, mortality rates remain high so far. Procalcitonin (PCT) repeated measurements have proven useful for assessing the prognosis and the antimicrobial treatment responsiveness in the patients with systemic bacterial infection. Little is known about it in the setting of candidemia. The PCT predictive value regarding the outcome of such patients was therefore addressed. Conclusion In this study, presepsin is the most valuable predictor about sepsis compared with PCT, IL-6, and CRP. Moreover, these results suggest that presepsin values can serve as a parameter that closely Methods A retrospective single-centre observational study. All patients with ICU-acquired pure candidemia between 2005 and 2011 were included. Baseline characteristics and both clinical and biological S12 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 who had suspected infection in the emergency room and ICU, but the clinical usefulness of measuring EAA in the diagnosis of sepsis in critically ill patients is not yet clear. P33 Serum CFD levels were measured upon admission and after 72 hours using the SYBR-Gold rapid direct fl uorescent assay [2]. Primary outcome was 28-day mortality. Logistic regression analysis of CFD quintiles adjusted for baseline comorbidities and severity of the disease was utilized. y Results Out of those diagnosed with severe sepsis, 28 (36.8%) have died either during hospitalization or within 28 days of admission to the ICU. Decedents had higher APACHE II score on admission (median 24.5 vs. 17.5, P = 0.140). Similarly their admission CFD levels were higher than in survivors (median 3,712 vs. 1,974, P = 0.001). Spearman’s correlation analysis showed signifi cant correlation between APACHE II score and CFD level on admission (ρ = 0.315, P = 0.007). ROC curve for APACHE II score and CFD level on admission for prediction of 28-day mortality showed area under the curve of 0.59, 95% CI 0.44 to 0.74 (P = 0.208), for APACHE II score; and area under the curve of 0.73, 95% CI 0.60 to 0.86 (P = 0.001), for CFD level on admission. The study group was divided into quintiles by CFD levels of admission. The 28-day mortality rate was 12.5% in the CFD lowest quintile and 60.9% in the highest quintile. Logistic regression analysis showed that adjusted for age, sex and APACHE II score CFD divided into quintiles was signifi cantly associated with death at 28 days, OR = 1.83 per quintile (95% CI 1.12 to 2.98, P = 0.015). g Reference Reference Methods We performed an observational cohort study in critically ill patients in the ICU of a tertiary care hospital. We investigated the correlation between EA levels and blood concentration of endotoxin measured by the chromogenic limulus amoebocyte lysate (LAL) assay, causative microorganism identifi ed in laboratory culture, procalcitonin (PCT), soluble CD14 subtype (named presepsin), IL-6, antithrombin, protein C, thrombomodulin, lactate, disseminated intravascular coagulation scores in both the Japanese Ministry of Health and Welfare and the Japanese Association for Acute Medicine, and severity of illness at ICU admission.i 1. Endo S, Yaegashi Y, Sato N, et al.: Comparative study of soluble CD14 and soluble CD14-subtype in sepsis. Med Postgrad 2006, 44:381-385. Reference 1. Marshall JC, et al.: J Infect Dis 2004, 190:527-534. Procalcitonin has a poor prognosis value in critically ill patients with candidemia refl ects the pathology. So we strongly suggest that presepsin will be not only a very useful new biomarker for a diagnosis of the sepsis, but also useful for monitoring the severity of the disease in the near future. Reference P34 Clinical usefulness of measuring endotoxin activity on ICU admission A Murai, H Ishikura, T Nishida, Y Irie, T Kamitani, R Yuge, T Kitamura, T Umemura Fukuoka University Hospital, Fukuoka City, Japan Critical Care 2012, 16(Suppl 1):P34 (doi: 10.1186/cc10641) g Results In our study population 5% had proven IA, 5% probable IA, 17.5% possible IA and 72.5% had no IA. We could not identify a GM threshold for IA. The best threshold was GM >1.1 for identifying patients with IA (proven + probable + possible) with a specifi city of 70.7% and negative predictive value of 76.6%. The ICU mortality was 41.9% and the hospital mortality was 58.1%. Patients who died in the ICU had higher APACHE, SAPS and SOFA scores (P <0.0001), and had a signifi cant increase in GM during their stay (0.27 ± 1.26 vs.–0.43 ± 1.7, P = 0.004). We observed higher mean GM values in nonsurvivors but Introduction According to the Surviving Sepsis Campaign, diagnosis of sepsis and infection is urgent, therefore rapid diagnostic tools play a major role in the management of septic patients. The endotoxin activity (EA) assay (EAA) is one of those tools based on the ability of antigen–antibody complexes to prime neutrophils for an augmented respiratory burst response [1]. EAA has been used widely in patients Introduction According to the Surviving Sepsis Campaign, diagnosis of sepsis and infection is urgent, therefore rapid diagnostic tools play a major role in the management of septic patients. The endotoxin activity (EA) assay (EAA) is one of those tools based on the ability of antigen–antibody complexes to prime neutrophils for an augmented respiratory burst response [1]. EAA has been used widely in patients S13 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 this was not statistically signifi cant. Patients who died in the hospital also showed a signifi cant increase in GM during their stay (0.11 ± 1.55 vs. –0.48 ± 1.51, P = 0.017). There was a trend towards higher GM values in patients treated with piperacillin/tazobactam (n = 34) but this was not statistically signifi cant. Neutropenic patients (n = 31) showed an increase in GM during their stay (0.32 ± 1.3 vs. –0.43 ± 1.7, P = 0.07). Patients on total parenteral nutrition (n = 125) had higher maximal GM levels (1.55 ± 1.94 vs. P34 0.88 ± 1.25, P = 0.058). Patients that were mechanically ventilated had signifi cantly higher mean (P = 0.038) and maximal (P = 0.007) GM levels. The presence of IA was associated with 100% hospital mortality. admission. Heart rate variability (HRV) is decreased in severe sepsis. The objective was to determine the ability of a panel of HRV indices to identify physiologically stable ED sepsis patients who will develop worsening organ failure. We hypothesized that patients meeting the outcome of progressive organ failure will have decreased HRV on initial presentation. Methods We performed a prospective observational study of adult ED patients admitted to the hospital for infection and treated with i.v. antibiotics. Patients in overt shock (vasopressor requirement or mechanical ventilation) at enrollment or with the inability to provide written informed consent were excluded. A panel of HRV indices was assessed over a 2-hour ED period using CIMVA (continuous individualized multiorgan variability analysis) software including standard deviation (SD), LF/HF ratio, Poincare SD, sample entropy, wavelet AUC, detrended fl uctuation analysis (DFA), correlation dimension, and the Lyapunov exponent. Patients were followed to assess the occurrence of the primary outcome of increased organ failure (SOFA score increase greater than 1 point at 24 hours), mechanical ventilation, vasopressor use, or in-hospital mortality. p y Conclusion The current GM threshold of 0.5 ng/ml does not allow one to discriminate between patients with and without IA. A threshold of 1.1 ng/ml had the best specifi city and negative predictive value for IA. There seems to be a correlation between GM levels and total parenteral nutrition due to interference with the ELISA test. p p y Results We enrolled 105 ED sepsis patients. Twenty patients were removed due to nonsinus cardiac rhythm or poor data quality of the telemetry signal. Complete HRV assessment was performed on 81 subjects with 17 patients removed who developed shock in the ED. The primary outcome was met in 44% (28/64) of the cohort. On HRV assessment, outcome patients had a lower LF/HF ratio (1.47 vs. 3.11, P = 0.009) and DFA (0.65 vs. 0.94, P = 0.04) compared with stable patients with no diff erences in other HRV indices. The overall mortality rate was 15%. Compared to stable patients, outcome patients had no diff erence in age, initial heart rate, systolic blood pressure, or serum lactate with similar initial SOFA scores that were higher at 24 hours (1.0 vs. P38 P38 Severe community-acquired pneumonia: risk factors for in-hospital mortality JM Pereira1, JA Paiva1, JP Baptista2, F Froes3, J Gonçalves-Pereira4 1Centro Hospitalar S. João, Porto, Portugal; 2Hospitais Universidade Coimbra, Portugal; 3Hospital Pulido Valente – CHLN, Lisbon, Portugal; 4Hospital S. Francisco Xavier, Lisbon, Portugal Critical Care 2012, 16(Suppl 1):P38 (doi: 10.1186/cc10645) Severe community-acquired pneumonia: risk factors for in-hospital mortality JM Pereira1, JA Paiva1, JP Baptista2, F Froes3, J Gonçalves-Pereira4 1Centro Hospitalar S. João, Porto, Portugal; 2Hospitais Universidade Coimbra, Portugal; 3Hospital Pulido Valente – CHLN, Lisbon, Portugal; 4Hospital S. Francisco Xavier, Lisbon, Portugal Critical Care 2012, 16(Suppl 1):P38 (doi: 10.1186/cc10645) f Conclusion The BDG profi le in ICU patients is similar to that of other inpatients. It can be useful in clinical practice if implemented in the proper setting and interpreted after consideration of the patient’s clinical status. Introduction Severe community-acquired pneumonia (SCAP) is an important cause of hospital mortality. The goal of this study was to identify variables associated with increased risk of in-hospital mortality at ICU admission. Introduction Severe community-acquired pneumonia (SCAP) is an important cause of hospital mortality. The goal of this study was to identify variables associated with increased risk of in-hospital mortality at ICU admission. P34 3.0), a higher ICU transfer rate (62 vs. 20%, P <0.001) and increased ICU length of stay. P36 Analysis of (13)β-D-glucan as a diagnostic adjunct for invasive fungal infections in the ICU setting N Yamada, K Shirai, T Doi, K Kumada, M Nakano, S Yoshida, I Toyoda, S Ogura Gifu University Hospital, Gifu, Japan Critical Care 2012, 16(Suppl 1):P36 (doi: 10.1186/cc10643) References 1. Committee for Guideline for Management of Deep-seated Mycoses 2007: 1. Committee for Guideline for Management of Deep-seated Mycoses 2007: Guideline for Management of Deep-seated Mycoses 2007. Tokyo: Kyowa kikaku; 2007. 1. Committee for Guideline for Management of Deep-seated Mycoses 2007: Guideline for Management of Deep-seated Mycoses 2007. Tokyo: Kyowa kikaku; 1. Committee for Guideline for Management of Deep-seated Mycoses 2007: G id li f M f D d M 2007 T k K kik k g p y Guideline for Management of Deep-seated Mycoses 2007. Tokyo: Kyowa kikaku; 2007. Guideline for Management of Deep-seated Mycoses 2007. Tokyo: Kyowa kikaku; 2007. Methods A prospective, multicentre, observational cohort study of all patients with SCAP consecutively admitted to 15 Portuguese ICUs during a 12-month period. Demographic characteristics, co-morbidities, general severity scores (SAPS II, SAPS3, total SOFA), microbiological data and initial empirical antibiotherapy were recorded. Logistic regression analysis was performed to identify predictors of in-hospital mortality. Results A total of 505 (14%) of the 3,572 enrolled patients had SCAP, mostly male (66%) with a median age 58 (29 to 82). Median general severity scores were: SAPS II 44 (21 to 80), SAPS3 65 (41 to 98) and total SOFA 8 (3 to 17). Comorbidities were present in 74% of the patients and the most frequent were: diabetes mellitus (22%), chronic respiratory failure (18%) and alcoholism (15%). Median Charlson’s comorbidity index was 4 (0 to 13). At ICU admission, 44% of SCAP patients had septic shock. Thirty-seven per cent of the cases were microbiologically documented (St. pneumoniae – 24%; infl uenza A 2. Drosos EK, et al.: β-D-glucan assay for the diagnosis of invasive fungal infections: a meta analysis. Clin Infect Dis 2011, 52:750–770. 2. Drosos EK, et al.: β-D-glucan assay for the diagnosis of invasive fungal infections: a meta analysis. Clin Infect Dis 2011, 52:750–770. Analysis of (13)β-D-glucan as a diagnostic adjunct for invasive fungal infections in the ICU setting Introduction Since invasive fungal infections are associated with high morbidity and increased mortality in the ICU, early diagnosis and treatment are essential. This study assesses the performance of an assay of serum (13)-D-glucan (BDG) concentration in patients admitted to the ICU. Conclusion While standard physiologic parameters in the ED were unable to diff erentiate sepsis patients who developed increased organ failure, a decreased LF/HF ratio and DFA, measurements of variability representing physiologic reserve, was associated with impending deterioration. The ability of decreased HRV to predict clinical outcomes in a high-risk yet physiologically identical population at presentation supports the need for continued studies into the predictive role of HRV assessment in the ED to supplement clinical decision-making in sepsis patients. Methods Patients admitted to our advanced critical care center from April 2007 to March 2011 with measurements of BDG were enrolled in this retrospective study. BDG was measured when invasive fungal infection was suspected based on the Japanese guidelines for diagnosis and treatment of invasive fungal infections. BDG levels were measured using the WAKO method. A BDG level greater than 11 pg/ml was considered to be positive. No gray zone was considered. Results Of the 872 patients enrolled in this study, there were 580 males and 292 females. The mean age was 60.7 years (range: 48 to 87). The mortality rate was 16.3%. We make a clinical diagnosis of invasive fungal infections according to Japanese guidelines for diagnosis and treatment of invasive fungal infections. The sensitivity of the BDG assay was 71.9% and the specifi city was 91.0%. There were signifi cant diff erences in sensitivity, specifi city, and optimal cut-off points among patients with diff erent clinical conditions (that is, trauma, burn, postoperative, and medical conditions).The area under the summary receiver operating characteristic curve was 0.82, but there were also diff erences across clinical categories. Characteristics of leptospirosis patients admitted to a tropical university hospital during the 2000 to 2010 period H Mehdaoui, E Caffi ot, R Theodose, R Valentino, D Resiere, C Chabartier, M Jonas Fort de France University Hospital, Fort De France, Martinique Critical Care 2012, 16(Suppl 1):P40 (doi: 10.1186/cc10647) Characteristics of leptospirosis patients admitted to a tropical university hospital during the 2000 to 2010 period H Mehdaoui, E Caffi ot, R Theodose, R Valentino, D Resiere, C Chabartier, M Jonas Fort de France University Hospital, Fort De France, Martinique Critical Care 2012, 16(Suppl 1):P40 (doi: 10.1186/cc10647) Introduction Leptospirosis is an endemic disease in the intertropical area. Most of the patients present with mild to moderate clinical forms, but leptospirosis may lead to multiple organ failure and death. Methods We retrospectively analyzed the characteristics of 113 patients with leptospirosis admitted to our emergency department. Results PCR and/or immunological investigations confirmed the Introduction Leptospirosis is an endemic disease in the intertropical area. Most of the patients present with mild to moderate clinical forms, but leptospirosis may lead to multiple organ failure and death. Introduction Leptospirosis is an endemic disease in the intertropical area. Most of the patients present with mild to moderate clinical forms, but leptospirosis may lead to multiple organ failure and death. Methods We retrospectively analyzed the characteristics of 113 patients with leptospirosis admitted to our emergency department. Results PCR and/or immunological investigations confi rmed the diagnosis for 88 patients. We compared the periods before and after PCR diagnosis implementation (2006), and determined the pattern of the most severe forms. Thirty-two patients were admitted to our ICU. Eight of the ICU patients died including four with confi rmed diagnosis. Nineteen patients were diagnosed before 2006, and 69 during the period to 2010. Patients were less frequently admitted to the ICU during the second period (29% vs 63% P = 0 013) ICU patients had a higher y g Methods We retrospectively analyzed the characteristics of 113 patients with leptospirosis admitted to our emergency department.i Conclusion Disease severity evaluated by SAPS II and sepsis staging score and inappropriate initial antibiotherapy were independent risk factors for in-hospital mortality. The use of a macrolide was independently associated with a reduced risk of death. Results PCR and/or immunological investigations confi rmed the diagnosis for 88 patients. We compared the periods before and after PCR diagnosis implementation (2006), and determined the pattern of the most severe forms. Thirty-two patients were admitted to our ICU. Eight of the ICU patients died including four with confi rmed diagnosis. Nineteen patients were diagnosed before 2006, and 69 during the period to 2010. Patients were less frequently admitted to the ICU during the second period (29% vs. P40 (H1N1) virus – 20%; Enterobacteriaceae – 18%) and 12% had secondary bacteremia. Antibiotics were administered in the fi rst 3 hours after hospital admission in 71% of the patients and 76% of them received combination therapy. Antibiotherapy was appropriate in 80% with a median duration of 8 days. Median ICU and hospital lengths of stay were 10 and 19 days respectively. Median ICU and hospital mortalities were 25% and 34% respectively. Variables independently associated with hospital mortality were: SAPS II score (OR 1.06; 95% CI 1.037 to 1.086), severe sepsis (OR 3.61; 95% CI 1.334 to 9.791), septic shock (OR 4.25; 95% CI 1.61 to 11.194), inappropriate antibiotherapy (OR 5.06; 95% CI 1.766 to 14.516) and the use of a macrolide (OR 0.40; 95% CI 0.203 to 0.809). Systemic corticosteroids for community-acquired pneumonia in adults RJ Pugh, N Roy Glan Clwyd Hospital, Rhyl, UK Introduction We aimed to evaluate evidence from randomised controlled trials (RCTs) investigating the eff ect of systemic corticosteroids in adults with community-acquired pneumonia (CAP). Observational data suggest that corticosteroids may decrease mortality in severe CAP [1], and several large RCTs have been published since the recent Cochrane review [2]. Conclusion The use of PCR dramatically improved the diagnosis of mild to moderate forms of leptospirosis and led to an apparent increase its incidence. Severe forms were less easy to assess as they occur later and we should have a more aggressive policy to improve the immunological diagnosis which was sometimes neglected since the implementation of PCR diagnosis. Severe forms have a more pronounced infl ammatory syndrome and diff use organ failure. Aggressive fl uid loading as recommended in septic states may have worsened hemodynamic and respiratory conditions in the ICU group. This is suggested by the hemodilution pattern found in this group. The association of renal, myocardial and respiratory failures in leptospirosis should lead to a careful monitoring of fl uid loading and myocardial status. Methods A systematic review of the literature: Cochrane Central Register for Controlled Clinical Trials, MEDLINE, EMBASE and SCOPUS, and reference lists of original studies and reviews. Data were collated and analysed using Review Manager v5.1. y g g Results A total of 254 RCTs were identifi ed. Seven met inclusion criteria, totalling 806 patients. Studies varied in methodology, participants, interventions, and outcome measures. Where meta- analysis was possible, data are presented in Table 1 (outcomes: hospital mortality, 30-day mortality, hospital length of stay, superinfection, hyperglycaemia). Excepting hyperglycaemia, eff ect estimates were not statistically signifi cant. Two small studies (n = 46 and n = 30) concentrated on severe CAP (using ATS and BTS criteria); one study found a statistically signifi cant reduction in mortality, lengths of stay and duration of mechanical ventilation in the steroid group, but similar improvements in the other study, and in a large subgroup of patients with severe CAP in another study (n = 93) were not found. Signifi cant reductions in infl ammatory markers in the week following initiation of steroid treatment were found in six studies. Characteristics of leptospirosis patients admitted to a tropical university hospital during the 2000 to 2010 period H Mehdaoui, E Caffi ot, R Theodose, R Valentino, D Resiere, C Chabartier, M Jonas Fort de France University Hospital, Fort De France, Martinique Critical Care 2012, 16(Suppl 1):P40 (doi: 10.1186/cc10647) 63%, P = 0.013). ICU patients had a higher heart rate (111 ± 28 vs. 93 ± 21, P = 0.001), and had more frequently jaundice (94% vs. 64%, P = 0.002) and oliguria (81% vs. 23%, P <0.001). Glycemia (8.7 ± 3.3 vs. 7.1 ± 3.4, P = 0.04), creatinin (530 ± 299 vs. 142 ± 113, P <0.0001), bilirubin (423 ± 251 vs. 69 ± 103, P <0.0001), CRP (325 ± 135 vs. 210 ± 127, P <0.0001), and WBCC (21.7 ± 9.5 vs. 9.7 ± 5.3, P <0.0001) were higher and protidemia (58 ± 15 vs. 68 ± 13, P = 0.002), hematocrit (24 ± 6 vs. 34 ± 6, P <0.0001), and P/F ratio (271 ± 127 vs. 352 ± 84, P = 0.036) were lower in the ICU group. Troponin was increased more frequently in the ICU group (44% vs. 9%, P = 0.0003) and ECG anomalies (78% vs. 52%, P = 0.02) were more frequent. Among the 22 early cardiac echographies performed in the ICU, 11 patients had LVEF <50%. P37 Impaired heart rate variability predicts clinical deterioration and progressive organ failure in emergency department sepsis patients R Arnold, G Green, A Bravi, S Hollenberg, A Seely P37 Impaired heart rate variability predicts clinical deterioration and progressive organ failure in emergency department sepsis patients R Arnold, G Green, A Bravi, S Hollenberg, A Seely Cooper University Hospital, Camden, NJ, USA Critical Care 2012, 16(Suppl 1):P37 (doi: 10.1186/cc10644) g Cooper University Hospital, Camden, NJ, USA g Cooper University Hospital, Camden, NJ, USA p y p , , , Critical Care 2012, 16(Suppl 1):P37 (doi: 10.1186/cc10644) p y p , , , Critical Care 2012, 16(Suppl 1):P37 (doi: 10.1186/cc10644) Introduction Emergency department (ED) sepsis patients without overt shock have a high incidence of clinical deterioration after S14 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P41 Prognostic impact of imported and newly-isolated methicillin-resistant Staphylococcus aureus in the ICU S Ohshimo, K Ota, T Tamura, Y Kida, J Itai, K Suzuki, K Kanao, Y Torikoshi, K Koyama, T Otani, T Sadamori, K Une, R Tsumura, Y Iwasaki, N Hirohashi, K Tanigawa Hiroshima University, Hiroshima, Japan Critical Care 2012, 16(Suppl 1):P41 (doi: 10.1186/cc10648) Prognostic impact of imported and newly-isolated methicillin-resistant Staphylococcus aureus in the ICU S Ohshimo, K Ota, T Tamura, Y Kida, J Itai, K Suzuki, K Kanao, Y Torikoshi, K Koyama, T Otani, T Sadamori, K Une, R Tsumura, Y Iwasaki, N Hirohashi, K Tanigawa Table 1 (abstract P39). Meta-analysis of clinical outcomes Outcome Number of studies Population Eff ect Hospital mortality 5 537 OR 0.65 30-day mortality 3 562 OR 0.90 Hospital LOS 2 244 MD –1.52 Superinfection 3 563 OR 1.24 Hyperglycaemia 2 517 OR 2.69* LOS, length of stay. P <0.0001. Table 1 (abstract P39). Meta-analysis of clinical outcomes Table 1 (abstract P39). Meta-analysis of clinical outcomes Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a leading pathogen of hospital-acquired pneumonia. The diff erence in outcome between patients with imported and newly-isolated MRSA in the ICU has not been well investigated. The aim of our study was to explore the incidence, risk factors and outcome in patients with imported and newly-isolated MRSA. Methods Patients admitted to the ICU in our university between April 2009 and May 2010 were prospectively studied. Nasal swabs were collected from all patients on admission and subsequently collected weekly during the ICU stay. When patients were intubated, intratracheal aspirates were concurrently collected. The correlations of positive culture of MRSA with clinical variables were analysed. Conclusion Systemic corticosteroid administration as adjunctive treatment for CAP does not appear to improve relevant clinical outcomes, regardless of severity, and is associated with signifi cantly increased incidence of hyperglycaemia. R f P43 467 females. Median age was 63 (1 to 97). Of these, imported MRSA was found in 124 (10%) patients, and newly-isolated MRSA in 57 (4%) patients. The incidence of imported MRSA was associated with the comorbidity of cardiovascular disease or malignancy and long hospital stay before admission to the ICU, whereas the incidence of newly- isolated MRSA was associated with the positive culture in intratracheal aspirates or blood/intravenous catheter, the comorbidity of shock, pneumonia or trauma, increased number of isolated sites, higher APACHE II score, prolonged ICU stay and higher mortality during the ICU stay. Although no statistical signifi cance was found in total patients, the subset analysis of the male patients demonstrated that the outcome of newly-isolated patients was signifi cantly poor compared with those of imported MRSA (P = 0.005). Multivariate analysis revealed that new isolation of MRSA in the ICU (P = 0.03; hazard ratio (HR), 2.62), negative culture of MRSA in nasal swab (P = 0.02; HR, 4.18), ≥2 isolated sites (P = 0.01; HR, 4.59) and comorbidity of ARDS (P = 0.002; HR, 4.63) were the independent poor prognostic factors. Predicting methicillin-resistant Staphylococcus aureus in critically ill patients with pneumonia presenting to the hospital AF Shorr1, DE Myers2, DB Huang3, BH Nathanson4, MF Emmons5 1Washington Hospital Centre, Washington, DC, USA; 2Pfi zer, Inc., Collegeville, PA, USA; 3VA NJ Healthcare System, East Orange, NJ, USA; 4OptiStatim, LLC, Longmeadow, MA, USA; 5Cerner LifeSciences, Beverly Hills, CA, USA Critical Care 2012, 16(Suppl 1):P43 (doi: 10.1186/cc10650) Introduction Methicillin-resistant Staphylococcus aureus (MRSA) represents an important pathogen in those presenting to the hospital with pneumonia and requiring ICU admission. However, empiric treatment against MRSA in those admitted to the ICU with severe non- nosocomial pneumonia could lead to overuse of anti-MRSA therapy. To address this concern, we sought to develop a simple clinical score for identifying ICU patients presenting to the hospital with pneumonia unlikely to be caused by MRSA. y y Methods We retrospectively identifi ed patients admitted to the ICU with community-acquired pneumonia (CAP) or healthcare-associated pneumonia (HCAP) between April 2007 and March 2009 at 62 hospitals in the USA. The diagnosis of pneumonia was based on ICD-9 codes. We only included patients with laboratory evidence of bacterial infection (for example, positive sputum, blood, pleural cultures or urinary antigen testing). P42 P42 Necrotizing pneumonia due to methicillin-sensitive Staphylococcus aureus secreting Panton-Valentine leukocidin: a review of case reports L Kreienbuehl1, E Charbonney2, P Eggimann3 1HUG, Geneva, Switzerland; 2Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, Canada; 3CHUV, Lausanne, Switzerland Critical Care 2012, 16(Suppl 1):P42 (doi: 10.1186/cc10649) Necrotizing pneumonia due to methicillin-sensitive Staphylococcus aureus secreting Panton-Valentine leukocidin: a review of case reports p L Kreienbuehl1, E Charbonney2, P Eggimann3 1HUG, Geneva, Switzerland; 2Li Ka Shing Knowledge Institute, St Michael’s Hospital, Toronto, Canada; 3CHUV, Lausanne, Switzerland Critical Care 2012, 16(Suppl 1):P42 (doi: 10.1186/cc10649) Introduction Community-acquired necrotizing pneumonia caused by Panton-Valentine leukocidin (PVL)-secreting Staphylococcus aureus is a highly lethal infection, which mainly aff ects healthy children and young adults [1,2]. This study focuses on necrotizing pneumonia due to methicillin-sensitive S. aureus strains, with the purpose to determine factors associated with outcome. pi y Conclusion The prevalence of MRSA in patients with CAP or HCAP requiring ICU care was high. A score to assess the risk for MRSA in these patients performed poorly but requires external validation. Given the high risk of MRSA in this setting along with the limited discriminatory power of our risk score, empiric therapy for MRSA in these patients seems appropriate. Methods We performed a systematic review of case reports on PVL-secreting MSSA necrotizing pneumonia and analyzed factors associated with outcome. Results A total of 32 patient descriptions were retained for analysis. Septic shock, infl uenza-like prodrome and the absence of a previous skin and soft tissue infection were associated with fatal outcome. In multivariate analysis, infl uenza-like prodrome (OR 7.44; 95% CI: 1.24 to 44.76; P = 0.028) and absence of previous skin and soft tissue infection (OR 0.09; 95% CI: 0.010 to 0.86; P = 0.036) remained signifi cant predictors of death. See Table 1. P44 P44 Predictors of multidrug-resistant Acinetobacter baumannii infections: a retrospective analysis in surgical ICU patients A Camkiran, A Kundakci, C Araz, A Pirat, P Zeyneloglu, H Arslan, G Arslan Baskent University, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P44 (doi: 10.1186/cc10651) Table 1 (abstract P42). Univariate analysis of mortality risk factors Univariate Died Survived analysis (n = 13) (n = 19) OR (95% CI) P value Flu-like prodrome 9/12 (75%) 4/16 (25%) 9.00 (1.60 to 50.7) 0.020 SSTI 1/13 (8%) 9/19 (47%) 0.09 (0.01 to 0.86) 0.024 Septic shock 11/11 7/15 (47%) 26.0 (1.30 to 522) 0.007 Leukocytopenia 9/11 (82%) 8/17 (47%) 5.06 (0.83 to 30.8) 0.115 Conclusion Infl uenza-like prodrome may be predictive of adverse outcome and previous skin and soft tissue infection may be associated with improved prognosis. References 1. Gillet Y, et al.: Lancet 2002, 359:753-759. 2. Gillet Y, et al.: Clin Infect Dis 2007, 45:315-321. Table 1 (abstract P42). Univariate analysis of mortality risk factors Univariate Died Survived analysis (n = 13) (n = 19) OR (95% CI) P value Flu-like prodrome 9/12 (75%) 4/16 (25%) 9.00 (1.60 to 50.7) 0.020 SSTI 1/13 (8%) 9/19 (47%) 0.09 (0.01 to 0.86) 0.024 Septic shock 11/11 7/15 (47%) 26.0 (1.30 to 522) 0.007 Leukocytopenia 9/11 (82%) 8/17 (47%) 5.06 (0.83 to 30.8) 0.115 Table 1 (abstract P42). Univariate analysis of mortality risk factors Introduction Multidrug-resistant Acinetobacter baumannii (MRAB) is an important cause of hospital-acquired infection and leads to an increasing morbidity and mortality in ICUs. The aim of this study was to investigate the predictors of MRAB infection in surgical ICU patients. Methods The charts of the patients who were admitted to the ICU between January 2008 and August 2010 were reviewed to identify patients with MRAB infection. Recorded data were as follows: age, sex, medical history, underlying surgical pathology, APACHE II score on ICU admission, days in hospital before ICU, presence of invasive procedures (intubation, tracheostomy, arterial, central venous lines, urinary and nasogastric catheters, enteral or parenteral nutrition and renal replacement therapy), days in the ICU and white blood cell (WBC) count on infection day, infection site, complications (such as organ/ system failure), length of stay (LOS) in the ICU and hospital, and fi nal outcome. P43 We determined, via logistic regression, variables independently associated with the presence of MRSA (two-thirds of cohort) and developed a risk score based on this. We then internally validated (one-third of cohort) the score. p p p g Conclusion The new isolation of MRSA during the ICU stay was associated with poor outcome compared with the imported MRSA. Clinicians should be aware of the high-risk group of MRSA infection. Strict hand hygiene plus a careful assessment of the patient, applying aggressive procedures such as patient isolation, staff cohorting, and active surveillance cultures should be indicated. Results The cohort included 957 patients (mean age 65.8 ± 16.4 years, 50.2% male, 43.7% HCAP). MRSA was identifi ed in 20.1%. The risk score assigned points as follows: 1 point – age <30 or >79 years, recent immunosuppression other than corticosteroids, shock; 2 points – admission from a skilled nursing facility, history of diabetes without coronary artery disease (CAD) or heart failure without CAD. The prevalence of MRSA increased with escalating score (P <0.001). We collapsed the score into three strata based on risk for MRSA (score of 0 to 1 (low), 2 to 4 (moderate), ≥5 (high)). The respective MRSA rates by strata equaled 15.2%, 24.7%, and 31.9%, (P <0.001). A score ≤1 as a screening test to exclude MRSA performed poorly (sensitivity 58.3%, specifi city of 53.3%). 1. Gillet Y, et al.: Lancet 2002, 359:753-759. References positive culture of MRSA with clinical variables were analysed. Results A total of 1,270 consecutive patients were enrolled. Median follow-up period was 404 (187 to 609) days. There were 803 males and 1. Garcia-Vidal et al.: Eur Respir J 2007, 30:951-956. 2. Chen et al.: Cochrane Database of Systematic Reviews 2011, 3. S15 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 2. Gillet Y, et al.: Clin Infect Dis 2007, 45:315-321. P45 P45 Risk factors for bronchial acquisition of resistant Gram-negative bacteria in critically ill patients and outcome I Papakonstantinou1, E Perivolioti1, C Vrettou2, I Baraboutis1, E Magira2, E Balioti1, D Panopoulou1, T Pitsolis1, C Routsi2, S Nanas2 1Evaggelismos Hospital, Athens, Greece; 2National and Kapodistrian University of Athens, Greece Critical Care 2012, 16(Suppl 1):P45 (doi: 10.1186/cc10652) Figure 1 (abstract P46). Introduction It has been advocated that resistant Gram-negative bacteria (RGNB) colonization of ICU patients is to some extent a result of increased use of antibiotics. The aim of our study was to investigate, in adjustment with patients’ characteristics, the impact of colonization status and antibiotic use during ICU stay on the impending acquisition of RGNB in the bronchial tree of newly intubated patients and to estimate the outcome. Methods Bronchial and pharyngeal surveillance cultures were obtained up to day 7 (d7) of ICU admission. RGNB considered for analysis on d7 were A. baumannii (RAB) and K. pneumoniae (RKP). Polymicrobial colonization with ≥2 RGNB (PMC) was also evaluated. To assess dependence between diff erent explanatory variables, multivariable logistic regression was used. Variables included in the model were: SOFA score, department prior to ICU admission, medical cause of admission, emergency surgery, CRF, prior aminoglycosides and tigecycline use during ICU stay and concurrent RAB or RKP pharyngeal colonization, respectively. To estimate outcome (death), variables included in multivariate model were: APACHE, SOFA score, department prior to ICU admission, medical cause of admission, emergency surgery, CRF and d7 RAB, RKP.i Figure 1 (abstract P46). Conclusion The need for a complex strategy to manage and eradicate an MRA outbreak in the ICU led to a clinically signifi cant decrease in antibiotic use and prescribing cost saving following eradication of MRA. Improved antibiotic stewardship is achievable through better infection control strategies. g y g y Results Ninety-fi ve eligible patients with bronchial colonization data on d7 were included for further analysis. In the case of RAB in multivariate model (R2 = 0.538), pharyngeal d7 RAB was the only predictor of d7 RAB bronchial colonization (OR 0.042, 95% CI 0.012 to 0.148, P <0.001). In the case of RKP in multivariate model (R2 =0.648), pharyngeal d7 RKP (OR 0.037, 95% CI 0.004 to 0.031, P = 0.004), aminoglycoside use (OR 0.094, 95% CI 0.015 to 0.573, P = 0.01) and SOFA score (OR 1.66, 95% CI 1.07 to 2.58, P = 0.023) characterized bronchial d7 RKP colonization. P45 Multivariate model for PMC (R2 = 0.49) revealed only d7 pharyngeal PMC as predictor of bronchial PMC (OR 0.12, 95% CI 0.026 to 0.50, P = 0.004). Department prior to ICU, medical cause of admission, CRF, and emergency surgery were not found to infl uence RGNB bronchial colonization. Outcome death increased with APACHE score (OR 0.84, 95% CI 0.76 to 0.94, P = 0.002) and bronchial d7 RKP colonization (OR 9.14, 95% CI 1.3 to 64.4, P = 0.026). Acknowledgements Thanks to Chelsea and Westminster Healthcare Charity, and ICU and microbiology staff . References Results During the study period 25 patients with MRAB infection were identifi ed. When compared with their matched control group (n = 25), 1. Gillet Y, et al.: Lancet 2002, 359:753-759. 2. Gillet Y, et al.: Clin Infect Dis 2007, 45:315-321. S16 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 patients with MRAB infection had a signifi cantly higher mean APACHE II score (P <0.001) and more frequently had an open wound (P = 0.002) or required mechanical ventilation (P = 0.005), arterial catheterization (P = 0.006), and central venous catheterization (P = 0.004). Multivariate logistic regression revealed that APACHE II score (OR, 1.19; 95% CI, 1.005 to 1.315; P = 0.043) and open wound (OR, 0.45; 95% CI, 0.003 to 0.587; P = 0.18) were predictors of MRAB infection in these patients. Compared to their controls, patients with MRAB infection had a longer LOS in the ICU (36.44 ± 30.44 days vs. 7.80 ± 8.13 days, P <0.001) and hospital (55.12 ± 40.81 days vs. 19.04 ± 13.44 days, P <0.001). In-hospital mortality rates for patients with MRAB infection and their controls were 56% and 32%, respectively (P = 0.154). Reference 1. Kollef M, et al.: Ann Internal Med 2001, 134:298-314. Improved antibiotic stewardship resulting from a multifaceted strategy implemented after an outbreak of multiresistant Acinetobacter baumannii in a university ICU M Beach, M Cohen, V Grover, J Ho, N Soni, B Azadian, S Singh Chelsea & Westminster Hospital, London, UK Critical Care 2012, 16(Suppl 1):P46 (doi: 10.1186/cc10653) Improved antibiotic stewardship resulting from a multifaceted strategy implemented after an outbreak of multiresistant Acinetobacter baumannii in a university ICU M Beach, M Cohen, V Grover, J Ho, N Soni, B Azadian, S Singh Chelsea & Westminster Hospital, London, UK Critical Care 2012, 16(Suppl 1):P46 (doi: 10.1186/cc10653) Introduction A 12-bed ICU experienced an outbreak of multiresistant Acinetobacter baumannii (MRA) from October 2009 to May 2010. A multifaceted strategy involving segregation, enhanced infection control procedures, and microbiological surveillance was implemented. We evaluated its impact on antibiotic stewardship. p y Conclusion Our results indicate that higher APACHE II scores and presence of an open wound are predictors of MRAB in ICU surgical patients. Patients with MRAB infection tended to have a higher mortality and had a longer LOS in the ICU and hospital than their controls. Methods A retrospective review of patient notes and results using AcuBase® was conducted: 90 consecutive patients before the outbreak (January to June 2008) and 91 thereafter (October 2010 to May 2011). Data included patient profi les, admission criteria, ICU survival, antimicrobials used, antibiotic days, number of patients on antibiotics, prescribing cost and the demographic of microbes isolated. Results Following the outbreak, enhanced infection control measures were implemented alongside the Matching Michigan protocols. Daily operational critical care and elective planning meetings and a staff education programme were undertaken. ICU mortality (31 (14%) vs. 43 (16%)) was unchanged. Microbiological isolates were overall similar, with a reduction in coagulase-negative Staphylococcus and Klebsiella and an increase in Enterobacter. The use of cefuroxime (3.2 vs. 2.3 antibiotic days/ patient) and quinolones (6 vs. 2) decreased. There was a reduction in average antibiotic days per patient episode (5.1 vs. 4.2) (P = 0.0291) and the prescribing cost savings were £13,558 (47%). See Figure 1. Predictive and prognostic factors of septic shock of nosocomial origin JP Quenot1, A Pavon1, C Binquet1, F Kara2, O Martinet3, F Ganster3, JC Navellou4, V Castelain5, D Barraud6, J Cousson4, JF Poussel7, P Perez8, K Kuteifan9, A Noirot2 y g y Results Fifty-one patients were included (30 with suspected IC and 21 with proven IC). At the onset of antifungal therapy, the Candida score was greater in the patients with suspected IC than in those with proven infection (3.7 ± 0.7 vs. 3.0 ± 0.8, P = 0.001) since multifocal colonization was more frequent in the former. In addition, the patients with suspected but unproven IC looked more seriously ill according to the SOFA score (8.3 ± 3.0 vs. 6.6 ± 3.5, P = 0.07). This mainly resulted from a greater level of hypotension as assessed through the SOFA score (2.8 ± 1.5 vs. 1.2 ± 1.5 points, P = 0.0006). Obviously, the clinical response to antifungal therapy was signifi cantly more consistent in the patients with unproven IC than in those with proven infection (P = 0.032). In addition, there was a trend toward an improved survival in the former patients (53 vs. 47%, P = 0.42). The only independent protective factor was echinocandin therapy duration (HR = 0.84 (95% CI 0.75 to 0.94), P = 0.0034).i 1University Hospital Bocage, Dijon, France; 2Centre Hospitalier, Haguenau, France; 3Nouvel Hopital Civil, Strasbourg, France; 4University Hospital, Besancon, France; 5Hopital Hautepierre, Strasbourg, France; 6Hopital Central, Nancy, France; 7Regional Hospital, Metz-Thionville, France; 8Hopital Brabois, Nancy, France; 9CHG, Mulhouse, France C iti l C 2012 16(S l 1) P49 (d i 10 1186/ 10656) Introduction The incidence of septic shock in intensive care in France is around 8 to 10%, with in-hospital mortality ranging from 55 to 60% [1]. Mortality increases by 10% when the infection causing septic shock is acquired in-hospital or in the ICU [1]. We aimed to determine predictive and prognostic factors for septic shock caused by a nosocomial infection (NI). Methods Subgroup analysis of a prospective, multicentre, observa- tional study performed between November 2009 and March 2011 in 14 ICUs from 10 university and community (nonacademic) hospitals in the northeast of France. This study was supported by the Collège Interrégional des Réanimateurs du Nord-Est. Patients were included if they were aged >18 years and had septic shock plus at least one criterion of hypoperfusion. Infection was classed as nosocomial if acquired in-hospital more than 48 hours after admission. References Methods A retrospective single-centre observational study including every patient with highly suspected but unproven IC (that is, Candida score >3, multifocal Candida sp. colonization, unresolved sepsis despite >2-day broad-spectrum antibiotics, negative blood culture) who received at least two doses of one echinocandin between 2008 and 2011. Patients with proven IC (that is, candidemia) over the same period were used as controls. These two groups of patients were compared regarding baseline characteristics and both clinical and biological follow-up data while receiving antifungal therapy. The clinical response to antifungal therapy was assessed through the SOFA score daily decrease from day 0 to day 3 in both groups and compared by repeated-measures ANOVA. Then, independent predictors of death in the ICU were determined by Cox regression analysis. 1. Gonçalves e Silva CR, Melo KE, Leão MV, Ruis R, Jorge AO: Relationship between Candida in vaginal and oral mucosae and salivary IgA. Rev Bras Ginecol Obstet 2008, 30:300-305. 2. Bai XD, Liu XH, Tong QY: Intestinal colonization with Candida albicans and mucosal immunity. World J Gastroenterol 2004, 10:2124-2126. 2. Bai XD, Liu XH, Tong QY: Intestinal colonization with Candida albicans and mucosal immunity. World J Gastroenterol 2004, 10:2124-2126. Empirical antifungal treatment in the critically ill patients: how does it impact on the outcome? R Bruyère, C Vigneron, J Quenot, M Hamet, F Dalle, S Prin, PE Charles University Hospital, Dijon, France R Bruyère, C Vigneron, J Quenot, M Hamet, F Dalle, S Prin, PE Charles University Hospital, Dijon, France Conclusion Of the parameters included in our model, concurrent d7 pharyngeal RAB and RKP, respectively, resulted eventually in bronchial colonization with the same pathogens. Of the overall antibiotics used only aminoglycosides had signifi cant correlation only for RKP colonization. y p j ical Care 2012, 16(Suppl 1):P47 (doi: 10.1186/cc10654) Introduction Given the high mortality attributable to invasive candidiasis (IC) and the lack of reliable diagnosis tool, antifungals S17 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion The above data show a slight reduction of Candida spp. colonization in septic shock patients treated with IgGAM therapy. Further studies are needed to confi rm this fi nding. are often started in high-risk patients with severe sepsis despite the absence of proven disease. According to current guidelines, echinocandins are the drugs of choice in this setting. However, the level of evidence supporting this statement is low. Conclusion The above data show a slight reduction of Candida spp. colonization in septic shock patients treated with IgGAM therapy. Further studies are needed to confi rm this fi nding. References Predictive and prognostic factors of septic shock of nosocomial origin Data control and statistical analysis were performed by the CIC-EC of Dijon University Hospital (INSERM Unit CIE1). Conclusion A signifi cant clinical improvement is achieved in patients with suspected but not proven IC receiving empirical antifungal therapy with an echinocandin. In contrast, the patients with proven IC are less responsive to therapy and are more likely to die in the ICU. Our data support the use of an echinocandin as empirical therapy in very high-risk patients. Reference Reference 1. Annane D: Am J Respir Crit Care Med 2003, 168:165. g References References 1. Pappas PG, et al.: Clin Infect Dis 2009, 48:503-535. 2. Leon C, et al.: Crit Care Med 2009, 37:1624-1633. 1. Pappas PG, et al.: Clin Infect Dis 2009, 48:503-535. 2 Leon C et al : Crit Care Med 2009 37:1624-1633 Results In total, 1,147 patients were included in the cohort, of whom 409 (35.6%) presented a NI (345/409 (84%) acquired in-hospital and 64/409 (16%) acquired in the ICU). The factors signifi cantly associated with NI (in-hospital or in-ICU) were: immunodepression, a Knaus score C to D, SAPS II score, and SOFA score. Other variables such as age, sex, type of admission and type of infection were not signifi cantly related to the nosocomial origin of infection. In-hospital mortality for community- acquired versus NIs was 40.8% vs. 53.5% respectively (P <0.01), and 46.9% vs. 62% respectively at 28 days (P <0.01). Relationship between polyclonal immunoglobulin therapy and colonization by Candida spp.i G Serafi ni, I Cavazzuti, C Venturelli, M Girardis University Hospital, Modena, Italy Critical Care 2012, 16(Suppl 1):P48 (doi: 10.1186/cc10655 Critical Care 2012, 16(Suppl 1):P48 (doi: 10.1186/cc10655) Conclusion Mortality of patients with septic shock of nosocomial origin is particularly high. Scores evaluating gravity of disease are also higher in patients with NI versus those with community-acquired infection. This could be explained by delayed presentation or diffi culties with management, but also by immunodepression and a poor state of prior health. It is likely that appropriate measures, particularly aimed at prevention, could help to reduce mortality in patients with septic shock caused by NI. Introduction Low IgA levels in blood serum and in saliva have been associated with an increased risk for Candida colonization and infec- tion [1,2]. In this retrospective cohort study, we aimed to evaluate the eff ects of an intravenous immunoglobulin preparation containing polyclonal IgG, IgM and IgA (IgGAM) on the prevention of Candida spp. colonization in patients with septic shock. p p Methods In this study we analyzed 69 patients with septic shock and without Candida spp. colonization before shock appearance admitted to the ICU of a university hospital from January 2008 to November 2011. All of the patients were treated in according to the Surviving Sepsis Campaign guidelines. In addition to standard therapy, 44 (64%) patients received IgGAM therapy (Pentaglobin® 38 g/l IgG, 6 g/l IgM, and 6 g/l IgA) within 24 hours from the diagnosis of septic shock at the dose of 250 mg/kg/day for 3 days. The colonization by Candida spp. was evaluated by analyzing the results of the microbiological surveillance cultures (two times per week) of pharyngeal swab, tracheal aspirate, urine and surgical drains between 48 hours and 21 days after the diagnosis of septic shock. P50 P50 Catheter-related bloodstream infection: factors aff ecting incidence K Boner1, M McGovern2, J Bourke1, C Walshe3, D Phelan1 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Harvard University, Cambridge, MA, USA; 3Beaumont Hospital, Dublin, Ireland Critical Care 2012, 16(Suppl 1):P50 (doi: 10.1186/cc10657) Introduction Catheter-related bloodstream infection (CRBSI), its associated morbidity, mortality and expense are the most important adverse eff ect of central venous catheters (CVCs) [1]. The objective of this study of a population in whom the rate of CRBSI fell signifi cantly g p Results In the IgGAM group, 11 patients (25%) developed Candida spp. colonization compared to nine patients (36%) of the control group. The Candida colonization index was similar in the two groups: 0.42 ± 0.16 in the IgGAM group and 0.45 in the control group. S18 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P <0.01); Knaus score C–D (OR 2.16, 95% CI 1.64 to 2.84, P <0.01); SOFA score (OR for an increase of 1 point 1.32, 95% CI 1.26 to 1.38, P <0.01); and infection acquired in the ICU (OR 1.86, 95% CI 1.03 to 3.37, P = 0.03). Protective factors were surgical admission (OR 0.61, 95% CI 0.41 to 0.89, P = 0.01) and urinary tract infection (OR 0.55, 95% CI 0.37 to 0.82, P <0.01). over 12 years [2] was to evaluate the infl uence of both patient and CVC factors on CRBSI rates in patients receiving total parenteral nutrition (TPN) in this time. Methods Set in a 525-bed university hospital providing acute and tertiary services. A prospective database was established in 1997, recording data on all patients with CVCs inserted for TPN administration. This database was examined up to 2009 to ascertain the eff ects of patient and CVC factors on CRBSI. Conclusion Our fi ndings are coherent with the literature. Multivariate analysis found nonmodifi able risk factors such as age, but also modifi able risk factors that warrant further investigation, such as infections acquired in-hospital or in the ICU. Future clinical studies in septic shock should take these fi ndings into account when selecting patients. p Results During the 12-year study period, 2,573 CVCs were inserted into 1,343 patients and 15,385 CVC days were accumulated. Overall, 13.8% of patients developed CRBSI throughout the study. Severe sepsis in the United Sates: a 5-year analysis J Knittel, S Quraishi Massachusetts General Hospital, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P52 (doi: 10.1186/cc10659) Massachusetts General Hospital, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P52 (doi: 10.1186/cc10659) Introduction We describe patient-level healthcare data related to severe sepsis over a 5-year period (2004 to 2008) in the United States. Methods We queried the largest all-payer inpatient care database in the United States to identify cases of hospital admissions between 2004 and 2008 with a primary diagnosis of severe sepsis (ICD 9: 995.92). This retrospective analysis was performed with data from the Healthcare Cost and Utilization Project National Inpatient Sample (NIS) repository. Data related to length of stay, in-hospital mortality, and hospital charge was extracted. The 2004 and 2008 data for these variables were compared and further analyzed by age and sex in SPSS v.19 (IBM Corporation, Amonk, NY, USA). Results are reported with ± standard error where applicable, and P <0.05 represented statistical signifi cance. Results Our query of the NIS data revealed a similar number of hospital admissions with a primary diagnosis of severe sepsis in 2004 versus 2008. Sex (male vs. female) and age group composition (18 to 44 vs. 45 to 64 vs. 65 to 85 vs. 85+) within these cohorts were similar. No signifi cant change in overall length of stay or in-hospital mortality rate was appreciated. However, a signifi cant increase in overall cost was appreciated ($67,670 ± 5,742 vs. $100,973 ± 10,525; P = 0.006), which outpaced healthcare-specifi c and general infl ation during this period. Sex did not infl uence length of stay or in-hospital mortality rate. Cost of care was higher for males versus females (2004: $78,361 ± 8,982 vs. $57,040 ± 5,959; P = 0.048 and 2008: $111,298 ± 13,835 vs. $90,730 ± 11,380; P <0.001). Age had a signifi cant infl uence on in- hospital mortality in 2004 and in 2008, with the highest percentage of in-hospital deaths in the 85+ category. Age also had a signifi cant infl uence on cost/day. Whereas in 2004 patients in the 85+ category represented the age subset with the lowest cost/day, in 2008 this age group witnessed a threefold increase in daily costs (P <0.001) and represented the highest cost/day subset. Conclusion CRBSI occurs commonly in TPN populations, but there are very limited published data as regards incidence or factors aff ecting incidence in this population. References 1. O’Grady NP, Alexander M, Dellinger EP, et al.; Healthcare Infection Control Practices Advisory Committee: Guidelines for the prevention of intravascular catheter-related infections. Infect Control Hosp Epidemiol 2002, 23:759-769. 2. Walshe CM, Boner K, Bourke J, et al.: Catheter related blood stream infection (CRBSI) in TPN patients. Benefi t of an educational programme using multimodal CRBSI expression. Clin Govern Int J 2010, 15:292-301. 2. Walshe CM, Boner K, Bourke J, et al.: Catheter related blood stream infection (CRBSI) in TPN patients. Benefi t of an educational programme using multimodal CRBSI expression. Clin Govern Int J 2010, 15:292-301. Severe sepsis in the United Sates: a 5-year analysis J Knittel, S Quraishi This large study of TPN patients provides prospective analysis of both patient and CVC factors infl uencing the development of CRBSI for the fi rst time. P50 In terms of patient factors aff ecting CRBSI rates, CRBSI was increased in patients with longer duration of TPN administration (where each additional day was associated with a relative risk ratio of 1.02, P <0.01), increased numbers of CVCs inserted (where each additional line was associated with a relative risk ratio of 1.21, P <0.01), and use of lipid formulation of TPN (58.9 vs. 49% use was associated with a relative risk ratio of 1.56, P <0.01). Overall 8.6% of CVCs inserted became infected. Hospital location of CVC insertion was an important risk factor for CRBSI. The most common site for insertion was the ICU (almost 40% of CVCs); however, compared to ICU insertion, insertion in the HDU was associated with an increased risk of CRBSI (a relative risk ratio of 1.75, P <0.01), as was insertion in the operating theatre for ward patients (a relative risk ratio of 2.08, P <0.01). CVC maintenance at ward level was associated with increased CRBSI rates, with a relative risk ratio of 2.06 (P <0.01). Reference Reference 1. Annane D: Am J Respir Crit Care Med 2003, 168:165. Prognostic factors of septic shock 1 1 2 JP Quenot1, A Pavon1, C Binquet2, F Kara3, O Martinet4, F Ganste JC Navellou5, V Castelain6, D Barraud7, J Cousson3, JF Poussel8, P Perez9, K Kuteifan3 JC Navellou5, V Castelain6, D Barraud7, J Cousson3, JF Poussel8, P Perez9, K Kuteifan3 1University Hospital Bocage, Dijon, France; 2CIC EC, Dijon, France; 3Centre Hospitalier, Haguenau, France; 4Nouvel Hopital Civil, Strasbourg, France; 5University Hospital, Besancon, France; 6Hopital Hautepierre, Strasbourg, France; 7Hopital Central, Nancy, France; 8Regional Hospitalier, Metz-Thionville, France; 9Hopital Brabois, Nancy, France p y Critical Care 2012, 16(Suppl 1):P51 (doi: 10.1186/cc10658) Conclusion Our data suggest that despite signifi cant increases in healthcare costs attributable to severe sepsis, survival and length of stay has not improved signifi cantly between 2004 and 2008. Dramatic increases in cost are particularly notable in males versus females and in patients who are 85 years old and over. Policies to control healthcare costs in the United States should focus on the root causes that lead to such signifi cant increases in cost without appreciable societal returns on investment. Introduction The incidence of septic shock in intensive care (ICU) in France is around 8 to 10%, with in-hospital mortality ranging from 55 to 60% [1]. The identifi cation of prognostic factors is essential to guarantee optimal management. Methods A prospective, multicentre, observational study was per- formed between November 2009 and March 2011 in 14 ICUs from 10 university and community (nonacademic) hospitals in the northeast of France. This study was supported by the Collège Interrégional des Réanimateurs du Nord-Est. Patients were included if they were aged >18 years and had septic shock plus at least one criterion of hypoperfusion. Data control and statistical analysis was performed by the CIC-EC of Dijon University Hospital (INSERM Unit CIE1). Univariate and multivariate logistic regression analysis was used to identify predictors of mortality at 28 days. P54 p Results Over a 24-month period we have a database with 1,571 patients. The results demonstrate that the median time to antibiotic administration is consistently near our target of 1 hour for all septic patients included in this pathway. Through continued refi nement and staged introduction the proforma and the process has demonstrated consistency from medical to surgical wards; introduction in new areas has rapidly improved results. See Figure 1 overleaf. Compliance with the sepsis resuscitation bundle in patients with severe sepsis and septic shock admitted to Scottish ICUs JA Davidson1, K Dunne2i Compliance with the sepsis resuscitation bundle in patients with severe sepsis and septic shock admitted to Scottish ICUs JA Davidson1, K Dunne2 1Victoria Infi rmary, Glasgow, UK; 2Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P54 (doi: 10.1186/cc10661) 1Victoria Infi rmary, Glasgow, UK; 2Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P54 (doi: 10.1186/cc10661) Introduction Severe sepsis is the second leading cause for admission to critical care and in spite of advanced care remains associated with a high mortality. When implemented the sepsis resuscitation bundle has been associated with a 20% reduction in mortality and is therefore recommended as standard care for all patients with severe sepsis [1]. Conclusion Our pathway has undergone a successful and dynamic development process guided by a multidisciplinary team. Compared with the usual audit process this has allowed rapid changes and improvements to take place and be tested. Further analysis of our database is ongoing, determining our impact on length of stay, mortality and intensive care admissions with a matched cohort. p p Methods All new admissions to seven west of Scotland ICUs were screened during a 12-week period for evidence of severe sepsis or septic shock. Those meeting the criteria were then assessed for sepsis bundle compliance. The Institute for Healthcare Improvement sepsis resuscitation bundle was taken as standard of care. This has a 6-hour time frame and includes measurement of serum lactate, blood cultures taken prior to antibiotics, antibiotics administered within 3 hours, fl uids of 20 ml/kg if hypotensive or hyperlatataemia and use of early goal-directed therapy in the event of persistent hypotension/ hyperlatataemia in spite of fl uid resuscitation.i P56 P56 Source-directed antimicrobials: a shot in the dark? Improving early administration of antibiotics: a ‘Plan Do Study Act’ approach Improving early administration of antibiotics: a ‘Plan Do Study Act’ approach A Revill1, N Wennicke2, J Tipping2, R Matull2 1Derriford Hospital, Plymouth, UK; 2Taunton Musgrove Park Hospital, Taunton, UK Critical Care 2012, 16(Suppl 1):P55 (doi: 10.1186/cc10662) Introduction Delayed administration of antibiotics is associated with an increased mortality in severe sepsis. The Surviving Sepsis Campaign advocates administering antibiotics to severely septic patients within 1 hour. Predicting the patients that will become severely septic is diffi cult, and therefore we have introduced a pathway via a unique care bundle to identify and treat all patients with suspected sepsis, prior to signifi cant organ dysfunction, and maintain a 1-hour target. Conclusion H1N1 pneumonia was associated with signifi cant morbidity and mortality requiring advanced multiorgan support in the majority of patients. Although the incidence of organ dysfunction in our cohort mirrored that found in the Swift study [1], in keeping with advances in management of H1N1-associated critical illness the mortality was lower in the current study. i y Methods In September 2009, we introduced an audit proforma and management tool into the medical admissions unit of our hospital. This was accompanied by an extensive education programme of all medical and nursing staff . The proforma consists of two parts, a recognition and intervention section. The process is triggered when the patient satisfi es two of the SIRS criteria and has symptoms consistent with an infection. All six management processes, including antibiotic administration, must then be completed within 1 hour of the trigger time. By using the ‘Plan Do Study Act’ cycle, we refi ned the proforma and streamlined the process and introduced it into emergency department and the surgical admissions unit. A dedicated multidisciplinary team was assigned to review and improve performance every 2 weeks by amending the form and processes. Methods In September 2009, we introduced an audit proforma and management tool into the medical admissions unit of our hospital. This was accompanied by an extensive education programme of all medical and nursing staff . The proforma consists of two parts, a recognition and intervention section. The process is triggered when the patient satisfi es two of the SIRS criteria and has symptoms consistent with an infection. Reference 1. Rowan KM, et al.: The Swine Flu Triage (SwiFT) study: development and ongoing refi nement of a triage tool to provide regular information to guide immediate policy and practice for the use of critical care services during the H1N1 swine infl uenza pandemic. Health Technol Assess 2010, 14:335-492. District hospital experience of organ support requirements for H1N1-associated pneumonia Results In total, out of 7,833 patients admitted to intensive care during the study period, 1,147 (14.6%) had septic shock. Factors signifi cantly associated with mortality at 28 days by logistic regression were: age >70 (OR 1.98, 95% CI 1.5 to 2.6, P <0.01); transfer (OR 1.42, 95% CI 1.04 to 1.95, P = 0.02); immunodepression (OR 1.91, 95% CI 1.41 to 2.57, Introduction The objective of our study was to describe the disease pattern, outcomes and organ support required in treating H1N1- associated pneumonia in a single-centre, district hospital ICU. S19 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods All of the patients with confi rmed H1N1 infection admitted to our ICU during the months of December 2010 and January 2011 were studied. The outcome measures were incidence, severity and support for organ dysfunction, length of stay in ICU and mortality. still a large proportion of patients not receiving aspects of the bundle in spite of being in a critical care environment. Reference still a large proportion of patients not receiving aspects of the bundle in spite of being in a critical care environment. Reference 1. Dellinger RP, Levy MM, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. Intensive Care Med 2007, 34:17-60. Results During the study period 27 patients were admitted. The mean age was 46.6 years (SD 13.6) with 20 (74%) patients being female, of whom two were pregnant. The mean APACHE scores were similar between survivors and nonsurvivors, 14.1 and 13.7 respectively. Twenty patients (74%) required invasive mechanical ventilation with median duration of 9 days (range 2 to 54 days). Advanced techniques like prone position ventilation and high-frequency oscillatory ventilation were required in 20% and 10% of these patients respectively. Two patients were referred for ECMO. Ventilator-associated pneumonia (VAP) ensued in 25% of invasively ventilated patients resulting in an increase in ventilator days (median) from 9 to 19 and ICU stay (median) from 15 to 23 days. Four (15%) required advanced cardiovascular support, 14 (52%) developed acute kidney injury (AKI) of which nine (33%) patients required renal replacement therapy. The ICU mortality was 11.1% and hospital mortality was 14.8%. The cohort who developed AKI had 21% mortality. The median ICU stay (range) was 15 days (2 to 68 days). P54 L Richardson1, GB McNeill2, S Gupta1 1University Hospital Southampton NHS Foundation Trust, Southampton, UK; 2Sheffi eld Teaching Hospitals NHS Foundation Trust, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P56 (doi: 10.1186/cc10663) l Results Of the 652 patients screened, 115 met the defi nition of severe sepsis or septic shock (17.6%). We collected full data from 108 patients, of which 69 patients (63.8%) had severe sepsis and 39 patients (36.1%) had septic shock. Full bundle compliance was 5.6%. Early ICU admission (within 6 hours) was associated with improved compliance with measured lactate (87.3% vs. 60.4%, P <0.01), and where indicated, vasopressor use (94.4% vs. 61.3%, P <0.01), CVP measurement (77.5% vs. 44.4%, P <0.01), and ScvO2 measurement (25.6% vs. 2.8%, P <0.01). ICU mortality was 12/64 patients (18.8%) with severe sepsis and 18/38 patients (47.4%) for those with septic shock. Full bundle compliance and mortality was not diff erent for those reaching ICU early compared with those who were admitted after 6 hours. Introduction The Surviving Sepsis Campaign advocates giving early empirical antibiotics directed against all likely pathogens [1]. The failure to instigate antimicrobials against a later confi rmed pathogen impacts negatively on mortality [2]. Many hospitals advise source- directed therapy from the beginning. Our project aims to elicit the proportion where the source of sepsis is initially predicted incorrectly thereby putting patients at risk. Methods A prospective cohort study was performed in two UK teaching hospitals of patients presenting with sepsis to critical care between May 2010 and March 2011. Hospital computer systems and patient notes were used to extract the initial suspected source of sepsis, and later verifi ed with true microbiology data. Overall mortality was measured and compared between correctly and incorrectly suspected source of sepsis patients. Conclusion At present the sepsis resuscitation bundle is not uniformly implemented. Although compliance with early goal-directed therapy and lactate measurement is better in those reaching ICU early, there is S20 Critical Care 2012, Volume 16 Suppl 1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 , pp http://ccforum.com/supplements/16/S1 Figure 1 (abstract P55). Median time to antibiotics. Figure 1 (abstract P55). Median time to antibiotics. We advise that in patients with severe sepsis or septic shock fi rst-line antibiotics should remain broad spectrum with rigorous follow up to de-escalate as early as possible. P54 References Results Of the 128 patients, the source of sepsis was wrongly identifi ed in 30% (38/128) (Southampton 28% 15/53, Sheffi eld 31% 23/75 respect- ively) (Figure 1). The most common source was the bowel, which was initially suspected as a respiratory source in most cases. Interestingly, the mortality was higher in the correctly identifi ed group (13%, 16/128 vs. 5%, 7/128). This probably refl ects the severity of illness where the diagnosis is sometimes more obvious. 1. Dellinger RP, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. 2. Kumar A, et al.: Initiation of inappropriate antimicrobial therapy results in fi vefold reduction of survival in human septic shock. Chest 2009, 136:1237-1248. 2. Kumar A, et al.: Initiation of inappropriate antimicrobial therapy results in fi vefold reduction of survival in human septic shock. Chest 2009, 136:1237-1248. 2. Kumar A, et al.: Initiation of inappropriate antimicrobial therapy results in fi vefold reduction of survival in human septic shock. Chest 2009, 136:1237-1248. Conclusion Good antimicrobial governance requires early adminis- tration of narrow-spectrum antibiotics as best guess source-directed therapy from the outset, because de-escalation is often not practical. Our data reveal that in 30% of cases we incorrectly guess the source. Figure 1 (abstract P56). Proportion of correctly and incorrectly identifi ed sources. P57 Relation between temperature in the initial 24 hours in patients with severe sepsis or septic shock with mortality and length of stay in the ICU Relation between temperature in the initial 24 hours in patients with severe sepsis or septic shock with mortality and length of stay in the ICU R Sanga, S Zanotti, C Schorr, B Milcareck, K Hunter, P Dellinger, J Parrilo Cooper University Hospital, Camden, NJ, USA Critical Care 2012, 16(Suppl 1):P57 (doi: 10.1186/cc10664) Relation between temperature in the initial 24 hours in patients with severe sepsis or septic shock with mortality and length of stay in the ICU P59 P59 Impact of antifungal treatment in ICU patients with Candida colonization: analysis of the EPIC II study population D Kett1, G Dimopoulus2, E Azoulay3, P Echeverria1, C De La Cuesta1, JL Vincent4 1University of Miami Miller School of Medicine, Miami, FL, USA; 2University Hospital ATTIKO Medical School, University of Athens, Greece; 3St-Louis Hospital and Paris VII University, Paris, France; 4Erasme University Hospital, Université Libre de Bruxelles, Belgium Critical Care 2012, 16(Suppl 1):P59 (doi: 10.1186/cc10666) in the ICU R Sanga, S Zanotti, C Schorr, B Milcareck, K Hunter, P Dellinger, J Parrilo Cooper University Hospital, Camden, NJ, USA Critical Care 2012, 16(Suppl 1):P57 (doi: 10.1186/cc10664) p y p Critical Care 2012, 16(Suppl 1):P57 (doi: 10.1186/cc10664) Introduction Fever is a common event (ranges from 25 to 70%) in patients admitted to the ICU. The usual clinical approach in most units is to treat the fever either with medications (acetaminophen, nonsteroid anti-infl ammatory drugs) or external measures, like cooling blankets. No studies assessed clinical evidence for these interventions. There is otherwise evidence that fever may be benefi cial, inducing heat shock proteins and decreasing NK-κB activation. Also treating fever can mask an important clinical sign and avoid early treatment in patients with severe sepsis. p Methods We did a case–control study using two available databases and collected 750 patients with the diagnosis of severe sepsis and septic shock. We collected age, sex, days on mechanical ventilation, APACHE II score, vasopressor use and correlated with the presence of hyperthermia (>101.3°F), hypothermia (<96.8°F) and normothermia Figure 1 (abstract P56). Proportion of correctly and incorrectly identifi ed sources. S21 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 in the initial 24 hours. We used a mean of the available temperature data. Then we used logistic regression (univariate and multivariate) to compare these temperatures with mortality and length of stay in the ICU. in the initial 24 hours. We used a mean of the available temperature data. Then we used logistic regression (univariate and multivariate) to compare these temperatures with mortality and length of stay in the ICU. Figure 2 (abstract P58). Indication for paracetamol administration (n = 152). Results Compared to patients with normal temperature the hyperthermic patients had a lower mortality (22.58% vs. 39.1%) in the univariate analysis (P <0.01). The patients with hypothermia had a mortality of 32.67% (NS). Length of stay was not signifi cantly diff erent between the groups. In the multivariate logistic regression the factors that were associated independently with mortality were age, APACHE II score, use of vasopressors, mechanical ventilation and temperature. Patients with T >101.3°F were 59% less likely to die when compared with patients with normal temperature. Conclusion The results of this study highlight the importance of investigating the real eff ects of fever in severe sepsis or septic shock. Is it necessary to treat when they are not causing harm to the patients? Are we delaying diagnosis of severe sepsis because of the lack of this important clinical sign? The next step should be a prospective trial of treatment versus no treatment of fever in the ICU. Figure 2 (abstract P58). Indication for paracetamol administration (n = 152). P58 y p Conclusion Pharmacological antipyretics are used regularly for pain management rather than fever management, with paracetamol the most common antipyretic therapy. The use of NSAIDS and physical cooling was rare. Noncore temperature measurements were common. g y y Critical Care 2012, 16(Suppl 1):P58 (doi: 10.1186/cc10665) Introduction Our primary aim was to determine the frequency of use of pharmacological and physical cooling strategies in ICU patients in current Australian and New Zealand (ANZ) practice. These patients had sepsis and infl ammation but did not have neurological injury or recent surgery. We also aimed to establish current indications for use of antipyretics in these patients, as well as information on the prevalence of fever and the methods to measure temperature. References 38°C. Paracetamol was used in 152/311 (48.8%), nonsteroidal anti- infl ammatory drugs (NSAIDS) in 2/311 (0.6%) and physical cooling in 3/311 (1.0%) (Figure 1). Paracetamol was administered for pain in 92/152 (60.5%) for both pain and fever in 26/152 (17.1%); and for fever alone in 14/152 (10%) (Figure 2). For the 40 patients who received paracetamol for an indication of fever, the peak recorded temperature was 38.3°C (SD 0.8°C). The peak temperature for patients receiving physical cooling was 39.2°C (SD 0.9°C). Temperature measurement were mainly noncore (n = 251/311) with axillary (37%; n = 116/311) and tympanic (35%; n = 110/311) the most common sites. 1. Marik P: Fever in the ICU. Chest 2000, 117:885-869. 1. Marik P: Fever in the ICU. Chest 2000, 117:885-869. 2. Levy M: Clinical review of fever in intensive care unit patients. Crit Care 2003, 7:221-225. 3. Dellinger P: Surviving Sepsis Campaign Guidelines. Crit Care Med 2008, 36:296-327. Cases of tetanus after the Japan crisis 2011 Cases of tetanus after the Japan crisis 2011 K Morino1, M Kobayashi2, Y Yamada3, S Yamanouchi4, Y Tsujimoto1, K Takeda1, S Sato1, S Kimura1, N Mita1, M Sato1, S Kushimoto4, S Endo3 1Yamagata Prefectural Medical Center for Emergency, Yamagata, Japan; 2Ishinomaki Red Cross Hospital, Ishinomaki, Japan; 3Iwate Medical University, Morioka, Japan; 4Tohoku University, Sendai, Japan Critical Care 2012, 16(Suppl 1):P62 (doi: 10.1186/cc10669) Cases of tetanus after the Japan crisis 2011 K Morino1, M Kobayashi2, Y Yamada3, S Yamanouchi4, Y Tsujimoto1, K Takeda1, S Sato1, S Kimura1, N Mita1, M Sato1, S Kushimoto4, S Endo3 1Yamagata Prefectural Medical Center for Emergency, Yamagata, Japan; 2Ishinomaki Red Cross Hospital, Ishinomaki, Japan; 3Iwate Medical University, Morioka, Japan; 4Tohoku University, Sendai, Japan Critical Care 2012, 16(Suppl 1):P62 (doi: 10.1186/cc10669) Methods Eight severe burn patients within 14 days after injuries (M:F = 5:3, 19 to 85 years old, 35 to 85% total body surface area) were treated with MCFG (200 to 300 mg, 3.45 to 4.49 mg/kg) once daily by intravenous infusion over 1 hour. The MCFG concentrations in the plasma at the end of the initial administration of MCFG (P1), just before the second dosing (T1), at the end of the fourth dosing (P4), and just before the fi fth dosing (T4) were determined, and were compared with the reported values in health volunteers [2]. MCFG concentrations in the burn eschar at T1 and T4 were also measured. Introduction Tetanus is an infectious disease caused by tetanus neurotoxin produced by Clostridium tetani [1]. This bacterium resides in the soil extensively and about 100 people contract this disease annually in Japan. Tetanus is prevented by vaccines. A 1968 law required universal DPT vaccination against diphtheria, pertussis and tetanus in Japan. The survival rate with intensive care has reached more than 90% in recent years. Tetanus is said to have been found to increase in natural disasters [2]. So we will describe cases in the aftermath of the 2011 Tohoku earthquake and tsunami. Results The plasma concentrations of MCFG per dose normalized with body weight (C/D) at P1, T1, P4, and T4 were 1.37 to 6.28, 0.51 to 1.38, 3.20 to 6.46, and 0.65 to 2.18 (μg/ml)/(mg/kg), respectively, indicating marked interindividual diff erences. These values were comparable with or slightly lower than the reported values in healthy volunteers (P1: 5.7, T1: 1.3, T4: 2.1). Pharmacokinetics of micafungin in patients with severe burn injuries j J Sasaki1, S Kishino2, N Aikawa1, S Hori1 1Keio University School of Medicine, Tokyo, Japan; 2Meiji Pharmaceutical University, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P60 (doi: 10.1186/cc10667) Conclusion The presence of bacteremia relates to the severity and the mortality of septic patients with high serum PCT in the ICU. Introduction Micafungin (MCFG), an echinocandin antifungal agent, exhibits more potent antifungal activity against a broad spectrum of clinically important Candida and Aspergillus species [1]. Few studies have reported the pharmacokinetics (PK) of antifungal agents in patients with burn injuries. A purpose of this study is to characterize the PK of MCFG in severe burn patients. References 1. Aikawa N, et al.: J Infect Chemother 2009, 15:219-227. 1. Aikawa N, et al.: J Infect Chemother 2009, 15:219-227. 2. Azuma J, et al.: Jpn J Chemother 2002, 50(Suppl 1):155-184. 2. Azuma J, et al.: Jpn J Chemother 2002, 50(Suppl 1):155-184. 2. Azuma J, et al.: Jpn J Chemother 2002, 50(Suppl 1):155-184. Cases of tetanus after the Japan crisis 2011 The MCFG concentrations in the burn eschar of three patients at T1 and T4 were <0.1 to 3.98 and 1.10 to 14.81 μg/ ml, respectively. Most of MCFG concentrations in the plasma and burn eschar were higher than the reported MIC90 of MCFG against clinically important Candida and Aspergillus species. There was no correlation between the laboratory parameters of liver/kidney function and the plasma C/D of MCFG. Methods We researched the case reports in a national database and a hospital database which could access patients’ exact data. We made and analysed these case profi les. Results We had nine tetanus cases in this crisis. This number was high compared with previous data. All patients lived in the Pacifi c coast of Tohoku districts and suff ered from the tsunami. Geographically, seven patients were in Miyagi prefecture, and Iwate Prefecture had two cases. Of the nine cases, we could examine seven cases in detail. Mean age was 67 years, two were male cases and fi ve women were injured on the day. Time to onset of symptoms such as trismus was an average of 12 days. The average was 3 days from symptom onset to medical consultation. All seven cases had some wounds, including minimal. Three had obvious wound infection. All patients had tetanus vaccine and tetanus immunoglobulin during their therapy but the time of injection was inconsistent because of the chaotic state. Four people were supported by mechanical ventilation with sedation and three out of four had tracheostomy. Three out of four with mechanical ventilation were treated with intravenous magnesium therapy to reduce spasticity. The average mechanical ventilation period was 23 days. We have no intravenous metronidazole preparation in Japan. No one had a reliable history of tetanus vaccine. No deaths were reported. Conclusion The plasma concentrations of MCFG in patients with severe burn injuries were comparable with or slightly lower than the reported values in healthy volunteers. In addition, MCFG seems to be capable of penetrating burn eschar. Bacteremia aff ects the mortality of septic patients with high serum procalcitonin level in the ICU M Kamochi, K Nagata, Y Isa, S Nihei, N Harayama, K Aibara, T Sata University Hospital of Occupational and Environmental Health, Kitakyush Japan M Kamochi, K Nagata, Y Isa, S Nihei, N Harayama, K Aibara, T Sata University Hospital of Occupational and Environmental Health, Kitakyushu, Japan Conclusion We reported nine tetanus patients and investigated seven cases in detail. Older people had developed an unknown vaccination history. So we should have more opportunity to give vaccinations to older people and be careful with tetanus in disasters. References p Critical Care 2012, 16(Suppl 1):P61 (doi: 10.1186/cc10668) p Critical Care 2012, 16(Suppl 1):P61 (doi: 10.1186/cc10668) Impact of antifungal treatment in ICU patients with Candida colonization: analysis of the EPIC II study population p Methods This point prevalence study was conducted on 17 November and 15 December 2010 in 38 ICUs in ANZ. We identifi ed a cohort of patients with sepsis and infl ammation without neurological injury or recent surgery. g y Results Of 506 patients surveyed on the point prevalence days, 311 were identifi ed to have sepsis in the absence of neurological injury or recent surgery. These patients had peak temperature of 37.3°C (SD 0.8°C). In 32.2% (n = 100/311) the peak temperature was above Introduction We wished to evaluate the impact of receiving antifungal therapy in ICU patients with Candida colonization. py p Methods EPIC II recruited 1,265 ICUs in 76 countries. Patient charac- teristics were collected on the study day. Outcome data were assessed at ICU and hospital discharge. Patients colonized with Candida spp. were classifi ed as having received antifungal treatment or not (P <0.05 compared between groups). Numerical values are reported as mean ± SD and length of stay (LOS) data as median (IQR). Figure 1 (abstract P58). Type of antipyretic and physical cooling used on the study day (n = 311). g y Results A total of 13,796 adult patients were in participating ICUs on the study day. Of these, 371 were classifi ed as colonized. Diff erences Table 1 (abstract P59). Patients with Candida colonization: characteristics and outcomes Therapy (n = 184) No therapy (n = 175) SAPS II 39 ± 15 41 ± 18 SOFA 7.6 ± 4.1 7.4 ± 4.4 MV 76% 63% Pressor 36% 32% ICU mortality 35% 22% Hospital mortality 41% 28% Table 1 (abstract P59). Patients with Candida colonization: characteristics and outcomes Figure 1 (abstract P58). Type of antipyretic and physical cooling used on the study day (n = 311). S22 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 in patient characteristics and outcomes are reported (Table 1). Baseline characteristics were similar in colonized patients treated with antifungal therapy compared to those that were untreated. Only a modest diff erence in the length of stay in the ICU prior to study day (25 (14, 40) vs. 21 (8, 43)) and utilization of mechanical ventilatory support (76% vs. 63%) was noted in the treated compared to the untreated patients with Candida colonization (P <0.05). Impact of antifungal treatment in ICU patients with Candida colonization: analysis of the EPIC II study population Despite the relatively similar baseline characteristics and equivalent severity of illness scores, treated patients had an increased ICU (35.3 vs. 22.3%) and hospital (41.0 vs. 27.7%) mortality (P <0.05). in patient characteristics and outcomes are reported (Table 1). Baseline characteristics were similar in colonized patients treated with antifungal therapy compared to those that were untreated. Only a modest diff erence in the length of stay in the ICU prior to study day (25 (14, 40) vs. 21 (8, 43)) and utilization of mechanical ventilatory support (76% vs. 63%) was noted in the treated compared to the untreated patients with Candida colonization (P <0.05). Despite the relatively similar baseline characteristics and equivalent severity of illness scores, treated patients had an increased ICU (35.3 vs. 22.3%) and hospital (41.0 vs. 27.7%) mortality (P <0.05). rapid diagnosis and immediate treatment are necessary to improve the survival of septic patients. However, the presence of bacteremia seems to relate to the severity and mortality of septic shock patients in the ICU. Methods The patients clinically suspected with sepsis were tested for serum procalcitonin level using a procalcitonin kit (BRAHMUS PCT Kit). The PCT test was performed 334 times from March 2008 to August 2010. Sixty-three adult patients showed high PCT level (>10 ng/ml). Thirty of 62 (48%) patients showed bacteremia. Sixteen of these bacteremic patients were Gram-negative bacteremia and 14 patients were Gram- positive bacteremia. The hemodynamic parameter, APACHE II score, SOFA score, serum lactate, some other laboratory data and mortality rate were compared between the patients with bacteremia and those without bacteremia. Statistical analyses were performed by chi-square test and Mann–Whitney U test. Conclusion As colonized patients receiving antifungal treatment had signifi cantly higher mortality, our data do not support the routine use of antifungal therapy in ICU patients based solely on colonization. Reference 1. Vincent JL, et al.: JAMA 2009, 302:2323-2329. 1. Vincent JL, et al.: JAMA 2009, 302:2323-2329. y Results The bacteremic patients with high serum PCT level showed signifi cant higher APACHE II score, SOFA score and serum lactate concentration than nonbacteremic patients. The mortality rate of bacteremic patients was signifi cantly higher than that of nonbacteremic patients (66% vs. 28.1%, P <0.01). There were no diff erences in the severity and the mortality between Gram-negative and Gram-positive bacteremia. P60 Pharmacokinetics of micafungin in patients with severe burn injuries J Sasaki1, S Kishino2, N Aikawa1, S Hori1 1Keio University School of Medicine, Tokyo, Japan; 2Meiji Pharmaceutical University, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P60 (doi: 10.1186/cc10667) p p References Introduction It is still controversial whether bacteremia aff ects the severity and the mortality of septic shock. Recent diagnostic criteria of septic shock do not include the presence of bacteremia, because Farrar JJ, et al.: J Neurol Neurosurg Psychiatry 2000, 69:292-301 Aceh Epidemiology Group: Glob Public Health 2006, 1:173-177. Farrar JJ, et al.: J Neurol Neurosurg Psychiatry 2000, 69:292-301 Aceh Epidemiology Group: Glob Public Health 2006, 1:173-177. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S23 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P63 Does the day of the week predict the presence of microbiologically confi rmed ventilator-associated pneumonia? C Linssen, H Van Dessel, W Van Mook Maastricht University Medical Centre, Maastricht, the Netherlands Critical Care 2012, 16(Suppl 1):P63 (doi: 10.1186/cc10670) Methods This study was a retrospective analysis of patients confi rmed as appendicitis pathologically from November 2009 to September 2010 at two hospitals. The delta neutrophil index was automatically calculated as a subset of routine complete blood count test. The diagnostic performance of the delta neutrophil index for perforated appendicitis was evaluated. Results During the study period, 308 patients were enrolled. Among them, 32 patients (10.4%) were confi rmed as perforated appendicitis. The delta neutrophil index was signifi cantly higher in the perforated group than the nonperforated group (4.8 ± 7.1% vs. 2.0 ± 2.0%, P <0.05). The sensitivity and specifi city of the delta neutrophil index for predicting perforated appendicitis was 25.0% and 96.7% respectively at a cutoff level of 5% with an area under the curve of 0.78 on the ROC curve. Introduction At our hospital, ventilator-associated pneumonia (VAP) is diagnosed by microbiological and cytological analysis of bronchoalveolar lavage fl uid (BALF). Opening hours of the in-house microbiological laboratory are between 8:00 am and 5:00 pm. During off -hours a laboratory technician is on call for urgent samples including BALF. The total laboratory work-up of the BALF takes 2 hours. The aim of the present study was to detect patterns in the submission time of BALF samples. Conclusion This study suggested that the delta neutrophil index is associated with perforated appendicitis. However, the sensitivity was not high enough to use as a clinical guidance. p Methods During a 60-month period (January 2006 to December 2010), the day and hour of submission of all consecutive BALF samples obtained from patients suspected of VAP were recorded. References 1. Nahm CH, Choi JW, Lee J: Delta neutrophil index in automated immature granulocyte counts for assessing disease severity of patients with sepsis. Ann Clin Lab Sci 2008, 38:241-246. Results A total of 376 BALF samples were included. On weekdays, on average a total of 59.8 ± 11.4 were submitted, compared to 34 and 43 samples on Saturdays and Sundays. For more than one-half (203, 54%) of the samples, the on-duty laboratory technician was required: 86 (23%) samples arrived within 1 hour before closing time, and an additional 117 (31%) were submitted thereafter. VAP was diagnosed in 149 (39.6%) samples, of which 79 (53%) after closing hours. BALF samples were obtained more frequently on Thursdays and Fridays (51 and 47 samples respectively) compared to Mondays and Tuesdays (64 and 76 samples). Interestingly, VAP was confi rmed proportionally more frequently on Mondays and Tuesdays (26/51 (51%) and 23/47 (49%)) compared to Thursdays and Fridays (20/64 (31%) and 26/76 (34%)). 2. Andersson RE: Meta-analysis of the clinical and laboratory diagnosis of appendicitis. Br J Surg 2004, 91:28-37. 3. Oliak D, Yamini D, Udani VM, et al.: Can perforated appendicitis be diagnosed preoperatively based on admission factors? J Gastrointest Surg 2000, 4:470-474. 4. Ansari-Lari MA, Kickler TS, Borowitz MJ: Immature granulocyte measurement using the Sysmex XE-2100. Relationship to infection and sepsis. Am J Clin Pathol 2003, 120:795-799. Conclusion The high number of BALFs processed after laboratory opening hours is of concern because of the suboptimal working conditions (fatigue, lack of supervision and experience). Technicians’ time spent on these samples puts a strain on the laboratory in terms of costs and absence of the technicians because of legal recuperation. A higher number of confi rmed episodes of VAP early in the week compared to just before the beginning of the weekend, combined with a larger number of BALF samples obtained on Thursdays and Fridays, may suggest that clinicians want to exclude VAP before the weekend resulting in a lower threshold for requesting a BALF. p p References VAP was microbiologically confi rmed if quantitative cultures were ≥104 cfu/ml and/or presence of ≥2% infected cells. P64 Assessing perforation of acute appendicitis using the delta neutrophil index refl ecting the peripheral immature granulocyte count Introduction The delta neutrophil index corresponds to the calculated immature granulocyte counts and the severity of sepsis. This study investigated the diagnostic value of the delta neutrophil index as a preoperative laboratory marker for appendiceal perforation in patients with acute appendicitis. Table 1 (abstract P65) Month Outcome June July August September Odds ratio (95% CI) P value ICU death, n (%) 100 (36.0%) 122 (38.5%) 137 (34.9%) 82 (24.9%) 0.83 (0.75; 0.93) 0.001 In-hospital death, n (%) 108 (38.6%) 125 (43.0%) 151 (38.5%) 96 (29.2%) 0.85 (0.76; 0.94) 0.002 Mean diff erence (95% CI) SMR, mean (SD) 1.25 (0.37) 1.63 (0.95) 1.31 (0.62) 0.89 (0.51) –0.15 (–0.28; –0.01) 0.04 CRBSI, mean (SD) 11.8 (19.7) 4.3 (11.9) 4.4 (5.4) 3.3 (6.0) –2.11 (–4.68; 0.45) 0.10 Eff ects of a multifaceted quality improvement intervention in reducing mortality and bloodstream infection in ICUs: insights from the QUALITI initiative AB Cavalcanti, JC Othero, JC Mouro, KN Silva, ES Victor, AA Kodama, O Berwanger, B Weber, LH Mota Research Institute – Hospital do Coração, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P65 (doi: 10.1186/cc10672) Introduction Our objective was to evaluate if the implementation of a multifaceted intervention program for quality improvement in ICUs of nonacademic hospitals would decrease mortality and the catheter- related bloodstream infection rate (CRBSI). Methods A clinical practice improvement program involving 17 Brazilian ICUs of nonacademic public hospitals located far from major economic centers under coordination of a not-for-profi t private hospital with the support of Brazilian Ministry of Health. We implemented the following interventions: (1) hospital visits to assess the facilities, human resources and processes; (2) workshop with hospital directors and ICU coordinators to elaborate improvement proposals based on the initial visit fi ndings; (3) multidisciplinary videoconference lectures every 3 weeks about critical care medicine assistance and quality issues; (4) website containing project educational material, videoconference recordings, and an evidence-based practice course; (5) subscription of an electronic clinical information resource for all participating hospitals (UpToDate®); (6) 3-day workshop to share the coordinating institution quality improvement practices with directors and ICU coordinators Ceftazidime dosage regimen recommendations in burn patients based on a Monolix population pharmacokinetic study Introduction The aim of our present work was to predict in burn patients the best adapted ceftazidime dosage regimen to obtain a serum target of 40 to 100 mg/l taking into account the infl uence of patients’ characteristics on ceftazidime pharmacokinetics (PK). Methods In a retrospective study we compared three groups of patients: patients with intermittent vancomycin (Intermittent group, n = 27) with target trough level of 5 to 10 mg/l, patients with continuous vancomycin (CV1 group, n = 24) with target trough level of 20 to 25 mg/l and patients with continuous vancomycin (CV2 group, n = 20) with target trough level of 15 to 20 mg/l. The demographic data, total and average vancomycin doses, target level achievement and side eff ects were analyzed. Methods A Monolix population PK model was developed and validated in 70 burn patients with Pseudomonas aeruginosa infection. Monte Carlo simulations (n = 1,000) were performed to explore the appropriateness of diff erent dosage regimens in burn patients. Target concentrations to achieve were defi ned as a 40 to 100 mg/l steady– state concentration interval. The recommended dosage was chosen as the minimum dose providing the maximum of patients in this interval. Results A two-compartment model described ceftazidime disposition. Serum creatinine and age were identifi ed as covariates of ceftazidime clearance. Age also infl uences the volume of distribution. The simulations showed that the common dosage regimens of 6 g/day did not allow achieving the desired target interval. This was achieved with continuous administration dosage regimens varying between 8 and 16 g/day in the youngest patients. Whatever the dosage regimen, the age and the serum creatinine, the mean highest percentage of patients reaching the 40 to 100 mg/l target interval was 76.43 ± 2.13% (range: 65.1 to 80.1%) (Table 1). f Results There was no diff erence in age, weight, surgical complexity and mortality between the groups. The average vancomycin daily dose (mg/kg) was the same in the Intermittent and CV2 groups, the dose was twofold higher in CV1 group (P <0.001) (Table 1). The CV2 group has less trough samples per day of treatment than the CV1 and intermittent groups (P = 0.015). Target levels were reached in 42.4%, 30.9%, and 42.9% samples in Intermittent, CV1 and CV2 groups, respectively (P <0.001). Below target were 9.8%, 38.5% and 16.7% samples in Intermittent, CV1 and CV2 groups, respectively. Ceftazidime dosage regimen recommendations in burn patients based on a Monolix population pharmacokinetic study There was no deep sternal infection in any patient. There was similar incidence of peritoneal dialysis in all three groups. No case of renal insuffi ciency was directly related to increased trough vancomycin level. Table 1 (abstract P67) Intermittent group CV1 group CV2 group TLC (n) 272 250 233 TLC per day (n) 1 (0.6 to 2.3) 0.92 (0.5 to 1.5) 0.81 (0.62 to 1.8) Daily dose 15 (7.6 to 45) 26.2 (7.6 to 54.3) 15.7 (5.9 to 37.3) CV, continuous vancomycin; TLC, trough level count. Table 1 (abstract P66). Recommended ceftazidime dosage regimen Target = 40 mg/l Creatinine 20 30 40 50 60 70 80 90 (μmol/l) years years years years years years years years 30 16 g 16 g 16 g 16 g 14 g 12 g 12 g 10 g 40 16 g 16 g 16 g 14 g 12 g 12 g 10 g 10 g 50 16 g 16 g 16 g 12 g 12 g 10 g 10 g 8 g 60 16 g 16 g 14 g 12 g 12 g 10 g 8 g 8 g 70 16 g 14 g 12 g 12 g 10 g 10 g 8 g 8 g 80 14 g 14 g 12 g 10 g 10 g 8 g 8 g 8 g 90 14 g 14 g 12 g 10 g 10 g 8 g 8 g 6 g 100 14 g 12 g 10 g 10 g 8 g 8 g 8 g 6 g 120 12 g 10 g 10 g 8 g 8 g 6 g 6 g 6 g 140 10 g 10 g 8 g 8 g 6 g 6 g 6 g 4 g 160 10 g 8 g 8 g 6 g 6 g 6 g 4 g 4 g Recommended ceftazidime dosage regimen after a 2 g loading dose required to reach a steady-state concentration between 40 and 100 mg/l in the highest percentage of typical burn patients in function of serum creatinine and age. Conclusion This study highlights the peculiarities of ceftazidime Table 1 (abstract P66). Recommended ceftazidime dosage regimen CV, continuous vancomycin; TLC, trough level count. Continuous versus intermittent vancomycin in children after cardiac surgery with delayed sternal closure Continuous versus intermittent vancomycin in children after cardiac surgery with delayed sternal closure P Skrak, L Hlinkova, L Kovacikova National Institute of Cardiovascular Diseases, Bratislava, Slovakia Critical Care 2012, 16(Suppl 1):P67 (doi: 10.1186/cc10674) Conclusion A multifaceted intervention program applied to a network of ICUs in nonacademic public hospitals reduced mortality. P Skrak, L Hlinkova, L Kovacikova National Institute of Cardiovascular Diseases, Bratislava, Slovakia Critical Care 2012, 16(Suppl 1):P67 (doi: 10.1186/cc10674) P66 Introduction Delayed sternal closure (DSC) is a technique used in patients with hemodynamic instability, lung dysfunction, edema or prolonged bleeding after cardiac surgery. This group of patients has signifi cant morbidity and mortality with fl uid overload and changes in renal function. Adequate antibiotic coverage is of great importance and vancomycin is used as a part of antibiotic prophylaxis in our department. The objective of our study was to compare the effi cacy and effi ciency of intermittent and continuous vancomycin in pediatric cardiac patients with DSC. P66 Ceftazidime dosage regimen recommendations in burn patients based on a Monolix population pharmacokinetic study S Ruiz1, JM Conil1, B Georges1, F Ravat2, T Seguin1, P Letocart1, O Fourcade1, S Saivin3 1Hôpital Rangueil CHU Toulouse, France; 2Centre hospitalier St Joseph et St Luc, Lyon, France; 3Institut Fédératif de Biologie CHU Toulouse, France Critical Care 2012, 16(Suppl 1):P66 (doi: 10.1186/cc10673) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Age and serum creatinine signifi cantly infl uence the ceftazidime disposition. These covariates must be used to propose the fi rst doses of ceftazidime. The required dosage regimens are higher than in other ICU patients and doses between 4 and 16 g/day are proposed. Age and serum creatinine signifi cantly infl uence the ceftazidime disposition. These covariates must be used to propose the fi rst doses of ceftazidime. The required dosage regimens are higher than in other ICU patients and doses between 4 and 16 g/day are proposed. from the 17 participant institutions; (7) 3-day nursing visits from the coordinating hospital to perform advice on care practice; (8) basic life support courses, 56 vacancies per hospital, and fundamentals of critical care support, 30 vacancies; and (9) implementation of a web-based system to collect ICU and hospital mortality, SAPS3, standardized- mortality ratio (SMR) and CRBSI after June 2011. We assessed variation of SMR and CRBSI on time using weighted linear regression, and variation of mortality on time using generalized-estimating equations. Results The results are presented in Table 1. Ceftazidime dosage regimen recommendations in burn patients based on a Monolix population pharmacokinetic study Conclusion In children after cardiac surgery with DSC both intermittent vancomycin with trough level of 5 to 10 mg/l and continuous vanco- mycin with trough level of 15 to 20 mg/l were comparable with regard to administered dose and target values achievement. There was signifi cantly higher daily dose and trough sample count below target values in patients with continuous vancomycin and target of 20 to 25 mg/l. Table 1 (abstract P65) S24 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 A post-authorisation survey to analyse the perioperative teicoplanin plasma concentrations in adult patients with chronic bone sepsis, who received loading doses of 12 mg/kg 12-hourly for 48 hours followed by 12 mg/kg once daily AJ Brink1, G Richards2, C Lautenbach1, N Rapeport1, V Schillack3, J Roberts4, J Lipman4 AJ Brink1, G Richards2, C Lautenbach1, N Rapeport1, V Schillack3, J Roberts4, J Lipman4 Results Our study population included fi ve patients (three female) with mean age 60.6 years. Median (range) colistin concentrations in ELF were 6.9 (6.2 to 13.9), 3.7 (2.7 to 11.6) and 2.1 (1.2 to 8.7) g/ml at 1, 4, and 8 hours, respectively, after nebulization. Colistin concentrations in serum were substantially lower than those observed in ELF with peak median (range) values 1.56 (1.19 to 2) g/ml. The estimated colistin mean half-life was 3.4 hours. p 1Milpark Hospital, Johannesburg, South Africa; 2University of Witwatersrand, Johannesburg, South Africa; 3Ampath National Referral Laboratory, Pretoria, South Africa; 4University of Queensland, Brisbane, Australia Critical Care 2012, 16(Suppl 1):P69 (doi: 10.1186/cc10676) Introduction To rapidly achieve teicoplanin trough (Cmin) concentrations ≥20 mg/l suggested for sternal sepsis, loading doses higher than 6 mg/ kg 12-hourly might be warranted [1]. Conclusion Administration of 1 million units of inhaled CMS resulted in high colistin concentrations in the ELF; moreover, concentrations were maintained for up to 8 hours in the majority of patients. This fi nding might support the use of inhaled CMS for the treatment of patients with VAP due to multidrug-resistant Gram-negative bacteria. Moreover, the low serum concentrations and the short half-life suggest that administration of inhaled colistin may be associated with less systemic toxicity. Methods Patients (n = 10) with deep-seated Gram-positive infections were enrolled perioperatively. During the fi rst 4 days of therapy teicoplanin loading doses of 12 mg/kg 12-hourly were administered for 48 hours and 12 mg/kg once daily thereafter. Surgical debridement was performed on D3. Samples were collected 15 minutes before and 30 minutes and 120 minutes after each teicoplanin administration. Total and unbound teicoplanin levels were determined using HPLC. P70 P70 Pharmacokinetics of inhaled colistin in critically ill patients with ventilator-associated tracheobronchitis Z Athanassa1, M Fousteri2, S Markantonis2, P Myrianthefs3, E Boutzouka3, E Tsigou3, A Tsakris4, G Baltopoulos3 1Hygeia Hospital, Marousi, Greece; 2Faculty of Pharmacy, University of Athens, Greece; 3Faculty of Nursing, University of Athens, Greece; 4Faculty of Medicine, University of Athens, Greece Critical Care 2012, 16(Suppl 1):P70 (doi: 10.1186/cc10677) 0 Pharmacokinetics of inhaled colistin in critically ill patients with ventilator-associated tracheobronchitis Introduction Although inhaled colistin is frequently used in ventilator- associated pneumonia (VAP), data regarding its pharmacokinetic properties are scarce [1-3]. The aim of this study was to describe colistin pharmacokinetics in critically ill patients after administration of a single dose of 1 million units of colistimethate sodium (CMS) via nebulization. Methods Patients with ventilator-associated tracheobronchitis dye to polymyxin-only susceptible Gram-negative bacteria were included in the study; patients receiving intravenous and/or nebulized colistin were excluded. CMS was administered at a dose of 1 million units every 8 hours for 7 days, via a vibrating-mesh nebulizer. Mini bronchoalveolar lavage was collected before and at 1, 4 and 8 hours post nebulization, while blood samples were collected before and at 0.16, 0.5, 1, 2, 4, and 8 hours post nebulization. Colistin concentrations in epithelial lining fl uid (ELF) and plasma were determined by high-performance liquid chromatography.i Conclusion Clearance of LZD in patients undergoing CVVHDF was signifi cantly lower than in patients with normal renal function. Pharmacokinetic data from CVVHDF patients demonstrated that fl ow rates signifi cantly infl uenced the effi ciency of LZD removal. The maintenance dose of LZD may need to be reduced in patients undergoing CVVHDF under reduced fl ow conditions. Reference 1. Meyer B, et al.: J Antimicrob Chemother 2005, 56:172-179. References 1. Ratjen F, et al.: J Antimicrob Chemother 2006, 57:306-311. 2. Marchand S, et al.: Antimicrob Agents Chemother 2010, 54:3702-3707. 3. Lu Q, et al.: Intensive Care Med 2010, 36:1147-1155. Results All patients had hypoalbuminemia (mean 20.2 g/l). The SS PK parameters of teicoplanin are described in Table 1. On D3 the median total and free Cmin were 14.66 (8.93 to 19.66) and 3.09 (0.0 to 6.4) mg/l, respectively. In a multivariate logistic regression model, total teicoplanin concentrations (P = 0.174) and serum creatinine concentration (P = 0.034) did not impact signifi cantly on free teicoplanin levels whereas, in contrast, albumin concentration did (OR 0.120, 95% CI 0.078 to 0.161, P <0.001). References i yp yil Results Fourteen CVVHDF patients and nine NRF patients were included into the study. CVVHDF was performed using polysulfone and triacetate membranes. Mean blood, dialysate and fi ltration fl ow rates were 79.3 ± 2.7 ml/minute, 8.7 ± 5.1 ml/minute and 5.5 ± 2.5 ml/minute, respectively. Sc was 0.86 ± 0.03. T–1/2 data (8.78 ± 3.74 vs. 5.54 ± 3.27 hours, P = 0.05) were signifi cantly longer in the CVVHDF compared with the NRF group, AUC data (247.9 ± 107.8 vs. 136.0 ± 84.9 g hour/ml, P = 0.02) were signifi cantly higher and CL (2.94 ± 1.38 vs. 5.92 ± 2.97 l/hour, P = 0.004) and Vd (31.0 ± 3.8 vs. 35.8 ± 3.3 l, P = 0.01) data were signifi cantly lower. LZD clearance was not correlated with the type of membrane used (polysulfone vs. triacetate: 2.8 ± 1.5 vs. 3.6 ± 1.2 l/hour, P = 0.39). P70 P68 Elimination of linezolid in patients undergoing low-fl ow continuous venovenous haemodiafi ltration teicoplanin Total Free Cmax 20.1 2.6 Cmin 6.7 2.3 AUC 137.9 28.6 CL 7.0 33.5 Vz 174.1 196.6 Methods LZD (600 mg) was administered intravenously every 12 hours in ICU patients on CVVHDF and NRF patients (creatinine clearance 50 ml/minute). Blood and fi ltrate samples were collected at 0, 1, 1.5, 2, 3 and 5 hours after infusion from both groups. The elimination half- life (T–1/2), maximum concentration, concentration time curve (AUC), volume distribution (Vd), clearance (CL) and sieving coeffi cient (Sc) were evaluated. Patient characteristics and CVVHDF parameters including the fi lter type, dialysate and fi ltration fl ow rates were recorded. P68 Elimination of linezolid in patients undergoing low-fl ow continuous venovenous haemodiafi ltration Recommended ceftazidime dosage regimen after a 2 g loading dose required to reach a steady-state concentration between 40 and 100 mg/l in the highest percentage of typical burn patients in function of serum creatinine and age. Introduction It has been reported that linezolid (LZD) is highly removed in patients undergoing high-fl ow continuous venovenous haemofi ltration (CVVH: blood fl ow and fi ltration rates were 186 ± 15 and 40 ± 8 ml/minute) compared with patients with normal renal Conclusion This study highlights the peculiarities of ceftazidime pharmacokinetics in burn patients with high interindividual variability. S25 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 References 1. Brink et al.: Int J Antimicrob Agents 2008, 32:455-458. 2. Mimoz et al.: Intensive Care Med 2006, 32:775-779. P70 Pharmacokinetics of inhaled colistin in critically ill patients with ventilator-associated tracheobronchitis Z Athanassa1, M Fousteri2, S Markantonis2, P Myrianthefs3, E Boutzouka3, E Tsigou3, A Tsakris4, G Baltopoulos3 1Hygeia Hospital, Marousi, Greece; 2Faculty of Pharmacy, University of Athens, Greece; 3Faculty of Nursing, University of Athens, Greece; 4Faculty of Medicine, University of Athens, Greece Critical Care 2012, 16(Suppl 1):P70 (doi: 10.1186/cc10677) d i l h h h l d l f l d l Table 1 (abstract P69). Steady-state pharmacokinetic parameters of teicoplanin Total Free Cmax 20.1 2.6 Cmin 6.7 2.3 AUC 137.9 28.6 CL 7.0 33.5 Vz 174.1 196.6 Table 1 (abstract P69). Steady-state pharmacokinetic parameters of teicoplanin function (NRF). It is generally considered that no adjustment of LZD dosage is needed in subjects undergoing CVVH. In Japan, continuous venovenous haemodiafi ltration (CVVHDF) has preferentially been administered under low fl ow rate. Investigating the eff ects of fl ow rate on LZD removal during continuous renal replacement therapy is essential to regulate therapeutic dosages. We aimed to investigate the pharmacokinetics of LZD in CVVHDF patients in this setting. function (NRF). It is generally considered that no adjustment of LZD dosage is needed in subjects undergoing CVVH. In Japan, continuous venovenous haemodiafi ltration (CVVHDF) has preferentially been administered under low fl ow rate. Investigating the eff ects of fl ow rate on LZD removal during continuous renal replacement therapy is essential to regulate therapeutic dosages. We aimed to investigate the pharmacokinetics of LZD in CVVHDF patients in this setting. Effi cacy of inhaled tobramycin in severe nosocomial pneumonia Both sampling methods demonstrated a gradation of cytokine level, with burns and ALI/ARDS having signifi cantly higher levels than patients with stable chronic lung disease or healthy controls. to estimate the effi cacy of inhaled tobramycin (IT) as an adjunct to systemic antibiotics in the treatment of severe NP. y Methods Twenty ICU patients with NP were enrolled in the study (all male, 49 ± 7.3 years old); primary reason for ICU stay – intraabdominal infections (60%), mediastinitis (10%), others (30%). Diagnosis of NP was made according to standard clinical and CPIS criteria. Associa- tions of multiresistant Gram-negative bacteria were detected in bronchoalveolar lavage (BAL) of all patients. Eighty percent of bacteria were sensitive to tobramycin. Patients were randomized into two groups – ‘IT’ (group 1, n = 10) + systemic antibiotics (carbapenems, amino glycosides, protected penicillins); ‘no IT’ (group 2, n = 10), only systemic antibiotics, same as in group 1. Groups were comparable in APACHE II and CPIS scores. IT (Bramitob) was administered 300 mg BID via nebulizer. y Conclusion The BMS probe was well tolerated and provided cytokine data comparable to that obtained by BAL in acute and chronic respiratory diseases. The BMS probe may have utility as a biomarker sampling modality in patients where clinicians have concerns over conventional BAL. Acknowledgements The BMS probes were provided by Olympus (Tokyo, Japan). Results Duration of IT use in group1 was 7.5 ± 2.5 days. There were no statistically reliable diff erences between groups detected due to the small number of patients enrolled. But it was clinically detected that treatment with IT in group1 was associated with a decrease of SIRS signs and CPIS scores and an increase of oxygenation index in 70% of patients. Positive dynamics in chest X-ray and computed tomography was detected in two patients of group 1 (20%; no dynamics in group 2). The titre of microbes in BAL decreased (100%) and their sensitivity to other groups of antibiotics, which they were previously resistant to, increased (40%) in group 1 patients after IT administration. Effi cacy of IT in patients with a registered resistance of microbes to tobramycin can be explained by a high local concentrations of tobramycin in lungs. The mortality in groups was similar (40% and 40%) and not related to a progression of NP. P72 Comparison of a bronchoscopic microsample probe with bronchoalveolar lavage to measure cytokine levels in critically ill patients V Grover, LE Christie, P Charles, P Kelleher, P Shah, S Singh Chelsea and Westminster Hospital, London, UK Critical Care 2012, 16(Suppl 1):P72 (doi: 10.1186/cc10679) Introduction The use of bronchoalveolar lavage (BAL) to investigate infl ammatory lung disease in the critically ill may not be tolerated in hypoxic patients. Furthermore, soluble protein analysis of BAL fl uid suff ers from inaccuracies related to saline dilution. The bronchoscopic microsample (BMS) probe allows absolute cytokine levels in epithelial lining fl uid (ELF) to be measured directly without lavage [1]. We compared cytokine levels from ELF obtained by the BMS probe with those from BAL, to verify its utility in critical illness. Table 1 (abstract P73) P value OR (95% CI) NIV success 0.005 0.1(0.01 to 0.4) SOFA score* 0.01 1.2(1 to 1.3) Male gender <0.001 14(5 to 39.4) Immunosuppressive status 0.001 4(1.7 to 9.6) Mean value. y y Methods We recruited 45 patients into fi ve groups in whom BMS and BAL were conducted sequentially: two ventilated with ALI/ARDS, six with burns inhalational injury (fi ve ventilated), 15 with COPD, 18 with interstitial lung disease and four healthy patients. The BMS probe was bronchoscopically inserted to the subsegmental level in order to contact the mucosa for 5 to 7 seconds, collecting approximately 20 μl ELF [1]. BAL was performed with 150 ml of 0.9% saline, discarding the fi rst 20 ml (bronchiolar fraction). We assayed IL-1, IL-6, IL-8, TNFα and G-CSF. Comparisons between paired cytokine ELF concentrations in BMS and BAL were analysed using the nonparametric Wilcoxon’s test and Spearman’s correlation coeffi cient. Conclusion VAP occurrence seems to be associated with increased morbidity and ICU mortality. NIV use, avoiding endotracheal intubation and invasive mechanical ventilation, has appeared to be eff ective in reducing the rate of VAP episodes, particularly in high-risk patients (severe immunosuppressed). The application of behavioural intervention bundles might represent the suitable preventive measure in settings where high rates of MDR pathogens limit the extensive use of pharmacological ones. fi Results The critically ill patients were aged 18 to 84 years (APACHE II 12 to 21). One patient had ARDS due to urinary tract infection and another related to pneumonia. No adverse incidents noted were noted. Overall, cytokine levels were all higher in the BMS group than Effi cacy of inhaled tobramycin in severe nosocomial pneumonia A Kuzovlev1, S Polovnikov2, V Stec2, V Varvarin2 1V.A. Negovsky Scientifi c Research Institute of General Reanimatology RAMS, Moscow, Russia; 2N.N. Burdenko Main Clinical Military Hospital, Moscow, Russia Critical Care 2012, 16(Suppl 1):P71 (doi: 10.1186/cc10678) A Kuzovlev1, S Polovnikov2, V Stec2, V Varvarin2 1V.A. Negovsky Scientifi c Research Institute of General Reanimatology RAMS, Moscow, Russia; 2N.N. Burdenko Main Clinical Military Hospital, Moscow, Russia Critical Care 2012, 16(Suppl 1):P71 (doi: 10.1186/cc10678) Conclusion The levels achieved on D3 in this study are similar to those achieved by Mimoz and colleagues using the same dosing schedule in ICU patients with VAP [2]. Only hypoalbuminemia impacted on the free levels of teicoplanin in this setting. High teicoplanin loading doses of 12 mg/kg 12-hourly should probably be extended beyond 48 hours, before major elective surgery for chronic bone sepsis. Introduction Nosocomial pneumonia (NP) is one of the most prevalent complications in ICUs. The effi cacy of inhaled antibiotics in treatment of NP was shown in several research works. The aim of this study was Introduction Nosocomial pneumonia (NP) is one of the most prevalent complications in ICUs. The effi cacy of inhaled antibiotics in treatment of NP was shown in several research works. The aim of this study was S26 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 BAL (P <0.0001), consistent with ELF dilution by saline lavage. The ratio of BMS-derived cytokine to BAL for each patient group did not diff er signifi cantly. Spearman coeffi cients (r) for IL-1, IL-6, IL-8, TNFα and G-CSF were 0.38, 0.52, 0.25, 0.38 and 0.40. All correlations were signifi cant (P <0.01) except for IL-8 (P = 0.05). Both sampling methods demonstrated a gradation of cytokine level, with burns and ALI/ARDS having signifi cantly higher levels than patients with stable chronic lung disease or healthy controls. to estimate the effi cacy of inhaled tobramycin (IT) as an adjunct to systemic antibiotics in the treatment of severe NP. BAL (P <0.0001), consistent with ELF dilution by saline lavage. The ratio of BMS-derived cytokine to BAL for each patient group did not diff er signifi cantly. Spearman coeffi cients (r) for IL-1, IL-6, IL-8, TNFα and G-CSF were 0.38, 0.52, 0.25, 0.38 and 0.40. All correlations were signifi cant (P <0.01) except for IL-8 (P = 0.05). Reference 1. Polovnikov SG, Kuzovlev AN, Iliychev AN: Case report of a successful treatment of severe nosocomial pneumonia with inhaled tobramycin. Pulmonologia 2011, 2:109-112. 1. Polovnikov SG, Kuzovlev AN, Iliychev AN: Case report of a successful treatment of severe nosocomial pneumonia with inhaled tobramycin. Pulmonologia 2011, 2:109-112. 1. Polovnikov SG, Kuzovlev AN, Iliychev AN: Case report of a successful treatment of severe nosocomial pneumonia with inhaled tobramycin. Pulmonologia 2011, 2:109-112. . Vincent JL, et al.: Drugs 2010, 70:1927-1944. Clinical and epidemiological risk factors for ventilator-associated pneumonia in a cohort of critically ill patients G D P l MA P i i V R i E Pi i i V B i i A O hi G De Pascale, MA Pennisi, V Raggi, E Piervincenzi, V Bernini, A Occhionero, P De Santis, A Moccaldo, S Cicconi, R Maviglia, M Tumbarello, M Antonelli Sacro Cuore Catholic University, Rome, Italy Critical Care 2012, 16(Suppl 1):P73 (doi: 10.1186/cc10680) Introduction Ventilator-associated pneumonia (VAP) represents a major infectious complication in the ICU. The aim of this study is to identify risk factors for VAP acquisition. y Methods All patients admitted to the 18-bed ICU of our university hospital between 1 October 2009 and 31 December 2010 were enrolled on the day of VAP diagnosis. Controls were selected by our computerized database. Statistical analyses were performed using the StataICl l program. fi y Conclusion Administration of IT as an adjunct to systemic antibiotics is effi cient and safe in treatment of severe nosocomial pneumonias caused by multiresistant Gram-negative bacteria. Profound randomized clinical trials on IT are required. p g Results Over the study period, among 902 admissions, 100 VAP occurred. The rate of multidrug resistance (MDR) was 23%. Development of VAP was associated with a signifi cantly longer duration of ICU stay (24 days (17 to 30) vs. 7 days (5 to 9); P <0.001) and mechanical ventilation (19 days (13 to 20) vs. 4 days (3 to 6); P <0.001). Overall ICU mortality was higher in the VAP population (41% vs. 29%; P = 0.09). Comparing patients aff ected by VAP with controls (100 matched patients), the former group was signifi cantly more likely to be male (P <0.001) and to be immunosuppressed (P = 0.004). In addition, VAP development was associated with higher rate of central venous catheter placements (P <0.001), higher mean SOFA score value (P <0.001) and previous exposure to antimicrobials (P = 0.004). Successful use of noninvasive ventilation, and trauma admission appeared as protective factors (P <0.001). Table 1 shows independent risk factors associated with VAP acquisition in multivariate analysis. Reference Effi cacy of inhaled tobramycin in severe nosocomial pneumonia Two patients of group 1 (20%) presented with hearing loss and tinnitus which revealed 3 months after the last IT administration. There were no cases of bronchospasm or renal insuffi ciency in group 1. y p Reference y p Reference 1. Ishizaka A, et al.: Crit Care Med 2001, 29:896-898. 1. Ishizaka A, et al.: Crit Care Med 2001, 29:896-898. 1. Ishizaka A, et al.: Crit Care Med 2001, 29:896-898. P73 Use of a ventilator-associated pneumonia (VAP) bundle to decrease the VAP rate in Syria Use of a ventilator-associated pneumonia (VAP) bundle to decrease the VAP rate in Syria R Alsadat1, M Mazloum2, A Alshamaa3, A Dakkak4, H Al-Bardan1, M Eltayeb2, A Marie2, F Esber1, O Naes3, M Shama5, I Betelmal5, M Kherallah6 1Al-Mouassat Hospital, Damascus, Syria; 2General Assembly of Damascus Hospital, Damascus, Syria; 3Al-Bassel Heart Institute, Damascus, Syria; 4Ibn Alnafees Hospital, Damascus, Syria; 5World Health Organization, Damascus, Syria; 6King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Critical Care 2012, 16(Suppl 1):P74 (doi: 10.1186/cc10681) R Alsadat1, M Mazloum2, A Alshamaa3, A Dakkak4, H Al-Bardan1, M Eltayeb2, A Marie2, F Esber1, O Naes3, M Shama5, I Betelmal5, M Kherallah6 1Al-Mouassat Hospital, Damascus, Syria; 2General Assembly of Damascus Hospital, Damascus, Syria; 3Al-Bassel Heart Institute, Damascus, Syria; 4Ibn Alnafees Hospital, Damascus, Syria; 5World Health Organization, Damascus, Syria; 6King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia p Results A total of 619 patients were enrolled in the study. During the baseline period (Phase 1), 238 patients with 2,456 catheter days were assessed, 30 patients developed a CRBSI. The CRBSI rate during this period was 12.2 per 1,000 catheter days. All nurses and principle doctors in the seven ICUs received training on the standard of care for catheter maintenance along with the introduction of Q-Syte™ and Posifl ush™. In Phase 2, following introduction of the interventions, 12 of 381 patients developed a CRBSI. Total catheter days during this period were 3,562. The CRBSI rate decreased to 3.4 per 1,000 catheter days. This was signifi cantly lower than during the baseline period (Wilcoxon nonparametric test, u = 4.36, P = 0.0003). Additional analyses demonstrated that patients were at higher risk for developing a CRBSI if associated with: a prolonged catheter dwell time, a higher number of insertion attempts, a blood infusion or an increased frequency of catheter connector changes. We also found that the patients who developed a CRBSI had prolonged hospital stay and signifi cantly added to the cost of treatment. Critical Care 2012, 16(Suppl 1):P74 (doi: 10.1186/cc10681) Introduction Implementation of a ventilator-associated pneumonia (VAP) bundle as a performance improvement project in the critical care units for all mechanically ventilated patients aiming to decrease the VAP rates over the study period at four major teaching hospitals in Damascus. Methods CDC criteria were used to defi ne VAP. Reference Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S27 maintenance standards in the baseline period (Phase 1). The bundle was introduced in Phase 2. CRBSI was determined according to US CDC diagnostic criteria. The rates of CRBSI before and after the introduction of the bundle of interventions were compared. Use of a ventilator-associated pneumonia (VAP) bundle to decrease the VAP rate in Syria VAP rates were calculated based on occurrences per 1,000 ventilator days, VAP rates were monitored on a monthly basis throughout the project period. VAP bundle elements included elevation of the head of the bed to between 30 and 45°, daily sedation vacation, daily assessment of readiness to wean, peptic ulcer disease prophylaxis and deep venous thrombosis prophylaxis if not contraindicated. Each hospital formed a task force with a team leader, one or two physicians and one or two nurses. Education took place at an initial conference and a follow-up meeting for the implementation process and frequent staff education session in individual units. Compliance with the VAP bundle was considered based on the implementation of all elements of the bundle. Statistical Control Chart (SPC) was used to monitor the compliance with the individual bundle elements as well the bundle as a whole. Conclusion Introduction of Q-Syte™ and Posifl ush™ and improved stan- d ards of practice for catheter maintenance can signifi cantly decrease CRBSI in the ICU. Reference Reference 1. O’Grady NP, Alexander M, Burns LA, et al.: Guidelines for the Prevention of Intravascular Catheter-Related Infections 2011 [www.cdc.gov/hicpac/bsi/ bsi-guidelines-2011.html] 1. O’Grady NP, Alexander M, Burns LA, et al.: Guidelines for the Prevention of Intravascular Catheter-Related Infections 2011 [www.cdc.gov/hicpac/bsi/ bsi-guidelines-2011.html] 1. O’Grady NP, Alexander M, Burns LA, et al.: Guidelines for the Prevention of Intravascular Catheter-Related Infections 2011 [www.cdc.gov/hicpac/bsi/ bsi-guidelines-2011.html] Results VAP bundle compliance rates were steadily increasing from 33 to 80% in Hospital 1, from 33 to 86% in Hospital 2 and from 83 to 100% in Hospital 3 during the study period. The VAP bundle was not applied in Hospital 4 and therefore no data were available. This correlated with a decrease in VAP rates from 30 to 6.4 per 1,000 ventilator days in Hospital 1, from 12 to 4.9 per 1,000 ventilator days in Hospital 3, whereas the VAP rate failed to decrease in Hospital 2 (despite better compliance) and it remained high around 33 per 1,000 ventilator days in Hospital 4 where the VAP bundle was not implemented or monitored.f P76 Wash your hands: simple measures save lives S Macedo, GV Bispo, LA Ferreira, TO Cavalcanti, PF Rosa, C Paiva, DR De Melo, LG Rezende São Jose do Avai Hospital, Itaperuna, Brazil Critical Care 2012, 16(Suppl 1):P76 (doi: 10.1186/cc10683) Introduction Sepsis is a challenge for the intensive therapy unit, being the principal cause of death during hospitalization. p Conclusion The VAP bundle is known to be an eff ective way to decrease VAP but has performed diff erently in diff erent hospitals in our study. Prevention of VAP requires concerted eff orts on the part of hospital administration, physicians, and ICU personnel. The program must be evidence-based, maintained, and accepted by ICU personnel. Monitoring and collection of data should be strict and objective. Continued education and feedback are crucial to maintain a low VAP rate. Other factors of healthcare infection prevention should also be taken into consideration. p p g p Methods We realized a longitudinal and individuated intervention authorized by the HSJA ethics committee applying the campaign ‘Simple Measures Save Lives’ in which 105 educational adhesives served as a guide for washing hands and fl ags for high-contaminated locations. A decontamination routine of monitors, control panels, fans and infusion bombs was established at each 12 hours; and continued education for the health team was intensifi ed during the intervention. Was separated two groups, patient enrollments in periods of 45 days before and after the intervention, with more than 24 hours of hospitalization: group A with 18 patients and group B with 15 patients. Results The hospital infection incidence decreased by 40% and VAP by 39.6%. Urine culture was positive in 33.3% of those patients (n = 5) in group A and in 16.7% (n = 1) in group B (a 50.1% decrease). The cultures of catheter tip were positive in 68.8% (n = 22) of catheters in group A, which used 32 catheters in total, and none in group B, which used 13 catheters. The sepsis incidence decreased by 39.6%. Septic shock was detected in 16.6% (n = 3) of patients in group A. There was a drop of the costs between groups (R4,479.28, 10.5%). The cost of campaign material was R$50.00. P76 Results Total number of patient consultations during morning ward round n = 99; total number of occasions a keyboard used n = 40; total number of times a keyboard used and keyboard required cleaning n = 37; keyboard used and cleaned when required prior to use n = 5. In total, a rate of compliance at 14% for cleaning keyboards when appropriate. Further observations over the same time period showed that keyboards were indicating they required cleaning on 96% of occasions observed. Introduction In a previous study we showed that cross-contamination with resistant bacteria occurred less frequently in a single-room (SR) ICU when compared to an open-plan (OP) ICU. We attempted to identify whether this was mediated by a change in human behavior; that is, whether hand hygiene (HH) practices were similar in the OP versus SR ICU. Methods The SR ICU comprises eight single-patient rooms. The OP ICU includes four beds in a common area. Covert HH observations were made of physicians and nurses in both ICU areas. Defi ned HH opportunities occurred before and after contact with the patient or their environment. Each observation session lasted 20 minutes. Compliance was defi ned as use of alcohol hand rub or chlorhexidine wash. Qualitative records were made of tasks preceding missed HH opportunities (patient contact, computer use, obtaining additional supplies or other). Conclusion The rate of compliance for cleaning bedside keyboards was 14%, below the standard set of 100%. This may be due to several factors, including lack of education and resistance to changes in behaviour which have previously been described in various models of human behaviour such as the theory for planned behaviour. This may refl ect compliance with other areas of infection control and have a detrimental eff ect on patient safety and health. This may also become a signifi cant issue in the future if compliance rates remain low as more IT and touchscreen equipment is employed in ICUs. The ICU has adjusted the induction programme for new medical staff to include education on infection control measures with bedside IT equipment. pp Results Observations sessions were completed on 34 and 35 occasions in the SR and OP ICUs respectively including 277 and 418 HH opportunities. The number of staff observed per session was 2.6 ± 0.7 in the SR ICU versus 2.1 ± 0.5 in the OP ICU (P = 0.01). P76 g Methods We realized a longitudinal and individuated intervention authorized by the HSJA ethics committee applying the campaign ‘Simple Measures Save Lives’ in which 105 educational adhesives served as a guide for washing hands and fl ags for high-contaminated locations. A decontamination routine of monitors, control panels, fans and infusion bombs was established at each 12 hours; and continued education for the health team was intensifi ed during the intervention. P75 A strategy for prevention and control of catheter-related bloodstream infection of ICU patients in China (Prevent CRBSI): a prospective, multicenter, controlled study G Cai, J Yan Zhejiang Hospital, Hangzhou, China Critical Care 2012, 16(Suppl 1):P75 (doi: 10.1186/cc10682) Introduction Catheter-related bloodstream infection (CRBSI) continues to be a key issue in ICUs despite recent improvements in the clinical technique, standardization of the CVC insertion protocol and hand hygiene. The impact of catheter maintenance on CRBSI rates in China needs to be further investigated. The objective of study is to evaluate a bundle of interventions for reducing CRBSI in ICUs. The bundle includes new technology (BD Q-Syte™ and BD Posifl ush™) in addition to updated standards of practice for catheter maintenance. Conclusion This intervention was a simple form to decrease the related number of infections in the neurovascular ICU, having spent irrelevant values when compared with treatment of these clinical tables. R f 1. Zanon F, Caovilla JJ, Michel RS, et al.: Sepsis in the intensive care unit: etiologies, prognostic factors and mortality. Rev Bras Terapia Intensiva 2008 20:128-134. 2. Silva E, Pedro MA, Sogaya ACB, et al.: Brazilian Sepsis Epidemiological Study. Crit Care 2004, 8:R251-R260. Methods This is a prospective, multicenter, controlled study. Patients receiving CVCs in the ICUs were eligible for inclusion. The study was performed in seven general and teaching hospitals from June 2010 to June 2011 in China. The clinicians conducted their original catheter 3. Chesley R: Getting to zero: an emerging policy framework for the elimination of hospital-associated infections. Infect Control Hosp Epidemiol 2008, 30:71-73. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S28 P77 Comparison of hand hygiene in single-room versus open-plan ICUs I Gork, S Benenson, M Brezis, CL Sprung, PD Levin Hadassah Hebrew University Medical Center, Jerusalem, Israel Critical Care 2012, 16(Suppl 1):P77 (doi: 10.1186/cc10684) equipment. A standard of 100% compliance for cleaning the keyboard appropriately when required was set for audit purposes. P76 There were fewer HH opportunities per session in the SR ICU (8.4 ± 3.3 vs. OP ICU 11.9 ± 5.2, P <0.001). HH compliance before patient contact was higher in the SR ICU than the OP ICU (1.8 ± 1.4 vs. 0.8 ± 1.1 episodes/session, P = 0.001), but similar after patient contact (2.6 ± 1.4 vs. 2.2 ± 1.5 episodes/ session, P = 0.29). Causes of missed HH opportunities were recorded on 98 and 140 occasions in the SR and OP ICUs. Comparing the SR to OP ICU: patient contact accounted for 21/98 (21%) versus 50/140 (36%, P = 0.02) missed HH opportunities respectively; use of the bedside computer 1/98 (1%) versus 14/140 (10%, P = 0.005); additional supplies (drugs, cleaning, dressing, and so forth) 9/98 (9%) versus 20/140 (14%, P = 0.24); and other 4/98 (6%) versus 15/140 (10%, P = 0.06). P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU A De Nicola, MJ Sucre San Leonardo Hospital, Castellammare di Stabia, Italy Critical Care 2012, 16(Suppl 1):P80 (doi: 10.1186/cc10687) P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU A De Nicola, MJ Sucre San Leonardo Hospital, Castellammare di Stabia, Italy Critical Care 2012, 16(Suppl 1):P80 (doi: 10.1186/cc10687) P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU A De Nicola, MJ Sucre San Leonardo Hospital, Castellammare di Stabia, Italy Critical Care 2012, 16(Suppl 1):P80 (doi: 10.1186/cc10687) Reduced air contamination in an ICU environment with a portable air purifi cation system air purifi cation system J Papaparaskevas1, V Papas2, M Pratikaki2, A Tsakris1, C Routsi2 1Medical School, University of Athens, Greece; 2Evangelismos Hospital, Athens, Greece Critical Care 2012, 16(Suppl 1):P79 (doi: 10.1186/cc10686) J Papaparaskevas1, V Papas2, M Pratikaki2, A Tsakris1, C Routsi2 1Medical School, University of Athens, Greece; 2Evangelismos Hospital, Athens, Greece Introduction Indoor air contamination has been implicated in hospital- acquiring infections, especially in immunocompromised patients. This implies that, along with other preventing measures, maintenance of good air quality in critical areas in hospitals is helpful to reduce the incidence of these infections. The objectives of this study were to evaluate the quality of an ICU air environment regarding total and fungal fl ora and the ability of a mobile air purifi cation system (Hegoa; ANEMO, Oullins, France). This device uses UVc technology (photocatalysis) to destroy a wide range of microorganisms, including fungi. Conclusion There were more HH opportunities in the OP ICU and HH compliance there was lower. The main diff erence in compliance occurred before patient care, with compliance after patient care being similar. This may refl ect ease of access from patient to patient in the OP ICU where turning around brings you easily from one patient to the next. In the SR ICU movement from patient to patient requires exiting one room and entering another with a clear end to patient care in one room and a beginning in the next. Patient contact and use of the bedside computer accounted for the majority of missed HH opportunities and present possibilities for interventions to improve HH compliance. Methods Air samples were obtained before and after the Hegoa air purifi cation system was started in seven ICU rooms, including a total of 10 beds, during a 24-hour period and at 3-hour intervals. From each room and time point, 200 l air samples were collected using a calibrated biocollector (Air Ideal; bioMerieux, Marcy L’Etoile, France). Cultures were performed on Triptycase Soy Agar and Sabouraud chloramphenicol agar plates, for the total and the fungal fl ora, respectively. Plates were incubated at 36°C and room temperature for a period of 7 days. P78 Compliance for decontamination of bedside computer keyboards on an ICU Results A total of 112 air samples from sampling sites in the ICU rooms were collected during the 24-hour study period. Before starting the air purifi cation unit, total fl ora ranged from 175 to 70 cfu/m3 and fungal fl ora from 30 to 35 cfu/m3. Total fl ora values were continuously decreasing and at 24 hours after air purifi cation onset were signifi cantly reduced to 30 to 50 cfu/m3 (72% reduction). Similarly, environment fungal levels were continuously decreased and at 24 hours after the start were undetectable. Introduction An audit to assess the compliance of decontamination of bedside computer keyboards by medical staff on an ICU. The topic was used for audit as previously identifi ed by the hospital infection control department as the worst-performing area on ‘clean trace’ scoring, and had been a regular topic of discussion between members of the medical team. Bedside keyboards automatically emit an alarm sound and small fl ashing LED light if cleaning is required prior to use. Discussion and hypothesis on reasons for rates of compliance follow this, including social/human factors that are barriers to compliance with infection control measures.f Conclusion The Hegoa mobile air purifi cation system shown a rapid lowering of contaminates with eventual elimination of fungal fl ora. Therefore, that equipment may provide an effi cient method of reducing air contamination into the ICU. Whether equipping ICU rooms with such devices could protect immunocompromised patients admitted to the ICU against fungal and microbial risk has to be examined. P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU CFU of sample sites before and after sanitation Sample sites Before UFC/cm2 After UFC/cm2 Mattress 104 <0.5 Vital parameters monitor 5,000 <0.5 Wall 2,000 <0.5 Bed rail <0.5 <0.5 Bed remote control <0.5 <0.5 Ventilator screen 5,000 <0.5 Ventilator chassis 104 <0.5 Infusion pump <0.5 <0.5 Table 1 (abstract P80). CFU of sample sites before and after sanitation Conclusion All-cause mortality in massive hemoptysis at our center was 18.8%. Lung cancer, necrotizing pneumonia and bronchiectasis carried signifi cantly higher mortality. BAE showed low mortality but required multiple interventions in nearly two-thirds of cases. Hence, surgery remains the intervention of choice in massive hemoptysis at our setup with acceptable mortality and outcome. p g p y C Wallace, S Cole, B McGuire Ninewells Hospital, Dundee, UK Critical Care 2012, 16(Suppl 1):P82 (doi: 10.1186/cc10689) Conclusion The destruction of the bacteria has practically taken place in all the points tested. The system has resulted to be compatible with the electronic equipment and a few minutes after the end of the procedure it is possible to use the area. The catalytic action of the silver atoms produces the tyndallisation of the surfaces, increasing the eff ectiveness of the sanitizer. Eight minutes after the end of the treatment, 98% of the OH – radicals have been destroyed and 95% of the dry cloud has been deposited, inhibiting the possibility of regeneration of any resistant microorganism. Introduction Almost 20% of adverse airway events reported to the Royal College of Anaesthetists 4th National Audit Project (NAP4) occurred in the ICU [1]. NAP4 commented that the failure to use capnography probably contributed to 77% of the ICU airway mortality. NAP4 subsequently made a number of recommendations pertaining to capnography use. We designed a survey to describe practice with regards to these. Methods A survey was sent to an intensivist at each of the 23 adult ICUs in Scotland. Results There was a 100% response rate. Nineteen (83%) units used capnography for all tracheal intubations on the unit, two (9%) in over three-quarters, one (4%) in under one-half and one (4%) unit reported never using it. For tracheal intubations prior to unit admission, the corresponding usage was three (13%) always, seven (30%) in over three-quarters, seven (34%) in over one-half and six (26%) in less than one-half of all intubations. Continuous capnography monitoring was in use on 54% of the intubated patients and 63% of the ventilator- dependent patients. P81 P81 Massive hemoptysis in a respiratory ICU: causes, interventions and outcomes – Indian study D Talwar, J Chudiwal, R Jain, S Kumar Metro Center for Respiratory Diseases, Noida, India Critical Care 2012, 16(Suppl 1):P81 (doi: 10.1186/cc10688) Introduction Massive hemoptysis carries high mortality and morbidity, requiring multidisciplinary management. In India, tuberculosis is a very common cause of severe hemoptysis and is being treated in tuberculosis hospitals where such an approach is not available. We evaluated the profi le of patients admitted with massive hemoptysis in a well-equipped Indian tertiary-care respiratory center. Conclusion UK Intensive Care Society (ICS) guidelines make strong recommendations for the use of capnography in all critically ill patients during intubation [2]. We show a reassuring compliance with those guidelines during tracheal intubations performed on ICUs. Compliance was much poorer with the guidelines for those intubations performed outside units. An AAGBI safety statement recommended that continuous capnography should be used in all patients with intubated tracheas, regardless of location [3]. This was not echoed in the 2009 ICS guidelines (although in the light of NAP4, these have been updated to support this). Despite the majority of units in Scotland having facilities to monitor patients using capnography, just over one-half were doing so routinely. Capnography monitoring will surely increase in the advent of NAP4 and because of the change to the ICS guidelines. References q pp y p y Methods Retrospective analysis of 376 patients admitted with hemoptysis to the respiratory ICU of the Metro Center for Respiratory Diseases, India was done. We identifi ed 90 patients with massive hemoptysis (>600 ml in 24 hours) between 2005 and 2011 and the results were analyzed. As per our protocol all patients had active medical management and those suitable for surgery underwent elective or emergent surgery. Unsuitable candidates underwent bronchial artery embolisation (BAE) or bronchoscopic interventions (BI) and if suitable were taken for surgery later. Results The mean age of patients was 49.5 ± 16.53 years with 73.33% (n = 66) being male. Mortality in male patients was signifi cantly higher than females (64.7 vs. 35.3%, P = 0.02). The mean length of stay in hospital was 10.44 ± 6.9 days and signifi cantly less (7.06 ± 4.8, P = 0.01) in the mortality group. Massive hemoptysis was due to tuberculosis (active and old) in 61%, pneumonia in 25.5%, bronchiectasis in 21.1%, aspergillus-releated disease in 11.1%. P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU Methods Observations and recording of medical staff and their compliance of cleaning bedside keyboards when required during a morning consultant-led ward round over a 10-day period. Further observations were taken at two other points in time during each day over the same time period. Observations were conducted by the author. Medical staff were informed at the beginning of the audit that this behaviour was being observed and staff were reminded of the principles of infection control, specifi cally maintaining clean IT f of the ICU of the ICU A De Nicola, MJ Sucre San Leonardo Hospital, Castellammare di Stabia, Italy Critical Care 2012, 16(Suppl 1):P80 (doi: 10.1186/cc1068 A De Nicola, MJ Sucre Introduction The ICU contains a large quantity of sensitive electrical equipment which must not be aff ected by any bio-decontamination S29 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P81). Types of management versus mortality in massive hemoptysis Table 1 (abstract P81). Types of management versus mortality in massive hemoptysis process. To disinfect the ICU environment we have used the original device, Medisize 99.99®, which releases a synergistic formulation of hydrogen peroxide with silver ions. The machine launches a dry cloud of 0.5 to 2 m particles which penetrates everywhere, without humidity or corrosive activity. process. To disinfect the ICU environment we have used the original device, Medisize 99.99®, which releases a synergistic formulation of hydrogen peroxide with silver ions. The machine launches a dry cloud of 0.5 to 2 m particles which penetrates everywhere, without humidity or corrosive activity. hemoptysis Management Total Mortality Percentage Medical 6 5 83.3 BI 35 7 20 BAE 19 1 5.2 Surgery 30 4 13.3 Multiple 21 2 9.5 y Methods The study has been conducted in the ICU area, just after the patients have been discharged, before and after the use of the Medisize 99.99® device. The overall number of samples taken has been 54 on three diff erent days. The sampling has been taken with Petri contact plates and incubated at 35°C for 48 hours, counting afterwards the CFU/plate. p Results We found the annulment of the contamination at all sites tested after sanitation (Table 1). CT chest in 65.5% and in 64.4% by fi ber optic bronchoscopy (FOB). However, combined FOB and CT scan could localize bleeding in 87.8%. See Table 1. Table 1 (abstract P80). 1. Cook TM, et al.: Br J Anaesth 2011, 106:617-631 and 632-642. 2. Thomas AN, et al.: Standards for Capnography in Critical Care. London: Intensive Care Society Standards and Guidelines; 2009. 3. The Association of Anaesthetists of Great Britain and Ireland: Safety Statement on Capnography Outside The Operating Theatre. London: AAGBI; 2009 [http:// www.aagbi.org/sites/default/fi les/AAGBI%20SAFETY%20STATEMENT_0] P80 Eff ectiveness of an innovative system for the bio-decontamination of the ICU Twelve (52%) units reported using capnography in all the intubated and ventilated patients. Of the units not using continuous capnography routinely, two (18%) had no equipment for continuous monitoring. P81 Lung cancer in 6.6% cases but this carried highest mortality. The bleeding site was identifi ed on 1. Cook TM, et al.: Br J Anaesth 2011, 106:617-631 and 632-642. 2. Thomas AN, et al.: Standards for Capnography in Critical Care. London: Intensive Care Society Standards and Guidelines; 2009. 3. The Association of Anaesthetists of Great Britain and Ireland: Safety Statement on Capnography Outside The Operating Theatre. London: AAGBI; 2009 [http:// www.aagbi.org/sites/default/fi les/AAGBI%20SAFETY%20STATEMENT_0] 1. Cook TM, et al.: Br J Anaesth 2011, 106:617-631 and 632-642. 2. Thomas AN, et al.: Standards for Capnography in Critical Care. London: Intensive Care Society Standards and Guidelines; 2009. 3. The Association of Anaesthetists of Great Britain and Ireland: Safety Statement on Capnography Outside The Operating Theatre. London: AAGBI; 2009 [http:// www.aagbi.org/sites/default/fi les/AAGBI%20SAFETY%20STATEMENT_0] 3. The Association of Anaesthetists of Great Britain and Ireland: Safety Statement on Capnography Outside The Operating Theatre. London: AAGBI; 2009 [http:// www.aagbi.org/sites/default/fi les/AAGBI%20SAFETY%20STATEMENT_0] S30 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 pressure support ventilation (PSV). This can be reduced with the application of an external positive end-expiratory pressure (PEEPe) [1]. However, an accurate measurement of PEEPi during PSV is challenging [2]. The aim of the present study is to investigate if the use of the electrical activity of diaphragm (EAdi) may yield the detection of PEEPi in patients undergoing PSV. We reasoned that if PEEPi was present the inspiratory airfl ow would start after EAdi had reached a given value (EAdi-threshold) necessary to generate the muscle pressure overcoming PEEPi. P83 Digitalized acoustic monitoring of lung congestion S Lev1, L Wolloch2, I Kagan1, M Grienv1, P Singer1 1Rabin Medical Center, Petah Tikva, Israel; 2Deep Breeze Ltd, Or-Akiva, Israel Critical Care 2012, 16(Suppl 1):P83 (doi: 10.1186/cc10690) Introduction Changes in lung water are known to change breath sound acoustics [1]. Using two pig models, we observed that continuous elevation of lung sound amplitude may indicate an increase in total lung water content [2]. Here we report three cases of ventilated patients in whom continuous acoustic monitoring was done during extravascular lung water (EVLW) measurements. Methods Ten patients with a clinical suspicion of PEEPi undergoing PSV were enrolled. Exclusion criteria were: age <18 years, hemodynamic instability, fever and PaO2/FiO2 <100 mmHg. All patients were tested during PSV for seven steps of 3 minutes each with increasing PEEPe (2, 4, 6, 8, 10, 12, 14 cmH2O). P81 At the end of each step, PEEPi was estimated with an end-expiratory occlusion maneuver. During the study, we continuously recorded airway pressure, fl ow, volume and EAdi wave- forms for off -line analysis. Data were analysed by linear regression and t test. P <0.05 was considered statistically signifi cant. Methods We retrospectively analyzed cases in which EVLWi (PiCCO) and other clinical parameters were measured, during continuous acoustic monitoring (VRI), using eight small sensors adhered to the anterior chest. A transmission factor (TF) was calculated, using the sound transfer function between diff erent sensors. The TF changes in correspondence to changes in tissue density [1]. The diff erence in TF was calculated between recordings when pulmonary edema was observed (>7 ml/kg threshold accompanied with an increase of 2 ml/kg in the EVLWi) and when absent. Statistical analysis was made using a t test. yi Results If PEEPi is present, EAdi-threshold is supposed to gradually decrease together with the raise of PEEPe; thus we divided patients into fi ve responders for whom EAdi-threshold was signifi cantly correlated with PEEPe, as opposed to fi ve nonresponders. In the group of responders we observed signifi cant correlations between the reduction of PEEPi and the increase of PEEPe (r2 = 0.86, P <0.01), and between EAdi-threshold and PEEPi at diff erent PEEPe levels (r2 =0.96, P <0.001). In the same group, respiratory rate (RR) decreased (r2 = 0.76, P = 0.01), tidal volume increased (r2 = 0.71, P = 0.02) and the peak of EAdi decreased (r2 = 0.94, P <0.001) at increasing levels of PEEPe. On the contrary, in the nonresponder group the increase of PEEPe was associated only with an increase of RR (r2 = 0.75, P = 0.01). Results A total of 336 continuous acoustic recordings in three patients (acoustic monitoring was applied together with EVLWi measurements) were analyzed (146 recordings when lung edema was present; 190 with no edema). In all patients, the acoustic profi le corresponded to changes in the clinical picture. In two of the cases, changes in acoustic profi le were similar to the ones in the EVLWi and other clinical parameters (Figure 1). In one case, where there was stability in lung sound acoustics, EVLWi and other clinical parameters were also stable. Signifi cant diff erences existed between recordings with edema (–3.61 ± 0.39) and without edema (–5.71 ± 0.15) (P <0.001). References 1. Donnerberg: Br J Dis Chest 1980, 74:23. 2. Lev: Crit Care 2011, 15:P174. P85 Adequate lung sliding identifi cation is not infl uenced by the level of academic or ultrasound training P85 Adequate lung sliding identifi cation is not infl uenced by the level of academic or ultrasound training E Piette1, R Daoust1, J Lambert2, A Denault3 1Hôpital du Sacré-Coeur de Montréal, Canada; 2Université de Montréal, Canada; 3Institut de Cardiologie de Montréal, Canada Critical Care 2012, 16(Suppl 1):P85 (doi: 10.1186/cc10692) Introduction Rapid confi rmation of the adequacy of endotracheal intubation is critical in the fi eld of emergency medicine (EM). Methods confi rming endotracheal tube (ET) position should have accuracy near 100%. Studies confi rming ET position using lung sliding (LS) identifi cation were done by physicians with extensive ultrasound (US) training using sometimes lengthy examination. These conditions are not easily reproduced in the emergency department. Our primary objective was to compare the accuracy of EM physicians with diff erent levels of academic and US training to correctly identify presence or absence of LS on random short sequences of lung US. Our secondary objective was to determine if results were better when participants had the choice to abstain themselves in uncertain cases. Conclusion Changes in lung water tend to result in changes in the sound TF, due to changes in the tissue’s density. These preliminary results indicate that monitoring lung sounds has the potential to monitor changes in lung water. Conclusion Changes in lung water tend to result in changes in the sound TF, due to changes in the tissue’s density. These preliminary results indicate that monitoring lung sounds has the potential to monitor changes in lung water. Methods We recorded in the operating room 280 short lung US sequences (one respiratory cycle), of present and absent LS of intubated patients and randomly presented them to two groups of EM physicians. Accuracy was calculated for diff erent academic and US training: none, basic Focused Assessment with Sonography in Trauma (FAST), FAST and advanced cardiac US, fellowship in EM US. We compared them using an ANOVA test. Only participants in the second group where instructed to abstain from answering in uncertain cases and accuracy was compared to the fi rst group using a Student’s t test. The project was approved by the research and ethics committees. References e e e ces 1. Mancebo J, et al.: Anesthesiology 2000, 93:81-90. 2. Marini JJ: Am J Respir Crit Care Med 2011, 184:756-762. 1. Mancebo J, et al.: Anesthesiology 2000, 93:81-90. 2. Marini JJ: Am J Respir Crit Care Med 2011, 184:756-762. P81 Conclusion In fi ve of 10 patients with clinical suspicion of PEEPi, when the PEEPe was increased we observed a decrease of EAdi-threshold, associated with improved respiratory mechanics, suggesting that EAdi- threshold could be a useful indicator for the presence of PEEPi. References Figure 1 (abstract P83). VRI (average ± SE) versus PiCCO and CXR. Figure 1 (abstract P83). VRI (average ± SE) versus PiCCO and CXR. P84 Usefulness of electrical activity of the diaphragm to detect intrinsic positive end-expiratory pressure during pressure support ventilation S Arrigoni, T Mauri, G Bellani, A Pradella, M Turella, V Sala, E Rezoagli, A Pesenti University of Milano-Bicocca, Monza, Italy 1. Donnerberg: Br J Dis Chest 1980, 74:23. Usefulness of electrical activity of the diaphragm to detect intrinsic positive end-expiratory pressure during pressure support ventilation positive end-expiratory pressure during pressure support ventilation S Arrigoni, T Mauri, G Bellani, A Pradella, M Turella, V Sala, E Rezoagli, A Pesenti University of Milano-Bicocca, Monza, Italy y , , y Critical Care 2012, 16(Suppl 1):P84 (doi: 10.1186/cc10691) Results Two medical students, 42 EM residents and 31 EM attendings participated. No diff erence in accuracy was shown between the subgroups of academic training with mean accuracies of 66.3% (medical Introduction Intrinsic positive end-expiratory pressure (PEEPi) may add a substantial workload on respiratory muscles of patients undergoing S31 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 students), 70.9% (residents) and 69.0% (attendings) (P = 0.361). No diff erence was shown between the subgroups of US training with means of 63.9% (no formation), 70.2% (FAST), 70.9% (FAST + advanced cardiac US), and 74.2% (fellowship) (P = 0.119). Accuracy was signifi cantly better when participants could abstain from answering in uncertain cases with means of 67.5% (95% CI: 65.7 to 69.4) in the fi rst group and 73.1% (95% CI: 70.7 to 75.5) in the second (P <0.001). students), 70.9% (residents) and 69.0% (attendings) (P = 0.361). No diff erence was shown between the subgroups of US training with means of 63.9% (no formation), 70.2% (FAST), 70.9% (FAST + advanced cardiac US), and 74.2% (fellowship) (P = 0.119). Accuracy was signifi cantly better when participants could abstain from answering in uncertain cases with means of 67.5% (95% CI: 65.7 to 69.4) in the fi rst group and 73.1% (95% CI: 70.7 to 75.5) in the second (P <0.001). medicine (EM) to diagnose pneumothorax as well as to evaluate the adequacy of endotracheal intubation. Presence of the Lung Pulse artefact (back and forth pleural motion induced by the heartbeat) as well as the underlying heart may aff ect correct identifi cation of LS in the left hemithorax, but this has never been studied. Our main objective was to evaluate the rate of correct identifi cation (accuracy) of the presence or absence of LS in the right and left hemithorax. p g Methods A total of 280 short lung US sequences (one respiratory cycle), recorded in the operating room, of presence and absence of LS in intubated patients were randomly presented to two groups of physicians (in total: two medical students, 42 EM residents and 31 EM attendings). Trans-thoracic echo evaluation before and during noninvasive ventilation y p g p g Results We included 21 patients (age (median) 38 years; male 71%). Most (80%) patients were admitted because of acute respiratory insuffi ciency by pneumonia. Seventeen (81%) had CD4 cell counts lower than 200/mm3. The SAPS 2 score was 47 points and the SOFA score on day 1 of admission was 6 points. Hospital mortality was 43%. All radiographic pneumonia images were viewed on lung US examinations. Possible and probable pneumonia by P. jiroveci was diagnosed in six patients; all of these patients presented diff use thin and/or gross B lines on both lungs. Bacterial (n = 7), mycobacterial (tuberculosis (n = 6) and Mycobacterium kansasii (n = 1)), and fungal (Aspergillus sp. (n = 1)) were diagnosed in other patients. Peripheral microabscesses were viewed on one patient with PCP and four patients with other etiologies (P = NS); pleural eff usions were present on US of seven patients with diverse etiologies (no PCP patient had pleural eff usions; P = 0.06); no pneumothorax was diagnosed in the study. Consolidation was present in one patient with PCP and 11 patients with bacterial, mycobacterial and fungal pneumonia (P = 0.05). There was a high degree of symmetry on lung US examinations of PCP patients, while there was always diff erences between the right and left hemithorax among other etiologic pneumonia (P <0.001).f ventilation L Vetrugno, M Costa, C Centonze, N Langiano, M Rojatti, G Della Rocca University Hospital of Udine, Italy Critical Care 2012, 16(Suppl 1):P88 (doi: 10.1186/cc10695) Introduction Over the last decade noninvasive ventilation (NIV) gained the dignity of fi rst-line intervention for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in the ICU. Its great interest is based on a lower complications rate compared with traditional invasive ventilation. However, the NIV application, although less invasive, cannot ignore its hemodynamic eff ect over the patient. This study evaluates the NIV eff ects on the left ventricle in terms of systolic and diastolic function through trans-thoracic echocardiography (TTE). We also try to obtain a preload value index equivalent of fl ow time corrected (FTc). p ql Methods Thirteen patients admitted to our ICU with ALI/ARDS underwent TTE before and during NIV. NIV was set as a 1 hour cycle with 5 to 7 cmH2O of PEEP and 5 to 7 cmH2O of pressure support ventilation. During NIV for a better patient compliance a continuous i.v. Usefulness of electrical activity of the diaphragm to detect intrinsic positive end-expiratory pressure during pressure support ventilation Sequences were divided equally between the right and left hemithorax. Each participant’s knowledge of the Lung Pulse artefact was noted. Only the second group was instructed not to answer in case of uncertainty. A Kolmogorov–Smirnov test showed the rate of correct LS identifi cation did not follow a normal distribution. Median rates are reported with interquartile range (IQR) and compared using a Mann– Whitney test. Conclusion Correct LS identifi cation on short lung US sequences is not infl uenced by the level of academic or US training. Accuracy is better when the possibility to abstain oneself from answering is given. LS identifi cation using one respiratory US sequences should be used with caution to confi rm adequacy of endotracheal intubation. P86 Lung ultrasound can diff erentiate Pneumocystis jiroveci versus other etiologies among critically ill AIDS patients with pneumonia A Japiassu, F Bozza IPEC-FIOCRUZ, Rio de Janeiro, Brazil Critical Care 2012, 16(Suppl 1):P86 (doi: 10.1186/cc10693) Trans-thoracic echo evaluation before and during noninvasive ventilation infusion of remifentanil was used (range 0.03 to 0.05 μg/kg/minute). At baseline (T0 = before NIV) and after 30 minutes of NIV (T1), the following data were recorded: respiratory – RR, SaO2%, PaO2, PaCO2, pH, BE, and HCO3 –; and cardiac – heart rate (HR), arterial blood pressure (systolic, diastolic and media), diastolic and systolic volume (EDV, ESV), ejection fraction (EF), stroke volume (SV), velocity time integral (VTI), FTc, E wave, deceleration time (Dt), A wave, ventricular fl ow propagation velocity (Vp). Conclusion We suggest that high-degree symmetric and diff use B lines, without pleural eff usions, are compatible with P. jiroveci as the etiology of recent diagnosed pneumonia in critically ill AIDS patients. P87 Diff erence in accuracy of lung sliding identifi cation between the right and left hemithorax R Daoust1, E Piette1, J Lambert2, A Denault3 1Hôpital du Sacré-Coeur de Montréal, Canada; 2Université de Montréal, Canada; 3Institut de Cardiologie de Montréal, Canada Critical Care 2012, 16(Suppl 1):P87 (doi: 10.1186/cc10694) Lung ultrasound can diff erentiate Pneumocystis jiroveci versus other etiologies among critically ill AIDS patients with pneumonia A Japiassu, F Bozza Results Knowledge of Lung Pulse was higher in the second group (55% vs. 21%, P <0.05). Globally, median accuracy of identifi cation of LS presence or absence was 74.0% (IQR: 48.0 to 90.0) in the fi rst group and 83.7% (IQR: 53.3 to 96.2) in the second (P = 0.006). For the fi rst group, median accuracy was 80.0% (IQR: 57.0 to 95.0) in the right hemithorax and 67.0% (IQR: 43.0 to 83.0) in the left (P <0.001). For the second group, median accuracy was 88.7% (IQR: 63.1 to 96.9) in the right hemithorax and 76.3% (IQR: 42.9 to 90.9) in the left (P <0.001). Introduction Lung ultrasound (US) can be applied as a point-of-care approach for diagnosis of pneumonia in AIDS patients. We compare US examinations of Pneumocystis jiroveci versus other etiologies of pneumonia in critically ill patients. p y p Methods Every HIV/AIDS patients admitted to the ICU with pneumonia was included. The fi rst US examination was performed until 72 hours after admission. Pneumonia was defi ned by clinical examination, laboratorial parameters and chest X-rays. Etiologic agents were defi ned according to appropriate cultures and serology. US was applied to four fi elds (apex, lateral middle third, anterior basal and posterior basal regions) for each hemithorax, with 2.5 MHz curved transducer. Three pneumonia patterns were defi ned: interstitial pneumonia, bronchopneumonia and pneumonia with consolidation. The presence of B lines, peripheral microabscesses (bronchopneumonia), consolidations and pleural eff usions were compared between the Pneumocystis pneumonia group (PCP) versus other etiologies. Conclusion Accuracy of identifi cation of LS presence or absence is higher in the right hemithorax. Our study is the fi rst to report this fi nding. Presence of the Lung Pulse artefact, as well as the underlying heart, probably explains the worse accuracy found in the left hemithorax. Caution should be taken in using LS identifi cation as a diagnostic tool in the left hemithorax and knowledge of the Lung Pulse artefact should be emphasized in chest US curriculum. P88 P89 P89 Listen to PaO2/FiO2 ratios: they tell us about length of stay V Inal, B Comert, L Yamanel GATA, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P89 (doi: 10.1186/cc10696) P89 Listen to PaO2/FiO2 ratios: they tell us about length of stay V Inal, B Comert, L Yamanel GATA, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P89 (doi: 10.1186/cc10696) Introduction Classifi cation of respiratory distress has been dependent on PaO2/FiO2; that is, <300 acute lung injury (ALI) and <200 acute respiratory distress syndrome (ARDS). In this study, PaO2/FiO2 was analyzed for predicting ICU patients’ length of stay (LOS). References References 1. Antonelli M, Pennisi MA, Montini L: Clinical review: Noninvasive ventilation in the clinical setting – experience from the past 10 years. Crit Care 2005, 9:98-103. References 1. Antonelli M, Pennisi MA, Montini L: Clinical review: Noninvasive ventilation in the clinical setting – experience from the past 10 years. Crit Care 2005, 9:98-103. 2. Shekerdemian L, Bohn D: Cardiovascular eff ects of mechanical ventilation. Arch Dis Child 1999, 80:475-480. 2. Shekerdemian L, Bohn D: Cardiovascular eff ects of mechanical ventilation. Arch Dis Child 1999, 80:475-480. Conclusion We concluded that the PaO2/FiO2 ratio was a powerful indicator for predicting ICU LOS in patients with RI. In addition there was no need to classify patients according to PaO2/FiO2 to predict LOS; any decreased ratio meant a longer LOS. However, this study was weak in power; it had a small sample, did not include comorbid conditions, did not account for accepted scoring systems, and did not include daily ABGA for prediction. On the other hand, these results are promising for future observations that ABGA taken in the ED would be a supplemental tool for the physician’s approach in the ICU. Diff erence in accuracy of lung sliding identifi cation between the right and left hemithorax On the other hand, these results are promising for future observations that ABGA taken in the ED would be a supplemental tool for the physician’s approach in the ICU. Age >300 (n) ALI (n) ARDS (n) (n) (years) LOS (days) LOS (days) LOS (days) Male 165 65 ± 8.2 47 8 ± 2.1 65 12 ± 3.4 53 16 ± 4.2 Female 108 69 ± 7.6 33 7 ± 2.9 43 11 ± 3.7 32 17 ± 3.8 Diff erence in accuracy of lung sliding identifi cation between the right and left hemithorax Results From T0 to T1 the following changes with Wilcoxon matched pairs test were statistically signifi cant (P <0.05). PaO2 (94 to 123 mmHg), SaO2 (87 to 97%) and PaO2/FiO2, RR (37 to 28/minute). At T0, EF was >55% in seven patients and <55% in six patients. In the group with EF <55% (T0) the EF increased at T1 (42 to 52%). Dt signifi cantly increased from T0 to T1 (182 to 198 cm/second). No signifi cant changes were observed in VTI, E/A ratio, Vp, and E/Vp ratio, from T0 to T1. g R Daoust1, E Piette1, J Lambert2, A Denault3 1Hôpital du Sacré-Coeur de Montréal, Canada; 2Université de Montréal, Canada; 3Institut de Cardiologie de Montréal, Canada Critical Care 2012, 16(Suppl 1):P87 (doi: 10.1186/cc10694) Introduction The fi eld of lung ultrasound (US) in critical care is in rapid expansion. Lung sliding (LS) identifi cation has been used in emergency Introduction The fi eld of lung ultrasound (US) in critical care is in rapid expansion. Lung sliding (LS) identifi cation has been used in emergency Conclusion Our study suggests that NIV improves cardiac function in patients with reduced EF, positioning the patients to a more favorable S32 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P89). Patient data Table 1 (abstract P89). Patient data point of the Frank Starling curve. In these patients we also showed an increase in FTc that seems to be aff ected by either preload or afterload reduction. ( ) Age >300 (n) ALI (n) ARDS (n) (n) (years) LOS (days) LOS (days) LOS (days) Male 165 65 ± 8.2 47 8 ± 2.1 65 12 ± 3.4 53 16 ± 4.2 Female 108 69 ± 7.6 33 7 ± 2.9 43 11 ± 3.7 32 17 ± 3.8 Conclusion We concluded that the PaO2/FiO2 ratio was a powerful indicator for predicting ICU LOS in patients with RI. In addition there was no need to classify patients according to PaO2/FiO2 to predict LOS; any decreased ratio meant a longer LOS. However, this study was weak in power; it had a small sample, did not include comorbid conditions, did not account for accepted scoring systems, and did not include daily ABGA for prediction. Reference 1. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N Engl J Med 2000, 342:1301-1308. 1. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N Engl J Med 2000, 342:1301-1308. 1. Pediatr Crit Care Med 2010, 11:12-17. Figure 1 (abstract P91). Oxygenation Index versus P/F ratio. The Oxygenation Index compared with the P/F ratio in ALI/ARDS M Van Haperen1, PH Van der Voort2, RJ Bosman2 1AMC, Amsterdam, the Netherlands; 2Olvg, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P91 (doi: 10.1186/cc10698) 1AMC, Amsterdam, the Netherlands; 2Olvg, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P91 (doi: 10.1186/cc10698) Introduction The usual way to describe the severity of pulmonary dysfunction in ventilated ICU patients is by using the PaO2/FiO2 ratio (PF). The PF may be adjusted by the ventilator pressure settings in order to reduce inspiratory oxygen fraction but the PF does not take the mean airway pressure (MAP) into account. In contrast, the Oxygenation Index (OI) is defi ned as the reciprocal of PF times MAP: OI = (FiO2×mean airway pressure) / PaO2. As such, the OI is a better representative of oxygenation dysfunction. The objective was to study the correlation between and the impact of the MAP on the PF and OI. p Results Seventeen patients with mean age of 70.2 years (SD 14.1) and median APACHE IV expected mortality of 31% (IQR 14 to 70), 10 admitted for medical reasons and seven for surgical reasons, and ventilated for 4 days median (IQR 3 to 6) fulfi lled inclusion criteria and were included in the study. Results of tidal volume measurements are shown in Table 1. Table 1 (abstract P92). Tidal volume measurements Table 1 (abstract P92). Tidal volume measurements Table 1 (abstract P92). Tidal volume measurements Table 1 (abstract P92). Tidal volume measurements Total number of aVt measurements 286 Number of aVt measurements per patient (IQR) 12 (4 to 20) aVt <6 ml/kg PBW 25 (9%) aVt 6 to 8 ml/kg PBW 156 (58%) aVt 8 to 10 ml/kg PBW 82 (29%) aVt >10 ml/kg PBW 23 (8%) Mean aVt per kg PBW (SD) 7.85 (1.23) aVt, actual tidal volume; PBW, predicted body weight. Methods We performed a retrospective analysis of 27 consecutive mechanically ventilated patients admitted to our ICU with bilateral interstitial/alveolar lung disease, defi ned as ALI or ARDS. The data of these patients were collected during a time period of maximum 30 consecutive days. Demographic data were recorded and the PF, OI and MAP were assessed daily at 6:00 am during the fi rst 30 days of admission. OI >8.1 is usually regarded as ARDS and >5.3 as ALI [1]. y g [ ] Results We included 27 patients, 25 were male, the mean APACHE II score was 22, the median length of stay on the ICU 11 days and the ICU mortality was 11/16 (69%). The mean PF was 165 (SD 83), the mean OI was 8.2 (SD 5) and the mean MAP was 16 cmH2O (SD 5). The 27 patients resulted in 364 measurements. Of these measurements 158 had OI >8.1, of which 157 had PF <200 and a mean MAP of 19.3 cmH2O. In one patient PF was >200 while OI was >8.1 with MAP 18 cmH2O. Of the 100 measurements with OI 5.3 to 8.1, 14 had PF 200 to 300 and 85 had PF <200. The MAP in these measurements was 17, 64 and 24 cmH2O respectively. Figure 1 shows the nonlinear relation between OI and PF. Conclusion In patients with ARDS, OI >8.1 is usually in agreement with PF <200. However, patients with ALI based on OI 5.3 to 8.1 frequently had PF <200. More studies are needed to determine the optimal level of OI for the diagnosis of ALI/ARDS. Reference Conclusion In this small single-centre study, the mean aVt is between 6 and 8 ml/kg PBW as prescribed, but only 58% of measured tidal volumes are indeed between 6 and 8 ml/kg PBW. P92 P92 Results Data were available for 815 patients (see Figure 1). Increasing OI was associated with increasing mortality (P <0.0001 chi-squared test for trend). Each step increase in OI was associated with approximately a 6% absolute increase in mortality. The OI was also associated with increasing Standardised Mortality Ratio (ICNARC model).i Do actual tidal volumes diff er from prescribed tidal volumes? R Kleijn, B Kalkman, N Verburg, H Oudijk, B Van Vondelen, M Luttmer, I Slagers, M Ruijters, I Meynaar Reinier de Graaf Groep, Delft, the Netherlands Critical Care 2012, 16(Suppl 1):P92 (doi: 10.1186/cc10699) g y Conclusion The highest OI occurring in the fi rst 24 hours of ventilation is an independent predictor of mortality. Collection of OI data may allow better prediction of outcome than P/F ratio data alone. References Introduction Studies have shown that the selection of incorrect tidal volume can cause ventilator-induced lung injury and increased mortality [1]. This study was done to determine if the actual tidal volume (aVt) diff ers from the prescribed tidal volume (pVt) based on predicted body weight (PBW). 1. Winter B, et al.: Management of Severe Refractory Hypoxia in Critical Care in the UK in 2010 Report from UK Expert Group [http://www.ics.ac.uk/ latest_news/management_of_severe_respiratory_failure_in_critical_care_] 2. Britos M, Smoot E, Liu KD, et al.: The value of positive end-expiratory pressure and FiO2 criteria in the defi nition of the acute respiratory distress syndrome. Crit Care Med 2011, 39:2025-2030. y Methods The ICU is a 10-bed intensivist-led unit in a 500-bed teaching hospital. All consecutive patients receiving invasive mechanical ventilation in June 2011 were included. Patients with noninvasive ventilation or with continuous positive airway pressure only were excluded. The ICU has a mechanical ventilation protocol that prescribes tidal volume to be between 6 and 8 ml/kg PBW. A table with prescribed tidal volumes based on PBW is available at the bedside throughout the ICU. All patients were ventilated with Drager Evita XL ventilators on pressure support (ASB) or pressure control mode (BIPAP). During the study period we compared the aVt with the pVt each day at 0, 6, 10, 14, 18 and 22 hours for all patients. P90 Worst Oxygenation Index during the fi rst 24 hours of ventilation predicts mortality y y Methods Data of 273 patients admitted to the ICU with RI were retrospectively analyzed for LOS in the ICU. Patients admitted to the emergency department (ED) with RI, documented arterial blood gas analysis (ABGA), and hospitalized in the ICU were eligible for this study within 4 years. The fi rst ABGA in ED PaO2/FiO2 were taken for predicting ICU LOS. Patients’ comorbid diseases, APACHE II/Glasgow scores, non/ invasive mechanical ventilation supports were not included in the analysis. Patients were classifi ed into three groups as: (1) >300 not having RI, (2) <300 ALI, (3) <200 ARDS; they were then compared for predicting ICU LOS, and also receiver operating curve (ROC) analysis and area under curve (AUC) were calculated.i RJ Jackson, TH Gould, MJ Thomas Bristol Royal Infi rmary, Bristol, UK Critical Care 2012, 16(Suppl 1):P90 (doi: 10.1186/cc10697) Introduction The ratio of PaO2 to FiO2 (P/F ratio) is often used to classify patients with hypoxic respiratory failure, and is recommended in guidelines from a UK expert group [1] but does not take airway pressures into account. A study found that adjusting for PEEP did not aff ect the predictive ability of the P/F ratio [2]; however, the mean airway pressure (MAP) may be a better indicator of lung recruitment. The Oxygenation Index (OI = (FiO2×MAP) / PaO2)) includes an adjustment for MAP. Results Analysis showed statistical signifi cance of P <0.01 for all groups pointing out that ED ABGA PaO2/FiO2 levels negatively aff ected patients’ LOS in the ICU. ROC analysis of PaO2/FiO2 for LOS showed signifi cant AUC: 0.917 levels, which was predicted as a powerful indicator. Patients’ data are presented in Table 1. 2 2 Methods We retrospectively assessed a computerised record (from 2008 to 2010) of ventilator parameters and identifi ed the highest OI for all ventilated patients from a general adult university teaching hospital ICU, during the fi rst 24 hours of ventilation. Patients were grouped according to highest OI, and mortality was calculated for subgroups. Figure 1 (abstract P90). Mortality and number of patients by Oxygenation Index. Figure 1 (abstract P90). Mortality and number of patients by Oxygenation Index. Figure 1 (abstract P90). Mortality and number of patients by Oxygenation Index. S33 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P93 P93 Intratracheal administration of siRNA targeting FAS reduces ischemia–reperfusion-induced lung injury L Del Sorbo, G Muraca, A Costamagna, G Rotondo, L Laudari, F Civiletti, E Tonoli, E Martin, V Fanelli, V Ranieri University of Turin, Italy Critical Care 2012, 16(Suppl 1):P93 (doi: 10.1186/cc10700) , , , University of Turin, Italy Critical Care 2012, 16(Suppl 1):P93 (doi: 10.1186/cc10700) Introduction Ischemia–reperfusion injury is one of the main causes of primary graft dysfunction after lung transplantation. Fas-mediated apoptosis plays a major role in the pathogenesis of ischemia– reperfusion injury. Exogenous administration of small interfering RNA (siRNA) is an eff ective strategy to specifi cally silence the expression of proteins through blocking the translation of mRNA. The aim of this study was to investigate in an ex vivo mouse model of lung ventilation and perfusion whether a specifi c siRNA targeting Fas is able to reduce ischemia–reperfusion injury. Figure 1 (abstract P91). Oxygenation Index versus P/F ratio. S34 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods C57BL/6 male mice were randomized to intratracheally receive a specifi c sequence of siRNA targeting FAS (siRNA-FAS) or a scrambled siRNA 48 hours before undergoing 6 hours of cold ischemic time (4°C) followed by 2 hours of ex vivo ventilation (peak inspiratory pressure = 7 cmH2O, PEEP = 2 cmH2O, respiratory rate = 100 breaths/minute, FiO2 = 100%) and reperfusion (4% bovine serum albumin RPMI medium with 10% fresh blood at 1 ml/minute fl ow rate) in a predisposed humidifi ed chamber at 37°C. At the end of the experiment, lung elastance, assessed through tidal volume, and total protein concentration in the bronchoalveolar lavage (BAL) fl uid were measured. A separate set of lungs were analysed by western blot before undergoing cold ischemia to assess the expression of FAS protein. Conclusion Acute lung injury induced by intratracheal hydrochloric acid instillation requires the function of TNFα receptor I and associates with activation of downstream proinfl ammatory signaling pathways p44/42 and c-Jun N-Terminal kinase. P96 g Methods Subjects were male and female C57Bl/6 mice, wild-type, TNFα knockout, TNFα receptor I knockout (n = 135). Hydrochloric acid was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage and lung tissue harvesting 24 hours post injection. The TNFα inhibitor etanercept was administered as pretreatment to a subset of mice prior to hydrochloric acid exposure.l A new miniaturized extracorporeal membrane oxygenator with integrated rotary blood pump (Ilias): fi rst results in a porcine mode of lung injury K Pilarczyk1, J Heckmann1, K Lyskawa1, A Strauß2, U Aschenbrenner2, H Jakob1, M Kamler1, N Pizanis1 1West German Heart Centre Essen, University Hospital, Essen, Germany; 2iliasmedical GmbH, Bochum, Germany Critical Care 2012, 16(Suppl 1):P96 (doi: 10.1186/cc10703) P93 P95 Retrieval of patients with severe respiratory failure on venovenous extracorporeal membrane oxygenation: an intensivist-led model A Burrell1, V Pellegrino1, D Pilcher1, S Bernard1, M Kennedy2 1The Alfred Hospital, Melbourne, Australia; 2Adult Retrieval Victoria, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P95 (doi: 10.1186/cc10702) g g p p Results The intratracheal administration of siRNA-FAS reduced the expression of FAS in the lung by 44% (siRNA-FAS 0.90 ± 0.11 vs. scrambled siRNA 1.61 ± 0.18 AU). Lung elastance and BAL total protein concentration were signifi cantly reduced in the siRNA-FAS group as compared to control in lungs exposed to 6 hours of cold ischemia followed by 2 hours of reperfusion. See Table 1. Introduction Patients with severe respiratory failure may require veno- venous extracorporeal membrane oxygenation (vv-ECMO). However, this treatment is only available in specialized centres. Previous reports of vv-ECMO cannula insertion and retrieval have included large teams of surgeons, perfusionists, physicians, retrieval doctors, paramedics and nurses. We hypothesized that an intensivist-led model for rapid response to a referring hospital, the insertion of vv-ECMO cannulae and subsequent retrieval would be safe and feasible. Table 1 (abstract P93) siRNA-FAS siRNA scrambled Elastance (cmH2O/ml) 11.34 ± 0.24 13.75 ± 0.99 BAL proteins (μg/ml) 529.1 ± 64.8* 928.5 ± 138.2 Data are mean ± SE. Comparison between groups was performed with the Student’s t test. P <0.05. Methods The Alfred Hospital ICU is the specialist centre for ECMO services for the states of Victoria and Tasmania in Australia. The intensivists in our ICU are trained to insert ECMO cannulae using a percutaneous femoral approach and manage the ECMO circuit during transport. A new ECMO retrieval service was set up in 2008 to allow the cannulation and retrieval of patients from other referring hospitals. The retrieval team comprises two intensivists to insert femoral cannulae and manage the ECMO circuit, a third physician to manage the ventilator and infusion pumps and a paramedic to manage the logistics of the patient transfer. We reviewed all consecutive patients from 2008 to 2011 with severe respiratory failure who received vv-ECMO and were retrieved to our specialist center. Conclusion The intratracheal administration of siRNA targeting FAS prevents the increase of the alveolar membrane permeability during ischemia–reperfusion injury. p j y cknowledgements Funded by PRIN and Regione Piemonte. P94 Acute lung injury in mice associates with p44/42 and c-Jun N-terminal kinase activation and requires the function of TNFα receptor I Critical Care 2012, 16(Suppl 1):P94 (doi: 10.1186/cc10701) Introduction Aspiration of hydrochloric acid-containing gastric juice leads to acute lung injury and hypoxemic respiratory failure due to an exuberant infl ammatory response associated with pulmonary edema from increased endothelial and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of TNFα in experimental acute lung injury from hydrochloric acid aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. g Conclusion An intensivist-led model of vv-ECMO cannulation and retrieval appears to be a safe and eff ective model for vv-ECMO retrieval. This model may lead to a more rapid and cost-eff ective response and is the subject of further study. P94 p Results There were 23 patients from 2008 to 2011. All cannulations were successfully performed percutaneously at the referring hospital by the intensivists. The underlying condition was H1N1 in 11 patients, bacterial pneumonia in six, acute lung injury in four, metastatic seminoma in one and multiple lung abscesses in one. The average age was 36 years (range 17 to 60 years). Males were 61%. Transport was by fi xed-wing aircraft in 35% and road ambulance in 65%. The retrieval distance averaged 76 km (range 7 to 1,770 km). During transport, there were two transient pump failures requiring hand cranking and one monitor failure. These resulted in no adverse clinical eff ects. The average ICU length of stay was 14 days. Overall survival to hospital discharge was 17/23 (74%). P94 Acute lung injury in mice associates with p44/42 and c-Jun N-terminal kinase activation and requires the function of TNFα receptor I N Maniatis1, A Sfi ka1, I Nikitopoulou1, A Vassiliou1, C Magkou1, M Kardara1, A Armaganidis1, C Roussos1, G Kollias2, S Orfanos1, A Kotanidou1 1University of Athens, Greece; 2‘Al. Fleming’ Biomedical Sciences Research Center, Vari, Greece Critical Care 2012, 16(Suppl 1):P94 (doi: 10.1186/cc10701) ECMO in nonintubated patients as a bridge to lung transplant: our experience M Chierichetti, A Santini, F Pagan, S Crotti, A Lissoni, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, Milan, Italy Critical Care 2012, 16(Suppl 1):P97 (doi: 10.1186/cc10704) Introduction Extracorporeal membrane oxygenation (ECMO) has now been used by an expanding number of centres for bridging to lung transplant (LTx) in patients with advanced cardiac and respiratory failure [1]. ECMO has been used for bridging to LTx almost exclusively in patients receiving mechanical ventilation. In order to avoid the drawbacks and complications associated with intubation and prolonged mechanical ventilation we hypothesized that the use of venovenous ECMO (VV-ECMO) in awake and spontaneously breathing patients might be an option for respiratory support in those patients who are severely deteriorating while waiting for lung transplant. Results A total of 19 trauma patients, 15 males and four females, underwent the ECLS technique. All of the following data are expressed as median and 25th and 75th percentiles are enclosed in parenthesis. Median age was 48 (31.8 to 63.8) years. Injury severity score was 59 (41.3 to 73.8). Thirteen patients had polytrauma with brain injury. Fourteen patients received va ECLS and fi ve patients vv ECLS. Indications to ECLS placement were: cardiac arrest in nine patients, severe bleeding shock in fi ve patients and acute respiratory failure in fi ve patients. In four patients, ECLS was placed in the shock room, two patients received ECLS in the operating room during damage control surgery and 13 patients in the ICU. Timing to ECLS from the trauma event was within 6 hours for six patients, between 6 and 24 hours for fi ve patients and over 24 hours for eight patients. Sixteen patients were admitted to the ICU. Five patients were discharged from the ICU. Brain death diagnosis as a consequence of traumatic injury was performed in six patients. In four of these patients organ donation was possible. Methods We performed a retrospective analysis of seven patients (three female, mean age 31.7 ± 12.1 years) who underwent lung transplant while on ECMO support between May 2009 and October 2011 and who had not been ventilated for more than 24 hours before the LTx. All patients were fully awake and they kept on receiving noninvasive ventilation for a variable amount of time per day after ECMO support was started, according to clinical evaluation. A new miniaturized extracorporeal membrane oxygenator with integrated rotary blood pump (Ilias): fi rst results in a porcine model of lung injury K Pilarczyk1, J Heckmann1, K Lyskawa1, A Strauß2, U Aschenbrenner2, H Jakob1, M Kamler1, N Pizanis1 1West German Heart Centre Essen, University Hospital, Essen, Germany; 2iliasmedical GmbH, Bochum, Germany Critical Care 2012, 16(Suppl 1):P96 (doi: 10.1186/cc10703) Results Hydrochloric acid instillation induced an infl ammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils and total protein, histologic lung injury score and respiratory system elastance, while TNFα receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55 kDa TNFα receptor I, but not by deletion of the TNFα gene. Hydrochloric acid induced a sixfold increase in apoptotic, caspase- 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNFα receptor I. In immunoblotting and immunohistochemistry studies, hydrochloric acid stimulated signaling via p44/42 and c-Jun N-Terminal kinase, which was blocked in TNFα receptor I knockout mice. Introduction Extracorporeal membrane oxygenation (ECMO) is used for most severe acute respiratory distress syndrome cases in specialized centres. However, critically ill patients fulfi lling ECMO criteria are often not suitable for transportation and currently available ECMO systems are not designed for emergency use or interhospital transfer. Therefore, a new miniaturized ECMO (Ilias; Figure 1) with only 5 kg weight was developed to reduce fi lling volume and simplify management. Introduction Extracorporeal membrane oxygenation (ECMO) is used for most severe acute respiratory distress syndrome cases in specialized centres. However, critically ill patients fulfi lling ECMO criteria are often not suitable for transportation and currently available ECMO systems are not designed for emergency use or interhospital transfer. Therefore, a new miniaturized ECMO (Ilias; Figure 1) with only 5 kg weight was developed to reduce fi lling volume and simplify management. Methods Acute lung injury was induced with repeated pulmonary saline infusion in 13 pigs until the Horowitz Index was <100 mmHg. Pigs were assigned to the following three groups: group 1 (n = 3), Methods Acute lung injury was induced with repeated pulmonary saline infusion in 13 pigs until the Horowitz Index was <100 mmHg. Pigs were assigned to the following three groups: group 1 (n = 3), Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum com/supplements/16/S1 S35 , pp http://ccforum.com/supplements/16/S1 Results All patients survived successfully until the transplant. Extracorporeal life support in major trauma: case series from a tertiary referral trauma 9 ECMO in nonintubated patients as a bridge to lung transplant: our experience M Chierichetti, A Santini, F Pagan, S Crotti, A Lissoni, L Gattinoni Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milano, Milan, Italy Critical Care 2012, 16(Suppl 1):P97 (doi: 10.1186/cc10704) Reference Reference 1. Bermudez CA, et al.: Extracorporeal membrane oxygenation as a bridge to lung transplant: midterm outcomes. Ann Thorac Surg 2011, 92:1226-1232. Figure 1 (abstract P96). The Ilias prototype. Extracorporeal life support in major trauma: case series from a tertiary referral trauma Extracorporeal life support in major trauma: case series from a tertiary referral trauma R Spina, S Biondi, A Circelli, G Cianchi, S Degl’Innocenti, M Bonacchi, A Peris Careggi Teaching Hospital, Florence, Italy Critical Care 2012, 16(Suppl 1):P98 (doi: 10.1186/cc10705) Results No device failed during the observation period. Oxygenation increased signifi cantly in both ECMO groups compared to baseline and to control (paO2 from 79 ± 8 before Ilias to 340 ± 108 mmHg and from 61 ± 12 mmHg to 309 ± 59 mmHg in the standard ECMO group). The CO2 elimination by the Ilias reduced arterial paCO2 from 134 ± 25 mmHg at baseline to 53 ± 7 mmHg. Hemodynamic instability, signifi cant activation of the plasmatic coagulation or platelet consumption was not observed. However, hemolyses were signifi cantly higher in the Ilias group compared to the Maquet group. Introduction Major trauma (MT) is a leading cause of death and disability worldwide. Immediate fatal complications are bleeding shock followed in the post-emergency phase by severe head and spine injury and post-traumatic respiratory failure. Venoarterial (va) or venovenous (vv) extracorporeal life support (ECLS) could represent a valid option to face these life-threatening trauma complications. Moreover, ECLS can potentially be employed to expand the pool of donors in patients with brain death diagnosis after trauma event. Conclusion The Ilias prototype provided excellent gas exchange with hemodynamic stability comparable to a standard ECMO system. Further development and design modifi cations (optimized rotation speed and surface coating of rotor) are already done and another experiment is projected to reduce hemolysis for clinical application. Methods Patient data were collected from January 2009 to November 2011. A multidisciplinary algorithm-based protocol was written by our ECLS Team. The va-ECLS indications were bleeding shock, with potential controllable bleeding sites, not responding to massive fl uid and blood resuscitation and to vasopressor support and cardiac arrest not responding after 10 minutes of cardiopulmonary resuscitation (CPR). vv-ECLS criteria establishment were severe hypoxia and/or hypercarbia due to acute lung failure not responding to conventional ventilatory strategies. The ECLS device is composed of a centrifugal pump and a hollow fi ber membrane oxygenator (Quadrox-D; Maquet, Germany). Coagulation status was controlled by the activated partial thromboplastin time every 4 hours and thromboelastography. P98 control group, conventional ventilation; group 2 (n = 5), standard venovenous ECMO (Maquet); group 3 (n = 5), Ilias group. Gas exchange, hemodynamics, hemolysis, and coagulation activation were examined over a period of 8 hours. A new miniaturized extracorporeal membrane oxygenator with integrated rotary blood pump (Ilias): fi rst results in a porcine model of lung injury All patients underwent BLTx on VV-ECMO support, three were converted to VA during transplant and then back to VV at the end of the procedure. One patient died after BLTx due to hemorrhagic complications. Mean ECMO support was BloodFlow 3.1 ± 0.8 l/minute, GasFlow 4.7 ± 2.5 l/ minute, no one needed mechanical ventilation before BLTx. After lung transplant fi ve patients remained intubated and they were ventilated for 13.9 ± 16.4 days. Mean duration of ECMO support after LTx was 4.7 ± 5.4 days. Mean ICU LOS after LTx was 18 ± 17.9 days. Among this population three patients developed hemorrhagic complication, two primary graft dysfunction, two neuromuscular dysfunction, while only one chronic renal failure. Results All patients survived successfully until the transplant. All patients underwent BLTx on VV-ECMO support, three were converted to VA during transplant and then back to VV at the end of the procedure. Figure 1 (abstract P96). The Ilias prototype. Conclusion Our experience shows that bridge to lung transplant with VV-ECMO in awake and spontaneously breathing patients is not only feasible but also successful. Survival to BLTx in our center was 100%, while survival after BLTx is comparable to that of patients who were not on ECMO support. ECMO in nonintubated patients as a bridge to lung transplant: our experience Mean blood gas values before ECMO support was started were: pH 7.26 ± 0.13, PaCO2 81.7 ± 31.6, PaO2 151.4 ± 164.2, PEEP 9 ± 4, FiO2 83 ± 20, mean time on ECMO before LTx 11.7 ± 17.7 days. p g p Conclusion ECLS in a multimodal approach in MT represents a challenging opportunity to face potential fatal complications. Moreover ECLS applied to selected trauma patients could expand the potential donor pool. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S36 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P99 Two years’ experience with bicaval dual lumen cannula for venovenous extracorporeal membrane oxygenation in adult refractory acute respiratory distress syndrome J Reeb, PE Falcoz, J Pottecher, X Delabranche, N Santelmo, A Steib, M Hasselmann, G Massard Hôpitaux universitaires de Strasbourg, France Critical Care 2012, 16(Suppl 1):P99 (doi: 10.1186/cc10706) trauma in 10 patients, sepsis in three patients, pulmonary hemorrhage in two patients and others in two patients. The overall hospital mortality rate was 39% (15/38). Compared with the nonsurvivors group, the survivors group was younger (33.3 ± 15.1 vs. 52.2 ± 18.1 years old, P = 0.001) and had lower APACHE II scores (18.7 ± 6.3 vs. 26.7 ± 6.6, P = 0.001) and SOFA scores (10.4 ± 2.7 vs. 12.7 ± 2.4, P = 0.014). Furthermore, the survivors group had early signifi cant resolution in the sequential SOFA scores compared with the nonsurvivors group. trauma in 10 patients, sepsis in three patients, pulmonary hemorrhage in two patients and others in two patients. The overall hospital mortality rate was 39% (15/38). Compared with the nonsurvivors group, the survivors group was younger (33.3 ± 15.1 vs. 52.2 ± 18.1 years old, P = 0.001) and had lower APACHE II scores (18.7 ± 6.3 vs. 26.7 ± 6.6, P = 0.001) and SOFA scores (10.4 ± 2.7 vs. 12.7 ± 2.4, P = 0.014). Furthermore, the survivors group had early signifi cant resolution in the sequential SOFA scores compared with the nonsurvivors group. g Conclusion Survivors had early improvements in SOFA scores after ECMO for severe ARDS patients. SOFA score evolution may be used for evaluating the eff ect of ECMO. Introduction Venovenous extracorporeal membrane oxygenation (ECMO) is a respiratory support increasingly used in adult refractory acute respiratory distress syndrome (ARDS). Corticosteroids for critically ill patients: an international survey of intensivists F Lamontagne1, N Adhikari2, D Cook3, KK Koo4, F Lauzier5, KE Burns2, I Douglas6, AF Turgeon5, H Quiroz1, Y Poulin1, K Choong3, N Ferguson2, ME Kho7, M Duff ett3, C Chant2, O Lesur1, MO Meade3 1Université de Sherbrooke, Canada; 2University of Toronto, Canada; 3McMaster University, Hamilton, Canada; 4University of Western Ontario, London, Canada; 5Université Laval, Québec, Canada; 6Denver Health Medical Center, Denver, CO, USA; 7Johns Hopkins University, Baltimore, MD, USA Critical Care 2012, 16(Suppl 1):P101 (doi: 10.1186/cc10708) y Methods A prospective single-center study between November 2009 and November 2011 including all medical and surgical adult patients receiving ECMO for refractory ARDS. All ECMOs were performed with DLC implanted percutaneously in the right internal jugular vein. Variables under study were: arterial blood gases, duration of ECMO support, activated cephalin time (TCA) values, number of blood products transfused, and patient’s outcome. Statistical test: Student’s t test.i p Results Twenty-fi ve ECMOs were performed in 24 patients (16 men and eight women). Mean age of patients was 52.2 years ± 17.5. All these patients had severe ARDS despite optimal medical therapy. At DLC implantation, mean pH, PaCO2, PaO2, and PaO2/FiO2 ratio were 7.25 ± 0.11, 60.5 ± 17.5 mmHg, 58.9 ± 13.6 mmHg, and 61 ± 14 respectively. Mean duration of respiratory support with ECMO was 9.5 ± 4.8 days and mean blood fl ow was 3.3 ± 0.6 l/minute. During ECMO, arterial blood gases were signifi cantly improved (P <0.05): mean PaCO2, and PaO2 were 39.9 ± 4.8 mmHg, and 92.7 ± 21.1 mmHg respectively. Concerning haemostasis and provision of blood products, mean TCA and mean pellets of red blood cells transfused were 53.6 ± 1.2 seconds and 10.7 ± 7 respectively. Eleven patients (46%) died under ECMO. Causes of death with ECMO support were: fi ve multiorgan failures, two septic shocks, two withdrawal of care, one hemodynamic shock, and one refractory hypoxemia. ECMO withdrawal was possible in 13 patients (twice in one patient) with PaO2/FiO2 ratio 258 ± 24 at withdrawal. Removal of the endotracheal tube was performed for eight patients (33%), 18.3 ± 6.2 days after DLC implantation. Eight patients (33%) were discharged alive from the ICU, 18.3 ± 6.3 days after DLC implantation. These eight patients were discharged alive from hospital. No adverse event related to the DLC was observed. Introduction We surveyed intensivists to evaluate their stated use of systemic steroids in the ICU. Resolution of organ functional scores to predict outcomes in severe acute respiratory distress syndrome patients receiving extracorporeal membrane oxygenation L Chiu, Y Chen, H Hu, C Huang, F Tsai, K Kao Chang Gung Memorial Hospital, Taoyuan, Taiwan Critical Care 2012, 16(Suppl 1):P100 (doi: 10.1186/cc10707) y Conclusion Certain clinical conditions may prompt intensivists to almost always prescribe systemic steroids and reduce equipoise for future placebo-controlled trials. Moreover, this survey shows that in selected academic centres a majority of intensivists do not prescribe corticosteroids for pneumonia, ALI and ARDS. Introduction Extracorporeal membrane oxygenation (ECMO) may be used as an alternative therapy for severe acute respiratory distress syndrome (ARDS) patients who have failed conventional mechanical ventilation. We undertook a study to investigate the determinants of mortality and the sequential evolution of organ failures in ECMO-treated ARDS patients. Methods This was a prospective observational study of severe ARDS patients who received venovenous ECMO in the ICU of Chang Gung Memorial Hospital between March 2006 and December 2010. We included data on all 38 consecutive patients who receive venovenous ECMO. Retrospective data included the following: demographics, primary diagnosis for ARDS, ventilator setting before ECMO, oxygenation, durations of ECMO, SOFA scores and outcome. l l f l bl Introduction Extracorporeal membrane oxygenation (ECMO) may be used as an alternative therapy for severe acute respiratory distress syndrome (ARDS) patients who have failed conventional mechanical ventilation. We undertook a study to investigate the determinants of mortality and the sequential evolution of organ failures in ECMO-treated ARDS patients. P102f P102 Eff ects of salbutamol on airway characteristics in mechanically ventilated adults without COPD J Van Rosmalen, QL Habes, I Havinga, F Van Beers, A Van Hees, D Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P102 (doi: 10.1186/cc10709) ECMO in nonintubated patients as a bridge to lung transplant: our experience Technological advances such as bicaval dual lumen cannula (DLC) allow one to decrease drawbacks associated with this cardiopulmonary bypass technique and to implement it in the ICU setting. We report our 2 years’ experience of using DLC for ECMO in adult refractory ARDS. Corticosteroids for critically ill patients: an international survey of intensivists The effi cacy of steroids in septic shock and ARDS remains uncertain and clinicians’ perceptions of competing indications and contraindications may jeopardize future randomized controlled trials (RCT). Knowledge of current practice will inform the design of future RCTs addressing the effi cacy of systemic steroids in septic shock and ARDS. Introduction We surveyed intensivists to evaluate their stated use of systemic steroids in the ICU. The effi cacy of steroids in septic shock and ARDS remains uncertain and clinicians’ perceptions of competing indications and contraindications may jeopardize future randomized controlled trials (RCT). Knowledge of current practice will inform the design of future RCTs addressing the effi cacy of systemic steroids in septic shock and ARDS. Methods We designed and conducted a self-administered survey of intensivists practicing in academic settings with expertise in ARDS clinical research. We generated questionnaire items in focus group sessions with content experts and refi ned them through a standardized process of clinical sensibility, pilot and intra-rater reliability testing. Respondents used a four-point scale to grade how frequently they would administer systemic steroids in a 14 diff erent clinical situations and reported their opinions of 16 near absolute indications or contraindications for systemic steroids. Local research staff distributed the survey to all intensivists practicing in the 11 centres (Canada and USA) with most patients enrolled in the OSCILLATE trial. Results In total, 103 of 125 potential respondents returned completed surveys (response rate 82%). A majority of respondents ‘almost always’ prescribe systemic steroids in the setting of recent systemic steroid use and low blood pressure (93%), signifi cant bronchospasm in a mechanically ventilated patient (93%) and vasopressor refractory septic shock (52%). A majority of respondents would ‘almost never’ prescribe steroids in severe community-acquired pneumonia (81%), ALI (76%) and ARDS (65%). One-half (50%) would ‘almost never’ prescribe steroids for severe ARDS (50%). The near absolute indications selected by a majority of respondents were ‘known adrenal insuffi ciency’ (99%) and ‘suspicion of cryptogenic organizing pneumonia’ (89%). The only near absolute contraindication selected by a majority of respondents was ‘systemic fungal infection’ (52%). Conclusion ECMO with a bicaval DLC is feasible in the ICU. It improves signifi cantly haemostasis parameters in patients suff ering from refractory ARDS. DLC also decreases drawbacks associated with the ECMO respiratory support. P100 Resolution of organ functional scores to predict outcomes in severe acute respiratory distress syndrome patients receiving extracorporeal membrane oxygenation L Chiu, Y Chen, H Hu, C Huang, F Tsai, K Kao Chang Gung Memorial Hospital, Taoyuan, Taiwan Critical Care 2012, 16(Suppl 1):P100 (doi: 10.1186/cc10707) P103 Respiratory system elastance monitoring during PEEP titration h 1 h 1 h 2 3 p p Methods In this prospective study, we enrolled 11 critically ill MV patients without COPD. These intubated patients were on volume- controlled ventilation (6 ml/kg/PBW). Exclusion criteria were the use of β-blockers, propofol or neuromuscular blockers. They received fi ve puff s of salbutamol (100 μg/puff ) delivered by metered dose inhaler via the adapter on the Y-piece. Ventilator settings and body position were unchanged during the study. Before and after salbutamol administration vital signs were recorded and lung mechanics were measured using the ventilator (Servo-i® or Hamilton-G5®) at –1, +1, +15, +30, +60, +90 and +240 minutes. Values after administration of salbutamol (T0) were compared to those before administration. Results are presented as mean ± SD. Data were evaluated by paired t test and P <0.05 was taken as statistically signifi cant. Introduction PEEP selection during mechanical ventilation (MV) for patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remains a challenge for clinicians. Clinicians often rely on experience and intuition in setting MV, resulting in a more variable treatment and outcome. We hypothesise that monitoring patient-specifi c respiratory system elastance (Ers) during PEEP change may provide an insight into the patient’s condition. y p g p Methods Thirteen patients with ALI/ARDS underwent a step-wise PEEP increase (5 cmH2O) recruitment manoeuvre (RM) until peak airway pressure reaches 45 cmH2O. Airway pressure and fl ow profi le were recorded using a pneumotachometer. The change of patient’s respiratory system elastance (Ers = 1 / compliance) and the end of expiratory lung volume (EELV) during RM were estimated and studied. The trials were approved by New Zealand South Island Regional Ethics Committee. y gi Results The study group consisted of seven men and four women, mean age 53 years. Underlying causes for ventilation were diverse. The median time spent on the ventilator before inclusion was 36 hours (6 to 151). After salbutamol administration inspiratory resistance and dynamic compliance decreased, but not signifi cantly. Expiratory resistance, dynamic compliance, elastance, SpO2 and EtCO2 did not change (Table 1).if g Conclusion There was no signifi cant eff ect of salbutamol inhalation on airway characteristics and vital signs in non-COPD patients on MV. Therefore standard salbutamol inhalation in MV patients without COPD can be aborted. Results The median (IQR) Ers over all patients was 34.0 cmH2O/l (26.1 to 51.0), refl ecting the heterogeneity of the patients and their response to PEEP. P103 Respiratory system elastance monitoring during PEEP titration h 1 h 1 h 2 3 This outcome supports the idea that MV/ PEEP should be individualised. During RM, patients’ Ers decreased with PEEP increase until a specifi c minimum and increase at higher PEEP. The decreased of Ers suggest alveolar recruitment whereas an increase of Ers at higher PEEP shows potential overinfl ation. An example is shown in Figure 1a. A clear infl ection/minimum Ers can be found in Figure 1a, indicating a Eff ects of salbutamol on airway characteristics in mechanically ventilated adults without COPD q g p Methods This was a prospective observational study of severe ARDS patients who received venovenous ECMO in the ICU of Chang Gung Memorial Hospital between March 2006 and December 2010. We included data on all 38 consecutive patients who receive venovenous ECMO. Retrospective data included the following: demographics, primary diagnosis for ARDS, ventilator setting before ECMO, oxygenation, durations of ECMO, SOFA scores and outcome. J Van Rosmalen, QL Habes, I Havinga, F Van Beers, A Van Hees, D Ramnarain, JA Van Oers St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P102 (doi: 10.1186/cc10709) Introduction In our ICU, salbutamol inhalation to prevent broncho- spasm is standard care in mechanically ventilated (MV) patients. In MV patients without COPD the eff ect of salbutamol remains unclear. Introduction In our ICU, salbutamol inhalation to prevent broncho- spasm is standard care in mechanically ventilated (MV) patients. In MV patients without COPD the eff ect of salbutamol remains unclear. Results A total of 38 severe ARDS patients receiving ECMO were eligible. The causes of ARDS in these 38 patients were pneumonia in 21 patients, S37 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Therefore we examined the eff ect of inhaled salbutamol on resistance and compliance in MV patients without COPD. Methods In this prospective study, we enrolled 11 critically ill MV patients without COPD. These intubated patients were on volume- controlled ventilation (6 ml/kg/PBW). Exclusion criteria were the use of β-blockers, propofol or neuromuscular blockers. They received fi ve puff s of salbutamol (100 μg/puff ) delivered by metered dose inhaler via the adapter on the Y-piece. Ventilator settings and body position were unchanged during the study. Before and after salbutamol administration vital signs were recorded and lung mechanics were measured using the ventilator (Servo-i® or Hamilton-G5®) at –1, +1, +15, +30, +60, +90 and +240 minutes. Values after administration of salbutamol (T0) were compared to those before administration. Results are presented as mean ± SD. Data were evaluated by paired t test and P <0.05 was taken as statistically signifi cant. Results The study group consisted of seven men and four women, mean age 53 years. Underlying causes for ventilation were diverse. The median time spent on the ventilator before inclusion was 36 hours (6 to 151). After salbutamol administration inspiratory resistance and dynamic compliance decreased, but not signifi cantly. Eff ects of salbutamol on airway characteristics in mechanically ventilated adults without COPD Expiratory resistance, dynamic compliance, elastance, SpO2 and EtCO2 did not change (Table 1). Conclusion There was no signifi cant eff ect of salbutamol inhalation on airway characteristics and vital signs in non-COPD patients on MV. Therefore standard salbutamol inhalation in MV patients without COPD can be aborted. Reference 1. Malliotakis P, et al.: Infl uence of respiratory eff orts on β2-agonist induced bronchodilation in mechanically ventilated COPD patients: a prospective clinical study. Respir Med 2007, 101:300-307. P103 Respiratory system elastance monitoring during PEEP titration YS Chiew1, JG Chase1, GM Shaw2, T Desaive3 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3University of Liege, Belgium Critical Care 2012, 16(Suppl 1):P103 (doi: 10.1186/cc10710) Introduction PEEP selection during mechanical ventilation (MV) for patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remains a challenge for clinicians. Clinicians often rely on experience and intuition in setting MV, resulting in a more variable treatment and outcome. We hypothesise that monitoring patient-specifi c respiratory system elastance (Ers) during PEEP change may provide an insight into the patient’s condition. Methods Thirteen patients with ALI/ARDS underwent a step-wise PEEP increase (5 cmH2O) recruitment manoeuvre (RM) until peak airway pressure reaches 45 cmH2O. Airway pressure and fl ow profi le were recorded using a pneumotachometer. The change of patient’s respiratory system elastance (Ers = 1 / compliance) and the end of expiratory lung volume (EELV) during RM were estimated and studied. The trials were approved by New Zealand South Island Regional Ethics Committee. Results The median (IQR) Ers over all patients was 34.0 cmH2O/l (26.1 to 51.0), refl ecting the heterogeneity of the patients and their response to PEEP. This outcome supports the idea that MV/ PEEP should be individualised. During RM, patients’ Ers decreased with PEEP increase until a specifi c minimum and increase at higher PEEP. The decreased of Ers suggest alveolar recruitment whereas an increase of Ers at higher PEEP shows potential overinfl ation. An example is shown in Figure 1a. Eff ects of salbutamol on airway characteristics in mechanically ventilated adults without COPD A clear infl ection/minimum Ers can be found in Figure 1a, indicating a Table 1 (abstract P102) T–1 T0 T+1 T+15 T+30 T+60 T+90 T+240 Rins (cm H2O/l/second) 14 (6) Salbutamol 14 (4) 13 (5) 13 (5) 12 (4) 12 (5) 12 (4) NS administration Rexp (cm H2O/l/second) 17 (7) 17 (6) 16 (6) 17 (8) 18 (10) 18 (8) 17 (7) NS Cdyn (ml/cm H2O) 44 (12) 42 (11) 42 (11) 37 (10) 36 (9) 38 (9) 38 (8) NS Cstat (ml/cm H2O) 49 (13) 49 (12) 47 (13) 43 (11) 44 (12) 45 (12) 43 (12) NS Elastance (cm H2O/l) 19 (3) 20 (4) 21 (4) 22 (4) 23 (4) 21 (4) 18 (8) NS Heart rate (beats/minute) 81 (22) 84 (24) 85 (23) 87 (22) 85 (22) 82 (18) 84 (20) NS SpO2 (%) 98 (1) 98 (2) 98 (2) 98 (1) 98 (2) 98 (2) 97 (2) NS EtCO2 (kPa) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.8) 4.7 (0.7) NS Figure 1 (abstract P103). Ers and EELV change with PEEP increase. Table 1 (abstract P102) T–1 T0 T+1 T+15 T+30 T+60 T+90 T+240 Rins (cm H2O/l/second) 14 (6) Salbutamol 14 (4) 13 (5) 13 (5) 12 (4) 12 (5) 12 (4) NS administration Rexp (cm H2O/l/second) 17 (7) 17 (6) 16 (6) 17 (8) 18 (10) 18 (8) 17 (7) NS Cdyn (ml/cm H2O) 44 (12) 42 (11) 42 (11) 37 (10) 36 (9) 38 (9) 38 (8) NS Cstat (ml/cm H2O) 49 (13) 49 (12) 47 (13) 43 (11) 44 (12) 45 (12) 43 (12) NS Elastance (cm H2O/l) 19 (3) 20 (4) 21 (4) 22 (4) 23 (4) 21 (4) 18 (8) NS Heart rate (beats/minute) 81 (22) 84 (24) 85 (23) 87 (22) 85 (22) 82 (18) 84 (20) NS SpO2 (%) 98 (1) 98 (2) 98 (2) 98 (1) 98 (2) 98 (2) 97 (2) NS EtCO2 (kPa) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.7) 4.7 (0.8) 4.7 (0.7) NS Therefore we examined the eff ect of inhaled salbutamol on resistance and compliance in MV patients without COPD. P103 Respiratory system elastance monitoring during PEEP titration YS Ch 1 JG Ch 1 GM Sh 2 T D 3 05 Flow-controlled expiration discloses PEEP-dependent dynamic hysteresis of the pressure–volume loop S Schumann1, L Vimlati2, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P105 (doi: 10.1186/cc10712) The relative hysteresis area was calculated as the quotient of hysteresis area divided by the rectangular area which is limited by the minima and maxima of pressure and volume of the respective pressure–volume loop [2].i P104 P104 Accuracy of the pressure–volume curve method compared to quantitative lung CT scan to assess the recruitable lung in patients with acute respiratory failure D Chiumello1, A Marino2, I Cigada2, F Menga2, M Brioni2, IR Piva2 1Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Università degli Studi di Milano, Milan, Italy Critical Care 2012, 16(Suppl 1):P104 (doi: 10.1186/cc10711) Introduction In patients with acute lung injury the knowledge of recruitable lung is useful for a physiological PEEP setting. The quantitative lung CT scan analysis remains the reference method [1]. However, it is time consuming and often it is not applicable in clinical management. The PV curve at two PEEP levels has been proposed as an alternative method [2]. The aim of this study was to evaluate the accuracy of these two methods in predicting the lung recruitability. Methods In fi ve Swedish Landrace Hybrid pigs weighing 26 ± 2 kg the lungs were ventilated in the volume-controlled mode. PEEP was set to 0, 6, 12 and 15 cmH2O. The fl ow-controlled expiration was realized by a computer-controlled expiratory resistance which was adjusted in a fashion that expiratory fl ow was strongly limited in the beginning and continuously facilitated towards the end of expiration. Using the gliding-SLICE method [1], intratidal inspiratory and expiratory compliance profi les were calculated from inspiration data only and from expiration data only, respectively. The dynamic hysteresis area was calculated as the area within the dynamic tracheal pressure– volume loop. The relative hysteresis area was calculated as the quotient of hysteresis area divided by the rectangular area which is limited by the minima and maxima of pressure and volume of the respective pressure–volume loop [2].i Methods Sedated and paralyzed patients underwent a PV curve using the low-fl ow method and whole-lung CT scan at 5 and 15 cmH2O of PEEP. The lung recruitability was defi ned as the decrease in the not aerated tissue by the quantitative lung CT analysis and as the diff erence between the lung volume computed on the two PV curves for an airway pressure of 20 cmH2O. 05 Flow-controlled expiration discloses PEEP-dependent dynamic hysteresis of the pressure–volume loop S Schumann1, L Vimlati2, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P105 (doi: 10.1186/cc10712) Critical Care 2012, 16(Suppl 1):P105 (doi: 10.1186/cc10712) Introduction Hysteresis of the pressure–volume loop is a measure for the additional energy that is required during inspiration to recruit and infl ate additional alveoli. The hysteresis area is usually constructed using data from a low-fl ow infl ation/defl ation maneuver; that is, from a quasi-static situation that the lung never sees during ongoing ventilation. However, during the dynamic conditions of mechanical ventilation the hysteresis area is biased by resistive pressure portions. Therefore we uncoupled fl ow and volume by linearizing expiratory fl ow (fl ow-controlled expiration). This enabled calculation of compliance separately for inspiration and expiration. We hypothesized that the volume-dependent intratidal compliance profi les diff er between inspiration and expiration, describing a dynamic hysteresis behavior.i Introduction Hysteresis of the pressure–volume loop is a measure for the additional energy that is required during inspiration to recruit and infl ate additional alveoli. The hysteresis area is usually constructed using data from a low-fl ow infl ation/defl ation maneuver; that is, from a quasi-static situation that the lung never sees during ongoing ventilation. However, during the dynamic conditions of mechanical ventilation the hysteresis area is biased by resistive pressure portions. Therefore we uncoupled fl ow and volume by linearizing expiratory fl ow (fl ow-controlled expiration). This enabled calculation of compliance separately for inspiration and expiration. We hypothesized that the volume-dependent intratidal compliance profi les diff er between inspiration and expiration, describing a dynamic hysteresis behavior. Methods In fi ve Swedish Landrace Hybrid pigs weighing 26 ± 2 kg the lungs were ventilated in the volume-controlled mode. PEEP was set to 0, 6, 12 and 15 cmH2O. The fl ow-controlled expiration was realized by a computer-controlled expiratory resistance which was adjusted in a fashion that expiratory fl ow was strongly limited in the beginning and continuously facilitated towards the end of expiration. Using the gliding-SLICE method [1], intratidal inspiratory and expiratory compliance profi les were calculated from inspiration data only and from expiration data only, respectively. The dynamic hysteresis area was calculated as the area within the dynamic tracheal pressure– volume loop. References 1. Gattinoni L, et al.: Lung recruitment in patients with the acute respiratory distress syndrome. N Engl J Med 2006, 354:1775-1786. 1. Gattinoni L, et al.: Lung recruitment in patients with the acute respiratory distress syndrome. N Engl J Med 2006, 354:1775-1786. i g p References 2. Ranieri VM, et al.: Volume–pressure curve of the respiratory system predicts eff ects of PEEP in ARDS: ‘occlusion’ versus ‘constant fl ow’ technique. Am J Respir Crit Care Med 1994, 149:19-27. 2. Ranieri VM, et al.: Volume–pressure curve of the respiratory system predicts eff ects of PEEP in ARDS: ‘occlusion’ versus ‘constant fl ow’ technique. Am J Respir Crit Care Med 1994, 149:19-27. 1. Schumann et al.: Anesthesiology 2011, 114:1111-1117. 2. Bachofen H, Hildebrandt J: J Appl Physiol 1971, 30:493-497. 1. Schumann et al.: Anesthesiology 2011, 114:1111-1117. 2. Bachofen H, Hildebrandt J: J Appl Physiol 1971, 30:493-497. 1. Schumann et al.: Anesthesiology 2011, 114:1111-1117. 2. Bachofen H, Hildebrandt J: J Appl Physiol 1971, 30:493-497. Figure 1 (abstract P104). Linear regression between quantitative CT scan analysis and PV curve method. 05 Flow-controlled expiration discloses PEEP-dependent dynamic hysteresis of the pressure–volume loop S Schumann1, L Vimlati2, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P105 (doi: 10.1186/cc10712) 2 Results Ten patients (mean age 65.4 ± 10.4 years, body mass index 24.0 ± 6.8 kg/m2, PaO2/FiO2 181 ± 37) were enrolled. The mean recruitable lung was 3.9 ± 6.3% of the total lung weight and 218 ± 266 ml for the quantitative lung CT scan and PV curve. The linear regression between the two methods (Figure 1) was not signifi cant (P = 0.338 and R2 = 0.115). Results Intratidal compliance profi les of inspiration and expiration diff ered strongly in mean value and slope at low PEEP. With increasing PEEP the inspiratory compliance profi le approximated closer to the expiratory compliance profi le. This was accompanied by a decreased relative hysteresis area by 26%. Conclusion The recruitable lung computed as the diff erence in not aerated tissue was not related to the diff erence in volume estimated by the PV curve. The role of the PV curve to estimate the lung recruitability remains to be elucidated. R f y y Conclusion Flow-controlled expiration allows for calculation of respira- tory system compliance separately for inspiration and expiration. This compliance displays the hysteresis behavior of the respiratory system during uninterrupted ventilation. Such analysis, which is similar to the time-honored quasi-static hysteresis area analysis, could be helpful for fi nding an optimal PEEP. References P105 potential method to optimise PEEP selection for a particular patient. Figure 1b shows the change of patient’s EELV with PEEP increase. As PEEP increases, the potentially recruitable collapsed lung decreases. Conclusion The change of patient-specifi c Ers and EELV during minimally invasive PEEP titration provides an insight into the patient’s lung condition, and thus could potentially be used as a method to individualise MV treatment and, in particular, PEEP selection. P105 Flow-controlled expiration discloses PEEP-dependent dynamic hysteresis of the pressure–volume loop S Schumann1, L Vimlati2, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P105 (doi: 10.1186/cc10712) Reference 1. Malliotakis P, et al.: Infl uence of respiratory eff orts on β2-agonist induced bronchodilation in mechanically ventilated COPD patients: a prospective clinical study. Respir Med 2007, 101:300-307. Figure 1 (abstract P103). Ers and EELV change with PEEP increase. Figure 1 (abstract P103). Ers and EELV change with PEEP increase. S38 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 potential method to optimise PEEP selection for a particular patient. Figure 1b shows the change of patient’s EELV with PEEP increase. As PEEP increases, the potentially recruitable collapsed lung decreases. Conclusion The change of patient-specifi c Ers and EELV during minimally invasive PEEP titration provides an insight into the patient’s lung condition, and thus could potentially be used as a method to individualise MV treatment and, in particular, PEEP selection. P108 Hemodynamics eff ects of recruitment maneuver k d k h Hemodynamics eff ects of recruitment maneuver D Protsenko, R Magomedov, O Ignatenko, AI Yaroshetskiy, B Gelfand Russian National Research Medical University, Moscow, Russia Critical Care 2012, 16(Suppl 1):P108 (doi: 10.1186/cc10715) Introduction Acute respiratory distress syndrome (ARDS) is a frequent complication in critically ill patients. Recruitment maneuver (RM) is a rescue procedure which improves oxygenation [1-3]. However, it is not clear whether improving oxygen delivery (DO2) exists after RM. The aim of this study was to evaluate the eff ects of RM on hemodynamics and DO2. Conclusion APRV settings and the protocol for sedation used in this trial allowed to reach suffi cient spontaneous breathing for the majority of the patients without any major complication and with a tidal volume below 8 ml/kg PBW. For a few patients, spontaneous breathing seems to be hard to obtain, especially within the fi rst 2 days. R f 2 Methods A prospective, randomized trial in ARDS patients (AECC criteria). The protocol was approved by the local ethics committee. Fifty-seven patients with extrapulmonary ARDS were randomized into three groups: group A (n = 17) – 40×40 RM (CPAP 40 cmH2O for 40 seconds), group B (n = 17) – PCV RM (PIP 40 to 50 cmH2O, PEEP 18 to 20 cmH2O for 120 seconds), and group C (n = 17) – stepwise PCV RM. Gas exchange and systemic hemodynamics by aortal blood fl ow (transesophageal Doppler; ARROW, USA) were measured before, after, 30 and 120 minutes after RM. 1. Mercat A, Richard JC, Vielle B, et al.; Expiratory Pressure (Express) Study Group: Positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial. JAMA 2008, 299:646-655. 1. Mercat A, Richard JC, Vielle B, et al.; Expiratory Pressure (Express) Study Group: Positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial. JAMA 2008, 299:646-655. Results In all groups we observed rapid increasing of paO2 (mmHg) from 65.9 ± 24.9; 77.2 ± 14.0; 87.0 ± 16.7 to 110.3 ± 38.7; 124.5 ± 45.5; 115.2 ± 32.6 (P <0.0001) after RM. We also observed signifi cant improvement of oxygenation 120 minutes after RM (95.6 ± 25.6; 99.3 ± 25.3; 108.1 ± 26.8). There was no statistical diff erence between groups. A device for ventilation-analogue mechanostimulation in vitro K Gamerdinger, M Schneider, E Smudde, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2012, 16(Suppl 1):P107 (doi: 10.1186/cc10714) y g, y Critical Care 2012, 16(Suppl 1):P107 (doi: 10.1186/cc10714) Introduction The mechanical stress–strain characteristics of most living tissues is nonlinear and frequency dependent. During spontaneous breathing the mechanical strain on the pulmonary tissue is akin to a sinusoidal profi le. In contrast, during mechanical ventilation the stimulation profi le of the lung tissue diff ers considerably from a sinusoidal pattern. While all in vitro experiments aiming at dynamic stimulation typically use sinusoidal patterns, we here describe the establishment of a new device aff ording a ventilation-analogue stimulation pattern, allowing a better imitation of the situation in vivo. The new device includes a linear motor connected to four piston pumps and it allows the identical stimulation of four probes at the same time. Here we show how we stressed four test samples with sinusoidal, rectangular and ventilation-analogue mechanostimulation and how we analyzed them for frequency contents by means of a fast- Fourier transform. p g p g Conclusion Three diff erent RMs increase oxygenation and decrease CO equally. But RM does not improve oxygen delivery due to decreasing CO. Reference 1. Schumann S, et al.: J Biomed Mater Res B Appl Biomater 2008, 86:483-492. Results At inclusion, baseline characteristics of the patients were the following: fi ve men and fi ve women, 59 years old on average (25 to 85), SAPS II score of 42 (20 to 71), PO2/FiO2 ratio of 107 (74 to 175) and respiratory system compliance (Cst,rs) of 25 ml/cmH2O (18 to 36). We did not observe any pneumothorax. Eight of patients had RASS ≥–4 during the 5 days of enrollment. From day 2, the level of spontaneous breathing ranged between 10 and 50% of total minute ventilation in eight patients. The tidal volume (spontaneous and mechanical) measured for 5 days for each patient was mainly distributed around 6 ml/kg PBW (Figure 1). The mean PO2/FiO2 ratio increased from 107 ± 29 at enrollment to 173 ± 91 at day 5 (P <0.05). Feasibility of early spontaneous breathing in acute respirator distress syndrome S Mortaza, A Mercat CHU Angers, France Critical Care 2012, 16(Suppl 1):P106 (doi: 10.1186/cc10713) Tidal volume distribution (ml/kg PBW) for 5 days for each patient. i Conclusion With our new mechanostimulation system we are able to confi gure the frequency content of the applied strain profi le and furthermore to identify the frequency content of the resulting stress on the tissue. of 6 ml/kg of predicted body weight (PBW), T high between 0.8 and 1 second, T low set to reach the same respiratory rate as in VAC. Patients were initially paralyzed. Then sedation was adapted to keep RASS ≥–4 and a level of spontaneous breathing between 10 and 50% of total minute ventilation. A computer was continuously connected to the ventilator for 5 days in order to record respiratory variables. 1. Marini JJ, Amato MB: Lung recruitment during ARDS. In Acute Lung Injury. Edited by Marini JJ, Evans TW. Berlin: Springer; 1998:236-257. 2. Odenstedt H, Lindgren S, et al.: Slow moderate pressure recruitment maneuver minimizes negative circulatory and lung mechanic side eff ects: evaluation of recruitment maneuvers using electric impedance tomography. Intensive Care Med 2005, 31:1706-1714. 3. Medoff BD, Harris RS, Kesselman H, et al.: Use of recruitment maneuvers and high-positive end-expiratory pressure in a patient with acute respiratory distress syndrome. Crit Care Med 2000, 28:1210-1216. P108 Contrarily, DO2 (ml/minute/m2) after RM statistically signifi cantly decreased from 709.5 ± 297.5; 804.9 ± 217.3; 811.7 ± 638.3 to 569.8 ± 211.9; 675.5 ± 244.7; 661.7 ± 421.3 (P = 0.053) and lasted more than 2 hours. The reason for this alteration was decreasing of cardiac output (CO) from 5.3 ± 2.5 l/minute to 3.6 ± 1.7 l/minute (P <0.0001) after RM. We hypothesized that the main reason for decreasing CO is rapid increasing of intrathoracic pressure during RM. Feasibility of early spontaneous breathing in acute respirator distress syndrome S Mortaza, A Mercat CHU Angers, France Critical Care 2012, 16(Suppl 1):P106 (doi: 10.1186/cc10713) S Mortaza, A Mercat Introduction Airway pressure release ventilation (APRV) is as a pressure preset mode that allows unrestricted spontaneous breathing throughout the entire ventilator cycle. In ARDS, this mode may decrease the need for sedation, increase alveolar recruitment and improve hemodynamic tolerance. The aim of this study is to assess the feasibility of a protocol combining APRV settings and sedation adaptation in order to obtain levels of spontaneous breathing between 10 and 50% of total minute ventilation. Methods We designed a monocentric study including 10 patients with early ARDS. We used a Dräger Evita XL ventilator. We initially used the volume-assist control (VAC) mode to set the PEEP, tidal volume and respiratory rate according to the increased recruitment strategy of the ExPress trial [1]. These settings were used to adjust the ventilator’s parameters in APRV mode: low pressure at the same level as the PEEP applied in VAC, high pressure (<32 cmH2O) set to reach a tidal volume Figure 1 (abstract P104). Linear regression between quantitative CT scan analysis and PV curve method. Figure 1 (abstract P104). Linear regression between quantitative CT scan analysis and PV curve method. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http //ccforum com/supplements/16/S1 S39 http://ccforum.com/supplements/16/S1 repetition rates ranging from 15/minute to 2,000/minute at amplitude volumes of 0.5 up to 2.8 ml, corresponding to a surface increase of 5% up to 100%, were used. The system was driven with sinusoidal, rectangular and ventilation-analogue profi les simulating the ventilatory pattern which is associated with the volume-controlled ventilation. Figure 1 (abstract P106). Tidal volume distribution (ml/kg PBW) for 5 days for each patient. Results The drive system allowed us to vary the amplitude from 0 up to 100% surface increase. At amplitudes of 0.5 and 1.0 ml we were able to apply a frequency range from 0 up to 2,000/minute, and at an amplitude of 2.0 ml a frequency range from 0 up to 800 sinusoidal defl ections per minute. We were able to apply the rectangular and the ventilation- analogue volume patterns to the probes. Close inspection of the pressure curves revealed that rapid volume increases were followed by peaks with subsequent relaxation decays when rectangular or ventilation-analogue stimulation patterns were applied. The frequency spectra of the pressure variation revealed side frequencies of up to 10 Hz for the rectangular mechanostimulation profi le. Figure 1 (abstract P106). P107 A device for ventilation-analogue mechanostimulation in vitro K Gamerdinger, M Schneider, E Smudde, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2012, 16(Suppl 1):P107 (doi: 10.1186/cc10714) References 1. Marini JJ, Amato MB: Lung recruitment during ARDS. In Acute Lung Injury. Edited by Marini JJ, Evans TW. Berlin: Springer; 1998:236-257. 2. Odenstedt H, Lindgren S, et al.: Slow moderate pressure recruitment maneuver minimizes negative circulatory and lung mechanic side eff ects: evaluation of recruitment maneuvers using electric impedance tomography. Intensive Care Med 2005, 31:1706-1714. g p y 3. Medoff BD, Harris RS, Kesselman H, et al.: Use of recruitment maneuvers and high-positive end-expiratory pressure in a patient with acute respiratory distress syndrome. Crit Care Med 2000, 28:1210-1216. Methods Silicone membranes serving as substitutes for biological tissue samples were placed inside a bioreactor [1] either in single-membrane or in double-membrane confi guration. Cyclic mechanostimulation at S40 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P109f Results The study comprised 20 patients with mean age of 58.9 ± 20.69 years, 11 men versus nine women (P >0.05). NT-proBNP was negatively correlated with PH on day 2 (P = 0.008, r = –0.53) and day 7 with (P = 0.02, r = –0.50). NT-proBNP was positively correlated with PEEP on day 2 (P = 0.05, r = 0.46) and day 7 (P = 0.035, r = 0.48). NT-proBNP was negatively correlated with the PaO2/FiO2 ratio on day 7 (P = 0.0035, r = 0.60). However, there was no signifi cant correlation between NT- proBNP and other respiratory indices including PaCO2, HCO3, PaO2, SaO2, FiO2, PAO2, P(A-a)O2 and a/A ratio (P >0.05). Neither troponin I nor troponin T showed any signifi cant correlation with any respiratory indices PH, PEEP, PaO2/FiO2, PaCO2, HCO3, PaO2, SaO2, FiO2, PAO2, P(A-a) O2 and a/A ratio on any day (P >0.05). None of the cardiac markers NT- proBNP, troponin I or troponin T showed any signifi cant correlation with the lung mechanics parameters Cdyn, Raw, Ceff , PIP, Pplat, and Pmean (P >0.05). Conclusion High NT-proBNP level was correlated with high PEEP, low PH and low PaO2/FiO2 ratio while troponin T and troponin I did not show signifi cant correlations with respiratory parameters in ARDS patients with structurally normal hearts. Early application of high-frequency oscillatory ventilation in H1N1 infl uenza-related severe ARDS is associated with better outcome S Jog, M Patel, D Patel Deenanath Mangeshkar Hospital and Research Centre, Pune, India Critical Care 2012, 16(Suppl 1):P111 (doi: 10.1186/cc10718) Results The groups were equal before and at the end of laparotomy. During the endotoxin infusion, PaO2/FiO2 was higher in groups I and II than in group III, whereas in pulmonary compliance or functional residual capacity no diff erences were found. In contrast, group I showed greater negative changes than group III in the circulatory variables; that is, arterial blood pressure, cardiac index, oxygen delivery and oxygen consumption. In all measured variables, group II showed an intermediate response to groups I and III, but no signifi cant diff erences were found between groups I and II. Groups I and II had slightly higher mean airway pressure at the end of the experiment than group III. However, this does not explain the circulatory diff erences since they occurred early in the course, temporally diff erent from the continuous slow increase of the airway pressures (P ≤0.01 ANOVA group by time interaction). Introduction High-frequency oscillatory ventilation (HFOV) is a promising rescue modality for refractory hypoxia and was used extensively in H1N1 infl uenza-related ARDS in 2009 and 2010. The aim of this study was to fi nd predictors of successful outcome of HFOV in H1N1 infl uenza-related severe ARDS [1].l l Methods Patients with H1N1 infl uenza-related severe ARDS by the new Berlin defi nition (applied retrospectively) receiving volume- controlled ventilation (VCV) as per the ARDSnet protocol with PO2/FiO2 ≤100 at PEEP ≥12 cmH2O and FiO2 ≥0.7 were connected to HFOV as a rescue therapy for refractory hypoxia. All patients were followed until discharge from the hospital (survivors) or death (nonsurvivors). Conclusion Low VT ventilation combined with higher PEEP in healthy animals exposed to laparotomy and subsequent experimental post- operative sepsis leads to a less prominent pulmonary dysfunction but to a more hypodynamic circulatory state compared to animals ventilated with a medium–high VT and lower PEEP. Reference Results About 80 parameters were evaluated as outcome predictors of HFOV like demographics, comorbidities, clinical features, laboratory parameters, X-rays, ventilatory and blood gas parameters and therapy- related complications. Previously collected data of 19 patients were analysed applying the new Berlin defi nition. Demographic, clinical, comorbidity, laboratory and radiological parameters were comparable in survivors and nonsurvivors. Table 1 shows comparison of survivors and nonsurvivors with respect mainly to ventilatory and gas exchange parameters before application of HFOV. References f P109 Diff erential pulmonary and circulatory eff ects of preventive lung protective ventilation in an experimental postoperative sepsis model J Sperber1, M Lipcsey2, A Larsson2, A Larsson2, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Eskilstuna, Sweden; 2Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P109 (doi: 10.1186/cc10716) Introduction It has been proposed that low tidal volume (VT) ventilation combined with higher PEEP should be used in patients with risk of developing postoperative lung injury instead of the commonly used VT of 10 ml/kg with lower PEEP [1]. Such a ventilatory mode would in theory reduce postoperative lung and organ dysfunction. However, this hypothesis has neither been tested clinically nor experimentally. Therefore we developed an experimental endotoxemic postoperative sepsis model to evaluate the eff ect of diff erent modes of ventilation. Methods Twenty-fi ve healthy pigs were randomized to three ventilation groups: I: PEEP 10 cmH2O, VT 6 ml/kg; II: PEEP 5 cmH2O, VT 10 ml/kg, changed to PEEP 10 cmH2O, VT 6 ml/kg at the end of laparotomy; III: PEEP 5 cmH2O, VT 10 ml/kg. For all groups the plateau pressure was kept below 28 cmH2O, normocapnia was reached by respiratory rate and FiO2 was adjusted to reach PaO2 >12 kPa. Laparotomy for 2 hours was performed to simulate a surgical procedure and then a continuous endotoxin infusion was started at 0.25 μg/kg/hour for 5 hours. Diff erences between groups were analyzed with ANOVA for repeated measures. Introduction It has been proposed that low tidal volume (VT) ventilation combined with higher PEEP should be used in patients with risk of developing postoperative lung injury instead of the commonly used VT of 10 ml/kg with lower PEEP [1]. Such a ventilatory mode would in theory reduce postoperative lung and organ dysfunction. However, this hypothesis has neither been tested clinically nor experimentally. Therefore we developed an experimental endotoxemic postoperative sepsis model to evaluate the eff ect of diff erent modes of ventilation.i Methods Twenty-fi ve healthy pigs were randomized to three ventilation groups: I: PEEP 10 cmH2O, VT 6 ml/kg; II: PEEP 5 cmH2O, VT 10 ml/kg, changed to PEEP 10 cmH2O, VT 6 ml/kg at the end of laparotomy; III: PEEP 5 cmH2O, VT 10 ml/kg. For all groups the plateau pressure was kept below 28 cmH2O, normocapnia was reached by respiratory rate and FiO2 was adjusted to reach PaO2 >12 kPa. Reference 1. Mitaka C, et al.: Increased plasma concentrations of BNP in patients with ALI. J Crit Care 1997, 12:66-71. P111 Early application of high-frequency oscillatory ventilation in H1N1 infl uenza-related severe ARDS is associated with better outcome S Jog, M Patel, D Patel Deenanath Mangeshkar Hospital and Research Centre, Pune, India Critical Care 2012, 16(Suppl 1):P111 (doi: 10.1186/cc10718) References Laparotomy for 2 hours was performed to simulate a surgical procedure and then a continuous endotoxin infusion was started at 0.25 μg/kg/hour for 5 hours. Diff erences between groups were analyzed with ANOVA for repeated measures. Reference 1. Mitaka C, et al.: Increased plasma concentrations of BNP in patients with ALI. J Crit Care 1997, 12:66-71. Early application of high-frequency oscillatory ventilation in H1N1 infl uenza-related severe ARDS is associated with better outcome S Jog, M Patel, D Patel Duration of conventional mechanical ventilation before HFOV, 1.4 ± 0.69 versus 3.66 ± 3.53 days (P = 0.03), was the only discriminating parameter between survivors and nonsurvivors. 1. Schultz et al.: Anesthesiology 2007, 106:1226-1231. 1. Schultz et al.: Anesthesiology 2007, 106:1226-1231. 1. Schultz et al.: Anesthesiology 2007, 106:1226-1231. P110 P110 High NT-proBNP level is correlated with high PEEP, low PH and low PaO2/FiO2 in ARDS Y Nassar, D Monsef, S Abdelshafy, G Hamed Cairo University, Giza, Egypt Critical Care 2012, 16(Suppl 1):P110 (doi: 10.1186/cc10717) Mechanical ventilation in intensive and critical care units of Russia: RuVent national epidemiologic study Results The gestational age more than 25 weeks was in 21 patients and between 20 and 25 weeks was in two patients. The mean age of these patients were 31 ± 5.7. The leading causes of ICU admission were obstetric emergency (26%), cardiovascular disease (26%) and infectious disease (26%) in these 23 patients. A total of 19 patients were respiratory failure and ARDS was diagnosed in nine of 19 patients. Of these nine ARDS patients, tidal volume (mean: 385 ± 31 ml), PaO2/FiO2 ratio (mean: 116 ± 47), positive end-expiratory pressure (PEEP) (mean: 13 ± 1.4 mmHg), peak airway pressure (mean: 34 ± 9.1 mmHg) and FiO2 (mean: 93 ± 7%). The intra-uterine fetal death ratio was 33% (3/9) and the Apgar score of the other six living births (6/9) was 7.8 ± 0.7. The hospital mortality rate of these ARDS patients was only 11% (1/9). p g y DN Protsenko1, AI Yaroshetskiy1, SG Suvorov2, AU Lekmanov2, BR Gelfand1 1Russian National Research Medical University, Moscow, Russia; 2Moscow Research Institute of Pediatrics and Child Surgery, Moscow, Russia Critical Care 2012, 16(Suppl 1):P114 (doi: 10.1186/cc10721) DN Protsenko1, AI Yaroshetskiy1, SG Suvorov2, AU Lekmanov2, BR Gelfand1 1Russian National Research Medical University, Moscow, Russia; 2Moscow Research Institute of Pediatrics and Child Surgery, Moscow, Russia Critical Care 2012, 16(Suppl 1):P114 (doi: 10.1186/cc10721) Introduction Experimental data have shown that mechanical ventilation can amplify or possibly trigger lung injury [1,2]. The biggest up-to-date clinical trial by the ARDS Network demonstrates reduction of mortality in ARDS patients with a protective lung strategy [3]. But we can see some gaps between international recommendations and real clinical practice [4,5]. y y Conclusion For pregnant ARDS patients, intensivists had a challenge for fetal and maternal life-threatening distress. In our study, early delivery combined with a lung-protective ventilation strategy may provide signifi cantly better fetal and maternal outcomes. References p Methods The multicenter clinical trial included 470 patients from 101 centers (ICUs) in Russia. Inclusion criteria were all patients without age restrictions ventilated for more than 12 hours for any reason from 14 to 18 February 2011. Recruitment of centers and data collection were made online. 1. Oram MP, et al.: Severe acute respiratory distress syndrome in pregnancy. Caesarean section in the second trimester to improve maternal 1. Oram MP, et al.: Severe acute respiratory distress syndrome in pregnancy. Caesarean section in the second trimester to improve maternal ventilation. P110 episodes increased by 8% and >15 day episodes decreased by 7% from 1999 to 2009. The overall reintubation rate was 13.8%. Less than 48-hour reintubation dropped from 14.4% in 1999 to 6.7 in 2009 while more than 48-hour reintubation remained similar (5.6% in 1999 and 6.2% in 2009). NIV use signifi cantly increased over this time – almost quadrupled (from 78 in 1999 to 315 in 2009). The most prominent increase was noted in the surgical and burn ICUs where, in 1999, NIV use was minimal (burn: 0, SICU: 6 and 16 and 131 in 2009). Medical and neuro ICUs doubled their use. A total 52.7% of NIV applications were associated with IV within 48 hours of NIV therapy. The sequence of IV–NIV revealed similar patterns through the years, overall, 58.4% of NIV application followed extubation, 24.2% preceded intubation and 17% was in between. P112 Outcomes of early delivery in pregnant patients with acute respiratory distress syndrome CY Hung, HC Hu, CH Chang, CC Huang, KC Kao Chang Gung Memorial Hospital, Taoyuan, Taiwan Critical Care 2012, 16(Suppl 1):P112 (doi: 10.1186/cc10719) References 1. Dreyfuss D, Saumon G: Am J Respir Crit Care Med 1998, 157:294-323. 2. Ignatenko O, Protsenko D, Yaroshetskiy A, Gelfand B: Crit Care 2010, 14(Suppl 1):P200. Methods Data were retrospectively collected using the Respiratory Care Services’ Database. The number of invasive and noninvasive MV services, their sequence if both were used for a given patient, the duration of the services, and reintubation episodes for years 1999 to 2009 were extracted. Four ICUs were included; surgical, medical, neuro and burn ICUs. If a patient was reintubated within 48 hours of extubation, the case was regarded as a single episode of MV and the duration was calculated accordingly. For NIV, if restarted within 48 hours, it was counted as a single episode as well.i 3. ARDS Network: N Engl J Med 2000, 342:1301-1 3. ARDS Network: N Engl J Med 2000, 342:1301-1308. 4 Esteban A et al : JAMA 2002 287:345 355 5. Esteban A, et al.: Am J Respir Crit Care Med 2008, 177:170-177. Mechanical ventilation demographics between 1999 and 2009 DS Sulemanji, E Burns, R Kacmarek Massachusetts General Hospital, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P113 (doi: 10.1186/cc10720) Mechanical ventilation demographi DS Sulemanji, E Burns, R Kacmarek DS Sulemanji, E Burns, R Kacmarek j Massachusetts General Hospital, Boston, MA, USA Conclusion Results of the RuVent study are comparable with international epidemiologic multicenter studies. Further investigations are needed for evaluation of the situation in ICUs which are a long distance from big medical centers. Critical Care 2012, 16(Suppl 1):P113 (doi: 10.1186/cc10720) Introduction Eff orts at many levels are being directed at decreasing the economic burden of mechanical ventilation (MV), its related complications and their consequences. Our aim was to determine the length of MV, reintubation rates and use of noninvasive ventilation (NIV), over a 10-year period. P112 Outcomes of early delivery in pregnant patients with acute respiratory distress syndrome p y y CY Hung, HC Hu, CH Chang, CC Huang, KC Kao Chang Gung Memorial Hospital, Taoyuan, Taiwan Critical Care 2012, 16(Suppl 1):P112 (doi: 10.1186/cc10719) Introduction Critical illnesses in pregnancy account for 0.11 to 0.89% of deliveries resulting in ICU admissions. The high rate of perinatal asphyxia in infants and high mortality rate in gravid patients supported a strategy of early delivery during the third trimester. The mortality rate of acute respiratory distress syndrome (ARDS) is high and varied from 15 to 72% among the studies. The present study reports the outcomes of early delivery within 48 hours after ICU admission in pregnant patients with ARDS. p Conclusion We found that the duration of MV decreased, reintubations within 48 hours decreased and the use of NIV increased over this 10- year period. The analysis of outcomes from our data has yet to be completed, but it would not be premature to speculate these results are related to the incorporation of SBT protocols and awaking trials, lesser use of neuromuscular blocking agents as well as extensive application of lung-protective ventilation strategies. p Methods A total of 23 pregnant patients with gestational age more than 20 weeks admitted to the ICU was recorded from January 2009 to November 2012. Emergent delivery was performed within 48 hours after ICU admission. The collected data included etiologies of ICU admission, patients’ characteristics and ventilator setting, infant and maternal clinical outcomes. P114 P110 Table 1 (abstract P111). Comparison of survivors and nonsurvivors Variable Survivors Nonsurvivors P value APACHE 13.3 ± 1.7 13.2 ± 2.2 0.14 Time VCV 1.4 ± 0.69 3.66 ± 3.5 0.03 PIP 35.6 ± 7.1 35.2 ± 5.1 0.44 PEEP 13.4 ± 2.0 13.4 ± 2.8 0.48 P/F 82.6 ± 31 68.8 ± 34 0.37 OI 36.08 ± 24 25.32 ± 7 0.1 Table 1 (abstract P111). Comparison of survivors and nonsurvivors Introduction Cardiac injury may occur in ARDS patients with structurally normal hearts and may be correlated with respiratory parameters [1]. We aimed at observing NT-proBNP, troponin I and troponin T relations with diff erent respiratory parameters in ARDS. f Methods Inclusion criteria were any adult patient diagnosed to have ARDS according to the criteria of the American–European Consensus Conference in 1994. Exclusion criteria were any structural heart disease by echo, pulmonary embolism, atrial fi brillation, renal insuffi ciency, and age <18. All patients benefi ted from a lung protective ventilation strategy. Plasma NT-proBNP, troponin I and troponin T were measured on day 0 and on day 2 and day 7 of ARDS diagnosis. PH, PaCO2, PaO2, P(A-a)O2 (alveolar–arterial gradient), PaO2/FiO2 ratio, a/A ratio, PEEP, PIP (peak airway pressure), Pmean, Pplat (plateau pressure) and Ceff (eff ective compliance), and Raw (airway resistance) were monitored daily. S41 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P112 episodes increased by 8% and >15 day episodes decreased by 7% from 1999 to 2009. The overall reintubation rate was 13.8%. Less than 48-hour reintubation dropped from 14.4% in 1999 to 6.7 in 2009 while more than 48-hour reintubation remained similar (5.6% in 1999 and 6.2% in 2009). NIV use signifi cantly increased over this time – almost quadrupled (from 78 in 1999 to 315 in 2009). The most prominent increase was noted in the surgical and burn ICUs where, in 1999, NIV use was minimal (burn: 0, SICU: 6 and 16 and 131 in 2009). Medical and neuro ICUs doubled their use. A total 52.7% of NIV applications were associated with IV within 48 hours of NIV therapy. The sequence of IV–NIV revealed similar patterns through the years, overall, 58.4% of NIV application followed extubation, 24.2% preceded intubation and 17% was in between. Mechanical ventilation in intensive and critical care units of Russia: RuVent national epidemiologic study Anaesth Intensive Care 2007, 35:975-978. Oram MP, et al.: Severe acute respiratory distress syndrome in pregn Results Total mortality was 35.1%, mortality in ARDS was 44.9%. Preva- lence of ARDS was 18.7%. Leading causes for initiation of respiratory support were pathology of the central nervous system (severe TBI 13.3%, stroke 15.7%, craniocephal tumors 5%), sepsis (8.3%), community- acquired pneumonia (8.8%) and ARDS (10.5%). Controlled modes of mechanical ventilation were predominant in our study (A/C 20.2%, SIMV 45.1%, BIPAP 12.6%), other modes includes pressure support ventilation, ASB and PAV. Prevalence of noninvasive respiratory support was only 1.1%. Mean tidal volume calculated by ideal body weight was 8.13 (6.84 to 9.35) for boys and men and 9.1 (7.6 to 10.9) for girls and women. Mean PEEP was 5 (4 to 8) in the whole study and 6 (5 to 9) for ARDS patients. ventilation. Anaesth Intensive Care 2007, 35:975-978. 2. Cole DE, et al.: Acute respiratory distress syndrome in pregnancy. Crit Care Med 2005, 33:S269-S278. 3. Bandi VD, et al.: Acute lung injury and acute respiratory distress syndrome in pregnancy. Crit Care Clin 2004, 20:577-607. P114 P114 Mechanical ventilation in intensive and critical care units of Russia: RuVent national epidemiologic study DN Protsenko1, AI Yaroshetskiy1, SG Suvorov2, AU Lekmanov2, BR Gelfand1 1Russian National Research Medical University, Moscow, Russia; 2Moscow Research Institute of Pediatrics and Child Surgery, Moscow, Russia Critical Care 2012, 16(Suppl 1):P114 (doi: 10.1186/cc10721) Eff ects of low and high tidal volume and pentoxifylline on intestinal blood fl ow and leukocyte–endothelial interactions in mechanically ventilated rats Eff ects of low and high tidal volume and pentoxifylline on intestinal blood fl ow and leukocyte–endothelial interactions in mechanically ventilated rats N Nakagawa1, P Aikawa1, HZ Zhang2, C Correia1, R Pazzeti1, C Valente Barbas1, T Mauad1, E Silva1, P Sannomiya1 1Faculdade de Medicina da Universidade de São Paulo, Brazil; 2University of Toronto and Saint Michael Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P116 (doi: 10.1186/cc10723) N Nakagawa1, P Aikawa1, HZ Zhang2, C Correia1, R Pazzeti1, C Valente Barbas1, T Mauad1, E Silva1, P Sannomiya1 1Faculdade de Medicina da Universidade de São Paulo, Brazil; 2University of Toronto and Saint Michael Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P116 (doi: 10.1186/cc10723) Table 1 (abstract P117) Male 1 Male 2 R estimated 3.7 ± 0.7 3.2 ± 0.7 R measured 5.2 ± 1.9 2.9 ± 1.2 C estimated 97.7 ± 20.6 85.4 ± 18.7 C measured 100.5 ± 21.9 76.5 ± 18.7 Mean ± SD of R in cmH2O/l/second and C in ml/mbar (measured = MLR, estimated = O+D). Introduction The combination of high positive end-expiratory pressure (PEEP) and low tidal volume (VT) decreases some risks of mechanical ventilation, including pulmonary overdistention, damage due to cyclic opening and closing of the alveoli, and infl ammatory responses that can lead to multiple-organ dysfunction. We hypothesized that high VT and high PEEP induce mesenteric microcirculatory disturbances and that those disturbances would be attenuated by pentoxifylline, which is anti-infl ammatory.l Mean ± SD of R in cmH2O/l/second and C in ml/mbar (measured = MLR, estimated = O+D). l y Methods We anesthetized (isofl urane 1.5%), tracheostomized, and mechanically ventilated 57 male Wistar rats with PEEP of 10 cmH2O and FIO2 of 0.21 for 2 hours. One group received low VT (7 ml/kg), another group received high VT (10 ml/kg), and a third group received high VT (25 mg/kg) plus pentoxifylline. We measured mean arterial pressure, respiratory mechanics, mesenteric blood fl ow, and leukocyte– endothelial interactions. Figure 1 (abstract P117). PTPinsp from measured Pdi (APdi) versus PTPinsp from reconstruction (ArPdi). Results The mean arterial pressure was similar among the groups at baseline (108 mmHg (IQR 94 to 118 mmHg)) and after 2 hours of mechanical ventilation (104 mmHg (IQR 90 to 114 mmHg)). P116f Results After validation with simulations, we used data from two healthy adults breathing at several levels of WOB. The occlusions caused the expected signals reproducing Pdi as desired with R and C values typical for healthy men (Table 1). Measured and assessed PTPinsp correlated well (R2 = 0.93 and 0.89) and had small mean diff erences (mean ± 2SD = 1.78 ± 3.81 and 0.27 ± 4.80 cmH2O.second) (Figure 1). P115 There were fewer adherent leukocytes (P = 0.005) and fewer migrated leukocytes (P = 0.002) in the low-VT group (5 cells/100 μm length (IQR 4 to 7 cells/100 μm length) and 1 cell/5,000 μm2 (IQR 1 to 2 cells/5,000 μm2), respectively) and the high-VT/pentoxifylline group (5 cells/100 μm length (IQR 3 to 10 cells/100 μm length) and 1 cell/5,000 μm2 (IQR 1 to 3 cells/5,000 μm2), respectively) than in the high-VT group (14 cells/100 μm length (IQR 11 to 16 cells/100 μm length) and 9 cells/5,000 μm2 (IQR 8 to 12 cells/5,000 μm2), respectively). and active breathing patients. The minute volume is adjusted according to end-tidal CO2 (ETCO2) information in passive breathing patients (and respiratory rate in active breathing patients), and oxygenation is adjusted according to SpO2 information. This study reports the ventilation and oxygenation delivered by IntelliVent-ASV® in long-term ventilated ICU patients. p Methods This prospective, observational study included 100 patients invasively ventilated using IntelliVent-ASV® from admission to weaning or death. The rate and reason for stopping automation were recorded. Settings automatically selected, delivered ventilation, respiratory mechanics and arterial blood gas results were collected once a day. Patients were categorized in diff erent lung conditions: normal lung, ALI/ARDS, COPD. Analysis of variance compared the ventilation-days for each type of lung condition for active and passive breathing patients. Results Patients (age 73 (64 to 79) years; SAPS II 56 (48 to 69)) were ventilated using IntelliVent-ASV® to weaning or death (31%) for a median duration of 3.0 (2.0 to 7.0) days without any safety issue. The ventilation controller was deactivated in two patients because of high PaCO2–ETCO2 gradient. Oxygenation controller was deactivated in seven patients for 1 day because of a poor SpO2 signal. In passive and active ventilation-days, minute volume, VT/PBW, respiratory rate, FiO2, and PEEP were statistically diff erent based on lung condition. In passive ALI/ARDS ventilation-days, VT/PBW was signifi cantly lower (7.5 (6.9 to 7.9) ml/kg) than passive normal lung (8.1 (7.3 to 8.9) ml/kg; P <0.05) and passive COPD patients (9.9 (8.3 to 11.1) ml/kg; P <0.05). In passive ALI/ARDS ventilation-days, FiO2 and PEEP were statistically higher than passive normal lung (35 (33 to 47)% vs. 30 (30 to 31)% and 11 (8 to 13) cmH2O vs. 5 (5 to 6) cmH2O, respectively; P <0.05). P115 In active normal lung ventilation-days, VT/PBW was not diff erent (8.4 (7.8 to 9.1) ml/kg) than in active ALI/ARDS (8.1 (7.5 to 9.3) ml/kg), and in active COPD (9.3 (8.6 to 11.6) ml/kg). In active ALI/ARDS and COPD ventilation-days, PEEP was signifi cantly higher than active normal lung (8 (5 to 10) cmH2O, 7 (5 to 10) cmH2O, and 5 (5 to 5) cm H2O, respectively; P <0.05). Methods This prospective, observational study included 100 patients invasively ventilated using IntelliVent-ASV® from admission to weaning or death. The rate and reason for stopping automation were recorded. Settings automatically selected, delivered ventilation, respiratory mechanics and arterial blood gas results were collected once a day. Patients were categorized in diff erent lung conditions: normal lung, ALI/ARDS, COPD. Analysis of variance compared the ventilation-days for each type of lung condition for active and passive breathing patients. Conclusion Low VT with high PEEP was lung-protective, and early pentoxifylline reduced the infl ammatory response to high VT with high PEEP (and presumed lung overdistention) during mechanical ventilation. yp g p g p Results Patients (age 73 (64 to 79) years; SAPS II 56 (48 to 69)) were ventilated using IntelliVent-ASV® to weaning or death (31%) for a median duration of 3.0 (2.0 to 7.0) days without any safety issue. The ventilation controller was deactivated in two patients because of high PaCO2–ETCO2 gradient. Oxygenation controller was deactivated in seven patients for 1 day because of a poor SpO2 signal. In passive and active ventilation-days, minute volume, VT/PBW, respiratory rate, FiO2, and PEEP were statistically diff erent based on lung condition. In passive ALI/ARDS ventilation-days, VT/PBW was signifi cantly lower (7.5 (6.9 to 7.9) ml/kg) than passive normal lung (8.1 (7.3 to 8.9) ml/kg; P <0.05) and passive COPD patients (9.9 (8.3 to 11.1) ml/kg; P <0.05). In passive ALI/ARDS ventilation-days, FiO2 and PEEP were statistically higher than passive normal lung (35 (33 to 47)% vs. 30 (30 to 31)% and 11 (8 to 13) cmH2O vs. 5 (5 to 6) cmH2O, respectively; P <0.05). In active normal lung ventilation-days, VT/PBW was not diff erent (8.4 (7.8 to 9.1) ml/kg) than in active ALI/ARDS (8.1 (7.5 to 9.3) ml/kg), and in active COPD (9.3 (8.6 to 11.6) ml/kg). P115 In active ALI/ARDS and COPD ventilation-days, PEEP was signifi cantly higher than active normal lung (8 (5 to 10) cmH2O, 7 (5 to 10) cmH2O, and 5 (5 to 5) cm H2O, respectively; P <0.05). P115 P115 Use of a fully closed-loop ventilation mode in long-term ventilated ICU patients: a prospective study J Arnal1, A Garnero1, M Wysocki2, D Demory1, G Corno1, A Berric1, S Donati1, L Ducros1, J Durand-Gasselin1 1Hôpital Ste Musse, Toulon, France; 2Hamilton Medical, Bonaduz, Switzerland Critical Care 2012, 16(Suppl 1):P115 (doi: 10.1186/cc10722) Use of a fully closed-loop ventilation mode in long-term ventilated ICU patients: a prospective study J Arnal1, A Garnero1, M Wysocki2, D Demory1, G Corno1, A Berric1, S Donati1, L Ducros1, J Durand-Gasselin1 1Hôpital Ste Musse, Toulon, France; 2Hamilton Medical, Bonaduz, Switzerland Critical Care 2012, 16(Suppl 1):P115 (doi: 10.1186/cc10722) Results A total of 19,734 IV and 2,472 NIV episodes were identifi ed during this period. The number of MV episodes increased from 1,660 in 1999 to 2,182 in 2009 with an increasing NIV/IV ratio (from 0.05 to 0.17). In the medical and surgical ICUs, median IV days decreased from 4 to 3 and 3 to 2 days respectively. Overall, 76% of IV episodes lasted <7 days, 14% between 8 and 14 days and 10% >15 days. The number of <7 day IV Introduction IntelliVent-ASV® is a closed-loop ventilation mode that automatically adjusts ventilation and oxygenation settings in passive S42 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 group (12.7 cmH2O (10.7 to 16.0 cmH2O)). There were fewer adherent leukocytes (P = 0.005) and fewer migrated leukocytes (P = 0.002) in the low-VT group (5 cells/100 μm length (IQR 4 to 7 cells/100 μm length) and 1 cell/5,000 μm2 (IQR 1 to 2 cells/5,000 μm2), respectively) and the high-VT/pentoxifylline group (5 cells/100 μm length (IQR 3 to 10 cells/100 μm length) and 1 cell/5,000 μm2 (IQR 1 to 3 cells/5,000 μm2), respectively) than in the high-VT group (14 cells/100 μm length (IQR 11 to 16 cells/100 μm length) and 9 cells/5,000 μm2 (IQR 8 to 12 cells/5,000 μm2), respectively). group (12.7 cmH2O (10.7 to 16.0 cmH2O)). A method for continuous noninvasive assessment of respiratory mechanics during spontaneous breathing K Lopez-Navas1, S Brandt2, H Gehring2, M Strutz1, U Wenkebach1 1Fachhochschule Lübeck, Germany; 2Universitätsklinikum Schleswig-Holstein, Lübeck, Germany y Critical Care 2012, 16(Suppl 1):P117 (doi: 10.1186/cc10724) Critical Care 2012, 16(Suppl 1):P117 (doi: 10.1186/cc10724) Introduction The proper assessment of patient’s work of breathing (WOB) is the key to a better or even automatic setting of ventilation parameters. We introduce the Occlusion+Delta method (O+D) to continuously determine resistance (R) and compliance (C), allowing one to assess noninvasively the inspiratory force. y p y Methods The O+D method uses a short expiratory occlusion producing immediate changes in airway pressure (Paw), fl ow (V’) and volume (V) but not in transdiaphragmatic pressure (Pdi). The diff erences between an occluded and an undisturbed cycle are related by V’R + V/C = Paw + Pdi. If both cycles are similar Pdi can be neglected, making its measurement unnecessary. Then R and C are derived from linear regression (MLR) and used to make a reconstruction of Pdi (rPdi). As control, R and C were calculated by MLR using the objectively measured (with balloon catheters) Pdi. The inspiratory pressure time product (PTPinsp) of measured Pdi (APdi) and reconstructed Pdi (ArPdi) were compared as expression of WOB. 2 2 y Conclusion IntelliVent-ASV® can be used safely in long-term ventilated ICU patients and selects automatically diff erent ventilation and oxygenation settings according to the lung condition, especially for passive breathing patients. P118l Introduction Severe cutaneous adverse reactions, such as Stevens– Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS) are uncommon but potentially critical ills. Pulmonary involvements in these severe cutaneous reactions patients are rare but are life-threatening complications. Therefore, we conducted a study to investigate the outcomes and risk factors of patients with severe cutaneous reactions with pulmonary complications. P118 Infl uence of catheter diameter, endotracheal tube diameter, suction pressure, and PEEP on the tracheal pressure and lung volume during endotracheal suctioning using a lung model KJ Snijders1, PE Spronk1, TW Fiks1, H Boon1, T Ten Kleij1, AA Becht1, FH De Jongh2 1Gelre Ziekenhuizen, Apeldoorn, the Netherlands; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P118 (doi: 10.1186/cc10725) y Methods This is a retrospective study conducted in a tertiary teaching hospital in Taiwan. Between September 2002 and June 2011, 23 consecutive patients admitted to our hospital under the diagnosis of severe cutaneous adverse reactions with pulmonary involvements were enrolled. The collected demographic data included gender, age and comorbidity. Laboratory data and possible off ending etiology also were collected by reviewing the medical records. Introduction Endotracheal suctioning (ETS) is frequently performed in the ICU for clearing bronchial secretions in intubated and ventilated patients. However, research shows that subatmospheric pressures in the trachea and decreases in lung volume are measured during ETS when unfavourable parameters are chosen, causing complications (for example, atelectasis) [1,2]. The aim of this study was to investigate the infl uence of the parameters: area ratio ‘catheter/endotracheal tube’, suction pressure, type of ventilation, and positive end-expiratory pressure (PEEP) on tracheal pressure and lung volume during ETS. Results A total of 21 severe cutaneous adverse reactions patients were eligible. In these 21 patients, 16 (76.2%) patients were SJS/TEN and fi ve (23.8%) patients were DRESS. Allopurinol was the most common culprit medicine (n = 9). There were 11 (52.4%) patients progressing to respiratory failure with mechanical ventilation. Among these 11 patients, one was upper airway obstruction, two patients were pneumonia, three patients were acute respiratory distress syndrome and the other fi ve patients were acute pulmonary edema. The overall hospital mortality rate was 47.6% (11/21). The survivors group was younger (51.5 ± 25.4 years vs. 70 ± 10.7 years, P = 0.046) and had less chronic kidney disease (9% vs. 60%, P = 0.021) compared with the nonsurvivors group. P119 Conclusion Our fi rst results demonstrate a great potential in the proposed method. A study with 30 volunteers is being carried out and results will be presented in 2012. P120 During spontaneous breathing cardiac output lacks major eff ect on pulmonary shunting in porcine lungs with partial collapse L Vimlati, A Larsson, G Hedenstierna, M Lichtwarck-Aschoff Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P120 (doi: 10.1186/cc10727) Introduction Spontaneous breathing (SB) improves oxygenation com- pared to mechanical ventilation (MV), and does so even without recruit- ing atelectasis [1,2]. Since it cannot be excluded that cardiac output (CO) impacts on pulmonary shunt, we investigated whether pulmonary shunt correlates with CO in a porcine model of lung collapse. 2 Conclusion During endotracheal suctioning the area ratio (between the catheter and the endotracheal tube) and the applied suction pressure should be minimal to avoid high pressure and lung volume losses. g Methods In 12 anaesthetized and relaxed supine piglets, lung collapse was induced by negative pressure application to the endotracheal tube during MV. Six animals resumed SB after 15 minutes; the other six P118l p ( ) p g g Methods A lung model (two intersurgical balloons of 2 litres each and an artifi cial trachea with a 25 mm internal diameter) for spontaneous breathing and pressure-controlled ventilation (PCV) was designed. Spontaneous breathing was simulated by varying the pressure inside the chamber in which the balloons were mounted by an electronically controlled syringe. During PCV, a Servo 300 ventilator was added. An open suction system (VBM, 5 mm suction gap) was used. After insertion of the catheter, suction (pressures ranged from 20 to 65 kPa) was applied for 15 seconds during withdrawal, as used in clinical practice. During spontaneous breathing the parameters pressure and area ratio (79%, 58%, 34%, 25%, 13%) were varied, while during PCV the PEEP was varied too. Each setting was repeated three times and the mean results were used for analysis. Conclusion Severe cutaneous adverse reaction with lung involvement may contribute a high mortality rate. Older age and comorbidity of chronic kidney disease were the risk factors of mortality in severe cutaneous adverse reactions patients. y Results For spontaneous breathing (n = 45) the mean tracheal pressure and lung volume decreased strongly when the area ratio and/or suction pressure increased (for example, mean tracheal pressure –13 ± 1.3 cmH2O compared to atmospheric pressure, and lung volume –524 ± 37 ml using 20 kPa suction pressure and area ratio 0.58), the fi rst having the greater infl uence. Similar results (n = 84) were found for PCV (for example, 22 ± 0.35 cmH2O and –536 ± 137 ml, using 20 kPa suction pressure, area ratio 0.58 and PEEP 20 cmH2O). P120 P120 During spontaneous breathing cardiac output lacks major eff ect on pulmonary shunting in porcine lungs with partial collapse L Vimlati, A Larsson, G Hedenstierna, M Lichtwarck-Aschoff Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P120 (doi: 10.1186/cc10727) Eff ects of low and high tidal volume and pentoxifylline on intestinal blood fl ow and leukocyte–endothelial interactions in mechanically ventilated rats Mesenteric blood fl ow was also similar between the groups: low VT 15.1 ml/ minute (IQR 12.4 to 17.7 ml/minute), high VT 11.3 ml/minute (IQR 8.6 to 13.8 ml/minute), high-VT/pentoxifylline 12.4 ml/minute (10.8 to 13.7 ml/minute). Peak airway pressure was lower (P = 0.03) in the low- VT group (10.4 cmH2O (IQR 10.2 to 10.4 cmH2O)) than in the high-VT group (12.6 cmH2O (10.2 to 14.9 cmH2O)) or the high-VT/pentoxifylline Figure 1 (abstract P117). PTPinsp from measured Pdi (APdi) versus PTPinsp from reconstruction (ArPdi). S43 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Our fi rst results demonstrate a great potential in the proposed method. A study with 30 volunteers is being carried out and results will be presented in 2012. Risk factors of mortality in severe cutaneous adverse reactions patients with pulmonary involvement Risk factors of mortality in severe cutaneous adverse reactions patients with pulmonary involvement P122 were kept on MV at a respiratory rate and tidal volume corresponding to SB. All animals were followed over 120 minutes, and repeated measurements were converted to the area under curve and analysed by Mann–Whitney test and linear regression. were kept on MV at a respiratory rate and tidal volume corresponding to SB. All animals were followed over 120 minutes, and repeated measurements were converted to the area under curve and analysed by Mann–Whitney test and linear regression. Oxygenation correlates with lung aeration during unsupported spontaneous breathing in porcine lung collapse model L Vimlati, A Larsson, G Hedenstierna, M Lichtwarck-Aschoff Uppsala University, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P121 (doi: 10.1186/cc10728) pp y pp Critical Care 2012, 16(Suppl 1):P121 (doi: 10.1186/cc10728) y gi Results Neither heart rate, mean arterial pressure nor respiratory rate showed any signifi cant change after IPV. Oxygen saturation improved immediately after IPV and was also present after 15 minutes. See Table 1. Introduction We investigated whether oxygenation correlates with lung aeration during unsupported spontaneous breathing (SB) and mechanical ventilation (MV) in a porcine lung collapse model. Table 1 (abstract P122). Values before, after and 15 minutes after therapy Table 1 (abstract P122). Values before, after and 15 minutes after therapy Heart Mean Respiratory Oxygen rate arterial pressure rate saturation Before 84.5 ± 18.2 86.6 ± 19.0 24.7 ± 5.6 93.9 ± 3.0 After 86.0 ± 17.6 87.4 ± 21.0 24.1 ± 6.7 95.8 ± 2.8 After 15 minutes 83.1 ± 16.7 85.4 ± 18.9 23.4 ± 6.0 95.5 ± 2.8* P <0.01. g Methods In 14 anesthetized supine piglets, lung collapse was induced by negative pressure application (NPA) to the endotracheal tube. Eight animals resumed SB 5 minutes after NPA, six animals were kept on MV at a respiratory rate and tidal volume corresponding to SB. Thoracic CTs and arterial blood gases were taken 2.5 and 30 minutes after NPA. Spearman rank correlation was used for testing; values are given as mean (95% CI). Results Thirty minutes after NPA the amount of lung tissue in collapsed regions was similar in both groups (MV: 40% (36 to 44), SB: 35% (26 to 43); P = 0.22). Resuming SB, PaO2/FiO2 improved signifi cantly more with less amount of collapsed lung tissue 2.5 minutes after NPA (r = –0.87, P = 0.033). During SB a signifi cant negative correlation between PaO2/ FiO2 and the amount of collapsed lung tissue (r = –0.76, P = 0.038) was observed; no such correlation could be seen during MV (r = –0.3, P = 0.2) (Figure 1). Conclusion We demonstrated that IPV is a safe therapy, and oxygen saturation improved after therapy with IPV. P123 Impact of an open lung approach on hemodynamic parameters after cardiac surgery A Leme, F Galas, M Volpe, J Fukushima, J Almeida, R Ianotti, L Hajjar, M Amato Heart Institute, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P123 (doi: 10.1186/cc10730) Safety and eff ect of intermittent intrapulmonary percussive ventilation on oxygen saturation and hemodynamic functions I Blum, R Janssen-Dean, A Van Overdijk, B Speelberg St Anna Hospital Geldrop, the Netherlands Critical Care 2012, 16(Suppl 1):P122 (doi: 10.1186/cc10729) Safety and eff ect of intermittent intrapulmonary percussive ventilation on oxygen saturation and hemodynamic functions I Blum, R Janssen-Dean, A Van Overdijk, B Speelberg St Anna Hospital Geldrop, the Netherlands Critical Care 2012, 16(Suppl 1):P122 (doi: 10.1186/cc10729) Results PaO2/FiO2 was higher and venous admixture (Qva/Qt) was lower in the SB group. Hemodynamics was stable and CO was similar in both groups. Qva/Qt correlated with CO (r = 0.83, P = 0.04) in the MV group, but not in the SB group (r = 0.08, P = 0.88) (Figure 1). Introduction Intrapulmonary percussive ventilation (IPV) is a ventilatory technique which is used to clear endobronchial secretions in patients. IPV uses a Phasitron, which delivers rapid, high-fl ow, mini- bursts of air mixed with oxygen to the patients. We investigated the safety of IPV on hemodynamic values and the eff ect of IPV on oxygen saturation and respiratory rate. g g g Conclusion SB achieves higher PaO2/FiO2 and lower Qva/Qt compared to MV. During SB, Qva/Qt seems to be unaff ected by CO. This lung collapse model has stable hemodynamics and gas exchange for at least 2 hours irrespective of the mode of ventilation. References Methods From April until August 2011 we investigated 42 consecutive patients admitted to our eight-bed adult general ICU with respiratory failure. Variables such as heart rate, mean arterial pressure, respiratory rate, and oxygen saturation were measured and compared at three diff erent time points: before starting IPV therapy, directly after and 15 minutes later. All patients received IPV using a Bird Intrapulmonary Percussionator Ventilator Model IPV-2C for a period of 20 minutes consisting of two cycles of 10 minutes. After the fi rst 10 minutes of IPV therapy in combination with chest compressions the frequency rate was reduced in order to suction the mobilized secretions. This cycle was then repeated. Statistical analysis was done with SPSS version 17. Student’s t test was used to compare values before therapy with directly after and after 15 minutes of therapy. P <0.05 was considered signifi cant. 1. Carvalho AR, Spieth PM, Pelosi P, et al.: Pressure support ventilation and biphasic positive airway pressure improve oxygenation by redistribution of pulmonary blood fl ow. Anesth Analg 2009, 109:856-865.f 2. Vimlati L, Kawati R, Hedenstierna G, Larsson A, Lichtwarck-Aschoff M: Spontaneous breathing improves shunt fraction and oxygenation in comparison with controlled ventilation at a similar amount of lung collapse. Anesth Analg 2011, 113:1089-1095. References 1. Wood CJ, et al.: Intensive Crit Care Nurs 1998, 14:124-136. 2 P d CM l I i C i C N 2009 25 21 30 1. Wood CJ, et al.: Intensive Crit Care Nurs 1998, 14:124-136. Figure 1 (abstract P120). Venous admixture (Qva/Qt) plotted against CO (pooled data for each group). Solid circles, mechanical ventilation (MV); open circles, spontaneous breathing (SB). Figure 1 (abstract P120). Venous admixture (Qva/Qt) plotted against CO (pooled data for each group). Solid circles, mechanical ventilation (MV); open circles, spontaneous breathing (SB). S44 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P125 respiratory parameters. Nevertheless, adverse hemodynamic eff ects can occur due to the RM technique. The aim of this study is to evaluate the eff ect of the RM on hemodynamic parameters in the immediate postoperative period after cardiac surgery. A rule for predicting the new equilibrated carbon dioxide partial pressure after changes in the ventilation frequency S Buehler, M Jensen, S Lozano, S Schumann, J Guttmann Uniklinik Freiburg, Germany Critical Care 2012, 16(Suppl 1):P125 (doi: 10.1186/cc10732) p p p g y Methods A total of 120 patients with PaO2/FiO2 ratio <250 was randomized to a conventional strategy of mechanical ventilation or open lung strategy. The open lung strategy was performed using RM with an inspiratory pressure amplitude of 15 cmH2O and PEEP of 30 cmH2O three times during 1 minute and setting PEEP after RM at 13 cmH2O. The conventional strategy was done using PEEP = 8 cmH2O and RM with CPAP = 20 cmH2O three times during 30 seconds and setting PEEP after RM at 8 cmH2O. The heart rate, systolic, diastolic and mean arterial blood pressures were recorded before, immediately and 5 minutes after RM. Respiratory mechanics and blood gas analysis were recorded before and after RM. Introduction In mechanical ventilation the arterial carbon dioxide partial pressure (PCO2) is one of the key parameters to control the ventilation frequency. Qualitatively, the eff ect of changes in the ventilation frequency on the arterial PCO2 level is well known. However, little is known about the time it takes for the PCO2 value to reach a new equilibrium after a change in the ventilation frequency (the period of latency), nor in what way the transition between two states of equilibrium takes place. Results The open lung group presented a higher variability on blood pressure immediately after RM compared to the conventional group. There were no diff erences in baseline blood pressure or 5 minutes after RM and heart rate between groups. The open lung group presented higher lung compliance (60 ± 17 vs. 48 ± 13 ml/cmH2O) and PaO2/FiO2 (431 ± 124 vs. 229 ± 68) ratio compared to the conventional group. Methods We carried out a clinical study on patients without any history of lung disease or intracranial surgery in order to determine these relations. Patient–ventilator asynchrony during conventional or automated pressure support ventilation in diffi cult-to-wean patients Introduction Cardiac surgical procedures are associated with a high incidence of postoperative complications, increasing costs and mortality. The aim of this study is to evaluate the eff ect of a strategy of protective ventilation on pulmonary complications after cardiac surgery. g y Methods We prospectively evaluated 120 patients immediately after cardiac surgery, presenting hypoxemia and PaO2/FiO2 <250. Patients were randomized to protective or conventional ventilation strategy. Protective strategy: PEEP = 13 cmH2O, recruitment maneuver (RM) with inspiratory pressure amplitude of 15 cmH2O and PEEP of 30 cmH2O. Conventional strategy: PEEP = 8 cmH2O and RM with CPAP = 20 cmH2O. Both patients were ventilated in pressure controlled at 6 ml/kg. Pulmonary mechanic and oxygenation parameters were collected at baseline, 15, 240 and 255 minutes after the start of treatment. Occurrence of respiratory complications was assessed in the fi rst 5 days according to the severity score 1 to 4. Introduction Patient–ventilator asynchrony, defi ned as a mismatch between patient’s inspiratory time and the ventilator insuffl ation time, occurs in nearly 25% of intubated patients. High asynchrony rates are associated with higher incidence of weaning failure and tracheostomy, and prolonged mechanical ventilation. The aim of this study was to compare the asynchrony rate during conventional pressure support ventilation (PSV) and automated PSV (SmartCare; Draeger) in diffi cult- to-wean patients. Methods A prospective, crossover study in diffi cult-to-wean patients (patients who required up to three spontaneous breathing trials (SBTs) or as long as 7 days to achieve successful weaning). Patients were ventilated with an Evita XL ventilator for two consecutive 3-hour periods applied in random order: with conventional PSV managed by the attending physicians; and with PSV managed by SmartCare. The periods were administered in the afternoon (3:00 to 9:00 pm) and in the night (12:00 pm to 6:00 am). In both periods, the starting PS level with either conventional or automated PSV was the basal level before enrolment. During every period, airway pressure, fl ow and volume signals were continuously recorded on a PC connected to the ventilator using dedicated software (VentView). These signals were analyzed offl ine by two clinicians. The asynchrony index was defi ned as Results The protective group compared to the conventional group had better lung compliance (60 ± 17 vs. 48 ± 13 ml/cmH2O, P <0.001) and higher PaO2/FiO2 (431 ± 124 vs. 229 ± 68, P <0.001) at 15 minutes after the start. 1. Amato MB, Barbas CS, Medeiros DM, et al.: Eff ect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998, 338:347-354. 1. Amato MB, Barbas CS, Medeiros DM, et al.: Eff ect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998, 338:347-354. P125 We collected data for the arterial PCO2 from blood gas analyses at discrete points in time as well as continuous end-tidal CO2 (etPCO2) and transcutaneous CO2 (PtcCO2) data and checked for the accuracy of the latter two. Least-squares fi tting and a statistical analysis were carried out. Conclusion An open lung approach after cardiac surgery improves lung compliance and the PaO2/FiO2 ratio with minimum hemodynamic detrimental eff ect. Results We determined a general rule to estimate the period of latency after a change in the ventilation frequency. Furthermore, we specifi ed the relation between a change in the ventilation frequency and the change in the PCO2 level. Last, the transition between two PCO2 levels was found to follow an exponential law and the fi tting resulted in a formula for the prediction of the new PCO2 level. The new equilibrium can be predicted with high confi dence in all cases after only 3 to 4 minutes using four data points while the period of latency lasts much longer, usually between 10 and 20 minutes. Reference 1. Amato MB, Barbas CS, Medeiros DM, et al.: Eff ect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998, 338:347-354. g y Conclusion The general rule for the period of latency allows an estimation of the amount of time it takes for the PCO2 value to stabilise again after a disturbance. A quantitative knowledge of the transition between two PCO2 equilibria allows for the prediction of the new PCO2 level long before the period of latency is over. Thus, with our relation between the change in ventilation frequency and the change in PCO2 at hand, an optimal PCO2 level can be aimed for at bedside in the shortest time span possible. A protective-ventilation strategy reduces pulmonary complications after cardiac surgery A protective-ventilation strategy reduces pulmonary complications after cardiac surgery g y F Galas1, A Leme1, J Almeida1, M Volpe2, R Ianotti1, J Fukushima1, L Hajjar1, M Amato3 1Heart Institute, São Paulo, Brazil; 2Federal University of Triângulo Mineiro Minas Gerais, Uberaba, Brazil; 3Hospital das Clinicas, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P124 (doi: 10.1186/cc10731) P126 P123 Impact of an open lung approach on hemodynamic parameters after cardiac surgery g Conclusion In porcine lung collapse PaO2/FiO2 correlates with lung aeration during unsupported SB, but not during MV at a similar breathing pattern. The less lung collapse the animals have, the more PaO2/FiO2 improves resuming SB. 2 2 p g Reference 1. Cressoni M, Caironi P, Polli F, et al.: Anatomical and functional intrapulmonary shunt in acute respiratory distress syndrome. Crit Care Med 2008, 36:669-675. 1. Cressoni M, Caironi P, Polli F, et al.: Anatomical and functional intrapulmonary shunt in acute respiratory distress syndrome. Crit Care Med 2008, 36:669-675. Introduction Lung recruitment maneuver (RM) has been associated with an increase of arterial oxygen saturation and improvement of Figure 1 (abstract P121). PaO2/FiO2 plotted against the proportion of atelectatic lung tissue. Open circles, SB; solid circles, MV. Figure 1 (abstract P121). PaO2/FiO2 plotted against the proportion of atelectatic lung tissue. Open circles, SB; solid circles, MV. Figure 1 (abstract P121). PaO2/FiO2 plotted against the proportion of atelectatic lung tissue. Open circles, SB; solid circles, MV. S45 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P124 P124 A protective-ventilation strategy reduces pulmonary complications after cardiac surgery F Galas1, A Leme1, J Almeida1, M Volpe2, R Ianotti1, J Fukushima1, L Hajjar1, M Amato3 1Heart Institute, São Paulo, Brazil; 2Federal University of Triângulo Mineiro Minas Gerais, Uberaba, Brazil; 3Hospital das Clinicas, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P124 (doi: 10.1186/cc10731) P126 Patient–ventilator asynchrony during conventional or automated pressure support ventilation in diffi cult-to-wean patients MM Bitondo1, HM Aguirre-Bermeo2, A Moccaldo1, P De Santis1, V Bernini1, A Tersali1, S Italiano2, DL Grieco1, FA Idone1, J Grandjean2, F Roche-Campo2, M Antonelli1, J Mancebo Cortes2, SM Maggiore1 1Catholic University of the Sacred Heart, Roma, Italy; 2San Pau University Hospital, Barcelona, Spain Critical Care 2012, 16(Suppl 1):P126 (doi: 10.1186/cc10733) Patient–ventilator asynchrony during conventional or automated pressure support ventilation in diffi cult-to-wean patients Also, the protective group had a lower incidence of complications after 5 days of follow-up (grade 2 = 47% vs. 55%, grade 3 = 9% vs. 13%, grade 4 = 0% vs. 3%, P = 0.045). Conclusion A protective-ventilation strategy after cardiac surgery reduces hypoxemia, increases lung compliances and results in less respiratory complications without adverse eff ects. Reference P127 Methods We performed a retrospective study of prospectively collected data involving 2,012 consecutive patients undergoing mechanical ventilation (MV) in a 16-bed university-affi liated hospital between 1 October 2005 and 31 August 2011. Eighty-fi ve patients with FE were matched 1:3 with successfully extubated patients (SE) using diagnostic category, age, Acute Physiology Score (APS) and duration of ventilation (DOV) before PE as matching criteria. P127 High levels of B-type natriuretic peptide predict weaning failure from mechanical ventilation in adult patients after cardiac surgery L Hajjar1, T Lara1, J Almeida1, J Fukushima1, C Barbas1, A Rodrigues1, E Nozawa1, JL Vincent2, F Jatene1, J Auler Jr1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Belgium Critical Care 2012, 16(Suppl 1):P127 (doi: 10.1186/cc10734) g Results Patients undergoing MV included 1,209 (60.1%) with SE; 224 (11.1%) died during ventilation (without prior FE); 206 (10.2%) were extubated to withdraw support; 180 (8.9%) were transferred from the ICU while ventilated; 81 (4.0%) were liberated from MV after tracheostomy; 85 (6.6%) failed PE. APS scores were higher (53 (42 to 69) vs. 43 (32 to 60), P <0.0001) and DOV before PE longer (1.8 (0.8 to 4.4) vs. 0.9 (0.4 to 2.6), P = 0.0001) in FE than in SE. There was 100% concordance of diagnostic category and no statistically signifi cant diff erences between the groups in regards to age, APS and DOV before PE. Table 1 illustrates the results of the case–control analysis. In addition, FE had more days in the hospital after ICU discharge than did SE: 11 (4 to 24) versus 5 (2 to 9), P <0.0001. Introduction Failure to wean from mechanical ventilation is related to worse outcomes after cardiac surgery. The aim of the study was to evaluate B-type natriuretic peptide (BNP) as a predictor factor of failure to wean from mechanical ventilation after cardiac surgery. Methods We conducted a prospective and observational cohort study of 101 patients that underwent on-pump coronary artery bypass grafting. BNP was measured postoperatively after ICU admission and at the end of a spontaneous breathing test (SBT). Demographic data, hemodynamic and respiratory parameters, fl uid balance, need for vasopressor or inotropic support, lengths of ICU and hospital stay were recorded. Weaning failure was considered as either the inability to sustain spontaneous breathing after 60 minutes or the need for reintubation within 48 hours.i Table 1 (abstract P128). Case–control study of failed extubation Introduction Failed extubation (FE), defi ned as reintubation within 48 hours of planned extubation (PE), is common. The literature suggests that FE complicates 10 to 20% of PE. The consequences of FE have not been well described, nor have its risk factors. Conclusion As compared with conventional PSV, Smartcare may reduce asynchronies in diffi cult-to-wean patients, possibly because of greater variability of the PS level. This needs to be further confi rmed. P128 Case–control study of failed extubation J Krinsley, P Reddy, A Iqbal Stamford Hospital, Stamford, CT, USA Critical Care 2012, 16(Suppl 1):P128 (doi: 10.1186/cc10735) Case–control study of failed extubation J Krinsley, P Reddy, A Iqbal Stamford Hospital, Stamford, CT, USA Critical Care 2012, 16(Suppl 1):P128 (doi: 10.1186/cc10735) P127 Case–control analysis of failed extubation: key outcomes Results BNP levels were signifi cantly higher both at ICU admission and in the end of breathing test in patients with weaning failure than in the other patients. A BNP concentration of 299 ng/l at the end of the SBT identifi ed weaning failure with 92% sensitivity and 87% specifi city, resulting in an area under the curve value of 0.91 (95% CI (0.86 to 0.97), FE SE P value ICU LOS 11.8 (7.7 to 17.5) 3.8 (2.1 to 7.5) <0.0001 VAP (%) 7.1 0.8 0.0043 Mortality (%) 23.5 10.2 0.0052 Figure 1 (abstract P127). Area under receiving operating characteristic curve for BNP-2 (at the end of spontaneous breathing test) to predict weaning failure. Conclusion FE is associated with increased ICU and hospital LOS, increased risk of VAP and increased mortality. Eff orts to prospectively identify patients at risk for FE may reduce its incidence and improve outcomes. Reference 1. Amato MB, Barbas CS, Medeiros DM, et al.: Eff ect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998, 338:347-354. S46 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P <0.001) (Figure 1). In a multivariate model, BNP level at the end of SBT was the only predictor of weaning failure from mechanical ventilation. Conclusion BNP was an independent predictor factor of failure to wean from mechanical ventilation after cardiac surgery, which suggests that optimization of the ventricular function must be a goal prior to liberation from mechanical ventilation. the number of asynchronies (wasted eff orts, double cycles, premature cycling off ) divided by the total respiratory rate (ventilator cycles + asynchrony events), multiplied by 100. the number of asynchronies (wasted eff orts, double cycles, premature cycling off ) divided by the total respiratory rate (ventilator cycles + asynchrony events), multiplied by 100. y y y Results Sixteen patients were enrolled (age 64 ± 11 years; SAPS II 66 ± 14; COPD 25%; days of mechanical ventilation before enrollment 9 ± 4, number of SBTs 3 ± 1). The asynchrony index was lower with Smartcare (10% vs. 14%, P = 0.01), but not diff erent between afternoon and night. Mean PS level (11 vs. 12 cmH2O) was not diff erent between conventional and automated PSV, although the coeffi cient of variability of PS level was greater with Smartcare (20% vs. 0%, P <0.01). No diff erences were observed in PaCO2 (36 vs. 36 mmHg), PaO2 (106 vs. 102 mmHg), total respiratory rate (22 vs. 23), and P0.1 (1.4 vs. 1.6 cmH2O) between conventional PSV and Smartcare. P128 Out-of-bed extubation: changing paradigms Out-of-bed extubation: changing paradigms Out o bed e tubat o : c a g g pa ad g s F Dexheimer Neto, R Cremonese, J Maccari, F Carlin, C Rodrigues, A Raupp, P Vesz, C Leaes, J De Andrade Hospital Ernesto Dornelles, Porto Alegre, Brazil Critical Care 2012, 16(Suppl 1):P129 (doi: 10.1186/cc10736) g g p g Dexheimer Neto, R Cremonese, J Maccari, F Carlin, C Rodrigues, p g Critical Care 2012, 16(Suppl 1):P129 (doi: 10.1186/cc10736) Introduction The position of the patient at the time of extubation is an important topic as several studies have shown that early mobilization is benefi cial for the critically ill patient and, generally, it occurs simultaneously with the weaning from mechanical ventilation (MV). Extubations are currently performed with the patient in a supine position (SP) with the head elevated, and there are no data available concerning the safety of removing the endotracheal tube of a patient seated in an armchair (SA). The aim of this study was to evaluate the safety of proceeding extubations in SA patients compared with those in a SP. Introduction The position of the patient at the time of extubation is an important topic as several studies have shown that early mobilization is benefi cial for the critically ill patient and, generally, it occurs simultaneously with the weaning from mechanical ventilation (MV). Extubations are currently performed with the patient in a supine position (SP) with the head elevated, and there are no data available concerning the safety of removing the endotracheal tube of a patient seated in an armchair (SA). The aim of this study was to evaluate the safety of proceeding extubations in SA patients compared with those in a SP. Methods A retrospective cohort study of a clinical and surgical 23-bed ICU, in a private hospital in Brazil – Hospital Ernesto Dornelles (Porto Alegre, RS, Brazil). Extubation success was the primary outcome – defi ned as tolerating removal of the endotracheal tube for at least g Methods A retrospective cohort study of a clinical and surgical 23-bed ICU, in a private hospital in Brazil – Hospital Ernesto Dornelles (Porto Alegre, RS, Brazil). Extubation success was the primary outcome – defi ned as tolerating removal of the endotracheal tube for at least Figure 1 (abstract P127). Area under receiving operating characteristic curve for BNP-2 (at the end of spontaneous breathing test) to predict weaning failure. Figure 1 (abstract P127). P130 Prediction of post-extubation failure by portable ICU ultrasound Y Sutherasan, P Theerawit, T Hongpanat, C Kiatboonsri, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P130 (doi: 10.1186/cc10737) Effi cacy of IAPS in each of these patient groups was studied. Methods We conducted a prospective, observational study from December 2010 to September 2011 using portable critical care ultrasound to examine air-column width diff erences of vocal cords before and after defl ation of a endotracheal cuff balloon. All patients also underwent cuff leak volume tests and vocal cord examination by direct video laryngoscopy. Results The average age was 64.3 ± 17.8 years, APACHE II score 21.0 ± 7.7, and SOFA score 8.4 ± 3.1. Six patients were diagnosed with A, three with B, two with C, and others had multiple diagnoses. Combinations with NPPV or cricothyroidotomy were also successful. Of the patients who required re-intubation, four were re-intubated for reasons other than aspiration. Two had possibly aspirated. Among patients receiving IAPS, the rate of re-intubation due to oropharyngeal aspiration was 8.3%. No major complication was observed. Results We enrolled 101 patients with planned extubation. The overall prevalence of post-extubation stridor and/or vocal cord oedema was 17%. Age, gender, duration of intubation and BMI were not diff erent between patients with and without post-extubation complications. The average sizes of endotracheal tubes were similar in both groups (No. 7.5). The mean diff erence of increasing of air-column width in patients without complications was considerably higher than those with complications (1.9 mm vs. 1.1 mm; P <0.001). The sensitivity and specifi city at air-column width diff erences ≥1.6 mm were 0.706 and 0.702 respectively. The positive predictive value and negative predictive value were 0.324 and 0.922. The area under the ROC curve of tracheal ultrasound was 0.823 (95% CI: 0.698 to 0.947) and that of the cuff leak volume test was 0.840 (95% CI: 0.715 to 0.964). Conclusion IAPS is a potential method for supraglottic airway management after extubation that may reduce the re-intubation risk. IAPS is a simple method requiring common instruments. Combined eff ects of IAPS with NPPV or cricothyroidotomy can modify airway management. IAPS is a temporary method in which the exact timing for re-intubation should not be missed. To successfully apply IAPS and reduce aspiration, the suctioning method, duration of application and position of the suctioning tube should be further optimized. f Conclusion Portable ICU ultrasound visualising air-column width diff erences between pre and post defl ation cuff balloon is a promising objective tool which aids in prediction of successful extubation. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 48 hours. All statistical analysis were done using SPSS version 16 and the diff erences between the groups were assessed using Student’s t test and the chi-square test. Figure 1 (abstract P131). Intermittent aspiration of pharyngeal secretion. Results Ninety-one patients were included in the analysis – from December 2010 to June 2011. Mean (± SD) age of the population was 71 ± 12 years, mean APACHE II score was 21 ± 7.6, mean duration of MV was 2.6 ± 2 days and mean number of spontaneous breathing trials was 1.3 ± 0.6. Extubation was performed in 33 SA patients (36%) and 58 SP patients (64%), with a similar success rate of 82% and 85%, respectively (P >0.05). Furthermore, no signifi cant diff erences between these groups were found in terms of APACHE II score, time of MV and postextubation distress or complications. Conclusion The outcomes of proceeding extubation in patients seated in armchairs are similar to those extubated in supine position with the head elevated. This new practice can be considered safe and allow extubations to be performed simultaneously with early mobilization. P130 Prediction of post-extubation failure by portable ICU ultrasound Y Sutherasan, P Theerawit, T Hongpanat, C Kiatboonsri, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P130 (doi: 10.1186/cc10737) 30 Prediction of post-extubation failure by portable ICU ultrasound Y Sutherasan, P Theerawit, T Hongpanat, C Kiatboonsri, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P130 (doi: 10.1186/cc10737) Prediction of post-extubation failure by portable ICU ultrasound Y Sutherasan, P Theerawit, T Hongpanat, C Kiatboonsri, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P130 (doi: 10.1186/cc10737) Figure 1 (abstract P131). Intermittent aspiration of pharyngeal secretion. oropharyngeal secretion, we devised a suctioning method: intermittent aspiration of pharyngeal secretion (IAPS). IAPS is a simple, low-cost technique utilizing an intermittent suction unit and a common suction tube (Figure 1), which may reduce the risk of re-intubation on extubated patients requiring supraglottic airway management. oropharyngeal secretion, we devised a suctioning method: intermittent aspiration of pharyngeal secretion (IAPS). IAPS is a simple, low-cost technique utilizing an intermittent suction unit and a common suction tube (Figure 1), which may reduce the risk of re-intubation on extubated patients requiring supraglottic airway management. Methods A retrospective study was performed on 24 patients who received IAPS after extubation from June 2009 to May 2011. A suction tube was placed in the pharynx after extubation. The same suction unit used in intermittent subglottic secretion drainage was applied. IAPS is eff ective for patients with large amounts of oropharyngeal secretion (A), patients with poor laryngopharyngeal function (B), and patients unable to expel viscous sputum (C). Effi cacy of IAPS in each of these patient groups was studied. Introduction Stridor and vocal cord oedema are common in ICU patients. Currently, the cuff leak volume test is a standard technique to assess these complications [1,2]; however, wide variations in terms of its sensitivity and specifi city have been demonstrated in many studies. Recently, ultrasound is a promising noninvasive method widely used in ICU patients and allows visualization of the vocal cords and larynx [3]. Thus, we would like to determine the diagnostic accuracy of portable ultrasound for detection of these post-extubation complications. Methods A retrospective study was performed on 24 patients who received IAPS after extubation from June 2009 to May 2011. A suction tube was placed in the pharynx after extubation. The same suction unit used in intermittent subglottic secretion drainage was applied. IAPS is eff ective for patients with large amounts of oropharyngeal secretion (A), patients with poor laryngopharyngeal function (B), and patients unable to expel viscous sputum (C). Effi cacy of biphasic cuirass ventilation in the critical care department 1. De Bast Y, De Backer D, Moraine JJ, et al.: The cuff leak test to predict failure of tracheal extubation for laryngeal edema. Intensive Care Med 2002, 28:1267-1272. T Yamashita1, Y Taniwaki2, H Takayama2, Y Sakamoto1 1Saga University, Saga, Japan; 2National Hospital Organization Nagasaki Medical Center, Omura, Japan Critical Care 2012, 16(Suppl 1):P132 (doi: 10.1186/cc10739) 2. Chung YH, Chao TY, Chiu CT, et al.: The cuff -leak test is a simple tool to verify severe laryngeal edema in patients undergoing long-term mechanical ventilation. Crit Care Med 2006, 34:409. Introduction Biphasic cuirass ventilation (BCV) assists ventilation by applying intermittent or continuous negative pressure to the thorax. BCV has been reported to improve lung function in various respiratory failures. However, to determine the therapeutic eff ect of BCV is diffi cult, because it is too diffi cult to include animal experiments. Therefore it is important to compile amounts of clinical cases for discussion. We have tried to fi nd a way of developing BCV in critical care. 3. Ding LW, Wang HC, Wu HD, et al.: Laryngeal ultrasound: a useful method in predicting post-extubation stridor. A pilot study. Eur Respir J 2006, 27:384. Introduction Biphasic cuirass ventilation (BCV) assists ventilation by applying intermittent or continuous negative pressure to the thorax. BCV has been reported to improve lung function in various respiratory failures. However, to determine the therapeutic eff ect of BCV is diffi cult, because it is too diffi cult to include animal experiments. Therefore it is important to compile amounts of clinical cases for discussion. We have tried to fi nd a way of developing BCV in critical care. Methods This is a retrospective, nonrandomized study. Before and after BCV, we compared pO2, pCO2, tidal volume, P/F ratio, respiratory index, A-aDO2, shunt ratio, dead space ventilation rate, and chest X-ray. We also performed a questionnaire study about BCV which focused on physicians and nurses working in the ICU. P130 Prediction of post-extubation failure by portable ICU ultrasound Y Sutherasan, P Theerawit, T Hongpanat, C Kiatboonsri, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P130 (doi: 10.1186/cc10737) Conclusion Portable ICU ultrasound visualising air-column width diff erences between pre and post defl ation cuff balloon is a promising objective tool which aids in prediction of successful extubation. References Conclusion Portable ICU ultrasound visualising air-column width diff erences between pre and post defl ation cuff balloon is a promising objective tool which aids in prediction of successful extubation. Referencesf P132fi Out-of-bed extubation: changing paradigms Area under receiving operating characteristic curve for BNP-2 (at the end of spontaneous breathing test) to predict weaning failure. S47 Intermittent aspiration of pharyngeal secretion for re-intubation prevention T Nakamura, O Nishida, J Shibata, N Kuriyama, Y Hara, M Yumoto Fujita Health University School of Medicine, Toyoake, Japan Critical Care 2012, 16(Suppl 1):P131 (doi: 10.1186/cc10738) Methods This is a retrospective, nonrandomized study. Before and after BCV, we compared pO2, pCO2, tidal volume, P/F ratio, respiratory index, A-aDO2, shunt ratio, dead space ventilation rate, and chest X-ray. We also performed a questionnaire study about BCV which focused on physicians and nurses working in the ICU. Introduction The inability of extubated patients to clear oropharyngeal secretion increases the risk of re-intubation. To eliminate excessive S48 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results From April 2008 to May 2010, BCV was performed by applying RTX (Medivent Ltd, London, UK) for 18 patients admitted to the ICU, National Hospital Organization Nagasaki Medical Center. All of them had acute respiratory failure, and 15 of them were intubated and mechanically ventilated. Thirteen were men, and the mean age was 68 years (1 to 82 years). One case could not continue the treatment due to discomfort of wearing the cuirass. We used the control mode (negative pressure –21 cmH2O, positive pressure +7 cmH2O, I:E ratio 1:1). It improved the tidal volume, P/F ratio, shunt ratio in all cases during BCV (P <0.05). Skin damage caused by the cuirass was observed in one case. According to the questionnaire survey, they had some problems about the durability of the urethane of the cuirass, too close to a thin body or deformation. Some of them had no confi dence because of unfamiliarity with the machine. Methods We used the Sensewear Armband (Bodymedia Inc., USA) to measure the Galvanic Skin Response (GSR) in 11 healthy volunteers (36 to 53 years). The 60-second averages of each test condition were made after 20 minutes of stabilization. Test conditions were pre and post baseline (no intervention), 10 cmH2O CPAP (Resmed, Sydney, Australia) and 15 LPM HFT (TNI, Würzburg, Germany) both in room air. Repeated ANOVA with P <0.05.if Results There were no statistically signifi cant diff erences in GSR between pre and post baselines. CPAP produced an increase in GSR compared to both baselines (45%; P <0.05) and to HFT (41%; P <0.05). HFT produced no signifi cant change in GSR compared to baseline. See Figure 1. P133f Introduction In emergency medicine, noninvasive ventilation (NIV) has grown up for COPD and acute pulmonary edema through the use of continuous positive airway pressure (CPAP). Recently, several studies have reported the use of NIV coupled with nebulized bronchodilators to optimize the management of acute asthma patients in emergency departments and ICUs. This has indicated an improvement in gas exchanges, decreased lung resistances and decreased work of breathing. The purpose of this study is to assess prehospital practices in CPAP for these patients, to target patients for its use, and to compare clinical data before and after achievement of CPAP with nebulization. Introduction In emergency medicine, noninvasive ventilation (NIV) has grown up for COPD and acute pulmonary edema through the use of continuous positive airway pressure (CPAP). Recently, several studies have reported the use of NIV coupled with nebulized bronchodilators to optimize the management of acute asthma patients in emergency departments and ICUs. This has indicated an improvement in gas exchanges, decreased lung resistances and decreased work of breathing. The purpose of this study is to assess prehospital practices in CPAP for these patients, to target patients for its use, and to compare clinical data before and after achievement of CPAP with nebulization. Methods We have conducted a retrospective, descriptive and observational study, by collecting all fi les (EMA, Dispatching Center) for each patient receiving CPAP associated with nebulization, for pulmonary bronchospasm (excluding acute pulmonary edema), and supported by the emergency medical service. Several data were analyzed: age, sex, history, severity signs, cardiac and respiratory rate, blood pressure, pulse oxymetry, need for intubation, nebulization of β2-agonists, anticholinergics, intravenous corticosteroids, and arterial blood gases. Intermittent aspiration of pharyngeal secretion for re-intubation prevention Conclusion GSR is a measurement of the sympathetic component of the autonomic nervous system. It is commonly referred to as the ‘Fight or Flight’ response, and when elevated indicates a state of psychological or physiological stress. Our data suggest that CPAP produces an increase in the GSR compared to rest, whilst TNI therapy produces no change in GSR compared to rest. This increased stress may lead to lower patient compliance when using CPAP therapy compared to TNI therapy, which has very high patient compliance rates. y Conclusion We conclude that BCV is also useful for respiratory care in the ICU. Further confi rmation is needed regarding problems such as the criteria to start and terminate BCV. References 1. Chari S, King J, Rajesh PB, Stuart-Smith K: Resolution of left lower lobe collapse postesophagectomy using the Medivent RTX respirator, a novel noninvasive respiratory support system. J Cardiothorac Vasc Anesth 2004, 18:482-485. 2. Dolmage TE, De Rosie JA, Avendano MA, Goldstein RS: Eff ect of external 2. Dolmage TE, De Rosie JA, Avendano MA, Goldstein RS: Eff ect of external chest wall oscillation on gas exchange in healthy subjects. Chest 1995, 2. Dolmage TE, De Rosie JA, Avendano MA, Goldstein RS: Eff ect of external chest wall oscillation on gas exchange in healthy subjects. Chest 1995, 107:433-439. 3. Ciesla ND: Chest physical therapy for patients in the intensive care unit. Phys Ther 1996, 76:609-625. Management of acute bronchospasm respiratory distress with CPAP ventilation associated with nebulization in the prehospital emergency setting y 4. Rocker GM, Mckenzie MG, Williams B, Logan PM: Noninvasive positive pressure ventilation:successful outcome in patients with acute lung injury/ARDS. Chest 1999, 115:173-177. Management of acute bronchospasm respiratory distress with CPAP ventilation associated with nebulization in the prehospital emergency setting J Cuny, C Berteloot, P Goldstein, E Wiel CHRU de Lille, France Critical Care 2012, 16(Suppl 1):P134 (doi: 10.1186/cc10741) g y g J Cuny, C Berteloot, P Goldstein, E Wiel CHRU de Lille, France Critical Care 2012, 16(Suppl 1):P134 (doi: 10.1186/cc10741) 5. Hill NS: Clinical applications of body ventilators. Chest 1986, 90:897. 5. Hill NS: Clinical applications of body ventilators. Chest 1986, 90:897. P135 Diff erence between continuous positive airway pressure via mask therapy plus chest physiotherapy (CPT) and incentive spirometry therapy plus CPT to treat or prevent acute atelectasis after cardiac surgery F ALMutairi1, S Fallows1, W Abukhudair2, B Islam2 1University of Chester, Manchester, UK; 2King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia Critical Care 2012, 16(Suppl 1):P135 (doi: 10.1186/cc10742) P135 Diff erence between continuous positive airway pressure via mask therapy plus chest physiotherapy (CPT) and incentive spirometry therapy plus CPT to treat or prevent acute atelectasis after cardiac surgery F ALMutairi1, S Fallows1, W Abukhudair2, B Islam2 1University of Chester, Manchester, UK; 2King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia Critical Care 2012, 16(Suppl 1):P135 (doi: 10.1186/cc10742) g y F ALMutairi1, S Fallows1, W Abukhudair2, B Islam2 1University of Chester, Manchester, UK; 2King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia Critical Care 2012, 16(Suppl 1):P135 (doi: 10.1186/cc10742) Introduction All types of therapy such as an incentive spirometry (IS) or continuous positive airway pressure (CPAP) have a valuable role to play in the prevention or the treatment of acute atelectasis. However, the type of therapy that should be used is not yet completely clear. This study aims to clarify the diff erence in eff ectiveness between CPAP therapy plus chest physiotherapy (CPT) and IS therapy plus CPT to treat or prevent acute atelectasis. Results The paO2 was highest under NIV with 129 ± 38 mmHg, followed by NHFO2 (101 ± 34 mmHg, P <0.01 vs. NIV) and VM (85 ± 21 mmHg, P <0.001 vs. NIV, P <0.01 vs. NHFO2, ANOVA). All other vital and blood gas parameters did not show signifi cant diff erences. Dyspnea rating on a 10-point Borg scale was signifi cantly better under NHFO2 (2.9 ± 2.1) and VM (3.3 ± 2.3) compared to NIV (5.0 ± 3.3) (P <0.05, vs. NHFO2 or VM). Comfort rating showed similar results: NHFO2 2.7 ± 1.8; VM 3.1 ± 2.8; NIV 5.4 ± 3.1 (P <0.05, NIV vs. NHFO2 or VM). In the fi nal global rating using German school grades from 1 to 6 NHFO2 also received the best rating (2.3 ± 1.4), followed by VM (3.2 ± 1.7, P = NS vs. NHFO2) and NIV (4.5 ± 1.7, P <0.01 vs. NHFO2 and P <0.05 vs. VM). For further treatment 10 patients chose NHFO2, three VM and one NIV. Methods Seventy-two patients who fi t the inclusive criteria (smoker, hemodynamically stable, normal lung and above 50 years old) participated in this study. The participants were divided randomly into two groups: the control group used IS 15 times per hour plus CPT 4 hours for 3 days, and the trial group used CPAP via mask therapy for half an hour every 2 hours plus CPT 4 hours. Short-term eff ect of humidifi ed high nasal fl ow oxygen in critically ill patients p F Van Beers, A Van Hees, J Van Rosmalen, D Ramnarain St Elisabeth Hospital Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P137 (doi: 10.1186/cc10744) if Conclusion Adding chest physiotherapy to CPAP via mask therapy had better outcomes to treat or prevent acute postoperative atelectasis. P136 Nasal high-fl ow oxygen in patients with hypoxic respiratory failure: eff ect on functional and subjective respiratory parameters compared to conventional oxygen therapy and noninvasive ventilation R Riessen, N Schwabbauer, B Berg, G Blumenstock, M Haap, J Hetzel University Hospital Tübingen, Germany Critical Care 2012, 16(Suppl 1):P136 (doi: 10.1186/cc10743) Introduction This study compared a nasal high flow oxygen therapy Figure 1 (abstract P135). Eff ect of adding CPT to CPAP via mask therapy to treat acute atelectasis. Figure 1 (abstract P135). Eff ect of adding CPT to CPAP via mask therapy to treat acute atelectasis. Figure 1 (abstract P135). Eff ect of adding CPT to CPAP via mask therapy to treat acute atelectasis. Introduction Recently, humidifi ed high-fl ow nasal cannula oxygen (HFNC) has gained popularity in treating patients with acute respiratory insuffi ciency. Studies have shown that HFNC generates a low level of positive airway pressure, reduction of airway resistance and fl ushes nasopharyngeal dead space leading to less work of breathing. However, in which type of patient HFNC could be of benefi t, the short-term as well as long-term eff ects, tolerance and outcome are unknown. We used HFNC in a variety of patients. We evaluated the short-term eff ect of HFNC. Methods We retrospectively studied respiratory, oxygen-derived and hemodynamic parameters before and 1 hour after start of HFNC in 50 patients during the past 12 months. All patients were treated in a mixed medical, surgical, neurological ICU of a teaching hospital. The HFNC used consisted of an air–oxygen blender with adjustable FiO2 (0.21 to 1.0), delivering a modifi able gas fl ow up to 60 l/minute (Optifl ow; Fisher & Paykel, Auckland, New Zealand). y Results Fifty patients were included, 29 men and 21 women, mean age 65 ± 14, mean APACHE II score on admission 19 ± 5.9. The mean duration of HFNC was 22 ± 21 hours. Diff erences in neurophysiologic eff ects between CPAP and a novel high-fl ow therapy systemfi fi Special Care Technologies, Banbury, UK y Critical Care 2012, 16(Suppl 1):P133 (doi: 10.1186/cc10740) Critical Care 2012, 16(Suppl 1):P133 (doi: 10.1186/cc10740) Introduction CPAP therapy for respiratory insuffi ciency is an established and accepted mode of therapy; however, patient compliance remains an issue. Recent studies have shown that high-fl ow therapy (HFT), which uses high fl ows of warmed and humidifi ed air/O2 mixtures through a nasal cannula, can also be eff ective in treating respiratory insuffi ciency. Although a nasal cannula is commonly preferred over a CPAP mask, patient comfort with HFT and CPAP has not been measured empirically. We sought to examine the autonomic neurophysiologic responses as a measure of comfort between these therapies. Methods We have conducted a retrospective, descriptive and observational study, by collecting all fi les (EMA, Dispatching Center) for each patient receiving CPAP associated with nebulization, for pulmonary bronchospasm (excluding acute pulmonary edema), and supported by the emergency medical service. Several data were analyzed: age, sex, history, severity signs, cardiac and respiratory rate, blood pressure, pulse oxymetry, need for intubation, nebulization of β2-agonists, anticholinergics, intravenous corticosteroids, and arterial blood gases. Figure 1 (abstract P133). g Results Over an 18-month period, 21 patients were enrolled: 38% for severe asthma, and 62% for COPD exacerbation. Regarding the history: 67% were under long-term corticosteroid, 48% smokers, 29% received antibiotics, and all of them presented a clinical bronchospasm, and severity criteria for respiratory distress. Sixty percent of patients were hypoxic (SpO2 <92%). All patients received salbutamol inhalation, associated with inhaled anticholinergic agent in 71.4%. Intravenous glucocorticoid drug was dispensed in 71.4% and intravenous salbutamol in 23.8%. None of the asthma patients was intubated, fi ve COPD patients (24.8%) were intubated. Twelve patients were admitted to the ICU (one with asthma and 11 with COPD). Comparison of clinical parameters between prehospital care and the emergency room shows a signifi cant diff erence (P <0.05) for respiratory rate (35.9 ± 7.48 vs. 24.95 ± 8.25) and pulse oxymetry (81.8 ± 15.8 vs. 96.4 ± 3.54). Conclusion NIV through CPAP associated with nebulizations appears to provide benefi t by reducing respiratory work (decreased respiratory rate) and improving alveolar ventilation (increased SpO2) in patients with asthma. However, in COPD patients, no improvement of symptoms has been observed. P135 Diff erence between continuous positive airway pressure via mask therapy plus chest physiotherapy (CPT) and incentive spirometry therapy plus CPT to treat or prevent acute atelectasis after cardiac surgery F ALMutairi1, S Fallows1, W Abukhudair2, B Islam2 1University of Chester, Manchester, UK; 2King Fahd Armed Forces Hospital, Jeddah, Saudi Arabia Critical Care 2012, 16(Suppl 1):P135 (doi: 10.1186/cc10742) The inspiratory capacity (IC) in liters was used to compare the two groups of therapy and it was measured by incentive spirometer after the operation as baseline test, after 12 hours, 24 hours, 48 hours and post therapy. At the same time, RR, HR and SpO2 were measured for both groups. Failure was defi ned as a need for advanced therapy. 2 Conclusion NHFO2 is a promising new device for oxygen supply in respiratory failure, off ering better oxygenation than the VM and better patient comfort and tolerance than NIV. Results Thirty-six patients participated in each group (57 male and 15 female). IC was increased signifi cantly in the CPAP group (P = 0.005) and SpO2 was decreased signifi cantly in the control group (P = 0.037). There were no signifi cant diff erences in RR and HR. See Figure 1. Diff erences in neurophysiologic eff ects between CPAP and a novel high-fl ow therapy systemfi Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S49 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P135f Methods We included 14 patients with hypoxic respiratory failure (paO2 <55 mmHg under room air). Exclusion criteria were ventilatory failure, hemodynamic instability, cardiogenic pulmonary edema, NIV contraindications and inability to cooperate. Patients were treated in a randomized order for 30 minutes each with NHFO2 (Optifl ow®; Fisher-Paykel), VM or NIV, using a FiO2 of 0.6. Every treatment phase was preceded by a 15-minute baseline phase in which the patients received oxygen via a standard nasal prong (SaO2 goal >88%). At the end of each treatment phase vital signs and blood gases were measured and patients rated their dyspnea and their general comfort on a 10-point scale. Finally, patients were ask for a global rating of all three devices ranging from 1 (very good) to 6 (failed) and could choose one device for further treatment. Short-term eff ect of humidifi ed high nasal fl ow oxygen in critically ill patients Indications for HFNC could be divided into fi ve categories: (1) no acceptance of noninvasive positive pressure ventilation (NPPV) (n = 8), (2) weaning from NPPV, (3) hypoxia (n = 14), (4) respiratory distress/discomfort (n = 9), and (5) other (n = 5). Despite the use of HFNC, in 15 patients intubation was unavoidable; group 1, n = 8, group 3, n = 6, group 4, n = 1. Oxygen saturation increased from 91 ± 7.2 to 97.5 ± 1.7 (P ≤0.05). PaO2/FiO2 ratio increased from 140 ± 79.1 to 169.8 ± 68 (P ≤0.05). PCO2 decreased from 6.5 ± 3.0 to 6.2 ± 2.9 mmHg (P ≤0.05). No signifi cant diff erences were seen in heart rate, blood pressure and respiratory rate. Ten patients died, in eight patients of which the policy was not to reanimate and not to be intubated due to extensive comorbidity. Two patients died during treatment in the ICU due to underlying disease. P139 The group that was in need of endotracheal intubation showed a less prominent response to 1-hour HNF therapy, expressed in PaO2 (13.2 ± 2.6 kPa vs. 16.1 ± 3.4 kPa, P = 0.548), saturation (94.4 ± 1.6% vs. 96.5 ± 0.8%, P = 0.228) and breathing frequency (25 ± 2.4/minute vs. 22 ± 2/minute, P = 0.357). The duration of HNF therapy was 26.1 ± 6.3 hours in the nonintubated group and 15.1 ± 9.8 hours for those who were intubated (P = 0.345). fi Methods A prospective observational study during a 6-month period in patients ≥18 years with acute hypoxic respiratory failure when conservative oxygen therapy (15 l/minute) failed. Arterial blood gas analysis was done before HNF therapy and after 1 hour on fl ow 50 l/ minute with FiO2 1.0. Breaths per minute and saturation were noted. When patients remained respiratory insuffi cient they were intubated. Methods All intubated patients who were admitted to the Ipswich Hospital ICU between April and December 2010 were identifi ed and data relating to basic demographics, airway management and the use of capnography were collected. An airway was classed as diffi cult if there were two or more attempts at intubation, a bougie was used, or it was Cormack–Lehane grade III/IV. Complications arising from airway intubation were also noted. Results A total of 139 intubations on 118 patients were identifi ed. Fifty-eight (42%) intubations occurred on the ICU, 41 (29%) in the emergency department (ED) and fi ve (4%) on the ward; 29 (21%) intubations occurred in theatre for surgery and six (4%) out of hospital. Of the 104 intubations on the ICU, ED or ward, nine (9%) were classed as diffi cult and there were 21 (20%) documented complications (hypoxia, hypotension, oesophageal intubation, cardiac arrest and aspiration). Complication rates were similar across junior trainees, senior trainees and consultants. Only 27% of all intubated patients received continuous capnography. g y Conclusion Our fi ndings are consistent with the NAP4 view that airway management outside the controlled confi nes of a theatre setting has the potential to be more diffi cult. Steps should be taken to minimise the risk associated with this procedure, including a thorough airway assessment, use of continuous capnography and the presence of suitably trained operators and assistants. P139 P139 An audit of airway complications in a district general hospital ICU JW Chan, KJ Turner, R Lloyd, R Howard-Griffi n Ipswich Hospital NHS Trust, Ipswich, UK Critical Care 2012, 16(Suppl 1):P139 (doi: 10.1186/cc10746) Good response on high nasal oxygen fl ow reduces the need for intubation in adult respiratory failure L Van Wagenberg, IM Hoekstra, GC Admiraal, M Slabbekoorn Medisch Centrum Haaglanden, Den Haag, the Netherlands Critical Care 2012, 16(Suppl 1):P138 (doi: 10.1186/cc10745) Good response on high nasal oxygen fl ow reduces the need for intubation in adult respiratory failure Introduction The 4th National Audit Project of The Royal College of Anaesthetists and The Diffi cult Airway Society (NAP4) highlighted the increased incidence of airway-related complications in an ICU setting [1]. The aim of this audit was to establish our local ICU airway intubation complication rate as well as our compliance with the NAP4 recommendation that continuous capnography should be used on all intubated patients. Introduction High nasal fl ow (HNF) therapy has proven its effi ciency in acute respiratory failure when compared to conservative oxygen therapy [1]. This study was performed to fi nd a responding and nonresponding group on HNF therapy in adults with hypoxic respiratory insuffi ciency measured by oxygenation and work of breathing. fi y y yg g Methods A prospective observational study during a 6-month period in patients ≥18 years with acute hypoxic respiratory failure when conservative oxygen therapy (15 l/minute) failed. Arterial blood gas analysis was done before HNF therapy and after 1 hour on fl ow 50 l/ minute with FiO2 1.0. Breaths per minute and saturation were noted. When patients remained respiratory insuffi cient they were intubated. Results A total of 20 patients was included. Mean age 63.95 ± 3 years and APACHE II score 23 ± 7. Mean PaO2/FiO2 (P/F) ratio on admission was 77.7 ± 4.2. A total of seven out of 20 patients (35%) needed endotracheal intubation. After 1 hour of HNF therapy PaO2 and saturation measured in arterial blood gas signifi cantly increased from respectively 8.9 ± 0.3 kPa to 16.1 ± 2.4 kPa (P = 0.023) and from 91.8 ± 1.2% to 96.5 ± 0.8% (P = 0.001). Work of breathing, measured by the frequency of breathing, signifi cantly decreased from 35 ± 3 times a minute to 22 ± 2 times a minute. Multidisciplinary care for patients with tracheostomy shortened time to decannulation A Van Hees, F Van Beers, J Van Rosmalen, D Ramnarain, W Van den Wildenberg St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P140 (doi: 10.1186/cc10747) A Van Hees, F Van Beers, J Van Rosmalen, D Ramnarain, W Van den Wildenberg St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P140 (doi: 10.1186/cc10747) Introduction Care for cannulated patients in our hospital is not uniform and clear, leading to an unnecessarily long period of cannulation and even unsafe situations. Therefore our hospital formed a specialized multidisciplinary cannula team (SMCT) consisting of an intensivist, three ventilation practitioners and a registered nurse. The aim of this team was to shorten the period of cannulation and to guarantee uniform care and safety around cannulated patients in our ICU and on the ward. Methods The study was conducted in the mixed medical and neurosurgical ICU of a teaching hospital with a duration of 22 months. Two groups of patients were studied. Group one received PDT before introduction of a SMCT (n = 49) and group two received PDT (n = 27) after introduction of a SMCT. During treatment in the ICU and after discharge, all patients were followed by the cannula team. This team eventually made the decision to decannulate the patients. We evaluated the results of a multidisciplinary cannula team (SMCT) during a follow-up period of 22 months. Results For patient data see Table 1. Seventy-six patients were included in this study. The study showed a reduction in time to decannulation after a mechanical ventilation period of 8.4 days. However clinically relevant, this was not statistically signifi cant. Methods The study was conducted in the mixed medical and neurosurgical ICU of a teaching hospital with a duration of 22 months. Two groups of patients were studied. Group one received PDT before introduction of a SMCT (n = 49) and group two received PDT (n = 27) after introduction of a SMCT. During treatment in the ICU and after discharge, all patients were followed by the cannula team. This team eventually made the decision to decannulate the patients. We evaluated the results of a multidisciplinary cannula team (SMCT) during a follow-up period of 22 months. Figure 1 (abstract P138). PaO2 after 1-hour HNF therapy. Figure 1 (abstract P138). PaO2 after 1-hour HNF therapy. Results For patient data see Table 1. Seventy-six patients were included in this study. P140 Multidisciplinary care for patients with tracheostomy shortened time to decannulation A Van Hees, F Van Beers, J Van Rosmalen, D Ramnarain, W Van den Wildenberg St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P140 (doi: 10.1186/cc10747) P139 The fi nding that complications occurred at a similar rate regardless of the seniority could be explained by more senior staff intubating the most unwell patients. Reference Conclusion All included patients did have a reduced P/F ratio and are therefore to be considered severely respiratory compromised. PaO2 and saturation increased with the use of HNF therapy, while work of breathing decreased. These changes were less prominent in the nonresponding group (Figure 1). The nonresponders, except one, were intubated within 15 hours after the start of HNF therapy. Reference 1. Cook TM, et al.: Br J Anaesth 2011, 106:632-642. 1. Cook TM, et al.: Br J Anaesth 2011, 106:632-642. 1. Roca et al.: Respir Care 2010, 55:408-413. 1. Roca et al.: Respir Care 2010, 55:408-413. Figure 1 (abstract P138). PaO2 after 1-hour HNF therapy. P136 P136 Nasal high-fl ow oxygen in patients with hypoxic respiratory failure: eff ect on functional and subjective respiratory parameters compared to conventional oxygen therapy and noninvasive ventilation R Riessen, N Schwabbauer, B Berg, G Blumenstock, M Haap, J Hetzel University Hospital Tübingen, Germany Critical Care 2012, 16(Suppl 1):P136 (doi: 10.1186/cc10743) P136 Nasal high-fl ow oxygen in patients with hypoxic respiratory failure: eff ect on functional and subjective respiratory parameters compared to conventional oxygen therapy and noninvasive ventilation R Riessen, N Schwabbauer, B Berg, G Blumenstock, M Haap, J Hetzel University Hospital Tübingen, Germany Critical Care 2012, 16(Suppl 1):P136 (doi: 10.1186/cc10743) Conclusion We used HFNC therapy for a variety of indications. In 70% of our study population HFNC was successful. Oxygen-derived parameters signifi cantly increased after 1 hour of HFNC. HFNC was successful and well tolerated in patients weaning from NPPV. After noncompliance of NPPV in 42% of patients in our population, intubation could be avoided with the use of HFNC. Introduction This study compared a nasal high-fl ow oxygen therapy (NHFO2) with conventional oxygen therapy via a Venturi mask (VM) or noninvasive ventilation (NIV) in patients with hypoxic respiratory failure. Study endpoints were functional respiratory parameters, dyspnea, patient comfort and a global rating by the patients. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S50 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P138 Infl uence of percutaneous tracheostomy on gas exchange in mechanically ventilated patients Introduction The infl uence of percutaneous tracheostomy on patients’ ventilator-dependency and clinical outcomes has been deeply investigated [1]. However, except for immediate intraprocedural variations [2], tracheostomy’s impact on gas exchange has scarcely been explored. The aim of the present study is to investigate the persisting eff ects of percutaneous tracheostomy on pulmonary function in a group of ICU-admitted patients. Conclusion PDT is an extremely safe procedure when performed by an experienced intensivist under bronchoscopic guidance. Our low complication rate is due to careful screening and selection of patients and being performed or supervised by an experienced intensivist under direct vision. Methods Clinical records of 107 patients from San Gerardo Hospital General and neurosurgical ICUs that underwent a percutaneous tracheostomy were retrospectively revised to compare ventilator settings, gas exchange and hemodynamic parameters on the day before and on the day after the procedure. For each parameter we averaged the values of three diff erent recordings during the day. A pre-established subgroup analysis on the hypoxemic (PaO2/FiO2 <300 mmHg) patients (n = 38) was performed. Analyses were performed by paired t test and linear regression; a level of P <0.05 was considered statistically signifi cant. P143 1. Terragni PP, et al.: JAMA 2010, 303:1483-1489. Risk factors for poor outcome in patients with osmotic demyelination syndrome y y MA Peters1, JG Van der Hoeven2, C Hoedemaekers2 1Canisius Wilhemina Hospital, Nijmegen, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P143 (doi: 10.1186/cc10750) y y MA Peters1, JG Van der Hoeven2, C Hoedemaekers2 1Canisius Wilhemina Hospital, Nijmegen, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P143 (doi: 10.1186/cc10750) MA Peters1, JG Van der Hoeven2, C Hoedemaekers2 1Canisius Wilhemina Hospital, Nijmegen, the Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P143 (doi: 10.1186/cc10750) y gi Results Among all analyzed patients, we found, after tracheostomy, a marginal decrease in PaCO2 (43 ± 9 vs. 42 ± 8 mmHg, before vs. after P <0.01) and increase in pH (7.43 ± 0.04 vs. 7.44 ± 0.03 mmHg, before vs. after P <0.01), with no variation in PaO2/FiO2. Considering the subgroup of hypoxemic patients, despite unchanged ventilator parameters, after the tracheostomy a higher PaO2/FiO2 (222 ± 60 mmHg vs. 256 ± 84 mmHg, before vs. after P <0.01) and a lower PaCO2 (46 ± 11 vs. 43 ± 9 mmHg, before vs. after P <0.01) were found. For hypoxemic patients, a positive correlation was found between PaCO2 on the day before tracheostomy and the decrease of PaCO2 (r2 = 0.29; P <0.01). Moreover, taking in account the subgroup of hypoxemic patients under pressure support ventilation (n = 28), the PaCO2 decrease was loosely but signifi cantly correlated with the pressure support level on the day before the procedure (r2 = 0.25; P <0.01). Introduction The osmotic demyelination syndrome (ODS) is a devastating complication of rapid correction of hyponatremia. The objective of this study was to identify prognostic factors that determine outcome in patients with ODS. outcome in patients with ODS. Methods We performed a literature search using MEDLINE and Embase. Case reports or case series were eligible for this study in cases of: (1) hyponatremia defi ned as a serum sodium ≤130 mEq/l on hospital admission or thereafter, but preceding the clinical signs of ODS; (2) a clear diagnosis of ODS, confi rmed by MRI scanning or pathology; and (3) a description of patient outcome. We defi ned a favourable outcome as a Glasgow Outcome Score >3 or a Modifi ed Rankin Scale <4. Results A total of 120 manuscripts were identifi ed describing 125 cases: 86/125 (69%) had a favourable outcome. 2. Benini A, et al.: Intensive Care Med 2002, 28:726-730. P142 Bronchoscope-guided percutaneous dilatational tracheostomy performed by an experienced intensivist: a 26-month experience at a tertiary care center in United Arab Emirates y M Rahman, R Ammar, D Abdullah, F Chedid, S Abuhasna Tawam Hospital, Al Ain, United Arab Emirates Critical Care 2012, 16(Suppl 1):P142 (doi: 10.1186/cc10749) Introduction Bedside percutaneous dilatational tracheostomy (PDT) is a safe procedure with an acute complication rate of 10 to 15%. Our hypothesis was that having an experienced person performing or supervising the procedure results in extremely low complications with PDT. We formed a tracheostomy team which always included at least a consultant or specialist experienced (at least 25 procedures) in performing the procedure. Methods A retrospective chart review of all patients who had PDT in a multidisciplinary adult medical surgical ICU during November 2008 to December 2010. The patients’ demographics, indications for intubation and PDT, early and late complications, date weaned off the ventilator, date of decannulation, discharge from ICU and hospital, and outcome of these patients in the hospital were noted. Conclusion With the introduction of a SMCT a clinically relevant reduction of cannulation period could be achieved. The group was small and probably underpowered to show a statistically signifi cant reduction in the cannulation period. p p Results Out of a total of 2,364 admission 57 patients underwent PDT, all with bronchoscopic guidance by an intensivist experienced in PDT (>25 procedures); there were 45 (78.9%) males and 12 (21%) females with the median age of 42 (range 18 to 90) years. The most common admission diagnosis was cardiac arrest n = 14 (24%) followed by severe head injury n = 13(23%) and cerebrovascular accident n = 8 (14%). The commonest indication for tracheostomy was airway protection n = 40 (73%) followed by prolonged mechanical ventilation n = 25 (45%). The median duration of intubation before PDT was 11 days (IQ 8 to 18). The median time elapsed between tracheostomy and weaning of ventilator was 1 day (IQ 1 to 3). However, the median time to decannulation was 37 day (IQ 10 to 136). Acute complication of paratracheal insertion occurred in n = 1 (1.8%) patient. No deaths were reported related to the procedure. However, n = 13 (22.8%) patients died during the hospital stay. No procedure was converted to surgical tracheostomy. The median duration between tracheostomy and discharge from ICU was 12 days (IQ 5 to 21). Chronic complication of subglottic stenosis occurred in n = 1 (1.8%) patient. Multidisciplinary care for patients with tracheostomy shortened time to decannulation The study showed a reduction in time to decannulation after a mechanical ventilation period of 8.4 days. However clinically relevant, this was not statistically signifi cant. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S51 Conclusion With the introduction of a SMCT a clinically relevant reduction of cannulation period could be achieved. The group was small and probably underpowered to show a statistically signifi cant reduction in the cannulation period. Table 1 (abstract P140). Results of comparative analysis between patients before and after the cannula team Pre-cannula Post-cannula team (n = 49) team (n = 27) Age (years) 52.2 ± 16.3 56.4 ± 16.8 APACHE II score 20.9 ± 5.2 21.4 ± 5.7 Intubated days before tracheostomy 12.8 ± 7.9 9.9 ± 7.3 Length of stay in ICU 34.1 ± 4.7 36.6 ± 28.9 Cannulation days 22 ± 15.4 20 ± 0.7 Mechanical ventilation after tracheostomy 9.3 ± 7.4 9.1 ± 9 Tracheostomy after mechanical ventilation 18.2 ± 27.7 9.8 ± 9.5 Table 1 (abstract P140). Results of comparative analysis between patients before and after the cannula team P142 Bronchoscope-guided percutaneous dilatational tracheostomy performed by an experienced intensivist: a 26-month experience at a tertiary care center in United Arab Emirates M Rahman, R Ammar, D Abdullah, F Chedid, S Abuhasna Tawam Hospital, Al Ain, United Arab Emirates Critical Care 2012, 16(Suppl 1):P142 (doi: 10.1186/cc10749) P142 Bronchoscope-guided percutaneous dilatational tracheostomy performed by an experienced intensivist: a 26-month experience at a tertiary care center in United Arab Emirates P142 Bronchoscope-guided percutaneous dilatational tracheostomy performed by an experienced intensivist: a 26-month experience at a tertiary care center in United Arab Emirates P141 Infl uence of percutaneous tracheostomy on gas exchange in mechanically ventilated patients A Pradella1, G Bellani1, S Abd El Aziz El Sayed Deab1, T Mauri1, E Rezoagli1, S Arrigoni1, F Leone1, G Citerio2, A Pesenti1 1University of Milano-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy Critical Care 2012, 16(Suppl 1):P141 (doi: 10.1186/cc10748) Risk factors for poor outcome in patients with osmotic demyelination syndrome The highest sodium concentration after correction was signifi cantly higher in the patients with a poor outcome (139.0 ± 9.3 vs. 134.0 ± 7.3, P = 0.003). Serum osmolality, and concentrations of potassium, chloride, creatinin and glucose were comparable between the outcome groups. The development of tetraparesis (55/125 (44%), P = 0.02) or a decreased level of consciousness (58/125 (46%), P <0.001) were associated with a poor outcome. In contrast, mutism or dysarthria (82/125 (66%), P = 0.002), tremors (29/125 (23%), P = 0.001) or ataxia (58/125 (46%), P <0.001) were associated with a favourable outcome. Conclusion The highest serum sodium concentration during sodium correction rather than the speed of sodium correction or severity of the hyponatremia is a determinant of outcome in patients with ODS. The development of tetraparesis and decreased consciousness are associated with a poor outcome in these patients. patients. We investigated the epidemiology of dysnatremia in a large cohort of surgical ICU patients and evaluated the possible infl uence of the time of acquisition of dysnatremia and fl uctuations in serum sodium concentrations on hospital mortality in these patients. out of 125 (34%) of cases were associated with alcohol abuse, 14/125 (11%) with malnutrition, 26/125 (21%) with use of diuretics and 9/125 (7%) with use of psychoactive medication; none of these characteristics were signifi cantly related to outcome. The sodium concentration on admission was 107.3 ± 9.6 in the patients with a favourable outcome versus 108.4 ± 9.4 in the patients with a poor outcome (P = 0.54). The speed of sodium correction was 1.12 ± 1.6 mmol/hour versus 1.16 ± 0.9 mmol/hour respectively in the favourable and poor outcome cases (P = 0.19). The highest sodium concentration after correction was signifi cantly higher in the patients with a poor outcome (139.0 ± 9.3 vs. 134.0 ± 7.3, P = 0.003). Serum osmolality, and concentrations of potassium, chloride, creatinin and glucose were comparable between the outcome groups. The development of tetraparesis (55/125 (44%), P = 0.02) or a decreased level of consciousness (58/125 (46%), P <0.001) were associated with a poor outcome. In contrast, mutism or dysarthria (82/125 (66%), P = 0.002), tremors (29/125 (23%), P = 0.001) or ataxia (58/125 (46%), P <0.001) were associated with a favourable outcome. sod u co ce t at o s o osp ta o ta ty t ese pat e ts. Impact of ketogenesis and strong ion diff erence on acid–base in our CICU The administration of large volumes of chloride-rich fl uids (as may occur during cardiac surgery to prime the cardiopulmonary bypass circuit or resuscitate the patient) is known to induce hyperchloraemic metabolic acidosis [1]. Using simplifi cations of the original Fencl–Stewart’s equations, it is possible to partition the base defi cit into its constituent parts, subsequently determining the relative contribution of chloride, albumin and unmeasured anions to acidosis [2,3]. Ketone production may contribute signifi cantly to the unmeasured anion component. Methods A prospective cohort analysis. Fifty postoperative cardiac patients were recruited. For each we measured urinary ketones three times per day for the fi rst 48 hours of their CICU admission. Arterial blood gas (ABG) data were recorded in conjunction each time. For each blood gas we partitioned the base defi cit into its constituent components using previously published equations [1-3]. Results A total of 231 ABGs were analysed Urinary ketones were Conclusion We found no evidence that increased ADH secretion would explain low sodium levels in Legionella patients, or other pneumonia patients, challenging the common believe of Legionella causing SIADH. Rather, ADH precursors were upregulated as a response to severe disease. Future studies continuing to explore the cause of sodium disturbance in Legionella are warranted. yi y Methods A prospective cohort analysis. Fifty postoperative cardiac patients were recruited. For each we measured urinary ketones three times per day for the fi rst 48 hours of their CICU admission. Arterial blood gas (ABG) data were recorded in conjunction each time. For each blood gas we partitioned the base defi cit into its constituent components using previously published equations [1-3]. Risk factors for poor outcome in patients with osmotic demyelination syndrome Mean age in the favourable outcome group was 44.7 ± 14.4 years versus 52.3 ± 13.6 years in the poor outcome group (P = 0.006). The ODS was exclusively pontine in 44/125 (35%), extrapontine in 34/125 (37%) and combined pontine and extrapontine in 47/125 (37%) of cases. The anatomical localisation of the lesion was not associated with outcome (P = 0.64). Forty-two Methods We performed a literature search using MEDLINE and Embase. Case reports or case series were eligible for this study in cases of: (1) hyponatremia defi ned as a serum sodium ≤130 mEq/l on hospital admission or thereafter, but preceding the clinical signs of ODS; (2) a clear diagnosis of ODS, confi rmed by MRI scanning or pathology; and (3) a description of patient outcome. We defi ned a favourable outcome as a Glasgow Outcome Score >3 or a Modifi ed Rankin Scale <4.i gi Results A total of 120 manuscripts were identifi ed describing 125 cases: 86/125 (69%) had a favourable outcome. Mean age in the favourable outcome group was 44.7 ± 14.4 years versus 52.3 ± 13.6 years in the poor outcome group (P = 0.006). The ODS was exclusively pontine in 44/125 (35%), extrapontine in 34/125 (37%) and combined pontine and extrapontine in 47/125 (37%) of cases. The anatomical localisation of the lesion was not associated with outcome (P = 0.64). Forty-two p Conclusion In a relatively large cohort of mechanically ventilated patients, percutaneous tracheostomy seems to increase the carbon dioxide elimination. This eff ect was even more pronounced in the subgroup of hypoxic patients, in whom also oxygenation improved. References S52 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 out of 125 (34%) of cases were associated with alcohol abuse, 14/125 (11%) with malnutrition, 26/125 (21%) with use of diuretics and 9/125 (7%) with use of psychoactive medication; none of these characteristics were signifi cantly related to outcome. The sodium concentration on admission was 107.3 ± 9.6 in the patients with a favourable outcome versus 108.4 ± 9.4 in the patients with a poor outcome (P = 0.54). The speed of sodium correction was 1.12 ± 1.6 mmol/hour versus 1.16 ± 0.9 mmol/hour respectively in the favourable and poor outcome cases (P = 0.19). Impact of ketogenesis and strong ion diff erence on acid–base in our CICU Impact of ketogenesis and strong ion diff erence on acid–base in our CICU T Clark, B McGrath, P Murphy, M Jayarajah Derriford Hospital, Plymouth, UK Critical Care 2012, 16(Suppl 1):P146 (doi: 10.1186/cc10753) yp g p Methods We measured CT-ProVasopressin and sodium levels in a prospective cohort of 925 pneumonia patients from a previous multicenter study with 31 patients having positive antigen tests for Legionella pneumophilia. We calculated Spearman rank correlations and multivariate regression models. T Clark, B McGrath, P Murphy, M Jayarajah Derriford Hospital Plymouth UK Results Legionella patients had higher rates of hyponatremia (sodium <130 mmol/l) (43% vs. 8%, P <0.01), but similar median CT- ProVasopressin levels (pmol/l) (20 (12 to 26) vs. 26 (13 to 53), P = 0.89) compared to pneumonia of other etiology. In Legionella patients, high CT-ProVasopressin was not associated with low sodium levels, but showed a positive correlation with sodium levels (r = 0.42, P <0.05). Independent of pneumonia etiology, CT-ProVasopressin were signifi cantly correlated with the pneumonia severity index (r = 0.56, P <0.05) and showed an association with risk for ICU admission (odds ratio per decile, 95% CI) (1.4, 1.2 to 1.6) and 30-day mortality (1.3, 1.2 1.4). Introduction Persistence of a mild metabolic acidosis or base defi cit was occasionally observed in our otherwise well patients post cardiac surgery, sometimes delaying discharge. We hypothesised that this metabolic abnormality may be due to either ketogenesis caused by a combination of starvation and the surgical stress response, or strong ion imbalances following fl uid administration. The administration of large volumes of chloride-rich fl uids (as may occur during cardiac surgery to prime the cardiopulmonary bypass circuit or resuscitate the patient) is known to induce hyperchloraemic metabolic acidosis [1]. Using simplifi cations of the original Fencl–Stewart’s equations, it is possible to partition the base defi cit into its constituent parts, subsequently determining the relative contribution of chloride, albumin and unmeasured anions to acidosis [2,3]. Ketone production may contribute signifi cantly to the unmeasured anion component. Introduction Persistence of a mild metabolic acidosis or base defi cit was occasionally observed in our otherwise well patients post cardiac surgery, sometimes delaying discharge. We hypothesised that this metabolic abnormality may be due to either ketogenesis caused by a combination of starvation and the surgical stress response, or strong ion imbalances following fl uid administration. Is inappropriate secretion of anti-diuretic hormone (SIADH) the cause of hyponatremia in Legionella pneumonia? P Schuetz, S Haubitz, B Mueller, for the ProHOSP Study Group Medical University Clinic, Kantonsspital Aarau, Switzerland Critical Care 2012, 16(Suppl 1):P144 (doi: 10.1186/cc10751) Is inappropriate secretion of anti-diuretic hormone (SIADH) the cause of hyponatremia in Legionella pneumonia? P Schuetz, S Haubitz, B Mueller, for the ProHOSP Study Group Medical University Clinic, Kantonsspital Aarau, Switzerland Critical Care 2012, 16(Suppl 1):P144 (doi: 10.1186/cc10751) Conclusion Dysnatremia was common in surgical ICU patients and was independently associated with an increased risk of in-hospital death in these patients. Dysnatremia at ICU admission was associated with a higher risk of death compared to ICU-acquired dysnatremia. Fluctuations in serum sodium concentrations were independently associated with an increased risk of in-hospital death, even in patients who remained normonatremic during the ICU stay. Introduction Medical textbooks list Legionella as a diff erential diagnosis for the syndrome of inadequate anti-diuretic hormone (ADH) secretion (SIADH), but empirical evidence supporting this association is largely lacking. Partly this is explained by the analytical challenges of ADH measurement. With the recent availability of an immunoassay that measures the more stable ADH precursor peptide (CT-ProVasopressin), we sought to investigate whether increased ADH levels would explain hyponatremia found in Legionella patients. P146 Risk factors for poor outcome in patients with osmotic demyelination syndrome Methods All patients admitted to the ICU between January 2004 and January 2009 were included retrospectively in this study. Hyponatremia was defi ned as a serum sodium concentration (sNa) <135 mmol/l and hypernatremia as a sNa >145 mmol/l. Hyponatremia was defi ned as a sNa less than 135 mmol/l and hypernatremia as a sNa greater than 145 mmol/l. Patients were classifi ed according to the onset of dysnatremia into those who had abnormal sodium concentrations in the initial blood sample, analyzed within 2 hours of admission to the ICU, or those acquiring dysnatremia thereafter. We performed a logistic regression multivariate analysis with hospital outcome as the dependent factor to investigate the possible infl uence of dysnatremia on hospital outcome. Results Of the 10,923 surgical ICU patients included in the study, 1,215 (11.2%) had hyponatremia and 277 (2.5%) hypernatremia at admission to the ICU. Among patients with normonatremia at admission to the ICU (n = 9,431), the incidence of ICU-acquired dysnatremia was 31.3%. Dysnatremia present at ICU admission (OR = 2.53; 95% CI: 2.06 to 3.12, P <0.001) and ICU-acquired dysnatremia (OR = 2.06; 95% CI: 1.71 to 2.48, P <0.001) were independently associated with an increased risk of in-hospital death. Dysnatremia at ICU admission (OR = 1.23; 95% CI: 1.01 to 1.50) was associated with a higher risk of in-hospital death, compared to ICU-acquired dysnatremia. Fluctuation in serum sodium concentration was also independently associated with an increased risk of in-hospital mortality; both in patients who remained normonatremic (>6 mmol/l/ICU stay) and those with dysnatremia (>12 mmol/l/24 hours or >12 mmol/l/ICU stay). Conclusion The highest serum sodium concentration during sodium correction rather than the speed of sodium correction or severity of the hyponatremia is a determinant of outcome in patients with ODS. The development of tetraparesis and decreased consciousness are associated with a poor outcome in these patients. Metabolic acid–base disturbances in patients in the emergency department EM Antonogiannaki, E Lilitsis, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2012, 16(Suppl 1):P149 (doi: 10.1186/cc10756) Metabolic acid–base disturbances in patients in the emergency department EM Antonogiannaki, E Lilitsis, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2012, 16(Suppl 1):P149 (doi: 10.1186/cc10756) p gy Methods The observational study included 50 children aged 12 months to 16 years (among them 27 boys) with metabolic acidosis after surgery- associated hemorrhage: 40% patients lost 58 ± 8.5% of total blood volume, 26% lost 150 ± 9.5% of total blood volume. Patients received 3.66% THAM infusion. The dose of THAM infusion was calculated as the dose administered (ml):negative standard BE (mmol/l)×kg body weight, and did not increase 1.5 ml/kg body weight every 24 hours. The following were analyzed: Na+, K+, ionized calcium, lactate, pH, pCO2, HCO3 and BE of arterial blood, before therapy, and after receiving a one- half dose and a full dose of THAM. The signifi cance of diff erences was assessed by Student’s t test, Mann–Whitney coeffi cient and chi-square test; P <0.05 was considered statistically valid.f y p Critical Care 2012, 16(Suppl 1):P149 (doi: 10.1186/cc1 Introduction The aim of the present study is to determine in unselected patients that visit the emergency department whether the physicochemical approach improves the ability to diagnose acid– base disorders compared with the two commonly used diagnostic approaches; one relying on the plasma bicarbonate concentration (HCO3 –) and anion gap (AG), and the other on the base excess (BE). 3 Methods A prospective observational study took place in the emergency department at a university hospital during the period of March to September 2011. Three hundred and sixty-fi ve patients were included. Arterial and venous samples were drawn for blood gases and a serum biochemical panel, respectively. The decision to collect arterial samples was made by the attending physician in the emergency department who was not involved in the study. Results There were no diff erences in the concentrations of electrolytes and lactate. At the stages of the research the following signifi cant dynamics have been noted: pH (7.27 ± 0.01; 7.31 ± 0.01; 7.35 ± 0.01; P <0,01), HCO3 (18.59 ± 0.26; 19.5 ± 0.3; 21.2 ± 0.41 mmol/l; P <0.01) and BE (–8.34 ± 0.3; –6.58 ± 0.37; –4.47 ± 0.45 mmol/l; P <0.01). PaCO2 tension did not change signifi cantly (38.9 ± 0.83; 37.3 ± 0.94; 37.5 ± 0.95 mmHg; P >0.05). Metabolic acid–base disturbances in patients in the emergency department EM Antonogiannaki, E Lilitsis, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2012, 16(Suppl 1):P149 (doi: 10.1186/cc10756) p y Results All patients were admitted to the hospital, while 103 of them (28%) were transferred directly to the ICU. Hypoalbuminemia (serum albumin ≤35 g/l) was observed in 191 patients (52%). The BE and HCO3 – were normal in 35% and 38% of the total patients, respectively. The corresponding values in patients admitted to the ICU were 41% and 28%. In a signifi cant proportion of patients in whom BE and/or HCO3 – were normal the physicochemical approach detected the presence of acidifying and/or alkalinizing disturbances. Hypoalbuminemia (metabolic alkalosis) was identifi ed in 45% of patients with normal HCO3 – and 48% of patients with normal BE. Strong ion diff erence (SID) acidosis (SID ≤36 mEq/l) was observed in 49% and 44% of patients with normal HCO3 – and BE, respectively. A high unmeasured strong ion concentration ([XA–] ≥8 mEq/l, metabolic acidosis) was observed in 48% of patients with normal HCO3 – and in 52% of patients with normal BE. Patients in whom hidden acidosis of high unmeasured strong anion type was observed were identifi ed by the common diagnostic approach only using the AG adjusted for hypoalbuminemia (AGadj ≥13 mEq/l). Patients who were admitted to clinical wards with acidosis, other than hyperchloremic, remained signifi cantly more days in the hospital than those without the disturbance.i Conclusion THAM infusion resulted in metabolic acidosis correction without the development of hypernatremia and increase of CO2 tension. However, the small number of observations does not allow one to assess accurately the clinical eff ect of THAM for these patients. References References 1. Taylor et al.: Intensive Care Med 2006, 32:295-301. 2. Story et al.: Br J Anaesth 2004, 92:54-60. 3. O’Dell et al.: Crit Care 2005, 9:R464-R470. References 1. Taylor et al.: Intensive Care Med 2006, 32:295-301. 2. Story et al.: Br J Anaesth 2004, 92:54-60. 3. O’Dell et al.: Crit Care 2005, 9:R464-R470. Conclusion Pyroglutamic acidosis is an uncommon condition, but should be considered in a high anion gap metabolic acidosis of unknown cause. The incidence in critical care may be more prevalent due to lack of screening currently. It is associated with sepsis, hepatic and renal dysfunction [3], and in patients who are receiving drugs such as paracetamol and fl ucloxacillin. If known precipitants are stopped, the condition can be rapidly reversed with full patient recovery. R f Buff er therapy in metabolic acidosis after surgery-associated hemorrhage in pediatric oncology N Matinyan, A Saltanov, I Letyagin, O Obukhova N.N. Blokhin Russian Cancer Research Center, Moscow, Russia Critical Care 2012, 16(Suppl 1):P147 (doi: 10.1186/cc10754) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods During a 12-month period, three patients on our ICU with unexplained high anion gap metabolic acidosis had their urine screened for organic acids. Methods During a 12-month period, three patients on our ICU with unexplained high anion gap metabolic acidosis had their urine screened for organic acids. contributed to greater than 75% of the BETOTAL, whilst in 74 (32%) of these the BECl was greater than the BETOTAL. In 18 ABGs a BECl of less than –2 caused a metabolic acidosis. contributed to greater than 75% of the BETOTAL, whilst in 74 (32%) of these the BECl was greater than the BETOTAL. In 18 ABGs a BECl of less than –2 caused a metabolic acidosis. Conclusion Our observation of persistent metabolic abnormalities in otherwise well postoperative cardiac patients may be due to iatrogenic strong ion imbalances caused by hyperchloraemic solutions. Ketogenesis was not a signifi cant contributing factor. The impact of relative hyperchloraemia on pH was buff ered by other counteracting metabolic factors (for example, hypoalbuminaemia), as in 74 ABGs the BECl was greater than the BETOTAL. Results All had chronic methicillin-sensitive Staphylococcus aureus infections treated with long-term paracetamol and fl ucloxacillin. All cases presented to intensive care with reduced level of consciousness after several weeks of treatment. In each case, common causes of high anion gap metabolic acidosis were excluded and urine specimens contained grossly elevated levels of pyroglutamic acid. Flucloxacillin and paracetamol were stopped and N-acetylcysteine commenced, which led to resolution of the metabolic acidosis within 48 hours. All three patients made full recoveries. The fi rst case has been previously described [2]. References References 1. Croal BL, et al.: Clin Chem 1998, 44:336-340. 2. Myall K, et al.: Lancet 2011, 377:526. 3. Peter J, et al.: Med J Aust 2006, 185:223-225. References 1. Croal BL, et al.: Clin Chem 1998, 44:336-340. 2. Myall K, et al.: Lancet 2011, 377:526. 3. Peter J, et al.: Med J Aust 2006, 185:223-225. Introduction Surgery in pediatric oncology is usually massive and traumatic and often leads to acute blood loss, which can result in metabolic acidosis. To treat acidosis, sodium bicarbonate is often used; however, its application has some side eff ects. In this situation tris- hydroxymethyl aminomethane (THAM) seems to be more eff ective. The objective of this study was to evaluate the eff ect of THAM for treating metabolic acidosis after surgery-associated hemorrhage in pediatric oncology. P145 P145 Fluctuations in serum sodium level are associated with an increased risk of death in surgical ICU patients Y Sakr, S Rother, AM Ferreira, C Ewald, P Dünich, K Reinhart Friedrich Schiller University Hospital, Jena, Germany Critical Care 2012, 16(Suppl 1):P145 (doi: 10.1186/cc10752) P145 Fluctuations in serum sodium level are associated with an increased risk of death in surgical ICU patients Y Sakr, S Rother, AM Ferreira, C Ewald, P Dünich, K Reinhart Friedrich Schiller University Hospital, Jena, Germany Critical Care 2012, 16(Suppl 1):P145 (doi: 10.1186/cc10752) p g p y p q Results A total of 231 ABGs were analysed. Urinary ketones were checked along with 181 of the ABGs. A total of 14 ketonuria checks were positive (8%) in 11 patients (22%). In nine ABGs ketonuria was associated with a signifi cant base defi cit, whilst in three it was also associated with a metabolic acidosis. The average starvation time was 39 hours (SD 11 hours). In 121 (52%) ABGs the chloride component of the base defi cit (BECl) was below –2. In 104 (45%) ABGs the BECl Introduction Dysnatremia may have an impact on outcomes in critically ill patients, but this has not been widely investigated in surgical ICU S53 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 An unusual cause of high anion gap metabolic acidosis: pyroglutamic acidosis RJ Wardell, LA Burrows, K Myall, A Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P148 (doi: 10.1186/cc10755) An unusual cause of high anion gap metabolic acidosis: pyroglutamic acidosis RJ Wardell, LA Burrows, K Myall, A Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P148 (doi: 10.1186/cc10755) Introduction Metabolic acidosis is a common acid–base disturbance in intensive care. A high anion gap indicates the presence of endogenous acids, which in critically ill patients are most commonly ketones, lactate and those accumulated in renal failure. However, excluding these causes means more rare forms of acid must be considered, including pyroglutamic acidosis. Pyroglutamic acidosis is caused by the accumulation of 5-oxoproline [1] due to the depletion of glutathione. This leads to loss of negative feedback and therefore the build-up of Y-glutamyl cysteine, which is converted to 5-oxoproline. Conclusion Hypoalbuminemia is a common fi nding in patients in the emergency department and complicates the interpretation of acid–base data using the common diagnostic approaches. A physico- chemical approach may better identify metabolic disturbances in this population. S54 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P150 Pre-admission baseline creatinine was available on all subjects. Logistic regression examined the RIFLE criteria outcome. Adjusted odds ratios (ORs) were estimated by multivariable logistic regression models. Estimates were adjusted for age, gender, race (white, nonwhite), Deyo– Charlson index, sepsis and patient type (surgical vs. medical).i P150 Aberrant bone metabolism in critical illness I Vanhees, L Solie, SJ Roberts, A Wauters, J Gunst, F Luyten, S Van Cromphaut, G Van den Berghe, HC Owen Katholieke Universiteit Leuven, Belgium Critical Care 2012, 16(Suppl 1):P150 (doi: 10.1186/cc10757) y g Results Pre-admission 25(OH)D defi ciency is predictive for acute kidney injury. Patients with 25(OH)D defi ciency have an OR for acute kidney injury of 1.73 (95% CI, 1.30 to 2.30; P <0.0001) relative to patients with 25(OH)D suffi ciency. The 25(OH)D defi ciency remains a signifi cant predictor of acute kidney injury following multivariable adjustment (adjusted OR 1.50; 95% CI, 1.42 to 2.24; P <0.0001). Patients with 25(OH) D insuffi ciency have an OR for acute kidney injury of 1.49 (95% CI, 1.15 to 1.94; P = 0.003) and an adjusted OR of 1.23 (95% CI, 1.12 to 1.72; P = 0.003) relative to patients with 25(OH)D suffi ciency. The vitamin D–acute kidney injury association is independent of the time between 25(OH)D draw and hospital admission.i Introduction Critically ill patients present with distinct alterations in bone metabolism. Concentration of major vitamins in critically ill patients Concentration of major vitamins in critically ill patients H Hayami, K Mizutani, M Shiota, N Nakayasu, T Masubuchi, M Idei, T Gotoh Yokohama City University Hospital, Yokohama, Japan Critical Care 2012, 16(Suppl 1):P152 (doi: 10.1186/cc10759) Introduction Commercially available vitamin solutions have been improved during the last decade, but there have been a few recent reports on defi ciency occurring in the ICU setting. In general, daily delivery of a comprehensive modern vitamin regimen will suffi ce and TPN solution which contains vitamins and trace elements is now widely used because of the view of convenience and infection control. But the dose of vitamins is determined by the American Medical Association (AMA) recommendation which is based on requirement by healthy subjects and it is not clear whether the dose is applicable for critically ill patients. We measured the concentration of major vitamins in 19 critically ill patients who stayed in the ICU and analyzed those treated for more than 3 weeks. f Results Circulating mononuclear precursors were increased in patients compared to healthy controls (99.1% vs. 83.9%; P <0.05). Patient PBMCs diff erentiated into mature actively resorbing osteoclasts in the presence or absence of osteoclastogenic factors (3.2-fold increase vs. healthy cells; P <0.01) and when cultured in PS this spontaneous osteoclast formation was increased further (2.3-fold; P <0.05). There were no diff erences in the osteogenic diff erentiation of hPDCs treated with PS, but there was a twofold (P <0.01) decrease in vascular endothelial growth factor receptor 1 expression. Scaff olds with patient serum-treated hPDCs displayed decreased vascularization and increased osteoclast activity leading to a 28.9% (P <0.001) decrease in bone formation. Methods Of 19 patients, 10 were treated for more than 3 weeks under artifi cial ventilation; seven of which received renal replacement therapy (RRT). Early enteral nutrition was established in six patients who were assessed to have normal intestinal function. For those patients who were diagnosed to have malfunction in intestine, nutrition was supplied via peripheral route for 1 week, and led to total parenteral nutrition after 1 week. Multivitamin product (Vit B1 3 mg, Vit B6 4 mg, Vit C 100 mg, Folate 400 mg, and so on) was administered from day 0. We measured the concentration of those vitamins every 7 days. Conclusion Circulating mononuclear precursors from critically ill patients seem prone to form osteoclasts both in the presence of osteoclastogenic factors and spontaneously. An unusual cause of high anion gap metabolic acidosis: pyroglutamic acidosis RJ Wardell, LA Burrows, K Myall, A Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P148 (doi: 10.1186/cc10755) We have previously reported a decrease in bone formation markers and a dramatic increase in bone resorption markers. In a rabbit model of critical illness, we observed signifi cantly lower bone mineral content in the trabeculae of critically ill rabbits compared to healthy controls. This suggested uncoupling between bone formation and degradation during critical illness, and could increase risk of fracture during rehabilitation or impaired healing of bone fractures. In this study, we investigated the eff ect of critical illness on bone metabolism at the tissue and cellular level. Conclusion Defi ciency of 25(OH)D prior to hospital admission is a signi- fi cant predictor of acute kidney injury in a critically ill patient population. Methods Circulating CD14/CD11b osteoclast precursors in peripheral blood samples of critically ill patients and healthy controls were measured by fl ow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and diff erentiated towards osteoclasts in vitro in 10% healthy (HS) or patient serum (PS) for 14 days. When analyzing bone formation, human periosteal-derived cells (hPDCs) were cultured in vitro in 10% HS or PS, and analyzed for osteoblast diff erentiation after 14 days. Bone formation was studied using serum-treated hPDCs implanted onto NuOss™ calcium phosphate scaff olds in a murine in vivo model. Low serum 25-hydroxyvitamin D levels and acute kidney injury in the critically ill Low serum 25-hydroxyvitamin D levels and acute kidney injury in the critically ill AB Braun1, AA Litonjua1, T Moromizato1, FK Gibbons2, E Giovannucci3, KB Christopher1 1Brigham and Women’s Hospital, Boston, MA, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3Harvard School of Public Health, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P151 (doi: 10.1186/cc10758) Introduction Given the importance of infl ammation in acute kidney injury and the relationship between vitamin D and infl ammation, we sought to elucidate the eff ect of vitamin D status on acute kidney injury. We hypothesized that defi ciency in 25-hydroxyvitamin D (25(OH)D) prior to hospital admission would be associated with acute kidney injury in the critically ill. g p g y p Conclusion In critically ill patients, especially those who received RRT, the concentration of water-soluble vitamins such as Vit C remained low even when they received the AMA recommended dose. Measurement of vitamins and additional administration will be needed as necessary depending on the disease condition. y j y y Methods We performed an observational study of patients treated in medical and surgical ICUs in two teaching hospitals in Boston, Massachusetts between 1998 and 2009. We studied 2,075 patients, age ≥18 years, in whom serum 25(OH)D was measured prior to hospitalization. The exposure of interest was pre-admission serum 25(OH)D and categorized a priori as defi ciency (25(OH)D ≤15 ng/ ml), insuffi ciency (25(OH)D 15 to 30 ng/ml) or suffi ciency (25(OH)D ≥30 ng/ml). The primary outcome was acute kidney injury defi ned as meeting RIFLE Injury or Failure criteria in the 7 days prior to critical care initiation and the 7 days following critical care initiation. We applied the serum creatinine criteria to determine the maximum RIFLE class. P151 y y Results Concentrations of Vit B1, Vit 12, and Folate were 37 ± 16 pg/ ml, 1,068 ± 1,702 pg/ml, and 9.9 ± 14.4 ng/ml on day 7, and there was tendency of increasing to normal range subsequently. On the other hand, the concentration of Vit C was low (2.5 ± 2.4 g/ml: median 1.75) on day 7, and it remained low through 3 weeks (median 2.0 g/ml on day 21). Especially, the concentration of Vit C was extremely low in seven patients who received RRT (median 1.0 g/ml) on day 7 compared with those without RRT (median 2.6 g/ml, P = 0.05). We administered a high dose of Vit C (ascorbic acid 1,000 mg/day) for three patients in this group but restoration to normal range was seen in only two patients. y y Results Concentrations of Vit B1, Vit 12, and Folate were 37 ± 16 pg/ ml, 1,068 ± 1,702 pg/ml, and 9.9 ± 14.4 ng/ml on day 7, and there was tendency of increasing to normal range subsequently. On the other hand, the concentration of Vit C was low (2.5 ± 2.4 g/ml: median 1.75) on day 7, and it remained low through 3 weeks (median 2.0 g/ml on day 21). Especially, the concentration of Vit C was extremely low in seven patients who received RRT (median 1.0 g/ml) on day 7 compared with those without RRT (median 2.6 g/ml, P = 0.05). We administered a high dose of Vit C (ascorbic acid 1,000 mg/day) for three patients in this group but restoration to normal range was seen in only two patients. Conclusion In critically ill patients, especially those who received RRT, the concentration of water-soluble vitamins such as Vit C remained low even when they received the AMA recommended dose. Measurement of vitamins and additional administration will be needed as necessary depending on the disease condition. Concentration of major vitamins in critically ill patients The murine in vivo model confi rmed an increase in osteoclastic resorption and a decreased vascularization, leading to decreased bone formation in patient scaff olds. These fi ndings will help to unravel the mechanisms behind bone loss during critical illness. Reduced cortisol metabolism drives hypercortisolism in critical illness E Boonen1, H Vervenne1, P Meersseman2, L Mortier3, YM Vanwijngaerden1, I Spriet2, L Langouche1, I Vanhorebeek1, G Van den Berghe1 1KU Leuven, Belgium; 2University Hospitals, Leuven, Belgium; 3Virga Jesse Hospital, Hasselt, Belgium Critical Care 2012, 16(Suppl 1):P155 (doi: 10.1186/cc10762) Results Vitamin D defi ciency was defi ned as a serum 25-OH vitamin D concentration <20 ng/ml. The study was conducted between February and August 2011. A total of 105 patients were included. Dosages were performed on day 3 (2, 4) (median, interquartiles). The number of patients with 25-OH vitamin D <10 ng/ml, between 10 and 20 ng/ml, between 20 and 30 ng/ml and >30 ng/ml was respectively 56, 26, 14 and 9. No diff erences were seen between defi cient and nondefi cient patients if we compare SAPS III (58 ± 13 vs. 60 ± 15), predicted mortality (34 ± 21% vs. 40 ± 25%), intra-ICU mortality (8.5 vs. 8.7%), intrahospital mortality (19.5 vs. 21.7%), mean length of stay in the ICU (10 days ± 8), and median SOFA score during the fi rst 5 days (5, 4, 4, 3, 3 vs. 4, 4, 3, 3, 4). A higher (but nonsignifi cant) prevalence of sepsis was found at admission in defi cient patients (42/82 patients vs. 8/23 patients). Eleven defi cient patients were treated with oral vitamin D (25,000 units/day) for 5 days. After treatment, 25-OH vitamin D was above 20 ng/ml in seven patients (31 ± 14 ng/ml). If we adjust groups for vitamin D post treatment, no diff erences were found if we compare defi cient versus nondefi cient patients for intra-ICU mortality (9.3% vs. 6.6%) and intra- hospital mortality (14.6% vs. 23.3%). Introduction Critical illness is hallmarked by elevated cortisol levels, refl ecting the severity of illness. Paradoxically, previous studies reported suppressed ACTH, implicating another mechanism driving elevated cortisol during critical illness. We hypothesized that cortisol metabolism is reduced in critical illness, in part via elevated bile acids, which may explain the paradoxical ACTH–cortisol dissociation by negative feedback inhibition. Methods In a fi rst clinical study (n = 59), we determined the time course of ACTH and cortisol levels during the fi rst week in the ICU. In a second study (n = 28), we calculated the plasma half-life of exogenous cortisol in critically ill patients. P155 The aim of our study was to evaluate the relationship between 25-OH vitamin D defi ciency at admission and the outcome in a medical ICU. Methods A prospective observational study in a 10-bed medical ICU at an inner-city hospital in Brussels. Patients with an expected stay in ICU >48 hours were included. P154 Plasma levels of Coenzyme Q10 are reduced in critically ill patients as compared to healthy volunteers and correlate with age A Coppadoro1, L Berra2, A Kumar2, M Yamada2, R Pinciroli2, E Bittner2, U Schmidt2, M Kaneki2 1University of Milan-Bicocca, Monza, Italy; 2Massachusetts General Hospital, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P154 (doi: 10.1186/cc10761) y Conclusion Reduced expression and activity of cortisol metabolizing enzymes, possibly driven by elevated bile acids, contributes to the hypercortisolism in the critically ill, which explains the increased cortisol plasma half-life and feedback-inhibited ACTH release. Reduced cortisol metabolism could inferentially suppress the cortisol response to an ACTH stimulation test, thereby reducing its diagnostic value for adrenal failure. Introduction The purpose of this study is to investigate Coenzyme Q10 (Q10) levels in critically ill patients as compared to healthy volunteers. Q10 is an essential cofactor for the electron transport chain reactions necessary for the aerobic cellular respiration. Q10 insuffi ciency, therefore, leads to mitochondrial dysfunction. It also acts as an antioxidant. Oxidative state is prominent in critically ill patients, favoring the production of oxygen-free radicals. A recent study showed reduced Q10 levels in septic shock patients [1]. Eff ect of low-dose hydrocortisone on the expression of glucocorticoid receptor alpha of the septic kidney in rats and its protective eff ect on kidney injury Eff ect of low-dose hydrocortisone on the expression of glucocorticoid receptor alpha of the septic kidney in rats and its protective eff ect on kidney injury DW Wu, HP Guo Qilu Hospital of Shandong University, Jinan, Shandong, China Critical Care 2012, 16(Suppl 1):P156 (doi: 10.1186/cc10763) No association between vitamin D defi ciency at admission and outcome in a medical ICU No association between vitamin D defi ciency at admission and outcome in a medical ICU M Claus, L Schmitz, A Roman, E Stevens, P Dechamps Hôpital Saint Pierre, Brussels, Belgium Critical Care 2012, 16(Suppl 1):P153 (doi: 10.1186/cc10760) M Claus, L Schmitz, A Roman, E Stevens, P Dechamps Hôpital Saint Pierre, Brussels, Belgium Critical Care 2012, 16(Suppl 1):P153 (doi: 10.1186/cc10760) Introduction Vitamin D defi ciency is associated with chronic illness and an excess in morbidity and mortality in the general population. Studies have found an even higher prevalence in ICU patients. Introduction Vitamin D defi ciency is associated with chronic illness and an excess in morbidity and mortality in the general population. Studies have found an even higher prevalence in ICU patients. S55 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 1. Donnino MW, et al.: Coenzyme Q10 levels are low and may be associated with the infl ammatory cascade in septic shock. Crit Care 2011, 15:R189. Reduced cortisol metabolism drives hypercortisolism in critical illness In a third clinical study (n = 51), urinary cortisol metabolites were quantifi ed to estimate the activity of cortisol metabolizing enzymes. In a fourth study (n = 64), we quantifi ed the major cortisol metabolizing enzymes in the liver and adipose tissue in relation to circulating cortisol and bile acids. We performed every study in a similar, heterogeneous ICU population, in comparison with a healthy control group matched for age, gender and BMI. Conclusion Our study confi rmed the high prevalence of vitamin D defi ciency in ICU patients but not the association with an excess of mortality. Reference y g p g g Results In the presence of elevated total cortisol, ACTH remained much lower in patients than in healthy controls (P <0.001), confi rming the ACTH–cortisol dissociation during critical illness. Cortisol half-life was substantially prolonged in patients compared to controls. Based on urinary metabolites, the activity of 5α-reductase and 5β-reductase was signifi cantly lower in patients than controls (P <0.0001). Furthermore, the calculated activity of 11-hydroxysteroid dehydrogenase type 2 was reduced (P <0.0001). In the liver, gene and protein expression of 5α-reductase and 5β-reductase was reduced (P <0.0001) and corre lated inversely with circulating cortisol. Moreover, the enzyme expression correlated inversely with circulating levels of conjugated bile acids, which were markedly elevated in patients [1] and which have been shown capable of suppressing expression and activity of cortisol metabolizing enzymes [2]. 1. Lee P, et al.: N Engl J Med 2009, 360:1912-1914. P154 References 1. Vanwijngaerden et al.: Hepatology 2011, 54:1741-1752. 2. McNeilly et al.: J Hepatol 2010, 52:705-711. Methods We recruited 18 healthy volunteers and 36 critically ill patients in the surgical ICU of the Massachusetts General Hospital. Ethical committee approval and written informed consent were obtained. At the moment of blood sampling, height, weight, and age as well as clinical data were collected. Plasma total Q10 concentrations were measured by high-performance liquid chromatography. The Assessment of Daily Living (ADL) score was obtained after discharge. Results Patients’ age and gender did not diff er as compared to healthy volunteers (P = NS). Plasma Q10 levels were lower in critically ill patients as compared to healthy volunteers (0.81 ± 0.22 vs. 0.50 ± 0.36 μg/ml, P <0.001). In critically ill patients, plasma Q10 levels inversely correlated with age (R = 0.40, P = 0.015). Lower levels of plasma Q10 (<0.4 μg/ ml, median) were associated with lower ADL score after discharge (P = 0.005). In our patient population, plasma Q10 levels were not related to PaO2/FiO2, septic shock, SAPS 2 at ICU admission, SOFA score or mortality (all P = NS). Methods We recruited 18 healthy volunteers and 36 critically ill patients in the surgical ICU of the Massachusetts General Hospital. Ethical committee approval and written informed consent were obtained. At the moment of blood sampling, height, weight, and age as well as clinical data were collected. Plasma total Q10 concentrations were measured by high-performance liquid chromatography. The Assessment of Daily Living (ADL) score was obtained after discharge.f Eff ect of low-dose hydrocortisone on the expression of glucocorticoid receptor alpha of the septic kidney in rats and its protective eff ect on kidney injury DW Wu, HP Guo Qilu Hospital of Shandong University, Jinan, Shandong, China Critical Care 2012, 16(Suppl 1):P156 (doi: 10.1186/cc10763) y Results Patients’ age and gender did not diff er as compared to healthy volunteers (P = NS). Plasma Q10 levels were lower in critically ill patients as compared to healthy volunteers (0.81 ± 0.22 vs. 0.50 ± 0.36 μg/ml, P <0.001). In critically ill patients, plasma Q10 levels inversely correlated with age (R = 0.40, P = 0.015). Lower levels of plasma Q10 (<0.4 μg/ ml, median) were associated with lower ADL score after discharge (P = 0.005). In our patient population, plasma Q10 levels were not related to PaO2/FiO2, septic shock, SAPS 2 at ICU admission, SOFA score or mortality (all P = NS). Qilu Hospital of Shandong University, Jinan, Shandong, China Critical Care 2012, 16(Suppl 1):P156 (doi: 10.1186/cc10763) Introduction Infl ammation out of control caused by sepsis can eventually lead to multiple organ dysfunction, of which the kidney is one of the most common injured organs. Sepsis-induced acute kidney injury (SI-AKI) can obviously increase the mortality of sepsis. At present, there are controversial views about the impact of exogenous glucocorticoid to SI-AKI on kidney pathological changes and glucocorticoid receptor (GR) expression. So, we want to investigate whether low-dose glucocorticoid has a protective eff ect on SI-AKI and what is the mechanism. y Conclusion Plasma Q10 levels are reduced in critically ill patients, suggesting reduced antioxidant capacity. Older patients seem to be more prone to exhibit low Q10 levels. Oral supplementation might be considered for those patients. 1. Vanwijngaerden et al.: Hepatology 2011, 54:1741-1752. 2. McNeilly et al.: J Hepatol 2010, 52:705-711. P159 Investigating diarrhoea on the ICU: a retrospective study N Tirlapur, M Kelsey, H Montgomery Whittington Hospital, London, UK Critical Care 2012, 16(Suppl 1):P159 (doi: 10.1186/cc10766) Results Hydrocortisone infusion was independently associated with mild hypoglycemia (APACHE II score <15, OR 2.40, 95% CI 2.01 to 2.85; APACHE II score 15 to 24, OR 1.53, 95% CI 1.44 to 1.62; APACHE II score >24, OR 1.10, 95% CI 1.05 to 1.15) and severe hyperglycemia in all APACHE II groups (APACHE II score <15, OR 3.26, 95% CI 2.59 to 4.10; APACHE II score 15 to 24 OR 1.45, 95% CI 1.33 to 1.68; and APACHE II score >24 OR 1.09, 95% CI 1.02 to 1.17). Hydrocortisone infusion was independently associated with mild hypoglycemia in patients with APACHE II score 15 to 24 (OR 1.74, 95% CI 1.42 to 2.13) and >24 (OR 1.64, 95% CI 1.42 to 1.90), but not in patients with APACHE II score <15 (OR 1.83, 95% CI 0.94 to 3.55). Introduction Diarrhoea is common in ICU patients, with a reported prevalence of 15 to 38% [1]. Many factors may cause diarrhoea, including Clostridium diffi cile, drugs (for example, laxatives, antibiotics), faecal impaction with overfl ow and enteral feeds. Diarrhoea increases nursing workload, impacts on patient dignity, increases costs and exacerbates morbidity through dermal injury, impaired enteral uptake and subsequent fl uid imbalance. We aimed to identify prevalence, yield of stool investigations and clinical impact of diarrhoea on our ICU. Introduction Diarrhoea is common in ICU patients, with a reported prevalence of 15 to 38% [1]. Many factors may cause diarrhoea, including Clostridium diffi cile, drugs (for example, laxatives, antibiotics), faecal impaction with overfl ow and enteral feeds. Diarrhoea increases nursing workload, impacts on patient dignity, increases costs and exacerbates morbidity through dermal injury, impaired enteral uptake and subsequent fl uid imbalance. We aimed to identify prevalence, yield of stool investigations and clinical impact of diarrhoea on our ICU. Methods A retrospective observational study of all ICU patients treated in a 15-bed district general hospital from 1 January 2010 to 31 December 2010 was performed. ICU patients from whom stool samples had been sent for microbiological analysis (including microscopy and C. diffi cile toxin (CDT)) were assumed to have suff ered diarrhoea. Stool sample results were compiled with patient demographics, ICU length of stay (LOS) and mortality data. Conclusion Hydrocortisone increases the risk of dysglycemia in critically ill patients. P158 SI-AKI model was reproduced using the cecum ligation and puncture method. Pathological changes of the kidney were detected by H & E staining. The expression of GRα and NF-κB in the kidney was detected by immunohistochemistry. The levels of IL-1, IL-6, TNFα and IL-10 in the plasma were detected by ELISA. Reference Referencei 1. Heymsfi eld SB, Baumgartner RN, Pan FS: Nutritional assessment of malnutrition by anthropometric methods. Treaty of Modern Nutrition in Health and Disease. Edited by Shils M, Olson JA, Shike M, Ross C. New York: Manole; 2003. 1. Heymsfi eld SB, Baumgartner RN, Pan FS: Nutritional assessment of malnutrition by anthropometric methods. Treaty of Modern Nutrition in Health and Disease. Edited by Shils M, Olson JA, Shike M, Ross C. New York: Manole; 2003. yp g y yp g y y p Methods Blood glucose measurements (n = 73,400) of patients admitted to the ICU from January 2007 to December 2009 (n = 2,167) were analyzed. Logistic regression was used to analyze the eff ect of hydrocortisone infusion on mild (blood glucose level ≥150 mg/dl) and severe hyperglycemia (≥180 mg/dl) and mild hypoglycemia (≤70 mg/ dl) separately. To adjust for severity of disease, patients were stratifi ed in APACHE II score groups (<15; 15 to 24; >24). P157 Hydrocortisone increases the risk of dysglycemia in critically ill patients RT Van Hooijdonk, JM Binnekade, RE Harmsen, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P157 (doi: 10.1186/cc10764) y y Conclusion The age, length of stay and diagnosis associated with the level of organ dysfunction are key factors to progress to the state of malnutrition. The multidisciplinary team has an ongoing role in controlling the supply of proteins and calories with essential nutrients in order to improve the provision, preventing complications and adverse outcomes. Introduction Hyperglycemia and hypoglycemia are independently associated with mortality and morbidity of critically ill patients [1,2]. Critically ill patients frequently receive hydrocortisone for refractory shock. While hydrocortisone infusion is associated with hyperglycemia [3], the eff ect of hydrocortisone on the incidence of hypoglycemia is uncertain. We hypothesized hydrocortisone infusion to increase the risk of hyperglycemia and hypoglycemia in critically ill patients. P159 Whether these dysglycemic eff ects diminish the benefi cial eff ects of hydrocortisone treatment should be investigated in future studies. References 1. Robter W, et al.: N Engl J Med 2004, 351:159-169. 2. Rittirsch D, et al.: Nat Protoc 2009, 4:31-36. 3. Leelahavanichkul A, et al.: Am J Physiol Renal Physiol 2008, 295:1825-1895. 3. Leelahavanichkul A, et al.: Am J Physiol Renal Physiol 2008, 295:1825-1895. Results The study included 247 patients hospitalized in ICUs, mean age 63 years and 60% (148 patients) were male. Sepsis was the most frequent cause of hospitalization at 57% and the average hospital stay was 16 days. The rate of albumin and mean hemoglobin level were respectively 2.1 and 9.5 g/dl. For those patients hospitalized over 10 days were observed average levels of 1.5 and 8.9 g/dl. For mechanical ventilation in patients with septic shock the results were 1.4 and 7.9 g/dl with a mean hospital stay of 14 days. The postoperative group was the highest level observed at 2.6 and 10.4 g/dl and mean total time of hospitalization of 5 days. The worst results based on diagnosis were respectively pulmonary septic shock, ischemic hemispheric brain stroke and cardiogenic shock. All patients with length of stay over 11 days resulted in a clinically malnourished state. Reference Reference Methods Healthy Wistar male rats were randomly divided into a sham group, SI-AKI group and SI-AKI hydrocortisone group (HC group). The 1. Donnino MW, et al.: Coenzyme Q10 levels are low and may be associated with the infl ammatory cascade in septic shock. Crit Care 2011, 15:R189. S56 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 2. Bagshaw SM, et al.: Crit Care Med 2009, 37:463-470. 3. Annane D, et al.: JAMA 2009, 301:2362-2375. Nutritional status of patients occupying ICUs in the state of Rio de Janeiro SO Oliveira1, R Goldwasses2, U Melo3, M Bandeira4, F Dessa5, R Almeida1, I Kouh6 1Albert Schweitzer State Hospital, Rio de Janeiro, Brazil; 2Healh Provider State Government, Rio de Janeiro, Brazil; 3Alberto Torres State Hospital, São Gonçalo, Brazil; 4Real Cordis Hospital, Rio de Janeiro, Brazil; 5Bangu Hospital, Rio de Janeiro, Brazil; 6UFRJ University Hospital, Rio de Janeiro, Brazil Critical Care 2012, 16(Suppl 1):P158 (doi: 10.1186/cc10765) p y Results The survival rate of the AKI group and HC group showed no statistical diff erence (P >0.05). H & E stain showed renal tubular epithelial cells swelling and falling off , and the tubular brush border disappeared and vacuolated in the AKI group. Pathological changes of the renal tubular could be alleviated after hydrocortisone treatment. Compared with the AKI group, immunohistochemistry showed that GRα expression was increased and NF-κB expression was decreased in the HC group (P <0.01). The level of TNFα, IL-6, and IL-1 were reduced and the level of IL-10 was increased in the HC group compared with the AKI group (P <0.01). Introduction Nutritional status and anemia infl uence the clinical course of hospitalized patients. Anemia appears in the fi rst days of hospitalization and can sustain itself and grow worse over time and is caused by a number of factors such as dilution secondary to fl uid replacement, hemolysis, abnormalities in iron metabolism, blood loss in the gastrointestinal tract and also due to decreased production of erythropoietin, a consequence of decreased erythropoiesis caused by the presence of infl ammatory cytokines. Conclusion Low-dose hydrocortisone can inhibit the NF-κB activity, possibly in part by increasing the expression of GRα in renal sepsis rats. Accordingly, it could reduce the production of infl ammatory factors participating in sepsis, eff ectively inhibit the infl ammation and extenuate the sepsis-induced renal pathological changes. l y y Methods A cross-sectional study on 30 November, patients >18 years. Evaluated characteristics of all patients admitted with age, sex, APACHE II score, mean length of stay, cause of hospitalization in mechanical ventilation, organ failure, sedation and analgesia, coma and underuse of vasoactive drugs. e e e ces 1. Krinsley JS, et al.: Crit Care Med 2007, 35:2262-2267. 2. Bagshaw SM, et al.: Crit Care Med 2009, 37:463-470. 3. Annane D, et al.: JAMA 2009, 301:2362-2375. 1. Krinsley JS, et al.: Crit Care Med 2007, 35:2262-2267. P160 P160 Preliminary report of surface electrogastrography in critically ill septic patients after resuscitation C Mancilla, R Galvez, G Landskron, E Tobar, A Madrid Hospital Clinico Universidad de Chile, Santiago, Chile Critical Care 2012, 16(Suppl 1):P160 (doi: 10.1186/cc10767) Table 1 (abstract P161). Nutritional adequacy and clinical endpoints in tolerant and intolerant EN patients % % Ventilator- Time to Calorifi c Protein free 60-day ICU stay discharge adequacy adequacy days mortality (days) alive Tolerant 64.3 63.7 11.2 26.2 11.3 25.2 Intolerant 55.5 55.6 2.5 30.8 14.4 31.1 Conclusion Intolerance is common amongst the EN ICU population and is associated with poor nutritional and clinical outcomes. Table 1 (abstract P161). Nutritional adequacy and clinical endpoints in tolerant and intolerant EN patients Introduction Impaired gastrointestinal motility is common in critically ill patients. Multiple conditions such as shock with diminished splanchnic perfusion, surgery, fl uid overload, intra-abdominal hypertension, and drugs are responsible for this phenomena. Assessing gastric motility in this setting is complex. Surface electrogastrography (sEGG) is a recent noninvasive technique that determines basal and postprandial gastric motility [1]. Our aim is to study basal gastric motility in critically ill septic patients in the post-resuscitative phase, by sEGG. y Methods Eligible patients were those admitted to the ICU with diagnosis of septic shock as stated by the Sepsis Conference 2001 [2]. At the moment of the study the patients were in the post-resuscitative phase, defi ned as normal clinical and laboratory perfusion parameters. sEGG is a noninvasive technique that uses skin abdominal electrodes to record myoelectrical stomach activity. The basal slow wave originates in the proximal stomach and propagates to the antrum with a frequency of approximately 3 cycles per minute (cpm). Basal activity below 2.4 cpm is defi ned as bradygastria and above 3.7 cpm as tachygastria [1]. Data were correlated with severity scores, lactate levels, and doses of sedatives. The study was approved by the Ethics Committee of the Hospital Clínico Universidad de Chile. P162 Gastric emptying assessment in critically ill patients with feed intolerance; comparison of 13C octanoic acid, paracetamol and 3-O-methylglucose absorption tests M Chapman1, R Fraser1, N Nguyen1, A Deane1, LS Vasist2, K Hacquoil2, M Barton2, GE Dukes2 1University of Adelaide, Australia; 2GlaxoSmithKline, Research Triangle Park, NC, USA Critical Care 2012, 16(Suppl 1):P162 (doi: 10.1186/cc10769) 1University of Adelaide, Australia; 2GlaxoSmithKline, Research Triangle Park, NC, USA Results We recruited 16 patients (10 females). 1. Chang F-Y: J Gastroenterol Hepatol 2005, 20:502-516. Frequency, determinants and impact of feed intolerance amongst the critically ill Frequency, determinants and impact of feed intolerance amongst the critically ill y U Gungabissoon1, K Hacquoil1, C Bains1, G Dukes2, M Irizarry3, D Heyland3 1GlaxoSmithKline, Stockley Park, UK; 2GlaxoSmithKline, Research Triangle Park, NC, USA; 3CERU, Queen’s University, Kingston, Canada Critical Care 2012, 16(Suppl 1):P161 (doi: 10.1186/cc10768) y U Gungabissoon1, K Hacquoil1, C Bains1, G Dukes2, M Irizarry3, D Heyland3 1GlaxoSmithKline, Stockley Park, UK; 2GlaxoSmithKline, Research Triangle Park, NC, USA; 3CERU, Queen’s University, Kingston, Canada Critical Care 2012, 16(Suppl 1):P161 (doi: 10.1186/cc10768) Introduction Provision of early and adequate enteral nutrition (EN) to critically ill patients is associated with improved clinical outcomes; however, 50 to 60% of prescribed EN is received. We aimed to characterise the incidence and determinants of intolerance and assess its infl uence on nutritional and clinical outcomes using the 2009 Critical Care Nutrition Survey (CCNS). y Methods The CCNS survey is a prospective observational cohort study of nutrition practices from over 150 ICUs around the world. Included patients were those that remained in ICU for ≥72 hours and were mechanically ventilated ≤48 hours of admission to ICU. We collected pertinent baseline and outcome data that included nutritional adequacy, ventilator-free days, 60-day mortality and ICU stay. Intolerance was defi ned as interruption of EN due to gastrointestinal (GI) reasons (high gastric residuals, increased abdominal girth/ abdominal distension, vomiting/emesis, diarrhoea or subjective discomfort). In the analysis of intolerance we included each potential eff ect into a logistic regression analysis to determine its signifi cance. g y Conclusion Diarrhoea was common on our ICU, its prevalence (17%) being consistent with established literature. It was associated with statistically increased ICU LOS and mortality, although any direction of causality remains to be established. A low stool investigation yield and low prevalence of C. diffi cile suggests that other noninfective causes of diarrhoea need excluding. Further research is required to establish the prevalence and pathogenesis of diarrhoea on UK ICUs, in order to develop evidence-based management plans for reducing its incidence, and its clinical and fi nancial impact. y Methods The CCNS survey is a prospective observational cohort study of nutrition practices from over 150 ICUs around the world. Included patients were those that remained in ICU for ≥72 hours and were mechanically ventilated ≤48 hours of admission to ICU. We collected pertinent baseline and outcome data that included nutritional adequacy, ventilator-free days, 60-day mortality and ICU stay. 1. Chang F-Y: J Gastroenterol Hepatol 2005, 20:502-516. 2 Levy MM et al : Crit Care Med 2003 31:1250-1256 References S57 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Of 782 patients (mean age ± 2SD 62.1 ± 37.1, 52.3% female) treated on our ICU, 334 stool samples were sent from 133 (17.0%) patients. Two samples (0.6%) yielded abnormal results: one out of 131 (0.8%) patients with CDT samples sent and one out of 108 (0.9%) patients with stool microscopy samples sent had a positive sample. The prevalence of C. diffi cile (1/782) and other organisms (1/782) was 0.1% and 0.1% respectively. In terms of diagnostic yields, positive fi ndings were found in one out of 191 (0.5%) CDT samples and one out of 141 (0.7%) stool microscopy samples (for Candida). When compared to patients without diarrhoea, suff erers were older (64.1 ± 33.2 vs. 61.7 ± 37.8 years, P = 0.16) with greater female preponderance (55.6% vs. 51.6%, P = 0.40). Suff erers experienced longer ICU LOS (16.3 ± 45.6 vs. 4.6 ± 19.4 days, P <0.0001) and greater ICU mortality (19.5% vs. 12.6%, P = 0.035) during the study period. Reference 1. Wiesen P, et al.: Curr Opin Crit Care 2006, 12:149-154. f g g y gi Results Data from 1,888 ICU patients receiving EN were analysed. The incidence of intolerance was 30.5%, and occurred after a median 3 days from EN initiation. Factors associated with intolerance were: diagnosis category (P = 0.0009) (GI, cardiovascular and sepsis categories with the highest risk), pre-emptive motility agent use (P = 0.0125), non- GI interruptions to feed (P = 0.0086) and global region (P = 0.0006). Intolerance was associated with poor nutritional adequacy, increased mortality, longer ventilator dependence and increased length of ICU stay (P <0.05) (Table 1). Poorer clinical outcomes were seen with increasing number of days of intolerance. 2. Levy MM, et al.: Crit Care Med 2003, 31:1250-1256. Frequency, determinants and impact of feed intolerance amongst the critically ill Intolerance was defi ned as interruption of EN due to gastrointestinal (GI) reasons (high gastric residuals, increased abdominal girth/ abdominal distension, vomiting/emesis, diarrhoea or subjective discomfort). In the analysis of intolerance we included each potential eff ect into a logistic regression analysis to determine its signifi cance. Results Data from 1,888 ICU patients receiving EN were analysed. The incidence of intolerance was 30.5%, and occurred after a median 3 days from EN initiation. Factors associated with intolerance were: diagnosis category (P = 0.0009) (GI, cardiovascular and sepsis categories with the highest risk), pre-emptive motility agent use (P = 0.0125), non- GI interruptions to feed (P = 0.0086) and global region (P = 0.0006). Intolerance was associated with poor nutritional adequacy, increased mortality, longer ventilator dependence and increased length of ICU stay (P <0.05) (Table 1). Poorer clinical outcomes were seen with increasing number of days of intolerance. Reference 1. Wiesen P, et al.: Curr Opin Crit Care 2006, 12:149-154. P160 Mean age 62 years (50 to 76) (P = 0.8). APACHE II score 25 (19 to 28) (P = 0.4) and SOFA score 9 (7 to 11) (P = 0.29). Lactate at admission 3.8 mmol/l (1.2 to 6.5) (P = 0.72). Fentanyl total dose 172.7 μg/kg (59 to 256.6) (P = 0.91) and midazolan total dose 3.4 mg/kg (0.1 to 3.1) (P = 0.07). We obtained a reliable register in all the patients and found six patients with bradigastria, three with tachygastria and nine with normal motility. In this small sample size study there was a trend to bradygastria in relation to high total doses of midazolam. P164 Preliminary experience with ketone-targeted treatment of diabetic ketoacidosis F Riccio, T Drake, S Mathieu, J Cranshaw Royal Bournemouth Hospital, Bournemouth, UK Critical Care 2012, 16(Suppl 1):P164 (doi: 10.1186/cc10771) Introduction In May 2011 the Joint British Diabetes Societies (JBDS) published new guidance for managing adult diabetic ketoacidosis. We developed a JBDS-based protocol that measured and treated capillary ketonaemia (not blood glucose) hourly with i.v. 0.1 IU insulin/kg/hour increased by 1 IU/hour if the ketone reduction was <0.5 mM/hour. The fi nal target was capillary ketonaemia <0.3 mM. To allow this insulin rate and avoid hypoglycaemia, 125 ml/hour of 10 or 20% dextrose was started when blood glucose was <14 mM (250 mg/dl). As the eff ects of this new protocol were unknown, all patients were managed in our high-dependency unit (HDU). We report our experience of the new protocol compared to our old ‘sliding scale’ insulin titration to blood glucose protocol. Table 1 (abstract P162). Correlations between GE parameters 13C GEC OMG AUC60 0.675 13C GEC OMG C60 0.758* 13C GEC PA AUC60 0.678* 13C GEC PA C60 0.590* PA AUC60 OMG AUC60 0.534* PA C60 OMG C60 0.553* P <0.0001. Table 1 (abstract P162). Correlations between GE parameters Table 1 (abstract P162). Correlations between GE parameters Methods We prospectively gathered results of the new protocol over 3 months and performed a chart review of the same results from patients admitted in the previous year managed on wards and the HDU. Results are expressed as median (range). Results Patients on the new protocol (n = 7) cleared ketones to <0.3 mM in 8 (7 to 20) hours. The insulin rate needed was 10 (6 to 17) IU/hour. Potassium during treatment was 4 (3.2 to 5.2) mM and required 35 (12 to 60) mM/hour to maintain the target 3.5 to 5 mM. No episodes of blood glucose <4 mM were recorded. Time to reach glucose <14 mM was 7 (1 to 15) hours with a fall rate of 3.7 (2.9 to 6.8) mM/hour. Patients on the old protocol (n = 39) were treated for 15 (5 to 20) hours with 3 (0.5 to 6) IU/hour. Potassium during treatment was 3.5 (2.8 to 5.5) mM and required 9 (6 to 16) mM/hour to maintain the target 3.5 to 5 mM. Time to reach glucose <14 mM was 3.5 (1 to 13) hours with a fall rate of 4.2 (0.3 to 13) mM/hour. Critical Care 2012, 16(Suppl 1):P162 (doi: 10.1186/cc10769) Introduction Delayed gastric emptying (GE) occurs frequently in critically ill patients and may result in impaired small intestinal delivery of drugs and nutrients. Use of direct methods of GE assessment (scintigraphy) in the ICU for clinical monitoring or research is challeng- ing. Indirect methods that utilize substances which rely on eff ective GE and rapid absorption from the small intestine off er a feasible estimate of GE. Three substances with these characteristics are 13C-octanoic acid (13C), paracetamol (PA) and 3-O-methylglucose (OMG). We have previously shown signifi cant correlation to scintigraphy for 13C (r = 0.63) and OMG absorption (r = –0.77 to –0.87). The current study examined the relationship between three indirect methods of GE assessment: 13C, PA, OMG. Conclusion sEGG is a feasible technique in critically ill septic patients. In the post-resuscitative phase 43.8% of patients present normal gastric motility, and 37% showed bradygastria. Future research is warranted in order to fi nd risk factors of gastrointestinal dismotility. References 1. Chang F-Y: J Gastroenterol Hepatol 2005, 20:502-516. 2. Levy MM, et al.: Crit Care Med 2003, 31:1250-1256. S58 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods GE was concurrently assessed in mechanically ventilated patients (n = 33) with enteral feeding intolerance (gastric residual volume >200 ml) on two occasions 24 hours apart. A test meal of 100 ml Ensure with 100 μl 13C, 1,000 mg PA, 3 g OMG was infused into the stomach over 5 minutes. Breath samples for 13C and plasma samples for PA, OMG determination were collected over the subsequent 4-hour period. Bivariate Pearson correlations were calculated between the following parameters; 13C (gastric emptying coeffi cient (GEC)), PA (concentration at 60 minutes (C60), AUC0–60 min), OMG ((C60), AUC0–60 min). See Table 1. Impact of blood glucose on blood lactate levels in a medical ICU: a retrospective cohort study G Adelsmayr, R Brunner, U Holzinger Medical University Vienna, Austria Critical Care 2012, 16(Suppl 1):P165 (doi: 10.1186/cc10772) Results In the 556 study patients, early enteral feeding was started in 379 patients (68.16%). Out of 379 patients, 100% calorie requirements were met only in 43 patients (7.73%). For the remaining study patients, more than 40%, 50% and 60% calorie goals were achieved in 115 (30.34%), 128 (33.77) and 93 (24.53%) patients respectively.f Introduction Although blood lactate and glucose both represent important markers in the intensive care setting, they have been considered quite independently. Especially, the ideal glucose target range has been the topic of recent studies with confl icting results [1,2]. Blood lactate is an acknowledged predictor of outcome in critically ill patients [3]. The aim of this study was to establish a possible correlation between elevated blood glucose and lactate levels in intensive care patients. p p y Conclusion The initiation of early enteral feeding is still far off for a signifi cant proportion of the ICU population despite evidence-based defi nite recommendations to improve ICU outcome. The calorie goal achievements were also very suboptimal. This important but still neglected nutritional therapy must be carefully looked at and implemented in all ICUs. P164 Preliminary experience with ketone-targeted treatment of diabetic ketoacidosis A total 0.02 results per hour <4 mM were recorded in the old protocol. Results Observed GE rates for all parameters were across the spectrum from fast to delayed (for example, with r range = 0.534 to 0.758, P <0.0001). ) Conclusion These three practical indirect methods of GE assessment may provide similar estimates of GE in critically ill patients. P163 Early enteral nutrition in the critically ill: a single-centre study A Gupta, E Rupert, R Sharma, D Mishra Rabindranath Tagore Hospital, Kolkata, India Critical Care 2012, 16(Suppl 1):P163 (doi: 10.1186/cc10770) P163 Early enteral nutrition in the critically ill: a single-centre study A Gupta, E Rupert, R Sharma, D Mishra Rabindranath Tagore Hospital, Kolkata, India Critical Care 2012, 16(Suppl 1):P163 (doi: 10.1186/cc10770) Conclusion The median insulin infusion rate in an individual patient and the range required to suppress capillary ketonaemia in all patients with diabetic ketoacidosis using this protocol was more than three times that in the old protocol and the amount of i.v. potassium required to maintain near-normal blood potassium during treatment was four times more. There was a slower correction of initial blood glucose. Blood glucose and potassium maintenance during treatment with this protocol would appear to require high-intensity nursing care to maintain patient safety. Introduction Early enteral nutrition in critically ill patients has been established as a valuable addition to improve overall outcome and mortality. The recommendation is to initiate feeding within 24 to 48 hours of admission and to meet the calorie goal within the next 48 to 72 hours. The purpose of this study was to fi nd out whether these guidelines are followed in the ICU as per the new protocol. 2. Kreyman KG, Berger MM, Deutz NE, et al.: ESPEN guidelines on enteral nutrition: intensive care. Clin Nutr 2006, 25:210-223. Reference Methods This is a prospective observational study done in a 32-bed mixed medical and surgical ICU over the period from March 2011 to August 2011. Consecutively, 575 patients admitted to this ICU were followed up. Nineteen patients were excluded from the study where enteral nutrition could not be commenced within 48 hours due to various reasons. The remaining 556 patients’ data were analyzed. Data were collected by interviewing the doctors and nurses as well as reviewing medical notes and all ICU charts. 1. Joint British Societies Guideline for the management of diabetic ketoacidosis. Diabet Med 2011, 28:508-515. Variability of insulin sensitivity during the fi rst 4 days of critical illness C Pretty1, A Le Compte1, JG Chase1, G Shaw2, JC Preiser3, S Penning4, T Desaive4 Methods We performed a retrospective study in 2,943 adult patients admitted to our ICU from 2004 until 2010. Glucose variability was calculated for all subjects as the LI [3] during the hospital stay on capillary, arterial and venous blood. The ROC curve was performed to verify discrimination of the LI towards mortality and ICU infections. 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3Hospital Erasme, Free University of Brussels, Belgium; 4University of Liege, Belgium Critical Care 2012, 16(Suppl 1):P167 (doi: 10.1186/cc10774) 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3Hospital Erasme, Free University of Brussels, Belgium; 4University of Liege, Belgium Critical Care 2012, 16(Suppl 1):P167 (doi: 10.1186/cc10774) y y Results There were 709 infections and 447 deaths. There was a signifi cant interaction between the LI and infections in patients. The LI had a great ability to predict hospital mortality (area under the curve = 0.62, 95% CI = 0.59 to 0.65, P <0.5; Figure 1) but moreover infections (area under the curve = 0.80, 95% CI = 0.78 to 0.82, P <0.5; Figure 2). Introduction Safe, eff ective tight glycaemic control (TGC) can improve outcomes in critical care patients, but is diffi cult to achieve consistently. Insulin sensitivity defi nes the metabolic balance between insulin concentration and insulin-mediated glucose disposal. Hence, variability of insulin sensitivity can cause variable glycaemia. This study investigates the evolution of insulin sensitivity level and variability in patients receiving TGC during the fi rst 4 days of their ICU stay. g Conclusion Glucose variability has ability to predict outcome but moreover infections in patients in the ICU, because it is a predictor of clinical outcomes in patients with hyperglycemia and diabetes. i y y Methods A retrospective analysis of patient data (n = 164 patients) from the SPRINT TGC study in the Christchurch Hospital ICU [1]. All patients commenced TGC within 12 hours of ICU admission and spent at least 24 hours on the SPRINT protocol. Model-based insulin sensitivity (SI) was identifi ed using a validated glucose–insulin system model developed for critical care patients. SI was identifi ed every hour for each patient using clinical data and the model. Level and hour-to- hour percentage changes in SI were assessed on cohort and per-patient bases. p References p Methods Blood gas data of 1,170 patients, admitted to the medical ICU of the Department of Medicine III, Medical University Vienna, between the years 2001 and 2009, were analysed retrospectively. The association of circulating blood glucose levels with corresponding lactate levels was investigated using a linear regression model. The impact of diff erent blood glucose intervals (<80, 80 to 120, 120 to 160, 160 to 200, >200 mg/dl) on blood lactate levels was analysed using ANOVA. The infl uence of blood glucose variability, expressed as the 1. McClave SA, Martindale RG, Vanek VW, et al.: Guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine (SCCM) and Americal Society for Parenteral and Enteral Nutrition (A.S.P.E.N.). JPEN J Parenter Enteral Nutr 2009, 33:277-316. 1. McClave SA, Martindale RG, Vanek VW, et al.: Guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine (SCCM) and Americal Society for Parenteral and Enteral Nutrition (A.S.P.E.N.). JPEN J Parenter Enteral Nutr 2009, 33:277-316. 2. Kreyman KG, Berger MM, Deutz NE, et al.: ESPEN guidelines on enteral nutrition: intensive care. Clin Nutr 2006, 25:210-223. 2. Kreyman KG, Berger MM, Deutz NE, et al.: ESPEN guidelines on enteral nutrition: intensive care. Clin Nutr 2006, 25:210-223. S59 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Strategies to reduce glucose variability should be studied to improve the outcomes in ICU patients. References 1. N Engl J Med 2001, 345:1359. 2. Crit Care Med 2008, 36:2316. 3. Diabetes 2004, 53:955. Figure 2 (abstract P166). blood glucose standard deviation, on mean lactate concentrations for the period of ICU stay was analysed using a linear regression model. To adjust for the severity of illness, a multivariate regression analysis was conducted including SAPS II and APACHE II scores. Figure 2 (abstract P166). g Results Blood glucose and lactate presented a U-shaped curve with a minimum blood lactate (1.5 mmol/l) between 80 and 120 mg/dl blood glucose. ANOVA and linear regression demonstrated a signifi cant infl uence of blood glucose and blood glucose variability on blood lactate (P = 0.0001). The identifi cation of this relation was supported by the result of a multivariate regression analysis, adjusting for severity of illness (P = 0.0001). P167 Introduction Hyperglycemia and glucose variability are important factors associated with morbidity and mortality in critically ill patients [1,2]. Our objective was to determine the association between the glucose Lability Index (LI), infections and outcome in critical illness. Methods We performed a retrospective study in 2,943 adult patients admitted to our ICU from 2004 until 2010. Glucose variability was calculated for all subjects as the LI [3] during the hospital stay on capillary, arterial and venous blood. The ROC curve was performed to verify discrimination of the LI towards mortality and ICU infections. Results There were 709 infections and 447 deaths. There was a signifi cant interaction between the LI and infections in patients. The LI had a great ability to predict hospital mortality (area under the curve = 0.62, 95% CI = 0.59 to 0.65, P <0.5; Figure 1) but moreover infections (area under the curve = 0.80, 95% CI = 0.78 to 0.82, P <0.5; Figure 2). Relationship between glycemic Lability Index, infections and outcome in critically ill patients Relationship between glycemic Lability Index, infections and outcome in critically ill patients A Donati, L Botticelli, R Castagnani, V Gabbanelli, E Damiani, R Domizi, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2012, 16(Suppl 1):P166 (doi: 10.1186/cc10773) References 1. N Engl J Med 2001, 345:1359. 2. Crit Care Med 2008, 36:2316. 3. Diabetes 2004, 53:955. Introduction Hyperglycemia and glucose variability are important factors associated with morbidity and mortality in critically ill patients [1,2]. Our objective was to determine the association between the glucose Lability Index (LI), infections and outcome in critical illness. , References 1. van den Berghe G, et al.: Intensive insulin therapy in the critically ill patients. N Engl J Med 2001, 345:1359-1367. 2. Finfer S, et al.: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283-1297. 3. Khosravani H, et al.: Occurrence and adverse eff ect on outcome of hyperlactatemia in the critically ill. Crit Care 2009, 13:R90. Strategies to reduce glucose variability should be studied to improve the outcomes in ICU patients. Strategies to reduce glucose variability should be studied to improve the outcomes in ICU patients. References 1. N Engl J Med 2001, 345:1359. 2. Crit Care Med 2008, 36:2316. 3. Diabetes 2004, 53:955. p References Conclusion The results demonstrate an infl uence of blood glucose and blood glucose variability on blood lactate, independent of severity of illness, in a medical ICU patient population. Variability of insulin sensitivity during the fi rst 4 days of critical illness Level and variability of SI were compared over time on 24-hour and 6-hour timescales for the fi rst 4 days of the ICU stay. Figure 1 (abstract P166). i y y Results Cohort and per-patient median SI levels increased by 34% and 33% (P <0.001) between days 1 and 2 of the ICU stay. Concomitantly, cohort and per-patient SI variability reduced by 32% and 36% (P <0.001). For 72% of the cohort, median SI on day 2 was higher than day 1. The day 1 and 2 results were the only clear, statistically signifi cant trends across both analyses. Analysis of the fi rst 24 hours using 6-hour blocks of SI data showed that most of the improvement in insulin sensitivity level and variability seen between days 1 and 2 occurred during the fi rst 12 to 18 hours of day 1. This rapid improvement was probably due to the decline of counterregulatory hormones as the acute phase of critical illness progressed.i Conclusion ICU patients have signifi cantly lower and more variable insulin sensitivity on day 1 than later in their ICU stay and particularly during the fi rst 12 hours. Clinically, these results suggest that while using Figure 1 (abstract P166). S60 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 TGC protocols with patients during their fi rst few days of ICU stay, extra care should be aff orded. Increased measurement frequency, higher target glycaemic bands, conservative insulin dosing and modulation of carbohydrate nutrition should be considered to safely minimize outcome glycaemic variability and reduce the risk of hypoglycaemia. Reference Methods We used the Eirus™ system (DipylonMedical, Solna, Sweden) based on microdialysis for CGM in three mechanically ventilated patients necessitating continuous intravenous insulin. The CGM system consists of a dedicated triple-lumen central venous catheter, a disposable sensor on a reusable sensor holder outside the patient, and a monitor producing a new measurement each minute. Calibrations were performed every 8 hours using arterial blood samples and a blood gas analyzer (Radiometer SAS, Neuilly-Plaisance, France). The attending nurses also performed intermittent blood glucose measurements with a point-of-care glucometer in order to set the insulin rate according to our glucose control method. Evaluation of a continuous blood glucose monitoring system using a central venous catheter with an integrated microdialysis function F Möller, J Liska, A Öwall, A Franco-Cereceda Evaluation of a continuous blood glucose monitoring system using a central venous catheter with an integrated microdialysis function F Möller, J Liska, A Öwall, A Franco-Cereceda Karolinska Institutet, Solna, Sweden Critical Care 2012, 16(Suppl 1):P170 (doi: 10.1186/cc10777) Introduction Glycemic control in critically ill patients has been debated over the last decade. An accurate glucose monitoring system is essential to understand and study this concern. We have evaluated the accuracy and technical feasibility of a continuous glucose monitoring system using intravascular microdialysis. p Results The best model for insulin secretion was based on blood glucose concentration alone. There was clear separation of secretion levels between normal glucose tolerant (NGT) and impaired glucose tolerant (IGT) patients. Hence, ISR was modeled as a constrained linear function of BG (in mmol/l) for NGT and IGT patients separately with R2 = 0.61 and 0.69 respectively. NGT: ISR = 893×BG – 2,996 (mU/hour). IGT: ISR = 296×BG – 1,644 (mU/hour). The glucose coeffi cients of 893 and 296 mU.l/mmol.hour were comparable to data published in a number of other studies for healthy and diabetic subjects. Methods Thirty patients undergoing cardiac surgery were monitored using a triple-lumen central venous catheter (Eirus TLC®; Dipylon Medical AB, Sweden) with an integrated microdialysis membrane. The catheter was placed with the tip in the superior vena cava, and functions both as a central venous catheter, enabling blood sampling and administration of medication, while simultaneously measuring Figure 1 (abstract P170). Clarke error grid analysis. Conclusion This work presents a simple model for pancreatic insulin secretion in critically ill patients based on clinical data. The model is a function of blood glucose level and glucose tolerance status and compares well with published data for healthy and diabetic subjects. This model can be incorporated into glucose–insulin system models and could potentially improve model-based glycaemic control. References 1. Chase JG, et al.: Crit Care 2008, 12:R49. 2. Van Cauter E, et al.: Diabetes 1992, 41:368-377. 3. Kjems L, et al.: Diabetes 2003, 52:380-386. Variability of insulin sensitivity during the fi rst 4 days of critical illness GV was assessed by the variability index defi ned as the mean of the absolute value of the fi rst derivative of the glucose signal during CGM. In order to simulate sCGM, we extracted CGM values every 15 minutes for calculating the corresponding variability index as the mean of the absolute value of the variation rate between two consecutive measurements. 1. Chase JG, et al.: Crit Care 2008, 12:R49. Endogenous insulin secretion in critically ill patients Endogenous insulin secretion in critically ill patients C Pretty1, A Le Compte1, J Lin1, G Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P168 (doi: 10.1186/cc10775) y p 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand y p 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Introduction Glucose–insulin system models can be used for improved glycemic control of critically ill patients. A key component of glucose– insulin models is pancreatic insulin secretion. There are limited data in the literature quantifying insulin secretion in critically ill patients at physiologic levels. This study presents a model of pancreatic insulin secretion in critically ill patients based on data from a critically ill population. Results The variability indexes were respectively 1.97, 1.65, 1.55 mmol/l/ hour for CGMS, and 1.07, 0.65, 0.83 mmol/l/hour for sCGMS. Conclusion sCGM in comparison with CGM may considerably under- estimate a marker of GV during glucose control in critical care patients. Reference 1. Mackenzie et al.: The metrics of glycaemic control in critical care. Intensive Care Med 2011, 37:435-443. p p Methods Samples were collected from 19 patients enrolled in a prospective clinical trial studying sepsis at the Christchurch Hospital ICU. Fifteen of the patients had confi rmed sepsis and three were diagnosed type 2 diabetics. All patients were on the SPRINT glycaemic control protocol [1]. Each patient had arterial blood samples assayed for insulin and C-peptide. Two sets of four samples were taken from each patient, with each set collected over 60 minutes. Blood glucose (BG) data were collected with a bedside glucometer. C-peptide data were deconvolved using the model and population parameter values of van Cauter and colleagues [2] to determine pancreatic insulin secretion rates (ISRs). Data from Kjems and colleagues investigating the potentiating eff ects of glucagon-like peptide-1 on insulin secretion [3] suggested a maximum secretion rate of 16 U/hour. A minimum rate of 1 U/hour was also adopted. Pilot trial of STAR in the medical ICU LM Fi k1 AJ L C 1 GM Sh 2 S P LM Fisk1, AJ Le Compte1, GM Shaw2, S Penning3, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3Université de Liege, Belgium Critical Care 2012, 16(Suppl 1):P172 (doi: 10.1186/cc10779) Introduction Medical ICU patients often develop stress-induced hyperglycemia. Regulating blood glucose (BG) levels in these patients using insulin can be diffi cult due to varying patient conditions and therapy, leading to increased risk of hypoglycaemia. This abstract describes a pilot trial of STAR, a computerised risk-management accurate glycemic control (AGC) protocol. g y p Methods Thirteen hyperglycaemic patients (BG >145 mg/dl) were consented from Christchurch Hospital ICU. The BG target range was 80 to 145 mg/dl or 108 to 162 mg/dl (chosen clinically). Model-based insulin sensitivity was calculated for every measurement and its variability for the next 1 to 3 hours forecast using stochastic models. These data and model were used to calculate new insulin/nutrition interventions for the next 1 to 3 hours, limiting risk of BG <80 mg/dl to a maximum of 5%. Nursing staff selected the BG measurement interval to manage workload. Insulin was delivered as boluses (max 6 U/hour; max increase +2U/intervention), with infusions up to 3 U/hour for highly resistant patients. Nutrition was 30 to 100% of clinical goal feed (max change ±30% per intervention) and constant rates were used when desired clinically. Limiting insulin/nutrition changes prevents over-response to erroneous BG measurements and results were resampled hourly for consistency. Approval was granted by the Upper South A Regional Ethics Committee (Christchurch, New Zealand). Figure 2 (abstract P170). Bland–Altman analysis. glucose. The patients were monitored for up to 48 hours postoperatively in the ICU. As reference, arterial blood samples were taken every hour. Results Data were available from all 30 patients. A total of 725 paired (arterial blood gas–microdialysis) samples were obtained. Glucose correlation coeffi cient was 0.87. Using Clarke error grid analysis, 100% of the paired samples were in region AB and 97.4% in region A (Figure 1). Mean glucose level was 8.6 mmol/l, bias –1.3% and mean absolute relative diff erence was 4.8%. A total 97.5% of the paired samples were correct according to ISO criteria. Bland–Altman analysis showed bias ± limits of agreement –0.11 ± 1.3 mmol/l (Figure 2). g Results Median BG was 109 mg/dl for 80 to 145 mg/dl target patients and 145 mg/dl for 108 to 162 mg/dl target patients. Impact of the type of glucose monitoring on the assessment of glycemic variability in critical care patients P Kalfon, M Chilles CH Chartres, France Critical Care 2012, 16(Suppl 1):P169 (doi: 10.1186/cc10776) P Kalfon, M Chilles CH Chartres, France Critical Care 2012, 16(Suppl 1):P169 (doi: 10.1186/cc10776) Introduction While minimizing glycemic variability (GV) during glucose control in critical care patients could become a new therapeutic goal, it is important to have a reliable assessment of GV. The aim of the study is to compare a real continuous glucose monitoring (CGM) system in comparison with a semi-continuous glucose monitoring (sCGM) system, with respect to the reliability of a marker of GV. Figure 1 (abstract P170). Clarke error grid analysis. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S61 variable and maximum BG levels was considered to link to better outcome. (3) On the other hand, part of the severe patients seemed to have the lowest of those variable and maximum BG levels, judging from two-dimensional or parabolic correlations between those BG parameters and SOFA scores. Figure 2 (abstract P170). Bland–Altman analysis. P172 P172 Pilot trial of STAR in the medical ICU LM Fisk1, AJ Le Compte1, GM Shaw2, S Penning3, T Desaive3, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand; 3Université de Liege, Belgium Critical Care 2012, 16(Suppl 1):P172 (doi: 10.1186/cc10779) Pilot trial of STAR in the medical ICU LM Fi k1 AJ L C 1 GM Sh 2 S P In total, 85.6% of time was in the specifi ed target band, with 1.18% of BG <72 mg/dl and 2.41% BG <80 mg/dl. BG measurement frequency was 13.3 measures/ day. Per-patient median carbohydrate intake was 10.7 g/hour (IQR: 4.0 to 11.9 g/hour) and median insulin usage was 2.5 U/hour (IQR: 1.75 to 3.5 U/hour). Requirements varied considerably by patient. Observed response to insulin varied by a factor of 14× between patients. Accurate control was maintained over a range of metabolic conditions, and STAR adapted safely to therapies including high-dose steroids, long-acting insulin (Glargine) and changing insulin response. Conclusion Central venous microdialysis is an accurate and reliable method for continuous blood glucose monitoring in critically patients. P171 P171 Variable and maximum blood glucose levels during the fi rst week after ICU admission are related to the severity of the patients M Hoshino1, Y Haraguchi2, K Oda1, S Kajiwara1 1Shisei Hospital, Saitama, Japan; 2Geriatric Health Services Facility, Kokubunnji-shi, Japan Critical Care 2012, 16(Suppl 1):P171 (doi: 10.1186/cc10778) Conclusion STAR provided AGC in a clinical setting. Tight and accurate control was able to be extended to patients with a range of metabolic requirements, and the risk-management approach proved capable of balancing clinical workload and risks presented by patient variability. Introduction Signifi cance of blood glucose (BG) control in ICU patients, especially in terms of mortality reduction, has been recognized in recent years. However, the relationship between BG profi le and the severity, as well as BG target, is not clearly elucidated. A preliminary study was performed in order to clarify the relationship between BG profi le and the severity of ICU patients. Glucometer accuracy and implications for clinical studies AJ Le Compte1, CG Pretty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P173 (doi: 10.1186/cc10780) P174 Initial experience with continuous intra-arterial fl uorescent glucose monitoring in patients in the ICU following cardiac surgery S Bird1, L Macken1, O Flower1, E Yarad1, F Bass1, N Hammond1, D LaCour2, P Strasma2, S Finfer1 1Royal North Shore Hospital, St Leonards, NSW, Australia; 2GluMetrics, Inc., Irvine, CA, USA Critical Care 2012, 16(Suppl 1):P174 (doi: 10.1186/cc10781) Introduction A need for continuous blood glucose monitoring has always been expressed by critical care practitioners. The results from several iterations of a novel optical fl uorescence-based intravascular blood glucose sensor were examined for correlation with an accepted laboratory assay. Ever since Van Den Berghe’s group demonstrated reductions in hospital mortality and morbidity from the application of tight glycaemic control [1], many groups have attempted to replicate those results with limited success. Practitioners have speculated upon the reasons behind this observation, and have cited manpower implications and incidence of hypoglycaemic episodes as contributing factors [2]. Investigators have speculated that a continuous blood glucose sensor might contribute towards safe eff ective glycaemic control [3]. Critical Care 2012, 16(Suppl 1):P174 (doi: 10.1186/cc10781) Introduction Continuous glucose monitoring (CGM) in ICUs has the potential to improve patient safety and outcomes. The GluCath Intravascular CGM System uses a novel quenched chemical fl uorescence sensing mechanism to measure glucose concentration (BG) in venous or arterial blood. This is the fi rst report of its use in cardiac surgery patients. p Methods This ongoing clinical study is evaluating the system deployed via a standard 20G radial artery catheter inserted for routine care in 20 patients undergoing cardiac surgery. Data are presented from fi ve run-in patients. Outcome measures are qualitative (ease-of-use, workfl ow fi t) and quantitative (accuracy vs. reference analyzer). Sensors were inserted shortly after ICU admission with placement confi rmed by ultrasound and in vivo calibration 30 minutes later. Clinical staff managed blood glucose according to usual protocols. Glucose values were recorded each minute for 24 hours; hourly reference samples from the same arterial catheter were analyzed on a Radiometer ABL Blood Gas Analyzer.i Methods A series of postoperative and direct admission ICU patients had an optical fl uorescence-based intravascular glucose sensor (GlySure Ltd, Abingdon, UK) placed into the left internal jugular vein on admission to the ICU. The sensor remained in situ throughout the ICU stay. Periodic blood samples and simultaneous real-time values of blood glucose measured by the sensor were recorded. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 patient. The defi ned cut-off for ‘good’ control for a patient was ≥70% of BG in 72 to 126 mg/dl (cTIB ≥0.7), and ‘poor’ as <70% (cTIB <0.7), based on original observed clinical BG. The number of true BG profi les that resulted in misclassifi cation between ‘good’ and ‘poor’ control for a patient was recorded over all Monte-Carlo runs. The maximum change in true and observed BG mean and standard deviation were used to evaluate potential worst-case scenarios. Figure 1 (abstract P174). ISO-modifi ed Bland–Altman plot. Results Good control was clinically measured in 76% of patients (24% with cTIB <0.7). Of these, 83% of ‘good’ and 64% of ‘poor’ control would never be misclassifi ed over all 100 runs due to sensor error. A total of 91% (good) and 87.5% (poor) could be misclassifi ed 10% of the time. Patients with cTIB near 0.7 were more likely to be misclassifi ed when accounting for glucometer error. Hence, a deadband around the cut-off would reduce this misclassifi cation. If ‘good’ cut-off was cTIB ≥0.5 (95% of clinical patients) then 100% correct classifi cation was 97% for good control patients, but fell to 40% of poor control patients. The median largest diff erence in observed and true mean BG across patients was –54 mg/dl (90th percentile: –21 mg/dl) and the standard deviation was 3.2 mg/dl (90th percentile: 1.8 mg/dl). g pi Patients with cTIB near 0.7 were more likely to be misclassifi ed when accounting for glucometer error. Hence, a deadband around the cut-off would reduce this misclassifi cation. If ‘good’ cut-off was cTIB ≥0.5 (95% of clinical patients) then 100% correct classifi cation was 97% for good control patients, but fell to 40% of poor control patients. The median largest diff erence in observed and true mean BG across patients was –54 mg/dl (90th percentile: –21 mg/dl) and the standard deviation was 3.2 mg/dl (90th percentile: 1.8 mg/dl). Figure 1 (abstract P174). ISO-modifi ed Bland–Altman plot. g g Conclusion Glucometers can distinguish between patients that received good and poor BG control but risk of misclassifi cation rises for patients nearer cut-off s. P174 The results were correlated with the results of blood sample analysed by a Yellow Springs Instrument glucose analyser. The sensor, which has a heparin coating on its surface, required no further heparinisation; a ‘keep vein open’ rate of normal saline infusion was maintained throughout the period of operation.i Results Sixteen patients received the current confi guration blood glucose sensor; during their combined length of stay, 296 paired values were obtained for correlation purposes. A total 99.6% of these values fall within the A+B areas of the Clarke error grid. All sensors continued to function throughout the length of stay, maximum 92 hours, and were withdrawn immediately prior to discharge from the ICU. Results The sensor was successfully deployed in all fi ve patients and did not interfere with clinical care, blood pressure monitoring or sampling. One patient suff ered a cardiopulmonary arrest; the sensor functioned successfully during resuscitation and urgent return to the operating room. One hundred and twenty reference samples ranging from 5.9 to 13.4 mmol/l were collected; 107/120 (89.2%) of GluCath measurements met ISO 15197 criteria (within ±20% of reference when BG >4.2 mmol/l; Figure 1). In Subject 1 the sensor was inadvertently retracted into the arterial catheter during the study, leading to measurement error from arterial fl ush solution contamination. In a sensitivity analysis excluding this patient, 89/95 (93.7%) of measurements met ISO 15197 with a mean absolute relative diff erence of 9.4%. Conclusion The pre-production intravascular blood glucose sensors successfully track blood glucose values, with improved insight into blood glucose variability in ICU patients. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Reliable classifi cation to associate with outcomes relies on the control protocol and cut-off choice to achieve suffi cient separation between groups so that device errors do not result in signifi cant misclassifi cation confounding the results. A deadband around cut-off values to eliminate patients at high risk of misclassifi cation may be required. 5 Preliminary ICU experience of a novel intravascular blood glucose sensor Preliminary ICU experience of a novel intravascular blood glucose sensor KP Mulavisala1, PB Gopal2, B Crane3, A Mackenzie3 1Axon Anaesthesia Associates Care Hospital Nampally, Hyderabad, India; 2Apollo Hospitals, Hyderabad, India; 3Glysure, Abingdon, UK Critical Care 2012, 16(Suppl 1):P175 (doi: 10.1186/cc10782) KP Mulavisala1, PB Gopal2, B Crane3, A Mackenzie3 1Axon Anaesthesia Associates Care Hospital Nampally, Hyderabad, India; 2Apollo Hospitals, Hyderabad, India; 3Glysure, Abingdon, UK Critical Care 2012, 16(Suppl 1):P175 (doi: 10.1186/cc10782) 1. Van den Berghe G, et al.: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359-1367. 2. Aragon D, et al.: Evaluation of nurse work eff ort and perception about blood glucose testing in TGC. Am J Crit Care 2006, 15:370-377. 3. Krinsley J, Preiser JC: Moving beyond TGC to safe eff ective glycemic control (SEGC). Crit Care 2008, 12:149. Glucometer accuracy and implications for clinical studies AJ Le Compte1, CG Pretty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P173 (doi: 10.1186/cc10780) pi y p Methods Forty-seven patients were studied. The following parameters were calculated during fi rst week after ICU admission. (1) Mean and maximum value of SOFA scores (SOFAm and SOFAmax, respectively). (2) Mean, standard deviation, maximum, minimum, and diff erence of BG levels (BGm, BGsd, BGmax, BGmin, BGd(BGmax – BGmin), respectively). BG levels were measured basically every 6 hours with capillary blood. (3) Correlation coeffi cients (r) between the abovementioned SOFA scores and BG parameters were calculated using two-dimensional and linear regression analysis (rt, rl, respectively). AJ Le Compte1, CG Pretty1, GM Shaw2, JG Chase1 1University of Canterbury, Christchurch, New Zealand; 2Christchurch Hospital, Christchurch, New Zealand Critical Care 2012 16(Suppl 1):P173 (doi: 10 1186/cc10780) Introduction Elucidating links between glycemic control and clinical outcome requires reliable discrimination between groups with diff erent target blood glucose (BG) cut-off s. Point-of-care glucometers are commonly used, but lower accuracy means BG errors will impact classifi cation and thus outcome analyses. This study reanalyses a BG control trial with an error model of a typical glucometer to assess the impact of sensor errors on interpretation of trial results.i Introduction Elucidating links between glycemic control and clinical outcome requires reliable discrimination between groups with diff erent target blood glucose (BG) cut-off s. Point-of-care glucometers are commonly used, but lower accuracy means BG errors will impact classifi cation and thus outcome analyses. This study reanalyses a BG control trial with an error model of a typical glucometer to assess the impact of sensor errors on interpretation of trial results. t l Results (1) rt and rl (rt/rl) between SOFAmax and BG parameters: BGsd (0.48/0.36), BGmax (0.47/0.33), BGd (0.47/0.35), BGm (0.30/0.22), BGmin (0.21/0.36). (2) (rt/rl) between SOFAm and BG parameters: BGsd (0.45/0.33), BGmax (0.45/0.28), BGd (0.45/0.30), BGm (0.28/0.17), BGmin (0.17/0.29).i Methods BG profi les from 301 patients (stay >24 hours) from the SPRINT trial with BG measurements (n = 25,000) using the Arkray SuperGlucocard II GT-1630. A model of sensor bias and variance (CV 2.7 to 3.5%, regression: y = 3.92 + 0.97x) was used to estimate possible ‘true’ BG profi les from measured BG and repeated 100 times for each Conclusion (1) Variable and maximum BG levels during the fi rst week (BGsd, BGd, BGmax), rather than mean and minimum BG levels (BGm, BGmin), were related to the severity. (2) Suppression of the higher S62 g References 1. Van den Berghe G, et al.: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359-1367. 2. Aragon D, et al.: Evaluation of nurse work eff ort and perception about blood glucose testing in TGC. Am J Crit Care 2006, 15:370-377. 3. Krinsley J, Preiser JC: Moving beyond TGC to safe eff ective glycemic control (SEGC). Crit Care 2008, 12:149. f Conclusion The GluCath System measured glucose concentration continuously in a cardiac surgery ICU without compromising arterial line function or patient care. In all patients the sensor operated without interruption for 24 hours following a single in vivo calibration. 2. Aragon D, et al.: Evaluation of nurse work eff ort and perception about blood glucose testing in TGC. Am J Crit Care 2006, 15:370-377. 3. Krinsley J, Preiser JC: Moving beyond TGC to safe eff ective glycemic control (SEGC). Crit Care 2008, 12:149. S63 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 in ‘tight’ glycaemic control [2]; however, recent negative studies have dampened this interest [3]. In view of more recent analyses, which off er possible explanations for equivocal results [4], it is possible there will be renewed interest in glycaemic control. The purpose of this survey is to assess the utilisation of tight glycaemic control protocols in a sample of ICUs in England, as a refl ection of current UK intensive care practice. Methods We identifi ed 171 large acute hospital trusts, of which 87 were randomly selected. Of these, 85 had ICUs, which were contacted by telephone. The senior nurse in charge at the time was asked whether their ICU used a protocol for the management of blood glucose, and what were the upper and lower target limits. P176 Does tight glycemic control positively impact on patient mortality? S Penning1, AJ Le Compte2, M Signal2, P Massion3, JC Preiser4, GM Shaw5, T Desaive1, JG Chase2 1Université de Liege, Belgium; 2University of Canterbury, Christchurch, New Zealand; 3CHU de Liège, Belgium; 4Erasme University Hospital, Brussels, Belgium; 5Christchurch Hospital, Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P176 (doi: 10.1186/cc10783) Introduction High and variable blood glucose (BG) levels have been associated with increased mortality. Tight glycemic control (TGC) aims at reducing BG levels to improve patient outcome and mortality. This research evaluates the impact of TGC on mortality. pp g Results A blood glucose protocol was used in 87.1% of ICUs surveyed. References y Conclusion Results show that, irrespective of TGC protocols, high cTIB and thus normoglycemia are associated with higher odds of living. This suggests that TGC positively infl uences patient outcome. 1. Van den Berghe G, et al.: N Engl J Med 2001, 345:1359-1367. 2. Mackenzie et al.: Intensive Care Med 2005, 31:1136. 2. Mackenzie et al.: Intensive Care Med 2005, 31:1136. 3. NICE-SUGAR Study Investigators: N Engl J Med 2009, 360:1283. 4. Mackenzie et al.: Intensive Care Med 2011, 37:435-443. 3. NICE-SUGAR Study Investigators: N Engl J Med 2009, 360:1283. 4. Mackenzie et al.: Intensive Care Med 2011, 37:435-443. g References Of these, the median lower limit of allowed blood glucose concentration was 4.0 mmol/l (range 3.0 to 7.0), with an upper limit of 8.0 mmol/l (range 6.0 to 12.0). Only 22 ICUs (25.9%) had a target range similar to the Leuven study. A further 34 ICUs used a lower limit similar to the Leuven study, of 4.0 to 4.5 mmol/l, but had a higher upper limit. This is refl ective of the general opinion from the nurses contacted, that a tight protocol is diffi cult to achieve, can result in hypoglycaemia, and has been recently relaxed in many departments. y Methods This study used glycemic data from 1,488 patients of two cohorts: Glucontrol (n = 704) and SPRINT (n = 784). TGC glycemic outcome is measured by cumulative time in the 4 to 7 mmol/l band (cTIB), defi ned daily for each patient. Each day, patients were divided into two groups: cTIB <70% and cTIB ≥70%. For each group, odds of living (OL = #lived / #died) was calculated. g Results OL for cTIB ≥70% patients tends to increase over time while OL for cTIB <70% patients decreases (Figure 1). On Day 1, OL for cTIB <70% patients and cTIB ≥70% patients are similar (OL = 5.1 and OL = 5.5 respectively). The diff erence between the two groups increases over the ICU stay. On Day 10, OL = 2.8 and OL = 10.5 for cTIB <70% and cTIB ≥70% patients respectively. These results suggest that survival rate is higher when cTIB ≥70% and thus when BG levels are tightly controlled around normoglycemia. The longer patients’ ICU stay, the lower survival rate they have when cTIB <70%. Conclusion Our data suggest that glycaemic control has, to a very large extent, been accepted as a standard of care in the UK, although in most ICUs this does not constitute tight glycaemic control. The full benefi t of tight glycaemic control, achieved by minimisation of mean glucose, glucose variability and episodes of hypoglycaemia, will not be achieved until robust techniques for continuous, or semi-continuous, blood glucose measurement are available. References P179 P179 Perioperative glycemic control with a computerized algorithm versus conventional glycemic control M Punke1, S Bruhn1, M Goepfert1, S Kluge1, H Reichenspurner2, A Goetz1, D Reuter1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2University Heart Center, Hamburg, Germany Critical Care 2012, 16(Suppl 1):P179 (doi: 10.1186/cc10786) P179 Perioperative glycemic control with a computerized algorithm versus conventional glycemic control M Punke1, S Bruhn1, M Goepfert1, S Kluge1, H Reichenspurner2, A Goetz1, D Reuter1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2University Heart Center, Hamburg, Germany Critical Care 2012, 16(Suppl 1):P179 (doi: 10.1186/cc10786) P179 Perioperative glycemic control with a computerized algorithm versus conventional glycemic control Introduction In critically ill patients, both hypoglycemia and hyperglycemia seem to infl uence outcome. Since hypoglycemia can lead to organ dysfunction, hyperglycemia seems to boost surgical site infections (SSI) [1]. It was shown that intensive insulin therapy (IIT) reduced mortality in critically ill patients [2]. Unfortunately several studies could not reproduce the eff ects [3,4]. In particular, IIT bears the risk of accidental hypoglycemia which could even have a negative eff ect on patient outcome [3,4]. In cardiac surgery, the use of blood cardioplegia for cardiopulmonary bypass frequently leads to high blood glucose levels during surgery. In particular, a computer- based algorithm that guides the insulin therapy might be benefi cial. We hypothesized that in patients undergoing major cardiac surgery with cardiopulmonary bypass and blood cardioplegia, the use of a computer-based algorithm for the application of insulin will lead to a tighter adherence of normoglycemia. Our primary study end-point was the duration, in which the patients fulfi lled the predefi ned target range of 80 to 150 mg/dl blood glucose. Patients with conventional blood glucose therapy served as controls.i Results The response of the MPC controller to measured deviations in glucose is shown in Figure 1. For glucose measurements below target, glucose is administered, while insulin administration is used to lower blood glucose from an elevated state to a desired target. The model parameter pG2, representing patient insulin sensitivity (insulin action on glucose uptake), was used by the MHE algorithm to tailor the model response to simulated patient dynamics. In response to pG2 changes in the simulated patient, MHE provided a 93% improvement in glucose reference tracking performance. g Conclusion The algorithm achieves tight glucose control in response to multiple measured and unmeasured disturbances. Furthermore, the MHE scheme updates patient parameters in real time in response to changing patient dynamics. 1. Roy et al.: Diab Tech Ther 2006, 8:617-626. P177 P178 Model-based regulation of glucose in critical care SP Gawel1, G Clermont2, RS Parker1 1University of Pittsburgh, PA, USA; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2012, 16(Suppl 1):P178 (doi: 10.1186/cc10785) Introduction Following van den Berghe’s landmark paper in 2001 (Leuven study) [1], the critical care community became very interested Introduction Glucose control in critical care has been shown to improve patient outcome, yet tight glucose control has led to increased Figure 1 (abstract P176). Whole-cohort odds of living over ICU stay. Figure 1 (abstract P176). Whole-cohort odds of living over ICU stay. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S64 Critical Care 2012, Volume 16 Suppl 1 htt // f / l t /16/S1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P178). Figure 1 (abstract P178). Figure 1 (abstract P178). P179 hypoglycemia in the clinic. We employed a systems engineering approach to assist clinicians in maintaining blood glucose within a desired target range while avoiding hypoglycemia in the critically ill. The long-term vision is a decision support system that provides recommended insulin and glucose administrations leading to patient- specifi c achievement of tight glucose control without hypoglycemia. Methods To achieve these goals, we employ a model predictive control (MPC) algorithmic platform using two control inputs: insulin for glucose control and glucose for hypoglycemia. The MPC controller is designed based on a nonlinear dynamic model of glucose–insulin–fatty acid interactions [1]. A moving horizon estimation (MHE) technique is used to alter the tissue sensitivity to insulin based on deviations between measurements and model predictions of glucose concentration as a mechanism for tailoring the controller model to individual patient dynamics. hypoglycemia in the clinic. We employed a systems engineering approach to assist clinicians in maintaining blood glucose within a desired target range while avoiding hypoglycemia in the critically ill. The long-term vision is a decision support system that provides recommended insulin and glucose administrations leading to patient- specifi c achievement of tight glucose control without hypoglycemia. hypoglycemia in the clinic. We employed a systems engineering approach to assist clinicians in maintaining blood glucose within a desired target range while avoiding hypoglycemia in the critically ill. The long-term vision is a decision support system that provides recommended insulin and glucose administrations leading to patient- specifi c achievement of tight glucose control without hypoglycemia. Methods To achieve these goals, we employ a model predictive control (MPC) algorithmic platform using two control inputs: insulin for glucose control and glucose for hypoglycemia. The MPC controller is designed based on a nonlinear dynamic model of glucose–insulin–fatty acid interactions [1]. A moving horizon estimation (MHE) technique is used to alter the tissue sensitivity to insulin based on deviations between measurements and model predictions of glucose concentration as a mechanism for tailoring the controller model to individual patient dynamics. References References 1. Ann Intern Med 2007, 146:233-243. 2. N Engl J Med 2001, 345:1359-1367. 3. N Engl J Med 2006, 354:449-461. 4. N Engl J Med 2009, 360:1283-1297. g p Methods All ICU patients from HUPR diagnosed with SCASTE within the fi rst 24 hours from January 2005 to December 2010 were included in this study and registered in the ARIAM-Andalucia Project. This database gathers the whole PAI from preadmission (PH), ER, ICU, hemodynamics laboratory and cardiology ward to discharge. Within these 6 years three main interventions were carried out: fi brinolysis protocol PH with ER and critical care unit EMS involving the ICU, continuous update of protocols based on AHA clinical guidelines, and 24-hour availability of the hemodynamic laboratory for primary coronary intervention (P-ICP available since 1 February 2007). Revascularization indexes are analyzed and grouped in 2-year periods (A, B, C), the time justifi ed as necessary for modifi cation after the intervention, attention times and PH action. The latter was measured by a score (aspirin, nitroglycerine, ECG, vein access, intravenous treatment and monitoring during transport) up to 6 points. A correct intervention must obtain at least 4 points. Statistical processing was by the R-UCA pack from R-Commander. 1. Ann Intern Med 2007, 146:233-243. 2. N Engl J Med 2001, 345:1359-1367. 3. N Engl J Med 2006, 354:449-461. 4. N Engl J Med 2009, 360:1283-1297. P180 Effi cacy of the novel heart attack centre extension pathway: a pilot study D Perera1, B Hoonjan1, K Krishnathasan1, M Selvanyagam1, H Neugebauer2 1Barts and The London School of Medicine and Dentistry, London, UK; 2Queens Hospital, Barking Havering and Redbridge NHS Trust, London, UK Critical Care 2012, 16(Suppl 1):P180 (doi: 10.1186/cc10787) Results A total of 590 patients were included in this study: 188 (A), 227 (B) and 175 (C). All groups were similar in mean age, gender, IAM location and origin. A statistically signifi cant increase was found in the revascularization and PHA attention between periods A versus C and B versus C with P <0.0001 and CI (0.15 to 0.42)/(0.17 to 0.45) and (0.2 to 0.6)/(0.11 to 0.39). No statistically signifi cant diff erence was found among groups A versus B. No signifi cant diff erence was observed in attention times. Prognostic value of Killip classifi cation in terms of health-related quality of life y A Ioannidis1, D Tsounis1, A Pechlevanis2, M Paraskelidou2 1HOU, Kalamaria, Greece; 2GHT ‘Agios Pavlos’, Kalamaria, Greece Critical Care 2012, 16(Suppl 1):P182 (doi: 10.1186/cc10789) A Ioannidis1, D Tsounis1, A Pechlevanis2, M Paraskelidou2 1HOU, Kalamaria, Greece; 2GHT ‘Agios Pavlos’, Kalamaria, Greece Critical Care 2012, 16(Suppl 1):P182 (doi: 10.1186/cc10789) Results The average time for patients to have an angiography via the IHT pathway was 5.5 days. Of the 33 patients (mean age 61 ± 15.2 SD) transferred via HACX, 30 patients (91%) were appropriately identifi ed for an angiogram. Seventeen patients (52%) required PCI, fi ve patients (15%) required CABG, four patients (12%) required nonsurgical intervention, and four patients (12%) required no treatment. Controls included 37 patients (mean age 71 ± 12.6 SD) of whom 17 patients (46%) required PCI, six patients (16%) required CABG, eight patients (22%) required nonsurgical intervention and six patients (16%) required no treatment. At 3-month follow-up, 32 patients (97%) in the HACX cohort and 36 patients (97%) in the IHT cohort were alive.fi Introduction The aim of the study was to evaluate the prognostic value of Killip classifi cation in terms of health-related quality of life (HRQoL). Methods The sample consisted of 112 patients treated for myocardial infarction (MI), as onset manifestation of coronary artery disease (CAD), during 2008/09 in a prefectural hospital in northern Greece. At 1-year follow-up visit, HRQoL was measured using a generic and a disease- specifi c instrument. The 15D consists of a visual analogue scale (VAS) and a total score. The scoring algorithm of the MacNew generates a global score, and three separate domains scores: emotional, physical and social. Introduction The aim of the study was to evaluate the prognostic value of Killip classifi cation in terms of health-related quality of life (HRQoL). Results Patients were grouped into the four Killip classes according to the degree of pulmonary congestion at admission (Table 1). Mean HRQoL for each group diff ered in the expected manner: the higher the class, the lower the HRQoL. Statistical signifi cant diff erences were observed in VAS of the 15D and the emotional and social domain scores of the MacNew. Accordingly, the majority of patients with no signs of pulmonary congestion at admission were classifi ed in NYHA functional class I at 1-year follow-up visit. Integral assistance process implantation for ST-elevated acute coronary syndrome Results There were no statistical diff erences between the groups regarding age, height, weight, premedical history or intraoperative amount of glucose administration during cardioplegia (33 ± 15 g). Blood glucose levels in groups 1 and 2 stayed signifi cantly longer in the target interval compared with group 3 (75 ± 19% vs. 72 ± 19%; vs. 50 ± 34%, P <0.01, n = 25, respectively). There was no signifi cant diff erence between the groups regarding ICU or hospital stay and SSI rates. JC Rodriguez-Yañez, M Celaya-Lopez, MJ Huertos-Ranchal, I Diaz-Torres, C Navarro-Ramirez, J Gomez-Ramos Hospital Universitario Puerto Real, Spain Critical Care 2012, 16(Suppl 1):P181 (doi: 10.1186/cc10788) JC Rodriguez-Yañez, M Celaya-Lopez, MJ Huertos-Ranchal, I Diaz-Torres, C Navarro-Ramirez, J Gomez-Ramos Hospital Universitario Puerto Real, Spain Critical Care 2012, 16(Suppl 1):P181 (doi: 10.1186/cc10788) Introduction The objective was to evaluate the implantation of assistance process implantation (PAI) for ST-elevated acute coronary syndrome (SCASTE) in our sanitary district. When we refer to PAI, we mean protocolysed assistance guidelines developed and published by Andalucia sanitary authorities that include recommendations to direct the assistance from the beginning of the process until patient discharge from the hospital. Conclusion Early computer-based insulin therapy allows one to better warrant normoglycemia in patients undergoing major cardiac surgery with the use of blood cardioplegia. References Introduction The Barts and the London Heart Attack Centre Extension (HACX) programme was introduced to provide a direct pathway for high-risk non-ST elevation myocardial infarction (NSTEMI) patients from the A&E of a district general hospital to a tertiary intervention centre. As a result, patients have earlier access to angiography and subsequent treatment, including percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG) or nonsurgical inter ven- tions. There is no research on the eff ectiveness of this novel HACX programme. Conclusion Coordination of the SCASTE attention, constant analysis by continuous registry of diff erent action levels (ARIAM-Andalucia registry), clinical guideline updates and adjustment to resources and environment, in this case a rural setting, meaning quality and a continuous improvement circle, reduce variability and lead undoubtedly to better assistance for our patients. Methods Over 3 months, 33 patients transferred via the HACX pathway and 37 patients transferred via the conventional interhospital transfer pathway (IHT) were followed up. All patients with acute coronary syndrome symptoms, relevant ECG changes (ST segment depression in two or more contiguous leads >1 mm, pathological T-wave inversion in V1 to V4, a GRACE score >88 and troponin I levels >0.1 ng/ml) were discussed with the cardiology team at the interventional centre prior to immediate transfer. We assessed patient suitability for angiography, post-angiography procedures, and 3-month mortality outcomes. Data were obtained from the hospital’s PAS computer system. P182 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Statistical analysis was performed with using ANOVA and the LPS post hoc test. Data shown are mean ± standard deviation, n = number of patients. P179 The adaptive MPC algorithm is currently being validated using a retrospective cohort of critically ill patients at the University of Pittsburgh Medical Center. Methods Seventy-fi ve patients were enrolled and randomized into three groups. Start of therapy was determined as the beginning of cardiopulmonary bypass. Group 1: therapy with computer-based blood glucose control (TGC System; Braun, Melsungen, Germany) and measurement of blood glucose every 30 minutes. Group 2: same therapy as group 1 and measurement of blood glucose every 15 minutes. Group 3: conventional therapy using a fi xed insulin dosing scheme. End of therapy was defi ned as discharge from the ICU. Acknowledgements Funded by NIH-R21-DK092813. Reference S65 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Prognostic value of Killip classifi cation in terms of health-related quality of life No diff erence was observed in the type Conclusion HACX is an eff ective pathway that accurately identifi es and rapidly transfers appropriate NSTEMI patients requiring early coronary revascularisation. However, there was no additional mortality benefi t at 3-month follow-up compared to the conventional IHT pathway. Further studies with larger patient cohorts and longer follow-up periods are required to substantiate the benefi ts of the HACX programme in order to consider whether this service could be implemented nationwide, or whether this is a service that does not need to exist at all. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S66 of MI, although patients in higher Killip classes had more aff ected arteries and were treated more often with CABG than PCI. Additionally, Killip class I patients were favored in a number of parameters including age, systolic blood pressure, heart rate, BMI, NT-proBNP level and LVEF. Conclusion Patients with MI as an onset manifestation of CAD present ith i d d f l ti Th ti l Table 1 (abstract P182). Prognostic value of Killip classifi cation in terms of health-related quality of life Methods In this retrospective study, the medical records of 619 patients with an admitting diagnosis of STEMI from Tan Tock Seng Hospital, Emergency Department between 1 January 2009 and 31 December 2009 were reviewed. We extracted data from the electronic records of the emergency case notes and inpatient discharge summaries. Results Among 619 patients, 363 (58.6%) arrived by emergency medical services (EMS) and 256 (41.4%) by self-transport. Three hundred and thirty (53.3%) patients underwent emergency angiography, of which 313 (94.9%) were treated with percutaneous coronary intervention (PCI), eight (2.4%) with coronary artery bypass grafting (CABG) and nine (2.7%) were conservatively managed. The D2B time was signifi cantly shorter in patients who arrived by EMS (60 vs. 82 minutes; P <0.001). There was no diff erence in D2B time between patients who arrived in the day (06:00 to 17:59 hours) or at night (18:00 to 05:59 hours). Chest pain, shortness of breath and diaphoresis were the three commonest presenting symptoms in patients with STEMI regardless of their mode of arrival. Previous myocardial infarction, PCI or CABG did not infl uence the mode of transport. Patients who arrived by EMS had a higher incidence of cardiogenic shock (20.7% vs. 11.7%; P = 0.020) and were signifi cantly older (63 vs. 59 years; P = 0.004) than patients who arrived by self-transport. Patients who arrived by EMS had a higher in-hospital mortality rate (12.1% vs. 5.1%; P = 0.003) and a longer mean length of stay compared to those who arrived by self-transport (6 vs. 4 days; P = 0.004). C l i I t d l ti ti t ith STEMI h d EMS Table 1 (abstract P182). Patient characteristics according to Killip classifi cation P183 Modes of arrival, door to balloon time and its impact on morbidity and mortality for ST elevation myocardial infarct YC Chia Tan Tock Seng Hospital, Singapore Critical Care 2012, 16(Suppl 1):P183 (doi: 10.1186/cc10790) P183 Modes of arrival, door to balloon time and its impact on morbidit and mortality for ST elevation myocardial infarct YC Chia Tan Tock Seng Hospital, Singapore Critical Care 2012, 16(Suppl 1):P183 (doi: 10.1186/cc10790) Introduction Timely reperfusion of the occluded coronary artery is crucial in reducing the amount of myocardial damage in patients with ST elevation myocardial infarct (STEMI). Prognostic value of Killip classifi cation in terms of health-related quality of life Patient characteristics according to Killip classifi cation Class I Class II Class III Class IV P value n (%) 52 (46.4) 40 (35.7) 14 (12.5) 6 (5.4) NA HRQoL (mean) 15D VAS 78.9 76.9 72.9 70.8 0.025 Total 0.844 0.842 0.823 0.803 0.478 MacNew Emotional 5.60 5.61 5.38 5.05 0.030 Physical 5.49 5.26 5.25 5.05 0.144 Social 5.72 5.47 5.30 5.14 0.014 Global 5.52 5.44 5.27 4.98 0.056 Age (years) 61.8 66.0 70.2 61.2 0.013 Gender (n, %) Male (85) 40 (47.1) 31 (36.5) 12 (14.1) 2 (2.4) 0.080 Female (27) 12 (44.4) 9 (33.3) 2 (7.4) 4 (14.8) ΒΜΙ 28.7 29.1 30.0 34.8 0.001 Systolic BP 127.5 122.5 115.7 140.0 0.000 Diastolic BP 75.8 73.6 70.7 76.7 0.173 Heart rate 64.9 68.7 72.3 71.7 0.030 CRP 3.6 4.9 6.0 3.8 0.087 NT-proBNP 741 1645 2193 1674 0.000 LVEF 61.9 55.2 49.0 45.0 0.000 MI type (n, %) STEMI (48) 24 (50.0) 18 (37.5) 4 (8.3) 2 (4.2) 0.638 NSTEMI (64) 28 (43.8) 22 (55.0) 10 (15.6) 4 (6.3) Aff ected arteries (n, %) 1 (38) 19 (50.0) 15 (39.5) 4 (10.5) 0 (0.0) 0.005 2 (30) 17 (56.7) 9 (30.0) 4 (13.3) 0 (0.0) 3 (44) 16 (36.4) 16 (36.4) 6 (13.6) 6 (13.6) Revascularization technique (n, %) None (14) 7 (50.0) 3 (21.4) 4 (28.6) 0 (0.0) 0.002 PCI (63) 35 (55.6) 22 (34.9) 6 (9.5) 0 (0.0) CABG (35) 10 (28.6) 15 (42.9) 4 (11.4) 6 (17.1) ΝΥΗΑ Ι (73) 42 (57.5) 21 (28.8) 8 (11.0) 2 (2.7) 0.013 ΙΙ (37) 8 (21.6) 19 (51.4) 6 (16.2) 4 (10.8) ΙΙΙ (2) 2 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) Rho 0.28 0.001 P183 Modes of arrival, door to balloon time and its impact on morbidity and mortality for ST elevation myocardial infarct YC Chia Tan Tock Seng Hospital, Singapore Critical Care 2012, 16(Suppl 1):P183 (doi: 10.1186/cc10790) Introduction Timely reperfusion of the occluded coronary artery is crucial in reducing the amount of myocardial damage in patients with ST elevation myocardial infarct (STEMI). This study aims to examine the common presenting symptoms of patients with STEMI, their modes of arrival at the emergency department (ED) and its impact on door-to- balloon (D2B) time and in-hospital morbidity and mortality. Prognostic value of Killip classifi cation in terms of health-related quality of life This study aims to examine the common presenting symptoms of patients with STEMI, their modes of arrival at the emergency department (ED) and its impact on door-to- balloon (D2B) time and in-hospital morbidity and mortality. Methods In this retrospective study, the medical records of 619 patients with an admitting diagnosis of STEMI from Tan Tock Seng Hospital, Emergency Department between 1 January 2009 and 31 December 2009 were reviewed. We extracted data from the electronic records of the emergency case notes and inpatient discharge summaries. g y p g Results Among 619 patients, 363 (58.6%) arrived by emergency medical services (EMS) and 256 (41.4%) by self-transport. Three hundred and thirty (53.3%) patients underwent emergency angiography, of which 313 (94.9%) were treated with percutaneous coronary intervention (PCI), eight (2.4%) with coronary artery bypass grafting (CABG) and nine (2.7%) were conservatively managed. The D2B time was signifi cantly shorter in patients who arrived by EMS (60 vs. 82 minutes; P <0.001). There was no diff erence in D2B time between patients who arrived in the day (06:00 to 17:59 hours) or at night (18:00 to 05:59 hours). Chest pain, shortness of breath and diaphoresis were the three commonest presenting symptoms in patients with STEMI regardless of their mode of arrival. Previous myocardial infarction, PCI or CABG did not infl uence the mode of transport. Patients who arrived by EMS had a higher incidence of cardiogenic shock (20.7% vs. 11.7%; P = 0.020) and were signifi cantly older (63 vs. 59 years; P = 0.004) than patients who arrived by self-transport. Patients who arrived by EMS had a higher in-hospital mortality rate (12.1% vs. 5.1%; P = 0.003) and a longer mean length of stay compared to those who arrived by self-transport (6 vs. 4 days; P = 0.004). Conclusion In our study population, patients with STEMI who used EMS tend to be older and arrived in cardiogenic shock. They therefore had a higher incidence of in-hospital mortality and morbidity although their D2B time was shorter compared to those who arrived by self-transport. Next-generation, fast and accurate point-of-care test for NT-proBNP based on Magnotech technology B Inçaurgarat1, J Nieuwenhuis2 1bioMérieux, Marcy L’Etoile, France; 2Philips, Eindhoven, the Netherlands Critical Care 2012, 16(Suppl 1):P184 (doi: 10.1186/cc10791) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Assay precision was characterised by CV levels of less than 10%. NT-proBNP results correlated well with VIDAS (r = 0.89), with a corresponding slope of the regression line of 1.12 (95% CI 1.01 to 1.22) and an intercept of 64.04 (95% CI –73.50 to 109.83). In the current format under development, the NT-proBNP assay time with plasma samples is only 5 minutes. We are in the process of adding a fi lter that will allow measurements from whole blood directly. Flow experiments show that the fi lling time of the cartridge with whole blood is less than 30 seconds, resulting in a total assay time of less than 6 minutes, and a time-to-result of less than 8 minutes. involve psychic or physical stressors such as a devastating disaster, but those clinical features have been not fully investigated. As Ibaraki prefecture suff ered from the Great East Japan Earthquake, we tried to clarify the characteristics of TC and investigate whether the Great East Japan Earthquake increased the occurrence of TC or not. involve psychic or physical stressors such as a devastating disaster, but those clinical features have been not fully investigated. As Ibaraki prefecture suff ered from the Great East Japan Earthquake, we tried to clarify the characteristics of TC and investigate whether the Great East Japan Earthquake increased the occurrence of TC or not. Methods Eleven consecutive patients with TC (fi ve men, six women) were enrolled between October 2009 and October 2011 in this study. Patients were diagnosed by echocardiography, left ventriculography, or nuclear scintigraphy. Absence of signifi cant coronary stenosis was confi rmed by coronary angiography or coronary computed- tomography angiography. Clinical characteristics (age, season, coronary risk factors, the condition that preceded onset as possible triggering factors and so on), laboratory data (troponin T, creatinine kinase, and so on) and data of electrocardiography (ECG) were obtained from reviewing medical records. p q Methods Eleven consecutive patients with TC (fi ve men, six women) were enrolled between October 2009 and October 2011 in this study. Patients were diagnosed by echocardiography, left ventriculography, or nuclear scintigraphy. Absence of signifi cant coronary stenosis was confi rmed by coronary angiography or coronary computed- tomography angiography. Right ventricular apical versus septal pacing: impact on left ventricular synchrony and function I Atteia, A Alazab, K Hussein, N Abeed, H Nagi Cairo University, Cairo, Egypt Critical Care 2012, 16(Suppl 1):P185 (doi: 10.1186/cc10792) Right ventricular apical versus septal pacing: impact on left ventricular synchrony and function I Atteia, A Alazab, K Hussein, N Abeed, H Nagi Cairo University, Cairo, Egypt Critical Care 2012, 16(Suppl 1):P185 (doi: 10.1186/cc10792) Introduction Right ventricular apical pacing alters the LV activation resulting in an adverse eff ect on LV function and synchrony. On the contrary, RV septal pacing results in narrower QRS and may be more physiological with less deleterious long-term eff ect on LV echocardiographic and hemodynamic parameters. y Methods Forty patients indicated for permanent DDD pacing were studied. All patients were subjected to transthoracic echocardiography calculating LVESD, LVEDD, EF% and CO together with tissue Doppler imaging (TDI) to detect LV dyssynchrony. Patients were randomly classifi ed into two groups, group I having RV apical pacing and group II having RV septal pacing. The acute threshold, R-wave sensing and fl uoroscopic time were measured in all patients and compared in both groups. Both groups were followed-up over a period of 6 months.i p Conclusion Although TC seems to mimic anterior STEMI, limb leads did not tend to show ST change in ECG in our cases. The Great East Japan Earthquake could increase patients with TC until the tremendous damage caused by the disaster will be over. Results QRS durations were signifi cantly narrower in group II patients (148 ± 6.9 vs. 162 ± 6 ms, P = 0.001). Electrical parameters at the time of implantation were satisfactory for all patients (acute stimulation threshold was 0.5 ± 0.18 V; R wave sensing was 11 ± 1.6 mV and ventricular impedance was 630 ± 90 Ohm). No single patient needed ventricular lead repositioning. The acute pacing threshold, R-wave sensing, ventricular impedance and fl uoroscopic time did not change signifi cantly in both groups. During follow-up, it was found that in group II patients with RV septal pacing there was signifi cantly lower LVESD (3.0 ± 0.6 vs. 3.4 ± 0.6 cm, P = 0.004), signifi cantly higher LVEF% (69 ± 13 vs. 61 ± 8, P = 0.01), signifi cantly higher CO (4.9 ± 0.3 vs. 4.5 ± 0.6 l), and signifi cantly lower septal to lateral wall delay in LV using TDI (72 ± 5 vs. 83 ± 6 ms, P = 0.001) if compared to group I patients with RV apical pacing. P187 p p References 1. Kutarski A, Ruciniski P, Sodolski T, Trojnar M: Factors infl uencing diff erences of RVA and RVOT pacing hemodynamic eff ects. Europace 2005, 7:288. doi:10.1016/j.eupc.2005.02.104. 2. Hafez M, Small GR, Hannah A, et al.: Impact of temporary right ventricular pacing from diff erent sites on echocardiographic indices of cardiac function. Europace 2011. doi: 10.1093/europace/eur 207. Results Out of 250 patients fi ve patients had preoperative CAD and were excluded. Seven patients (incidence 2.8%) were diagnosed to have SC. Five out of seven (71.4%) patients were ethanolic and vasopressor requirement was high in all these patients (Figure 1). Echocardiography revealed global hypokinesia with left ventricular ejection fraction between 10 and 25%. They were managed with inotropic support and four patients required an intraaortic balloon pump (IABP). Two patients succumbed to cardiogenic shock on the second day (mortality 28.5%). IABP was weaned between 7 and 9 days. Patients had normal cardiac status at the time of discharge around the fourth week post liver transplant. Stress cardiomyopathy after live donor liver transplantation: incidence, risk factors and mortality Introduction The incidence of cardiac complications in the post live donor liver transplantation (LDLT) period has been reported to be nearly 70% [1]. Stress cardiomyopathy (SC) is a severe complication which has varied presentation and has grave prognosis if not diagnosed and managed aggressively. g gg y Methods Data for 250 LDLTs (June 2010 to July 2011) were collected to assess incidence, risk factors and mortality due to SC. Diagnostic criteria [2] for SC were taken as: global hypokinesia or new ST segment elevation or T-wave inversion in absence of coronary artery disease (CAD) or pheochromocytoma. Etiologies of chronic liver disease and preoperative cardiac status along with intraoperative vasopressor use and dosages were noted. Conclusion Long-term RV septal pacing is feasible, and reliable with less adverse eff ects on LV synchrony and function when compared to RV apical pacing. R f P187 Stress cardiomyopathy after live donor liver transplantation: incidence, risk factors and mortality S Gupta, D Govil, S Bhatnagar, S Patel, S Srinivasan, P Pandey, M Sodhi, J KN, P Singh, S Saigal, A Soin, V Vohra, Y Mehta Medanta – The Medicity, Gurgaon, India Critical Care 2012, 16(Suppl 1):P187 (doi: 10.1186/cc10794) Next-generation, fast and accurate point-of-care test for NT-proBNP based on Magnotech technology B Inçaurgarat1, J Nieuwenhuis2 1bioMérieux, Marcy L’Etoile, France; 2Philips, Eindhoven, the Netherlands Critical Care 2012, 16(Suppl 1):P184 (doi: 10.1186/cc10791) Introduction In the emergency care setting, where time is of the essence, there is a need for fast and reliable information on NT-proBNP levels for diagnosis and management of acute dyspnea. Rapid NT- proBNP testing near the patient has the potential to streamline the process of care, but only if it is robust, fast and accurate enough to operate safely at the point of care (POC). Here we report on a novel NT-proBNP POC test which can be entirely carried out in a handheld device. This test has the potential to be rapid (<8 minutes), easy to use (fi ngerprick sampling), and with good accuracy compared to state-of- the-art automated laboratory assays. y y Methods This new NT-proBNP POC test is based on Magnotech technology. A one-step sandwich immunoassay is performed in a compact plastic disposable cartridge with on-board dry reagents and magnetic nanoparticles. After a short incubation step the amount of bound nanoparticles, proportional to the concentration of NT-proBNP in the sample, is detected optically [1]. The precision of the assay was determined for plasma samples with NT-proBNP levels at clinically relevant values of 125 ng/l and 411 ng/l (10 replicates). Assay accuracy was determined by measuring 104 patient samples (lithium heparin plasma, NT-proBNP levels from 20 to 5,000 ng/l) on both the handheld device and the bioMérieux VIDAS laboratory system, and comparing results by Passing and Bablok regression analysis. S67 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Clinical characteristics (age, season, coronary risk factors, the condition that preceded onset as possible triggering factors and so on), laboratory data (troponin T, creatinine kinase, and so on) and data of electrocardiography (ECG) were obtained from reviewing medical records.i Conclusion In its current implementation the Magnotech-based NT- proBNP assay shows promising performance for rapid, reliable NT- proBNP testing at the POC in emergency settings. Development work is presently focused on the integration of a blood fi lter into the cartridge, to allow fi ngerprick tests. Results The number of cases of TC after the earthquake was fi ve for 7 months and that of before is six for 17 months. The occurrence rate of TC seemed to increase after the earthquake. Reviewing all of our cases, 45.5% (n = 5/11) of patients have TC in the autumn, 72.7% (n = 8/11) of patients suff ered from a physical stressor, and 27.3% (n = 3/11) of patients a psychic stressor. No obvious stressor was found in only one patient. The patients complained of chest pain or dyspnea (54.5% each). The rate of coronary risk factors were; family history, 10% (n = 1/10); smoking, 60% (n = 6/11); diabetes, 57.1% (n = 4/7); hypertension, 63.6% (n = 7/11); dyslipidemia, 44.4% (n = 4/9); and obesity, 22.2% (n = 2/9). Laboratory data showed that elevated troponin T was observed in 60% (n = 6/10), high CK and CK-MB were 45.5% (n = 5/11) and 100% (n = 9/9), respectively. ECG fi ndings of all of the patients; ST elevation was observed in precordial leads of V2 to V4 (27.3%, 54.5% and 27.3%, respectively) and ST depression was in V5 (36.4%). Reversed r progression was observed in 18.2%, poor r progression was 27.3%, abnormal Q was 18.2%, long QT interval was 72.7% and negative T was 63.6% of TC patients. Reference 1. Bruls DM, et al.: Lab Chip 2009, 9:3504-3510. 1. Bruls DM, et al.: Lab Chip 2009, 9:3504-3510. P188 Sh Short-term hemodynamic eff ects of nebivolol in acute decompensated heart failure: a randomized clinical trial R Puig, M Ochiai, J Cardoso, K Vieira, E Brancalhao, M Lima, A Pereira Barretto Hospital Auxiliar de Cotoxo – InCor – HCFMUSP, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P188 (doi: 10.1186/cc10795) Introduction The objective was to analyze clinical characteristics of patients with infective endocarditis (IE) requiring surgery when the disease is diagnosed. Introduction Acute decompensation heart failure in patients in use of β-blocker has become frequent and maintenance of this drug remains controversial, mainly in low cardiac output. Nitric-oxide-dependent vasodilation of nebivolol could be useful in this situation. Methods A retrospective study of all patients, during 5 years in a tertiary hospital in Spain, which required admission to the ICU with the diagnosis of IE (Duke criteria modifi ed) and required surgery at the same time. We compiled demographics, clinical characteristics and complications. Data were analyzed with SPSS 17.i Methods We evaluated hospitalized patients with acute decompen- sated heart failure, NYHA IV, EF <0.45, in use of dobutamine and carvedilol. Intervention: patients were randomly assigned to carvedilol maintenance or exchange to nebivolol according to Table 1. Hemodynamic parameters were compared using a noninvasive model fl ow technique (Nexfi n®; BMEYE), 24 hours before, 6 and 24 hours after the randomization. P <0.05 was signifi cant. p y Results We had 73 patients, 79% male, mean age 65. Forty-fi ve percent had previous heart disease. Eighty-four percent presented with fever, 56.5% general syndrome, 56.2% heart failure, 19.2% pain, and 7% coma. The duration of the clinic before diagnosis was mainly between 7 and 30 days (32%), followed by more than 30 days (27%). Less than 3 days duration represented 13%. Blood cultures were positive in 82%. The most common agent was Streptococcus (39%), followed by Staphylococcus aureus MS (16%), SCN (12%), Enterococcus (12.3%), S. aureus MR (1.4%), Escherichia coli (1.4%), Pseudomonas Aeruginosa (1.4%), Aspergillus (1.4%), and polymicrobial (1.4%). Twelve percent were negative cultures. The valve more frequently aff ected was aortic. In all cases TTE was carried out for diagnosis. In 69 cases TEE was performed. The principal echo fi ndings were: vegetation (42%), new insuffi ciencies (26%), and also stenosis, perivalvular abscess and normal echo. Fifty-eight percent of patients had no distal emboli. Other localizations: splenic (11%), hepatic (2.7%), bones (2.7%), brain (4%), lung (5%) and more than one (11%). Consecutive case series of Takotsubo cardiomyopathy: a disease potentially triggered by the Great East Japan Earthquake Mito Kyodo General Hospital, University of Tsukuba, Mito City, Japan Critical Care 2012, 16(Suppl 1):P186 (doi: 10.1186/cc10793) Mito Kyodo General Hospital, University of Tsukuba, Mito City, Japan Critical Care 2012, 16(Suppl 1):P186 (doi: 10.1186/cc10793) Introduction Takotsubo cardiomyopathy (TC) is a rare disease that mimics ST elevated myocardial infarction (STEMI). TC is known to p Conclusion Our incidence was 3%. SC generally presents on the second to third postoperative day and usually recovers by the second Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S68 Critical Care 2012, Volume 16 Suppl 1 http //ccfor m com/s pplements/16/S1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P187). Figure 1 (abstract P187). Figure 1 (abstract P188). Systemic vascular resistance change: nebivolol high×nebivolol low or carvedilol. Figure 1 (abstract P188). Systemic vascular resistance change: nebivolol high×nebivolol low or carvedilol. week. Ethanolics and patients who require high vasopressor support intraoperatively are more prone to develop SC. References 1. Therapondos G, et al.: Cardiac morbidity and mortality related to orthotopic liver transplantation. Liver Transpl 2004, 10:1441-1453. 1. Therapondos G, et al.: Cardiac morbidity and mortality related to orthotopic liver transplantation. Liver Transpl 2004, 10:1441-1453. 2. Bybee KA, et al.: Systematic review: Transient left ventricular apical ballooning: a syndrome that mimics ST-segment elevation myocardial infarction. Ann Intern Med 2004, 141:858-865. Figure 1 (abstract P188). Systemic vascular resistance change: nebivolol high×nebivolol low or carvedilol. 2. Bybee KA, et al.: Systematic review: Transient left ventricular apical ballooning: a syndrome that mimics ST-segment elevation myocardial infarction. Ann Intern Med 2004, 141:858-865. 2. Bybee KA, et al.: Systematic review: Transient left ventricular apical ballooning: a syndrome that mimics ST-segment elevation myocardial infarction. Ann Intern Med 2004, 141:858-865. P189 P189 Patients with infective endocarditis patients in the ICU: how are they? P Fernandez Ugidos1, R Gomez Lopez1, P Vidal Cortes1, AV Aller Fernandez2, JM Lopez Perez2 1Complejo Hospitalario Universitario Ourense, Spain; 2Complejo Hospitalario Universitario A Coruña, Spain Critical Care 2012, 16(Suppl 1):P189 (doi: 10.1186/cc10796) Malperfusion and branch compromise in acute type A aortic syndrome syndrome R Gomez Lopez1, P Fernandez Ugidos1, P Vidal Cortes1, J Lopez Perez2, J Priego Sanz1, M Bouza Vieiro2, A Aller Fernandez2, L Seoane Quiroga2, S Fojon Polanco2 1Complexo Hospitalario Universitario de Ourense, Spain; 2Complexo Hospitalario Universitario de A Coruña, Spain Critical Care 2012, 16(Suppl 1):P190 (doi: 10.1186/cc10797) i y Conclusion ECI ≥7 determines a poor CAD prognosis of coronary ischemic events. Furthermore, ECI ≥7 may serve as a marker of the content of wall calcium, obstruction level and composition of the plaques. ECI seems to provide prognostic information as well as providing information about the characteristics of the plaque of atheroma. Introduction Malperfusion is a factor associated with higher risk of death and complications in patients with acute type A aortic syndrome (AAAS). Our objective is to determine the incidence and characteristics of this disease in our population and to verify the relevance in morbidity and in-hospital mortality. Methods A historical cohort study that includes all patients with AAAS admitted to the ICU after surgical management in a single institution from January 2000 to July 2010. Anatomical, clinical, biochemical, electrocardiographic and echocardiographic signs of ischemia were considered. The events of interest were death or major complication (neurological damage, multiorgan failure (MOF), acute lung injury (ALI), postoperative hemorrhage) during hospitalization. P192 Echocardiography in the ICU: an audit of 3 years practice A Hall, J Walker, I Welters Royal Liverpool Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P192 (doi: 10.1186/cc10799) p p g g p Results A total of 65 patients were identifi ed (24.6% women, 61.86 ± 12 years old, APACHE II score 12.9 ± 7.2, EuroSCORE 7.4 ± 2.6). Thirty-three (50.8%) presented branch compromise, aff ecting coronary arteries in 12 patients (18.4%) (symptomatic (S) seven (10.5%), asymptomatic (A) fi ve (7.7%)), nine (13.8%) carotid (S fi ve (7.7%), A four (6.1%)), 28 (43%) brachiocephalic or subclavian (S 17 (26.1%), A 11 (16.9%)), 15 (22.8%) mesenteric (S seven (10.5%), A eight (12.3%)), 13 (20%) renal (S nine (13.8%), A four (6.1%)), and 31 (47.7%) iliac (S 16 (24.6%), A 15 (23%)). Twenty-eight (43.1%) showed clinical ischemia of at least one system and 54 (83%) clinical signs of global hypoperfusion. Comparing patients with and without data of hypoperfusion, diff erences in incidence of death (45.5% vs. 18.8%, P = 0.03), neurological complication (35.7% vs. 10.8%, P = 0.03), MOF (16.6% vs. 25%. P = 0.07) and ALI (21.3% vs. Prognostic value of the echocardiographic-derived calcium index in coronary artery disease y y J Jimenez, J Iribarren, J Lacalzada, A De La Rosa, R Juárez, A Barragán, J Bonilla, G Blanco, R Pérez, M Brouard, I Laynez Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2012, 16(Suppl 1):P191 (doi: 10.1186/cc10798) Conclusion Echocardiography in the ICU patient relies on numerous factors including skill and equipment availability and patient windows [1]. Our results confi rm that there is a role for echo in these patients, important in a population where assessment of cardiac output and fi lling status is notoriously diffi cult. Our results also show that TTE performed by ICU physicians with basic training provides very useful information for the management of patients. This makes the focused courses on echocardiography very important [2,3]. Limitations of the study: an unknown amount of missing data and a likelihood of patient and operator bias as to which patients had echocardiography. In conclusion, echocardiography is a useful tool in the management of the haemodynamically unstable patients. R f Introduction Calcifi cation of diff erent cardiac structures is associated with atherosclerotic risk factors. The aim of this study is to determine whether the echocardiography-derived calcium index (ECI) assessed by transthoracic echocardiography (TTE) predicts cardiovascular events, besides determining the coronary artery calcium score (CACS), the presence of obstructive coronary artery disease (CAD) and the composition of plaques, all of which determined by multidetector computed tomography (MDCT). Methods We carried out a prospective study of 82 consecutive patients, with an intermediate likelihood for CAD, who were evaluated by noninvasive coronariography by MDCT. ECI was blindly assessed by TTE. A 36-month follow-up was conducted to detect cardiovascular events. Methods We carried out a prospective study of 82 consecutive patients, with an intermediate likelihood for CAD, who were evaluated by noninvasive coronariography by MDCT. ECI was blindly assessed by TTE. A 36-month follow-up was conducted to detect cardiovascular events. Results The area under the ROC curve (AUC) of the Agatston score scale as a predictor of signifi cant obstruction identifi ed by MDCT was 0.80 (95% CI: 0.68 to 0.91); P <0.001. The optimal cut-off was 239. P191 Prognostic value of the echocardiographic-derived calcium index in coronary artery disease J Jimenez, J Iribarren, J Lacalzada, A De La Rosa, R Juárez, A Barragán, J Bonilla, G Blanco, R Pérez, M Brouard, I Laynez Hospital Universitario de Canarias, La Laguna, Spain Critical Care 2012, 16(Suppl 1):P191 (doi: 10.1186/cc10798) P188 Sh This corresponds with our patients: one-third of cases need urgent surgery, 56% have shock, about 60% ARF, and mortality reaches 30%. atrioventricular block, stroke, perioperative MI, and liver failure. The ICU stay was 33 days (median 6). The hospital mortality was 31.5%. Conclusion IE has high morbi-mortality. The subgroup of patients requiring early surgery presents the most severe disease. This corresponds with our patients: one-third of cases need urgent surgery, 56% have shock, about 60% ARF, and mortality reaches 30%. Agatston score ≥239 has a sensitivity (Se) of 60.6% (95% CI: 0.42 to 0.77), specifi city (Sp) of 97.8% (95% CI: 0.88 to 0.99), positive predictive value (PPV) of 95.2% and negative predictive value (NPV) of 77.2%. The AUC of ECI to predict an optimal cut-off value for Agatston score was 0.90 (95% CI: 0.83 to 0.96); P <0.001. ECI ≥7 had a Se of 59.1% (95% CI: 0.36 to 0.79), a Sp of 93.3% (95% CI: 0.83 to 0.98), PPV of 76.5% and NPV of 86.2%. There was a signifi cant linear trend of ECI, and ECI ≥7 has in MDCT a greater presence of both severe calcifi ed wall and obstructive CAD, number of aff ected vessels, and mixed/calcifi ed plaques (all P <0.001). There were 23 coronary ischemic events. The AUC of ECI as a predictor of adverse cardiac events post MDCT was 0.92 (95% CI: 0.852 to 0.987); P <0.001. ECI ≥7 had a Se of 77.3% (95% CI: 54.6 to 92.2), a Sp of 90% (95% CI: 79.5 to 96.2), PPV of 73.9% and NPV of 91.5%. The Kaplan–Meier survival analyses show a statistically signifi cant diff erence between patients with VCSI ≥7 or not regarding an ischemic event (χ2: 52, P <0.001). This accumulation of risk occurs mainly in the fi rst 2 years after the determination of ECI. atrioventricular block, stroke, perioperative MI, and liver failure. The ICU stay was 33 days (median 6). The hospital mortality was 31.5%. atrioventricular block, stroke, perioperative MI, and liver failure. The ICU stay was 33 days (median 6). The hospital mortality was 31.5%. Conclusion IE has high morbi-mortality. The subgroup of patients requiring early surgery presents the most severe disease. This corresponds with our patients: one-third of cases need urgent surgery, 56% have shock, about 60% ARF, and mortality reaches 30%. P188 Sh Forty-one percent of patients required ICU admission before surgery with an average stay of 5.6 days. A total of 31.5% suff ered multiorgan failure. Antibiotics were given 17 days before surgery. In 6.8% it was not possible to give them preoperatively. Eighty-two percent of patients took combination therapy (19% four). Cephalosporins, aminoglucosids and vancomycin were the most used. Two patients died before surgery. Thirty-fi ve percent of the interventions were urgent. In 16.4%, reoperation was necessary, mainly for bleeding, followed by prosthetic dysfunction, recurrent IE, mediastinitis and pseudoaneurysm repair. A total 56% of patients presented postoperative shock. MV was needed during 5 days (range 0 to 53). Acute renal failure post surgery was present in 58%. Other complications were secondary infection, ventricular dysfunction, Table 1 (abstract P188) Carvedilol Nebivolol 6.25 mg/bid 2.5 mg/qd 12.5 mg/bid 5.0 mg/qd 25.0 mg/bid 10.0 mg/qd Results We selected 30 patients, 75% men, age 56.0 (SD = 13.0) years, ejection fraction 23.4 (SD = 7.2)%, ischemic myocardiopathy present in 16.7%, Chagas disease in 40% and 43.3% of patients were nonischemic/ non-Chagas. Baseline indexed systemic vascular resistance was 2,255.9 (SD = 792.4) dynes.second/cm5/m2, and cardiac index was 2.7 (SD = 0.6) l/minute/m. In the nebivolol group (n = 15) the indexed systemic vascular resistance reduced 0.6% and in the carvedilol group (n = 15) it reduced 5.0% in 24 hours (mean diff erence 4.4%; 95% CI: –12.6 to 21.4%; P = 0.513). The cardiac index maintained unchanged (P = 0.274). Comparing patients that received a high dose of nebivolol (5 to 10 mg/ day) to those with a low dose (<5 mg/day) or carvedilol, we observed a tendency to superiority of high dose in reduction of systemic vascular resistance, although not statistically signifi cant (Figure 1). Conclusion Short-term nebivolol use in decompensated heart failure was hemodynamically safe. Further studies should be done to clarify this matter. Table 1 (abstract P188) Carvedilol Nebivolol 6.25 mg/bid 2.5 mg/qd 12.5 mg/bid 5.0 mg/qd 25.0 mg/bid 10.0 mg/qd S69 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 atrioventricular block, stroke, perioperative MI, and liver failure. The ICU stay was 33 days (median 6). The hospital mortality was 31.5%. Conclusion IE has high morbi-mortality. The subgroup of patients requiring early surgery presents the most severe disease. 1. Orme R, et al.: Br J Anaesth 2009, 102:340-344. 2. Vieillard-Baron et al.: Intensive Care Med 2008, 34:243-249. 3. Jensen et al.: Eur J Anaesth 2004, 21:700-707. Malperfusion and branch compromise in acute type A aortic syndrome 29.6%, P = 0.09) were found.f Introduction Assessment of the haemodynamically unstable patient is a core part of ICU management and relies predominantly on a combination of clinical skill and measurement of physiological variables. Echocardiography in the ICU has become increasingly popular as a tool for assessment of cardiac output, fl uid status and ventricular function. Traditionally transoesophageal echo (TOE) has been favoured due to the belief that it gave superior images [1]. Transthoracic echo (TTE) is not often performed as it relies on 24-hour availability of trained personnel, availability of equipment and good patient windows [1]. There was also a perceived lack of benefi t; however, recent studies have shown good or adequate images in over 85 to 90% of patients resulting in a change of management in 48% [1]. g g Methods Data were collected prospectively in all patients undergoing echocardiography in a teaching hospital ICU from January 2008 to January 2011. The main focus of our investigation was to ascertain the clinical questions to be answered and the outcome of echo on management. Conclusion More than 80% of the patients with AAAS suff ered mal per- fusion in our series. They had a higher risk of death and neurological complication during hospitalization. Results A total of 238 echoes were performed on 216 patients with an average age of 59.75 years (TTE: 198, TOE: 19, and both: 14). The most commonly asked questions were on fi lling status and contractility (40%) and left ventricular function (33%). Ninety percent of clinical questions asked were answered fully (74%) or partially (16%) by echo. Sixty-one percent of echoes resulted in a change of management (5% of which were to continue with increased confi dence). TTE performed by operators with basic training resulted in a 54% change in management. Changes included more fi lling (39%) and changes in inotropes or diuretics. References 1. Orme R, et al.: Br J Anaesth 2009, 102:340-344. 2. Vieillard-Baron et al.: Intensive Care Med 2008, 34:243-249. 3. Jensen et al.: Eur J Anaesth 2004, 21:700-707. Results The area under the ROC curve (AUC) of the Agatston score scale as a predictor of signifi cant obstruction identifi ed by MDCT was 0.80 (95% CI: 0.68 to 0.91); P <0.001. The optimal cut-off was 239. 1. Orme R, et al.: Br J Anaesth 2009, 102:340-344. 2. Vieillard-Baron et al.: Intensive Care Med 2008, 34:243-249. 3. Jensen et al.: Eur J Anaesth 2004, 21:700-707. S70 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P193 Figure 1 (abstract P194). Characteristics of the data diff erences between (a) free and (b) hold breathing. Figure 1 (abstract P194). Characteristics of the data diff erences between (a) free and (b) hold breathing. P193 Left ventriculum diastolic dysfunction in pediatric septic shock M Georgiyants, V Korsunov Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2012, 16(Suppl 1):P193 (doi: 10.1186/cc10800) Kharkov Medical Academy Post-Graduate Education, Kharkov, Ukraine Critical Care 2012, 16(Suppl 1):P193 (doi: 10.1186/cc10800) Introduction One of the causes of septic mortality is a low cardiac output secondary to preload failure. Same patients demonstrate preload failure after aggressive volume replacement [1]. Methods Ultrasound impulse-wave Doppler evaluation of transmitral fl ow: VmaxE, VmaxA, ejection time E,A; DT E wave, IVRT of LV. Ultrasound evaluation of end-diastolic and end-systolic LV volume, stroke volume (LVEDV, LVESV, SV) on Teichholz L. EDLVP = 1.06 + 15.15×VTI peakA/ VTI peakE. Coronary perfusion pressure (CPP) = EDLVP – diastolic BP. We evaluate these parameters in 34 patients (age 28.1 ± 8.0 months) with septic shock (SS) diagnosed according to Consensus 2002. Control (C) – 44 healthy children (age 40.7 ± 8.5 months). Statistical analyses with t criteria. Figure 1 (abstract P194). Characteristics of the data diff erences between (a) free and (b) hold breathing. improve the accuracy and reliability of diagnosis on early stages [2]. If the device can be designed as portable, then it can be used by heart disease patients for daily monitoring to avoid or minimize heart attack accidents. To improve the accuracy of heart auscultation analysis, usually the lung sound must be minimized, or vice versa. It is very diffi cult. This study tried to use heart and lung interference sounds as physiological parameters. P194 Existence of interference between the heart and respiratory sounds: preliminary report 4. Mrowka R, Cimponeriu L, Patzak A, Rosenblum MG: Am J Physiol Regul Integr Comp Physiol 2003, 285:R1395-R1401. Brawijaya University, Malang East Java, Indonesia j y y g Critical Care 2012, 16(Suppl 1):P194 (doi: 10.1186/cc10801) 6. Toledo E, et al.: Med Eng Phys 2002, 24:45-52. 7. Darowski M: Front Med Biol Eng 2000, 10:157-165. Introduction Heart diseases still persist as one of the fi rst-ranked causes of mortality in the world and Indonesia. Currently, mortality from coronary heart diseases is estimated to reach 53.5 per 100,000 population [1]. Auscultation is a fundamental diagnostic method for heart disease, noninvasive and inexpensive [2], but highly dependent on the expertise and experience of the listener. Improved accuracy of diagnosis is usually then performed through further examination using the electrocardiogram, magnetic resonance imaging and the computed tomography scan. Unfortunately, these tools require very expensive investment costs that are only available in large hospitals [3]. This is the main reason for supporting the development of computer- based auscultation technique tools that are cheaper and are able to References So this preliminary research aims to prove that interference does occur between heart and respiratory sounds. This interference sound will be used as an analysis technique to improve the accuracy of a new auscultation device. Results The increase of VmaxA and decrease of VmaxE in patients of SS are demonstrated. IVRT and DT are less than in control group. We evaluated a decrease in E/A proportion. EDLVP in patients was more, and CPP lower, than in controls. See Table 1. Table 1 (abstract P193). Diastolic function in pediatric septic shock Table 1 (abstract P193). Diastolic function in pediatric septic shock Value SS C P v Table 1 (abstract P193). Diastolic function in pediatric septic shock Value SS C P value VmaxA 81 65 <0.05 VmaxE 99 108 >0.05 ETA 80 102 0.01 ETE 102 149 0.001 DTE 51 93 0.001 IVRT 48 87 0.01 E/A 1.3 1.7 0.01 EDLVP 23 9.8 <0.001 CPP 22 50 0.001 EDLVV 46 65 <0.001 SV 14 26 0.001 Methods This research was conducted on nine randomly chosen volunteers whose heart sounds were recorded in two conditions: 30 seconds free and hold breathing. The heart sound recording process is done electronically using a modifi ed standard stethoscope to generate digital data. Modifi cations were performed using a mic condenser combined with a voice processing system based on Windows XP. Accuracy of the equipment is ensured by the noise–signal ratio test. Methods This research was conducted on nine randomly chosen volunteers whose heart sounds were recorded in two conditions: 30 seconds free and hold breathing. The heart sound recording process is done electronically using a modifi ed standard stethoscope to generate digital data. Modifi cations were performed using a mic condenser combined with a voice processing system based on Windows XP. Accuracy of the equipment is ensured by the noise–signal ratio test. Results Generally, it can be seen (Figure 1) that there are pronounced diff erences in heart sound data recorded in the conditions of free and hold breathing. This means that the respiration process is likely to aff ect the heart sounds heard on the chest surface. The diff erences that appear are in the form of nodes and amplitude. Diff erences in the form of a node indicate a diff erence in frequency of sounds and color (timbre), while the amplitude diff erences may indicate diff erences in strength and speed of sound propagation. References In general, the number of diff erences in the recording position is close to the number of respiratory cycles so that it is possible these diff erences are caused by respiratory processes. Conclusion The diff erences that can be noticed from the graphical visualization of recorded sounds are in the form of nodes and amplitude. These diff erences that indicate the frequency, sound color, strength and speed of sounds improve the existence of an interference wave between the heart and respiratory sounds. These characteristics will be used to design the new portable auscultation device. References Conclusion Pediatric SS accompanied with LV diastolic dysfunction, which decreases the eff ectiveness of volume restoration therapy, reduces preload and cardiac output. f 1. Jardin F, et al.: Persistent preload defect in severe sepsis despite fl uid loading. A longitudinal echocardiographic study in patients with septic shock. Chest 1999, 116:1354-1359. 1. Jardin F, et al.: Persistent preload defect in severe sepsis despite fl uid loading. A longitudinal echocardiographic study in patients with septic shock. Chest 1999, 116:1354-1359. 1. Persatuan Perawat Nasional Indonesia [http://inna-ppni.or.id/html] 2. Javed F, Venkatachalam PA, Fadzil A: J Phys Conf Ser 2006, 34:1098-1105. 3. Stasis A, et al.: A Multiple Decision Tree Architecture for Medical Diagnosis: The Diff erentiation of Opening Snap, Second Heart Sound Split and Third Heart Sound. CMS Springer-Verlag; 2004:254-274. 4. Mrowka R, Cimponeriu L, Patzak A, Rosenblum MG: Am J Physiol Regul Integr Comp Physiol 2003, 285:R1395-R1401. 5. Schikowski T, et al.: Respir Res 2007, 8:1-11. 6. Toledo E, et al.: Med Eng Phys 2002, 24:45-52. 7. Darowski M: Front Med Biol Eng 2000, 10:157-165. 1. Persatuan Perawat Nasional Indonesia [http://inna-ppni.or.id/html] 2. Javed F, Venkatachalam PA, Fadzil A: J Phys Conf Ser 2006, 34:1098-1105. 3. Stasis A, et al.: A Multiple Decision Tree Architecture for Medical Diagnosis: The Diff erentiation of Opening Snap, Second Heart Sound Split and Third Heart Sound. CMS Springer-Verlag; 2004:254-274. 2. Javed F, Venkatachalam PA, Fadzil A: J Phys Conf Ser 2006, 34:1098 1105. 3. Stasis A, et al.: A Multiple Decision Tree Architecture for Medical Diagnosis: The Diff erentiation of Opening Snap, Second Heart Sound Split and Third Heart Sound. CMS Springer-Verlag; 2004:254-274. P195 Eff ects of the intravenous administration of purine nucleosides guanosine or inosine against hemorrhagic shock in pigs Eff ects of the intravenous administration of purine nucleosides guanosine or inosine against hemorrhagic shock in pigs g Schmidt, D Otsuki, DO Souza, J Auler Jr Faculdade de Medicina da Universidade de São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P195 (doi: 10.1186/cc10802) aculdade de Medicina da Universidade de São Paulo, Brazil , ritical Care 2012, 16(Suppl 1):P195 (doi: 10.1186/cc10802) Introduction Hemorrhagic shock leads to the appearance of substances in plasma that depress Na/K ATPase activity, an eff ect that could be related to signifi cant morbidity and mortality. Recently, some fi ndings S71 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 function, under similar hemodynamic conditions. Hepatic, renal and myocardial respiration of the measured mitochondrial complexes did not signifi cantly diff er between the two treatment groups, except for renal Complex I, State 4 respiration. indicated that purine nucleosides such as guanosine, inosine or adenosine might prolong survival in shocked rats, an eff ect potentially related to the stimulation of Na/K ATPase activity. This study aimed to evaluate the eff ects of intravenous administration of guanosine or inosine combined with lactate Ringer solution (LR) on hemodynamic and oxygenation parameters and survival in an experimental model of hemorrhagic shock (HS). P197 P197 Goal-directed hemodynamic resuscitation in high-risk patients undergoing cardiac surgery: a randomized controlled trial – preliminary data (GRICCS STUDY) E Osawa1, A Rhodes2, J Fukushima1, J Almeida1, F Jatene1, R Nakamura1, M Sundin1, J Auler Jr1, R Kalil Filho1, F Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Charing Cross Hospital, Imperial College NHS Trust, London, UK Critical Care 2012, 16(Suppl 1):P197 (doi: 10.1186/cc10804) g Methods HS was induced in 24 pigs (25 to 30 kg) by blood removal for 20 minutes to target a mean arterial pressure (MAP) of 40 mmHg, which was maintained for 60 minutes with additional blood removal or retransfusion. Animals were treated with LR alone (three times the volume of blood withdrawn) or associated to 1 mmol/l guanosine or 1 mmol/l inosine. Hemodynamic and oxygenation parameters were evaluated at baseline, after HS, immediately after fl uid resuscitation, and 30, 60, 120, 240 and 360 minutes after fl uid resuscitation. Primary outcome was post-shock survival. Statistical analysis of parametric data was performed with one-way ANOVA for repeated measures followed by Student–Newman–Keuls. Goal-directed hemodynamic resuscitation in high-risk patients undergoing cardiac surgery: a randomized controlled trial – preliminary data (GRICCS STUDY) p y E Osawa1, A Rhodes2, J Fukushima1, J Almeida1, F Jatene1, R Nakamura1, M Sundin1, J Auler Jr1, R Kalil Filho1, F Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Charing Cross Hospital, Imperial College NHS Trust, London, UK Critical Care 2012, 16(Suppl 1):P197 (doi: 10.1186/cc10804) Introduction Low cardiac output is a frequent clinical circumstance after cardiac surgery and results in higher morbidity and mortality rates. Goal-directed therapy (GDT) is a validated design that has been proved to reduce the number of perioperative outcomes. We investigated the results of a cardiac index optimization protocol through the use of the LiDCO rapid device. y Results The hemodynamic and oxygenation parameters were not signifi cantly diff erent among pigs treated with RL alone or in combi- nation with guanosine or inosine. No eff ects on post-shock survival were observed in any group. Methods A prospective study that randomized 34 high-risk patients (EuroSCORE higher than 6 or LVEF lower than 45%) to a GDT protocol or a conventional hemodynamic therapy. Patients from the GDT group were resuscitated to a cardiac index higher than 3 l/minute/m2 through the implementation of a three-step approach: (1) fl uid challenge of 250 ml aliquots, (2) dobutamine infusion up to a dose of 20 μg/kg/minute, and (3) blood transfusion to reach a hematocrit higher than 28%. The control group was managed according to institutional protocol. Categorical variables were compared using Fisher’s exact test and categorical variables were compared using the Mann–Whitney U test. Conclusion The actual preliminary results did not demonstrate any additional improvement induced by guanosine or inosine on the hemodynamic and oxygenation parameters or on the post- shock survival during HS. These fi ndings need to be confi rmed in a larger group of animals and further investigation with cellular and biochemical analysis may help to elucidate the eff ects of guanosine and inosine during HS. g Acknowledgments Supported by FAPESP and CNPq. References Acknowledgments Supported by FAPESP and CNPq. g y Results Sixteen patients from the GDT group were compared with 18 patients from the control group. There was a tendency towards reduction in ICU stay in patients from GDT group in relation to the control group (7 days vs. 6 days, P = 0.18). Economic evaluation of early-goal directed therapy for high-risk surgical patients g Methods In 16 anesthetized pigs, evolving septic shock after 12 hours of fecal peritonitis was randomly treated with either norepinephrine (0.8 ± 0.6 μg/kg/minute; mean ± SD) or angiotensin II (0.31 ± 0.37 μg/ kg/minute; n = 8, each) and fl uids for 48 hours. Organs were harvested at the end of the experiment, and mitochondria isolated by tissue homogenization and diff erential centrifugation. Mitochondrial oxygen consumption (VO2) was measured by high-resolution respirometry (Oroboros Instruments, Innsbruck, Austria). Groups were compared using Mann–Whitney U test. In addition, mitochondrial respiration was also compared to a similarly instrumented control group without fecal peritonitis (n = 8; Kruskal–Wallis test). g p C Ebm, M Cecconi, H Aya, M Geisen, A Rhodes, M Grounds St George’s Healthcare Trust, London, UK C Ebm, M Cecconi, H Aya, M Geisen, A Rhodes, M Grounds St George’s Healthcare Trust, London, UK Critical Care 2012, 16(Suppl 1):P198 (doi: 10.1186/cc10805) Introduction Early goal-directed therapy (EGDT) has been shown to reduce postoperative morbidity and length of hospital stay. Our objective was to analyse the cost-eff ectiveness of early goal-directed proactive therapy versus standard reactive care in patients at high risk of developing postoperative complications. p g p p p Methods Patient-level outcome data used were based on a previous randomised, controlled trial. A Markov decision model was constructed to analyse costs and outcomes associated with the use of EGDT. Outcomes assessed were postoperative complications, mortality, quality-adjusted life expectancy (QALY) and incremental costs/QALY. Results The main analysis, based on 28-day survival data of 122 patients, revealed an incremental cost-eff ectiveness ratio of EGDT of £280.15 per patient. Additional costs of £525.43 per patient associated with EGDT were mainly due to costs related to monitor acquisition and staffi ng (two additional nurses). These costs were balanced by savings due to the signifi cant reduction in length of stay in the hospital and in the ICU and lower complication rates in the GDT arm (mean expenditures/patient £4,511.25 vs. £5,218.75). This outcome was Methods Patient-level outcome data used were based on a previous randomised, controlled trial. A Markov decision model was constructed to analyse costs and outcomes associated with the use of EGDT. Outcomes assessed were postoperative complications, mortality, quality-adjusted life expectancy (QALY) and incremental costs/QALY. Norepinephrine versus angiotensin II in septic shock: eff ects on isolated kidney, heart and liver mitochondrial respiration V Jeger, M Vuda, T Correa, J Takala, S Djafarzadeh, SM Jakob Inselspital University Hospital Bern, Switzerland Critical Care 2012, 16(Suppl 1):P196 (doi: 10.1186/cc10803) Norepinephrine versus angiotensin II in septic shock: eff ects on isolated kidney, heart and liver mitochondrial respiration V Jeger, M Vuda, T Correa, J Takala, S Djafarzadeh, SM Jakob Inselspital University Hospital Bern, Switzerland Critical Care 2012, 16(Suppl 1):P196 (doi: 10.1186/cc10803) Conclusion Goal-directed hemodynamic resuscitation with the use of a minimally invasive device seems to be a promising perioperative strategy aimed at reducing the rates of worse outcomes and the ICU stay after cardiac surgery. p y p Critical Care 2012, 16(Suppl 1):P196 (doi: 10.1186/cc10803) Introduction Mitochondrial dysfunction has been proposed to infl uence organ function and outcome in sepsis. Both vasopressor agents norepinephrine and angiotensin II can interfere with mitochondrial function. The aim of this study was to compare mitochondrial respiration after exposure of septic animals to either of these two drugs. Goal-directed hemodynamic resuscitation in high-risk patients undergoing cardiac surgery: a randomized controlled trial – preliminary data (GRICCS STUDY) Comparison of the primary endpoint variable (composite of death or major postoperative complications within 30 days after surgery or before discharge) between groups showed a reduced complication rate in the GDT group (52.2% vs. 45.6%, P = 0.12), mainly attributed to worse acute renal failure RIFLE criteria in the control group. 1. Darlington DN, Gann DS:. J Trauma 2005, 58:1055-1060. 2. Schmidt AP, et al.: Pharmacol Ther 2007, 116:401-416. 1. Darlington DN, Gann DS:. J Trauma 2005, 58:1055-1060. 2. Schmidt AP, et al.: Pharmacol Ther 2007, 116:401-416. P195 Eff ects of the intravenous administration of purine nucleosides guanosine or inosine against hemorrhagic shock in pigs Kruskal–Wallis followed by the Dunn test was used for analysis of nonparametric data. The post-shock survival was evaluated by the Kaplan–Meier curve. 1. Darlington DN, Gann DS:. J Trauma 2005, 58:1055-1060. 2. Schmidt AP, et al.: Pharmacol Ther 2007, 116:401-416. What matters during a hypotensive episode: fl uids, vasopressors, or both? What matters during a hypotensive episode: fl uids, vasopressors, or both? J Lee1, R Kothari2, JA Ladapo3, DJ Scott1, LA Celi1 1Massachusetts Institute of Technology, Cambridge, MA, USA; 2Mount Sinai School of Medicine, New York City, NY, USA; 3New York University School of Medicine, New York City, NY, USA Critical Care 2012, 16(Suppl 1):P199 (doi: 10.1186/cc10806) J Lee1, R Kothari2, JA Ladapo3, DJ Scott1, LA Celi1 1Massachusetts Institute of Technology, Cambridge, MA, USA; 2Mount Sinai School of Medicine, New York City, NY, USA; 3New York University School of Medicine, New York City, NY, USA g Conclusion In septic patients with CVP >12 mmHg after resuscitation, micro circulatory fl ow was signifi cantly lower as compared to patients with CVP ≤12 mmHg, whereas capillary density did not diff er between groups. y Critical Care 2012, 16(Suppl 1):P199 (doi: 10.1186/cc10806) Introduction The objective of this retrospective study was to investigate the relationships between fl uid and vasopressor interventions and patient outcomes. In intensive care, it is imperative to resolve hypotensive episodes (HEs) in a timely manner in order to minimize end-organ damage. The current clinical practice is fi rst to attempt fl uid resuscitation and then to follow with vasopressor therapy if fl uid resuscitation is unsuccessful. However, the eff ects of fl uid and vasopressor interventions on patient outcomes have not been clearly established.i Figure 1 (abstract P200). Boxplots of microvascular fl ow index (MFI) in patients with a central venous pressure (CVP) ≤12 mmHg or >12 mmHg. p y Methods Hypotension was defi ned as MAP below 60 mmHg. The primary outcome was in-hospital mortality. Secondary outcomes included ICU LOS, HE duration, Hypotension Severity Index (HSI) (MAP curve area below 60 mmHg during the HE), and rise in serum creatinine. The patient cohort included patients in the MIMIC-II database [1] who experienced a single HE. Multivariate logistic regression and propensity score analysis were employed. Sensitivity analyses were conducted in subpopulations stratifi ed by treatment type and diagnosis. p pi y yp g Results A total of 3,163 patients in MIMIC-II met the inclusion criteria. The multivariate regression results showed that fl uid resuscitation was signifi cantly associated with shorter ICU LOS (OR = 0.71, P = 0.007) and greater HSI (OR = 1.26, P = 0.04). What matters during a hypotensive episode: fl uids, vasopressors, or both? Vasopressor administration signifi cantly decreased HE duration (OR = 0.29, P <0.001) and HSI (OR = 0.72, P = 0.002) but was correlated with increased in-hospital mortality risk (OR = 2.86, P <0.001) (even after propensity adjustment; OR = 2.44, P <0.001), prolonged ICU LOS (OR = 1.29, P = 0.04), and rise in serum creatinine (OR = 1.44, P = 0.002). Sensitivity analyses in treatment- specifi c and diagnosis-specifi c subpopulations corroborated the relationship between vasopressors and increased in-hospital mortality. Conclusion Regarding the relationship between vasopressor therapy and in-hospital mortality, similar fi ndings have been reported in previous studies analyzing sepsis [2], cardiac surgery [3], and heart failure [4]. We speculate that benefi ts of vasopressor use may be restricted to subsets of patients with specifi c conditions. This study illustrates the utility of electronic medical records in research when randomized controlled trials are diffi cult to conduct. f Figure 1 (abstract P200). Boxplots of microvascular fl ow index (MFI) in patients with a central venous pressure (CVP) ≤12 mmHg or >12 mmHg. P199 g y y Results A total of 345 measurements in 70 patients (APACHE II 21 (6.5) (mean (SD))) were included. MFI in patients with CVP >12 mmHg was signifi cantly lower than in CVP ≤12 mmHg (1.83 (0.92 to 2.75) vs. 2.25 (1.35 to 2.90) (median (IQR)), P = 0.032), whereas TVD in both groups did not diff er signifi cantly (14 (12.84 to 15.75) vs. 14.3 (13 to 15.8) mm/ mm2, P = 0.38). See also Figure 1. P201 Human protein C concentrate to restore physiological values in adult septic shock patients: eff ects on microcirculationf Human protein C concentrate to restore physiological values in adult septic shock patients: eff ects on microcirculationf Economic evaluation of early-goal directed therapy for high-risk surgical patients Results Achieved blood pressure levels and cardiac output were not diff erent between the two septic groups, and both groups received the same amount of fl uids (norepinephrine: 1.6 ± 0.5 ml/kg/hour, angiotensin II: 1.3 ± 0.8 ml/kg/hour; P = NS). Compared to controls, mitochondrial VO2 was not diff erent in septic animals. The only diff erence between the two septic groups was higher renal Complex I, State 4 respiration in norepinephrine-treated (median (range): 309 (164 to 415) pmol/(secondmg)) versus angiotensin-II-treated animals (210 (89 to 273) pmol/(secondmg); P = 0.05).if Results The main analysis, based on 28-day survival data of 122 patients, revealed an incremental cost-eff ectiveness ratio of EGDT of £280.15 per patient. Additional costs of £525.43 per patient associated with EGDT were mainly due to costs related to monitor acquisition and staffi ng (two additional nurses). These costs were balanced by savings due to the signifi cant reduction in length of stay in the hospital and in the ICU and lower complication rates in the GDT arm (mean expenditures/patient £4,511.25 vs. £5,218.75). This outcome was Conclusion We found no signifi cant eff ects of septic shock treated with either angiotensin II or norepinephrine and fl uids on mitochondrial Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S72 Table 1 (abstract P198) Outcome Unit GDT Standard Ward stay (days) 11 (7 to 15) 14 (11 to 27) Incr. costs (£) 525.43 – Inc. eff ect (QALY) 1.88 – ICER (£/QALY) 280.15 – ICER, incremental cost-eff ectiveness ratio. P200 Elevated central venous pressure in septic patients is associated with impairment of microcirculatory blood fl ow N Vellinga1, C Ince2, EC Boerma1 1Medisch Centrum Leeuwarden, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P200 (doi: 10.1186/cc10807) P200 Elevated central venous pressure in septic patients is associated with impairment of microcirculatory blood fl ow N Vellinga1, C Ince2, EC Boerma1 1Medisch Centrum Leeuwarden, the Netherlands; 2Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P200 (doi: 10.1186/cc10807) Critical Care 2012, 16(Suppl 1):P200 (doi: 10.1186/cc10807) Introduction The microcirculation plays a pivotal role in oxygen delivery to the tissue. Microcirculatory alterations have been observed to occur independently of the major infl ow variable for microcirculation: mean arterial pressure. According to physiological theory, the microcirculation is considered to be a low-pressure compartment. Maximum optimal central venous pressure (CVP) according to Surviving Sepsis Campaign (SSC) guidelines is 12 to 15 mmHg in mechanically ventilated patients. We hypothesized that a CVP >12 mmHg would hamper microcirculatory perfusion but not diff usion, by acting as outfl ow obstruction. robust to variations in treatment eff ect (probability of morbidity and mortality) and sensitive to implementation costs of EGDT. See Table 1. Conclusion The implementation of EGDT appears clinical and cost- eff ective. Additional implementation costs will be off set by savings due to a marked decrease in complication rates and hospital length of stay. We conclude that GDT provides signifi cant benefi ts with respect to both clinical and fi nancial outcomes. Reference Methods We retrospectively analyzed combined measurements of CVP and sidestream dark-fi eld derived sublingual microcirculatory variables in patients with severe sepsis or septic shock. Measurements were made 0, 0.5, 2, 12 and 24 hours after resuscitation in accordance with SSC guidelines. Diff erences in small vessel microvascular fl ow index (MFI) and total vessel density (TVD) between two groups (CVP ≤12 mmHg and CVP >12 mmHg) were analyzed with a Mann–Whitney U test. 1. Pearse R, Dawson D, Fawcett J, Rhodes A: Early goal directed therapy after major surgery reduces complications and duration of hospital stay. A randomized, controlled trial. Crit Care 2005, 9:R687-R693. P201 Human protein C concentrate to restore physiological values in adult septic shock patients: eff ects on microcirculation A Morelli 1, A Donati2, A Di Russo1, F D’Ippolito1, C Raff one1, A D’Egidio1, MR Lombrano2, S Tondi2, E Damiani2, V Cecchini1, A Orecchioni1, P Pietropaoli1 1University La Sapienza, Rome, Italy; 2Marche Polytechnique University, Ancona, Italy Critical Care 2012, 16(Suppl 1):P201 (doi: 10.1186/cc10808) References 1. Saeed M, et al.: Crit Care Med 2011, 39:952-960. 2. Dunser M, et al.: Crit Care 2009, 13:R181. 3. Shahin J, et al.: Crit Care 2011, 15:R162. 4 Thackraya S et al : Eur J Heart Fail 2002 4:515-529 1. Saeed M, et al.: Crit Care Med 2011, 39:952-960 P202 Heart rate reduction with esmolol in septic shock: eff ects on microcirculation A Morelli1, A Donati2, A Di Russo1, F D’Ippolito1, A Carsetti2, R Domizi2, A D’Egidio1, C Raff one1, C Scarcella2, C Ertmer3, S Rehberg3, P Pietropaoli1, M Westphal3 1University La Sapienza, Rome, Italy; 2Marche Polytechnique University, Ancona, Italy; 3University Hospital of Muenster, Germany Critical Care 2012, 16(Suppl 1):P202 (doi: 10.1186/cc10809) Heart rate reduction with esmolol in septic shock: eff ects on microcirculation A Morelli1, A Donati2, A Di Russo1, F D’Ippolito1, A Carsetti2, R Domizi2, A D’Egidio1, C Raff one1, C Scarcella2, C Ertmer3, S Rehberg3, P Pietropaoli1, M Westphal3 p 1University La Sapienza, Rome, Italy; 2Marche Polytechnique University, Ancona, Italy; 3University Hospital of Muenster, Germany Critical Care 2012, 16(Suppl 1):P202 (doi: 10.1186/cc10809) Results Infusion of E. coli resulted in a hypodynamic state of sepsis despite fl uid administration. Signifi cant decreases in MFI and PPV of small vessels were in sublingual, conjunctival, jejunal and rectal lodges 3 and 5 hours after the start of E. coli infusion in comparison to baseline variables. Correlation between sublingual and conjunctival (r = 0.80, P = 0.036), sublingual and jejunal (r = 0.94, P = 0.005), sublingual and rectal (r = 0.79, P = 0.03) MFI was observed 3 hours after onset of sepsis. There was no correlation in change of MFI and PPV between sublingual mucosa and other evaluated regions. However, the sublingual mucosa exhibited the most pronounced alterations of microcirculatory fl ow in comparison to conjunctival, jejunal and rectal mucosa microvasculature (P <0.05). Introduction Preclinical and clinical studies report that β-blockers may be an interesting option to attenuate the deleterious eff ects of prolonged catecholamine exposure during septic shock. Nevertheless, there are concerns that β-blockers may have negative chronotropic and inotropic eff ects leading to inappropriately low cardiac output. The objective of the present study was therefore to elucidate whether a reduction in heart rate (HR) with esmolol may negatively aff ect microcirculation in patients with septic shock who remained tachycardic after hemodynamic optimization. Conclusion Microcirculatory alterations were observed in all investi- gated lodges, including sublingual, jejunal and rectal mucosa, and conjunctiva of the eye at the same time point during experimental sepsis. There is a clear association between sublingual microcirculation and conjunctival, jejunal or rectal microcirculation in the very early course of an extreme hypodynamic state of sepsis. Methods After 36 hours of initial hemodynamic stabilization, 11 septic shock patients with HR >95 bpm and requiring norepinephrine (NE) to maintain mean arterial pressure (MAP) between 65 and 75 mmHg, despite adequate volume resuscitation, received a continuous esmolol infusion to maintain HR between 94 and 80 bpm. NE was titrated to achieve a MAP between 65 and 75 mmHg. Data from right heart catheterization and sidestream dark-fi eld imaging were obtained at baseline and after 24 hours. Early course of microcirculatory perfusion in the eye and digestive tract during experimental sepsis A Pranskunas1, R Rasimaviciute1, E Milieskaite1, A Vitkauskiene1, P Dobozinskas1, V Veikutis2, Z Dambrauskas1, D Vaitkaitis1, V Pilvinis1 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2012, 16(Suppl 1):P203 (doi: 10.1186/cc10810) Table 1 (abstract P201). Microcirculatory variables Baseline 24 hours 48 hours 72 hours MFIs Treated 2.8(2.6; 3) 3 (2.7; 3) 2.9 (2.8; 3) 3 (2.9; 3) Controls 2.8 (2.1; 2.9) 2.8 (2.1; 2.8) 2.8 (2.2; 3) 3 (2.6; 3) PVD Treated 17.8 (16.5; 22.2) 19.7 (17.4; 22.5) 19.7 (18.1; 23) 19.9 (17; 22.2) Controls 20.2 (17.4; 23.5) 18.8 (17.6; 20.2) 19.4 (17.5; 20.7) 18.7 (17.5; 21.2) Conclusion The administration of human PC concentrate did not i fl i i l bl d fl i i h k i Table 1 (abstract P201). Microcirculatory variables Introduction Studies show that sublingual mucosa is a reproducible part for small intestine mucosal microcirculatory perfusion in sepsis, when they are not exposed by local factors. However, it is of great interest how sublingual microcirculation can refl ect other beds of microcirculation. The aim of the study is to evaluate and compare the microcirculatory perfusion of potentially available parts of the body, such as sublingual mucosa, conjunctiva of the eye, mucosa of jejunum and rectum, at the same time points during experimental sepsis. p g p p Methods Pigs were randomly assigned to sepsis (n = 9) and sham (n = 4) groups. The sepsis group received a fi xed dose of live Escherichia coli infusion over 1 hour. Animals were observed 5 hours after the start of E. coli infusion. In addition to systemic hemodynamic assessment, we performed conjunctival, sublingual, jejunal and rectal evaluation of microcirculation using sidestream dark-fi eld videomicroscopy at the same time points: at baseline, 3 and 5 hours after the start of live E. coli infusion. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)) and diff usion distance (perfused vessel density, total vessel density) was done using a semiquantitative method. References y Critical Care 2012, 16(Suppl 1):P201 (doi: 10.1186/cc10808) References 1. Saeed M, et al.: Crit Care Med 2011, 39:952-960. 2. Dunser M, et al.: Crit Care 2009, 13:R181. 3. Shahin J, et al.: Crit Care 2011, 15:R162. 4. Thackraya S, et al.: Eur J Heart Fail 2002, 4:515-529. Introduction We investigated whether human protein C (PC) concen- trate to restore physiological values in adult septic shock patients can infl uence microcirculatory blood fl ow. S73 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods We enrolled 36 septic shock patients with plasma protein C activity <60%. Patients were randomly allocated to be treated with either a continuous infusion of PC concentrate at 3 UI/kg/hour for 72 hours to reach plasma protein C activity between 70 and 120% or a standard treatment (control; each n = 18). In both groups, NE was titrated to achieve a MAP between 65 and 75 mmHg. Data from right heart catheterization and sidestream dark-fi eld imaging were obtained at baseline and after 24, 48 and 72 hours.if the small vessels (MFIs) and norepinephrine requirements did not vary during the 24-hour observational period. Results are summarized in Table 1. Conclusion In patients with established septic shock who remained tachycardic after hemodynamic optimization in accordance with the current guidelines, titration of esmolol to reduce the HR to a predefi ned threshold did not aff ect microcirculatory blood fl ow. Results For the same MAP and cardiac output, no signifi cant diff erences were found between groups in terms of microvascular fl ow index of the small vessels (MFIs) and perfused vessel density (PVD). Results are summarized in Table 1. P203 P203 Early course of microcirculatory perfusion in the eye and digestive tract during experimental sepsis A Pranskunas1, R Rasimaviciute1, E Milieskaite1, A Vitkauskiene1, P Dobozinskas1, V Veikutis2, Z Dambrauskas1, D Vaitkaitis1, V Pilvinis1 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2012, 16(Suppl 1):P203 (doi: 10.1186/cc10810) P204 Results Apart from a statistically signifi cant decrease in HR and cardiac index (CI) (P <0.05), stroke volume (SV), microvascular fl ow index of P204 Microcirculation and blood transfusion: eff ects of three diff erent types of concentrated red blood cells – preliminary results A Donati, E Damiani, R Domizi, C Scorcella, A Carsetti, MR Lombrano, V Fiori, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2012, 16(Suppl 1):P204 (doi: 10.1186/cc10811) Table 1 (abstract P202) Baseline 24 hours HR 119 ± 12 85 ± 9 CI 4.4 ± 1 3.1 ± 1* SV 81 ± 35 80 ± 23 MFIs 2.6 ± 0.6 2.8 ± 0.3 NE 0.7 ± 0.7 0.5 ± 0.4 P <0.05. Introduction Red blood cell (RBC) transfusions are used to increase oxygen delivery; however, a restrictive transfusion strategy (predefi ned hemoglobin threshold of 7 g/dl) was demonstrated to be associated with lower mortality and incidence of nosocomial infections than a liberal one [1,2]. This may be related to the storage process, which could aff ect the ability of RBCs to transport and delivery oxygen, or to immunomodulating eff ects of cytokines from residual leukocytes [2]. Introduction Red blood cell (RBC) transfusions are used to increase oxygen delivery; however, a restrictive transfusion strategy (predefi ned hemoglobin threshold of 7 g/dl) was demonstrated to be associated with lower mortality and incidence of nosocomial infections than a liberal one [1,2]. This may be related to the storage process, which could aff ect the ability of RBCs to transport and delivery oxygen, or to immunomodulating eff ects of cytokines from residual leukocytes [2]. S74 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 explores the relationship between peripheral perfusion parameters and macrohemodynamic, metabolic, hepatosplanchnic, and micro- circulatory-related parameters during hyperdynamic septic shock. Figure 1 (abstract P204). De Backer score pre and post transfusion in group 2. Methods Thirty-nine sets of parallel assessments of hemodynamic or perfusion-related parameters were performed in 13 hyperdynamic (cardiac index >2.5 l/minute/m2) septic shock patients (age 68 ± 18 years, APACHE II score 26 ± 6, SOFA score 11 ± 4, ICU mortality 2/13) during the fi rst 24 hours of resuscitation. P204 Assessment included: echocardiographic and pulmonary artery catheter-derived parameters; indocyanine green plasma disappearance rate (ICG-PDR, Limon) and gastric tonometry; metabolic parameters (lactate, SvO2 and p(v-a) CO2); sublingual microcirculatory assessment (SDF); thenar StO2 and vascular occlusion test (VOT) derived parameters (NIRS); peripheral perfusion parameters including capillary refi ll time and central to toe temperature diff erence. Results Peripheral perfusion was normal in 22 sets (56%) and abnormal in 17 (44%). A normal peripheral perfusion was associated with lower APACHE II scores (23.2 ± 3 vs. 28.4 ± 7, P = 0.009), better metabolic parameters (lactate: 2.3 ± 0.6 mmol/l vs. 3.5 ± 1.1 mmol/l, P = 0.002 and SvO2: 78.1 ± 6% vs. 73.9 ± 5%, P = 0.049), and better StO2 recovery slope after VOT (3.54 ± 1.4 vs. 0.94 ± 0.5%/second, P <0.001) as compared with an abnormal one. No correlation could be demonstrated with macrohemodynamic parameters, hepatosplanchnic perfusion para meters (gastric tonometry and ICG-PDR), or with sublingual microcirculatory parameters. p Conclusion A normal peripheral perfusion is associated with normal metabolic perfusion parameters and less impaired micro- vascular reactivity. No relation between peripheral perfusion and hepatosplanchnic or sublingual microcirculatory fl ow could be established in this study.i Figure 1 (abstract P204). De Backer score pre and post transfusion in group 2. The aim of the study is to evaluate the eff ects, on microcirculation of septic patient, of three types of RBCs. y ClinicalTrials.gov Identifi er: NCT01271153 y ClinicalTrials.gov Identifi er: NCT01271153 i owledgements Supported by grant FONDECYT 1100610 (Ch i Acknowledgements Supported by grant FO Methods A controlled randomized prospective study on 45 patients with sepsis, severe sepsis or septic shock requiring RBC transfusion. Patients are randomized into three groups receiving: (1) fresh standard RBCs (storage <10 days); (2) leukodepleted RBCs; and (3) old standard RBCs (storage >20 days) respectively. Before and 1 hour after the transfusion, microcirculation is evaluated using sidestream dark-fi eld imaging [3] and near-infrared spectroscopy with a vascular occlusion test. We also monitor temperature, heart rate, mean blood pressure, hemochrome, blood gases, blood lactates and SOFA score. P206 P206 Hyperoxia aff ects peripheral tissue microcirculation in patients with pulmonary arterial hypertension S Dimopoulos, G Tzanis, C Manetos, A Tasoulis, A Mpouchla, E Tseliou, I Vasileiadis, N Diakos, J Terrovitis, S Nanas University of Athens, Greece Critical Care 2012, 16(Suppl 1):P206 (doi: 10.1186/cc10813) g Results Preliminary data on 18 patients, six for each group: before and after transfusion, in group 2, but not in groups 1 and 3, there is a trend to an increase in MFIs (P = 0.09), DeBacker score (Figure 1, P <0.05), PPV (P = 0.07) and PVD (P = 0.07). No relevant diff erences for other parameters. Introduction Pulmonary microcirculation abnormalities play a central role in pulmonary arterial hypertension (PAH) pathophysiology. We hypothesized that PAH patients also have systemic muscle microcirculation alterations compared to healthy subjects. The aim of this study was to investigate peripheral muscle microcirculation by near-infrared spectroscopy (NIRS) in PAH patients and to test the eff ects of hyperoxia into their tissue microcirculation. p Conclusion After transfusion, microcirculation seems to be improved in the leukodepleted RBC group with a signifi cant improvement of De Backer score and a trend to improve the other microcirculatory parameters, while in the other three groups there was not this trend. References f yp Methods Eight PAH patients and eight healthy subjects matched for age, gender and body mass index underwent NIRS evaluation. Tissue O2 saturation (StO2, %), defi ned as the percentage of hemoglobin saturation in the microvasculature compartments, was measured on the thenar muscle. Subsequently, the 3-minute brachial artery occlusion technique was applied before, during, and after 15 minutes of 100% of O2-breathing. Main measurements included the oxygen consumption rate (OCR, %/minute), the reactive hyperemia time (RHT, minutes), and the time needed for StO2 to reach its baseline values after the release of the occlusion. 1. Hebert PC: N Engl J Med 1999, 340:409-417. 1. Hebert PC: N Engl J Med 1999, 340:409-417. 2. Rosemary L: Blood Transfusion 2010, 8(Suppl 3):S26. 2. De Backer D: Crit Care 2007, 11:R101. P207 Supraclavicular ultrasound-guided subclavian vein cannulation in infants under 5 kg P Kenderessy Faculty Children Hospital, Banska Bystrica, Slovakia Critical Care 2012, 16(Suppl 1):P207 (doi: 10.1186/cc10814) 16%). All right-sided catheters lay at an angle <30°. However, 38% (14/37) of left-sided catheters had not crossed the midline, and 59% (22/37) lay at an angle >30° to the vertical. Only 11% (4/37) of left-sided catheters had crossed the midline and lay at an angle of <30°, and all of these lay below the level of the carina. No immediate complications of insertion were identifi ed. See Table 1. Table 1 (abstract P208). Site of CVC insertion (n = 137) Internal jugular Subclavian Right 95 5 Left 32 5 Introduction Central venous cannulation is at some point diffi cult in small children and is associated with many complications especially in multiple-attempt cases. Various techniques exist to achieve successful cannulation. Ultrasound (US)-guided techniques are reported to be safe and reduce the rate of complications for internal jugular vein (IJV) cannulation. We describe an US-guided supraclavicular approach to another central vein – the subclavian vein (SCV). The supraclavicular approach to the SCV with anatomical landmarks was described by Yoff a, but physicians are hesitant to use this technique because of the short distance to pleura.i Conclusion There was a wide variation of catheter tip placements accepted without re-positioning. Left-sided catheter tips are more at risk of less precise (and thus potentially nonoptimal) placement. Our results indicate that a clearer placement strategy is required. References p Methods The principle of the US-navigated technique is to fi nd the SCV at the supraclavicular level and to obtain a longitudinal view of the SCV and to allow access to the vein in-plane view (absolute control of the needle). The ultrasound probe (2.5 cm, 6 to 13 MHz) was placed above the clavicle to visualize the IJV and tilted showing the subclavian artery and SCV in longitudinal view. This view permitted an in-plane puncture of the vein avoiding arterial or plural hit. 1. Stonelake PA, et al.: The carina as a radiological landmark for central venous catheter tip position. Br J Anaesth 2006, 96:335-340. p p 2. Bodenham A: Reducing major procedural complications from central venous catheterization. Anaesthesia 2011, 66:1-9. Results Seventy-eight infant and newborns under 5 kg (1.2 to 5 kg) and 83 SCV cannulations were enrolled in this observational study during a period of 11 months (January 2011 to November 2011). Central venous catheter placement: where is the end of the line? K Tizard, I Welters Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P208 (doi: 10.1186/cc10815) Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P208 (doi: 10.1186/cc10815) Conclusion Power-injectable PICCs have many advantages in the ICU: they can be used as multipurpose central lines for any type of infusion including high-fl ow infusion, for hemodynamic monitoring, and for high-pressure injection of contrast media during radiological procedures. Their insertion is successful in 100% of cases and is not associated with signifi cant risks, even in patients with coagulation disorders. Their maintenance is associated with an extremely low rate of infective and noninfective complications. Introduction There is still controversy regarding safe placement of central venous catheters (CVCs) as to where the tip should lie to avoid mechanical complications whilst maintaining eff ective use [1,2]. The carina has previously been suggested as a useful landmark to avoid intracardiac placement and its associated risks, and also that the catheter tip should lie within the superior vena cava parallel to its walls [1,2]. However, this has been disputed and there remains no consensus as to optimal tip placement. To gauge our current practice we performed a retrospective review of CVCs placed via the internal jugular or subclavian route in intensive care patients to assess where CVC tips were placed. P209 Power-injectable peripherally inserted central catheters in intensive care patients MG Annetta, C Marano, A Brutti, D Celentano, M Pittiruti Catholic University, Rome, Italy Introduction In ICUs, peripherally inserted central catheters (PICCs) may be an alternative option to standard central venous catheters, particularly in patients with coagulation disorders or at high risk for infection. Some limits of PICCs (such as low fl ow rates) may be overcome by the use of power-injectable catheters. Methods We have retrospectively reviewed all of the power-injectable PICCs inserted in adult and pediatric patients in the ICU during a 12-month period, focusing on the rate of complications at insertion and during maintenance. All PICCs were inserted by specifi cally trained nurses, using ultrasound guidance and the microintroducer technique, according to a specifi c insertion protocol. p p Conclusion A supraclavicular US-guided approach to SCV cannulation is safe and eff ective possibility for central vein cannulation in small infants. More studies are needed to establish a learning curve for pure paediatric intensivists without experience with US navigation. Results We have collected 89 power-injectable PICCs (65 in adults and 24 in children), 4 to 6 Fr, both multiple and single lumen. All insertions were successful. There were no major complications at insertion and no episodes of local infection or catheter-related bloodstream infection. Noninfective complications during management were not clinically relevant. There was one episode of symptomatic thrombosis during the stay in the ICU and one episode after transfer of the patient on a nonintensive ward. P205 P205 Peripheral perfusion is correlated to metabolic perfusion parameters and microvascular reactivity but not with hepatosplanchnic or microcirculatory fl ow parameters in hyperdynamic septic shock G Hernandez1, T Regueira1, A Bruhn1, P Mcnab1, E Veas1, C Pedreros1, A Fuentealba1, E Kattan1, G Bugedo1, M Rovegno1, R Castro1, C Ince2 1Pontifi cia Universidad Catolica de Chile, Santiago, Chile; 2University of Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P205 (doi: 10.1186/cc10812) Results PAH patients had a signifi cantly lower resting StO2 (65.8 ± 14.9 vs. 82.1 ± 4.0, P = 0.01), a lower OCR (35.3 ± 9.1 vs. 43.4 ± 19.7) and a higher RHT (3.0 ± 0.6 vs. 2.0 ± 0.3, P <0.001) compared to controls. Hyperoxic breathing increased StO2 (65.8 ± 14.9 to 71.4 ± 14.5, P <0.05) in PAH patients, while OCR was reduced (35.3 ± 9.1 to 25.1 ± 6.6, P <0.05) and RHT was further increased (3.0 ± 0.6 to 4.2 ± 0.7, P <0.01). Conclusion PAH patients present a signifi cant impairment of peripheral tissue microcirculation as assessed by the NIRS occlusion technique. Acute hyperoxic breathing aff ects peripheral microcirculatory function in PAH patients, possibly due to oxidative stress and evoked vasoconstriction. Introduction Peripheral perfusion assessment is increasingly being recognized as a potential surrogate of global perfusion parameters during septic shock resuscitation. Nevertheless, its correlation with other perfusion parameters is not well established. This study S75 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P207 Supraclavicular ultrasound-guided subclavian vein cannulation in infants under 5 kg P Kenderessy Faculty Children Hospital, Banska Bystrica, Slovakia Critical Care 2012, 16(Suppl 1):P207 (doi: 10.1186/cc10814) P207 Supraclavicular ultrasound-guided subclavian vein cannulation infants under 5 kg P Kenderessy Faculty Children Hospital, Banska Bystrica, Slovakia Critical Care 2012, 16(Suppl 1):P207 (doi: 10.1186/cc10814) P207 Supraclavicular ultrasound-guided subclavian vein cannulation in infants under 5 kg P Kenderessy Faculty Children Hospital, Banska Bystrica, Slovakia Critical Care 2012, 16(Suppl 1):P207 (doi: 10.1186/cc10814) All cannulations were performed by a single anesthesiologist trained for ultrasound in central line cannulation with established eye–hand coordination (5 years experience with peripheral blocks under US). For all cases the SCV was easily and quickly visualized, one case had an extremely narrow SCV. The US window for cannulation was always established for free in-plane placement of the needle. The overall success rate for puncture was 100% and for cannulation was 98%. In the case with an extremely narrow vein (because of oedema and stricture) the SCV was punctured but it was impossible to pass the catheter in. The success rate of puncture at fi rst attempt was 97%, at second attempt was 100%. A second attempt was necessary in two cases because needle visualization and angle of the needle movement were not considered correct. No complication was reported. P212 in pediatric cardiac surgery in terms of success rate and mechanical and infectious complications. Errors in the arterial blood pressure measurement F Franchi1, V De Palo1, A Faltoni1, S Cecchini1, L Cubattoli2, P Giomarelli1 1University of Siena, Italy; 2Hospital of Siena, Italy Critical Care 2012, 16(Suppl 1):P212 (doi: 10.1186/cc10819) Methods After Ethics Committee approval and written informed consent from the parents of the children were obtained, 200 children who were scheduled for cardiac surgery were randomly allocated to IJV (n = 100) and SV (n = 100) groups. Introduction The artefacts aff ecting arterial wave morphology may compromise recorded values of arterial blood pressure (ABP) and can lead to therapeutic errors. The aim of this study is to evaluate the errors between invasive and noninvasive arterial pressure values, the incidence of artefacts due to an inadequate dynamic response of the transducer-tubing system, and their detection by the ICU staff . g p Results The mean age was 37 months (95% CI, 29 to 45 months) in group IJV and 35 months (95% CI, 29 to 42 months) in group SV (P = 0.619). The 95% CI for weight in groups IJV and SV were 10.4 to 14.2 kg and 10.2 to 13.0 kg, respectively (P = 0.595). The CVC success rates at fi rst attempt for groups IJV and SV were 67% and 70%, respectively (P = 0.761). An alternative location was required to perform CVC in 90 patients in group IJV and in 92 patients in group SV (P = 0.806). The overall frequency of mechanical complications during the catheter insertion and its use was 26% in group IJV and 28% in group SV (log-rank test: P = 0.753). Signifi cantly more arterial punctures occurred in group IJV than in group SV (14% vs. 4%, P = 0.024). Catheter tip misplacement was observed more frequently in group SV than group IJV (12% vs. 1%, P = 0.003). Catheter colonization rates were signifi cantly higher in group IJV than group SV (15% vs. 5%, log-rank test: P = 0.020). There was no diff erence in bloodstream infection per 1,000 catheter days between group IJV and group SV (3.4 vs. 1.4, respectively: P = 0.319). Methods Seventy-fi ve consecutive patients (50 male, mean age 55 ± 18) admitted to the ICU for heterogeneous pathologies were enrolled. The T-Line TL-200 system for continuous noninvasive blood pressure measurement in medical ICU patients The T-Line TL-200 system for continuous noninvasive blood pressure measurement in medical ICU patients The T-Line TL-200 system for continuous noninvasive blood pressure measurement in medical ICU patients B Saugel, F Fassio, A Hapfelmeier, AS Meidert, RM Schmid, W Huber Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P213 (doi: 10.1186/cc10820) B Saugel, F Fassio, A Hapfelmeier, AS Meidert, RM Schmid, W Huber Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P213 (doi: 10.1186/cc10820) Introduction The T-Line TL-200 (Tensys Medical Inc., San Diego, CA, USA) is a noninvasive arterial blood pressure (BP) monitoring system allowing continuous beat-to-beat monitoring of systolic arterial pressure (SAP), mean arterial pressure (MAP), and diastolic arterial pressure (DAP). It provides a real-time BP waveform like that obtained using an arterial catheter for BP monitoring. The aim of this study was to compare BP measurements obtained using the T-Line TL-200 system with simultaneous invasive BP measurements using a femoral arterial catheter in unselected critically ill medical patients. Results A total of 856 lines performed in 602 patients were evaluated. In 607 US-guided cannulating internal jugular veins with only four cases of malposition, there were no cases of pneumothorax recorded. A total of 161 subclavian veins were cannulated with no US, of which six cases of pneumothorax were reported; two cases needed intercostal tube insertion. Eighty-eight femoral vein cannulations with no US were performed and no complications were recorded. Conclusion Chest X-ray is not necessary after US-guided CVL place- ment. Cutting out the chest X-ray procedure post insertion proved to be cost-eff ective. y Methods In 28 patients treated in a medical ICU of a German university hospital, BP values were simultaneously obtained using a femoral arterial catheter and the T-Line TL-200 device. All recorded data were included in the fi nal analysis. For comparison of BP measurements, Bland–Altman analysis accounting for repeated measurements was performed. P213 Methods A retrospective study of 856 lines placed in 602 patients being evaluated over a period of 11 months. All cases were performed in a controlled ICU environment. Chest X-rays were performed 30 minutes post-insertion in the D0 adult ICU unit in a tertiary medical center in Abu Dhabi, UAE. The D0 ICU has a capacity of 24 beds with an average admission rate of 55 to 60 patients per month. Records were assessed and evaluated, and data collected and statistically studied. Ultrasound-guided central venous line placement in critically ill patients: is chest X-ray needed to assess post-insertion pneumothorax? Ultrasound-guided central venous line placement in critically ill patients: is chest X-ray needed to assess post-insertion pneumothorax? O Samir Abdel Gelil Kotb, A Ali Abel Aziz, Y Awad Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2012, 16(Suppl 1):P211 (doi: 10.1186/cc10818) Ultrasound-guided central venous line placement in critically ill patients: is chest X-ray needed to assess post-insertion pneumothorax? p O Samir Abdel Gelil Kotb, A Ali Abel Aziz, Y Awad Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2012, 16(Suppl 1):P211 (doi: 10.1186/cc10818) Introduction Critically ill patients, mostly on positive pressure ventilation, are at higher risk of pneumothorax as well as their need for a central venous line (CVL) to optimize fl uid status, CVP measurement, and so forth, and where the CVL is not being placed in the best circumstances with the patients being critically ill, unstable and with higher chances of error predisposed by pre-existing lung disease, obesity or whatever the admitting diagnosis. Before CVL placement was a blind technique relying on the anatomical positions identifying the position of major blood vessels and thus post-insertion X-ray was needed to confi rm correct placement and to assess for pneumothorax. But with ultrasound (US) being more widely available, and most CVLs placed as US guided, the ultimate question develops: is post-insertion chest X-ray still needed? Conclusion The bias between invasive and noninvasive ABP measure can be relevant and mislead in the therapeutic management. These errors can be avoided by identifying the artefacts that aff ect arterial signal and so the ICU staff must pay attention to the recognition of arterial dumping in critically ill patients. P212 Inclusion criteria were: the presence of an intra-arterial catheter (IAC) for invasive blood pressure monitoring, and age >18 years. Pregnancy was excluded. At admission and every time the IAC was replaced we acquired invasive systolic, diastolic, and medium arterial pressure values (I-SP, I-DP, I-MP) during hemodynamic stability (variations of mean arterial pressure <10%); at the same time, noninvasive systolic and diastolic arterial pressure values (Ni-SP, Ni-DP) were measured with a sphygmomanometer at the same arm of the IAC. Noninvasive medium arterial pressure (Ni-MP) was calculated as follows: (SP + 2DP) / 3. At every time of the study, before ABP value acquisition, medical and nursing staff answered a questionnaire on the reliability of the arterial waveform. The staff could perform the fast fl ush test if considered appropriate. However, the fast fl ush test was executed by the main investigator at the end of questionnaire in all patients. Bland–Altman analysis was performed. Conclusion In pediatric cardiac surgery patients, IJV and SV catheters had similar success rates as well as overall mechanical complication rates. Although the catheter colonization rate was signifi cantly higher with IJV than SV, both access routes had similar rates of bloodstream infection. y p Results We compared 130 pairs of Ni-SP, Ni-DP and Ni-MP and I-SP, I-DP and I-MP. The mean bias between Ni-SP and I-SP was –11 mmHg (limit of agreement (LoA) –43.6 to 21.4 mmHg). The mean bias between Ni-DP and I-DP and between Ni-MP and I-MP was 6.1 mmHg (LoA –15.5 to 27.7 mmHg) and 0.37 mmHg (LoA –21.0 to 21.7 mmHg), respectively. We performed the fast fl ush test 130 times; an inadequate dynamic response of the transducer-tubing system was observed 55 times: in 45 cases the arterial signal was underdumped and in 10 cases was overdumped. The arterial dumping was correctly detected by the medical staff in 95% of cases, by nursing staff and postgraduates in 35% of cases. Reference . Pickering TG: Principles and techniques of blood pressure measurement. Cardiol Clin 2002, 20:207-223. Comparison of internal jugular and subclavian access for central venous catheterization in pediatric cardiac surgery A Pirat1, A Camkiran1, P Zeyneloglu1, M Ozkan1, E Akpek2, G Arslan1 1Baskent University, Ankara, Turkey; 2Acibadem University, Istanbul, Turkey Critical Care 2012, 16(Suppl 1):P210 (doi: 10.1186/cc10817) Comparison of internal jugular and subclavian access for central venous catheterization in pediatric cardiac surgery A Pirat1, A Camkiran1, P Zeyneloglu1, M Ozkan1, E Akpek2, G Arslan1 1Baskent University, Ankara, Turkey; 2Acibadem University, Istanbul, Turkey Critical Care 2012, 16(Suppl 1):P210 (doi: 10.1186/cc10817) Methods We retrospectively reviewed the chest radiographs of 197 consecutive intensive care patients admitted on and before 30 June 2011. A total of 101 patients had evidence of 137 new CVCs. For each new catheter the Picture Archiving & Communication System was used to record the tip position (after any repositioning) in relation to the carina and the degree of angulation from the vertical.i Introduction Central venous catheterization (CVC) is an essential com- po nent of perioperative care in pediatric cardiac surgery. Traditionally the internal jugular vein (IJV) is used for CVC in cardiac surgery. The aim of this study was to compare IJV and subclavian vein (SV) routes for CVC Introduction Central venous catheterization (CVC) is an essential com- po nent of perioperative care in pediatric cardiac surgery. Traditionally the internal jugular vein (IJV) is used for CVC in cardiac surgery. The aim of this study was to compare IJV and subclavian vein (SV) routes for CVC Results Twenty-fi ve per cent (34/137) of all catheter tips lay >10 mm below the carina, therefore potentially increasing the likelihood of intracardiac placement. This was reduced for left-sided catheters (6/37; S76 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P216 i Results Mean age was 69 ± 13 years, 14 patients received noradrenaline, eight patients dobutamine, and nine patients volume expansion. During the whole record, the number of episodes of Ov or At ranged from 0 to 15 with a duration of 0 to 6 hours: 17 patients had at least one episode of Ov and/or At tracing, 10 patients had at least two episodes, eight patients had at least fi ve episodes. Seven episodes lasted more than 20 minutes and three more than 1 hour. During the fi rst hour, sAP was overestimated by 5.0 ± 1.4 mmHg (P <0.0001) (range: 0.3 to 5.9) or by 4.3 ± 0.9% (range: 0.4 to 15.9%), raw PPV was 9.5 ± 7.3 versus 10.0 ± 7.8 for the corrected PPV (range from –2.6 to 4.3); raw dP/dt was overestimated by 134 ± 47 mmHg/second (P <0.0001) (range: –13 to 353) or by 24 ± 6%. 1. Wax DB, et al.: Anesthesiology 2011, 115:973-978. P215 Results A total of 76,826 pairs of BP measurements (each consisting of SAP, MAP, and DAP) were analyzed. For MAP, Bland–Altman analysis revealed a mean diff erence of +0.47 mmHg (95% limits of agreement: –16.53 to +17.46 mmHg). For SAP and DAP, the bias and 95% limits of agreement were –9.01 mmHg (–37.47 to +19.45 mmHg) and +5.22 mmHg (–13.50 to +23.94 mmHg), respectively. Right/left ventricular area ratio does not correlate with right ventricular impedance F Paalvast1, D Reis Miranda1, M Knook2, A Rossi1, J Van Bommel1, D Gommers1 1Erasmus Medical Centre, Rotterdam, the Netherlands; 2Reinier de Graaf Ziekenhuis, Delft, the Netherlands Critical Care 2012, 16(Suppl 1):P215 (doi: 10.1186/cc10822) Reliability of radial arterial pressure monitoring after cardiac surgery C Lavault1, MC Fevre2, A Hebrard2, M Durand2, F Grimbert1, Y Lavault1, P Albaladejo2 1UJF – Grenoble1/CNRS/TIMC-IMAG UMR 5525 (Equipe PRETA), Grenoble, France; 2CHU Grenoble, France Critical Care 2012, 16(Suppl 1):P214 (doi: 10.1186/cc10821) Introduction Invasive monitoring in critically ill patients allows a continuous measurement of arterial pressure, cardiac output, and the derivation of dynamic predictors of fl uid responsiveness. However, the pressure signal may be altered by the dynamic characteristics of the fl uid-fi lled tubing. The aim of the present study was to evaluate the reliability of radial artery blood pressure measurement and derived indexes during the early period after cardiac surgery. Methods After IRB approval, 30 patients admitted to the ICU after elective cardiac surgery (CABG: 16, valve surgery: 11; combined: 3) with a radial artery catheter were included. In the ICU, an independent continuous recording of arterial pressure during at least 18 hours was started via a double-head pressure transducer (Flotrac; Edwards Lifesciences, Irvine, CA, USA) for a retrospective analysis and three fast fl ushes were performed. First, the whole record was examined for episodes of overdamping (Ov) or attenuation (At). Ov was defi ned as a decrease in systolic (sAP), an increase diastolic (dAP), and an unchanged mean pressure (mAP). At was defi ned as a decrease in sAP, dAP and mAP. Second, three periods of 10 minutes during the fi rst hour were analysed assuming that the dynamic characteristics remained constant. This allowed the correction of the distorted raw signals and the study of the consequences of an underdamped signal on sAP, pulse pressure variation (PPV) and dP/dt as an estimate of left ventricular contractility. A paired t test was used for statistical comparison, P <0.05 was considered statistically signifi cant. Results Cardiac output dropped from 4.4 ± 0.8 to 1.7 ± 0.9 l/minute. The relations between occluder resistance and RV/LV area ratio during diastole and during systole were not signifi cant (r = 0.48 resp. r = 0.54). Conclusion The RV/LV area ratio does not correlate with right ventricular impedance in this closed pericardium model. References 1. Bouferrache K: Acute respiratory distress syndrome, mechanical ventilation, and right ventricular function. Curr Opin Crit Care 2011, 17:30-35. 2. Mansencal N: Comparison of diff erent echocardiographic indexes secondary to right ventricular obstruction in acute pulmonary embolism. Am J Cardiol 2003, 92:116-119. Right/left ventricular area ratio does not correlate with right ventricular impedance F Paalvast1, D Reis Miranda1, M Knook2, A Rossi1, J Van Bommel1, D Gommers1 1Erasmus Medical Centre, Rotterdam, the Netherlands; 2Reinier de Graaf Ziekenhuis, Delft, the Netherlands Critical Care 2012, 16(Suppl 1):P215 (doi: 10.1186/cc10822) D Gommers1 1Erasmus Medical Centre, Rotterdam, the Netherlands; 2Reinier de Graaf Ziekenhuis, Delft, the Netherlands Critical Care 2012, 16(Suppl 1):P215 (doi: 10.1186/cc10822) g g p y Conclusion The T-Line TL-200 system allows determination of MAP with a satisfactory agreement when compared to invasive assessment of MAP using a femoral arterial catheter in unselected critically ill medical patients. Higher mean diff erences and 95% limits of agreement for SAP and DAP measurements might be explainable by limited comparability of central (femoral) and peripheral (radial) SAP and DAP measurements. Introduction The right ventricular/left ventricular (RV/LV) area ratio is often used to titrate airway pressure during mechanical ventilation [1]. This ratio has only been validated for detecting pulmonary embolism [2]. The purpose of this study was to evaluate the relationship between RV/LV area ratio and RV impedance in a pig model with a closed pericardium. P214 Reliability of radial arterial pressure monitoring after cardiac surgery C Lavault1, MC Fevre2, A Hebrard2, M Durand2, F Grimbert1, Y Lavault1, P Albaladejo2 1UJF – Grenoble1/CNRS/TIMC-IMAG UMR 5525 (Equipe PRETA), Grenoble, France; 2CHU Grenoble, France Critical Care 2012, 16(Suppl 1):P214 (doi: 10.1186/cc10821) Methods Eight anesthetized pigs were instrumented with closed pericardium for the measurement of arterial blood pressure, central venous pressure, RV and pulmonary pressure. On the main pulmonary artery, an ultrasonic fl owprobe (MA14PAX; Transonic) was positioned to obtain pulmonary fl ow. Distally, a balloon occluder was positioned facilitating gradual constriction of the pulmonary artery. To obtain a stepwise pressure diff erence increment over the banding we gradually infl ated the balloon occluder. Occluder resistance is computed as the systolic right ventricle pressure minus systolic pulmonary pressure divided by cardiac output times 79.9. An ECG-gated CT scan of the heart was performed, 10 minutes after each banding. The RV/LV area ratio was computed by reconstructing the CT images to a typical echocardiographic four-chamber view and dividing the RV area by the LV area. All measurements were performed in triplicate and averaged. As repeated measurements in eight independent animals were used, a signifi cant relation was sought between RV/LV area ratio and occluder resistance with ANOVA. Correlation was obtained by a Spearson’s correlation. f References 1. Ultrasound detects central line placement and postprocedure 1. Ultrasound detects central line placement and postprocedure pneumothorax. Emerg Med 2009. pneumothorax. Emerg Med 2009. 2. Ultrasound-guided central line placement: no X-ray needed. Emerg Med 2011. S77 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Eff ects of cardiac output levels on the measurement of transpulmonary thermodilution cardiac output in patients with acute lung injury Figure 1 (abstract P218). K Kao, H Hu, C Hung, C Chang, C Huang probe (CardioQ™; Deltex Medical, UK) was inserted, connected to the CardioQ-ODM™ monitoring system and correct positioning verifi ed. Simultaneous determination of cardiac output by ODM and ECOM™ was performed before and after cross-clamping of the aorta. Results Cardiac output ranged from 1.4 to 13.1 l/minute. Linear regression is represented by the equation y = 0.30x + 2.2 and the correlation coeffi cient r2 = 0.15. The bias was +1.5 l/minute with 95% limits of agreement between –2.1 and 5.1 l/minute (Figure 1). Conclusion Using the CardioQ™ as a reference, the ECOM™ system cannot be recommended as a clinical cardiac output measurement technique in abdominal aortic surgery, due to its poor correlation and wide limits of agreement. probe (CardioQ™; Deltex Medical, UK) was inserted, connected to the CardioQ-ODM™ monitoring system and correct positioning verifi ed. Simultaneous determination of cardiac output by ODM and ECOM™ was performed before and after cross-clamping of the aorta. Introduction Transpulmonary thermodilution cardiac output (CO) correlates closely with pulmonary artery (PA) thermodilution CO. Levels of CO may contribute varying amounts of thermal indicator loss and recirculation during thermodilution CO measurement. This study aimed to investigate the eff ects of CO levels on the agreement between transpulmonary and PA thermodilution CO in acute lung injury (ALI) patients. p p g Results Cardiac output ranged from 1.4 to 13.1 l/minute. Linear regression is represented by the equation y = 0.30x + 2.2 and the correlation coeffi cient r2 = 0.15. The bias was +1.5 l/minute with 95% limits of agreement between –2.1 and 5.1 l/minute (Figure 1). p p g Results Cardiac output ranged from 1.4 to 13.1 l/minute. Linear regression is represented by the equation y = 0.30x + 2.2 and the correlation coeffi cient r2 = 0.15. The bias was +1.5 l/minute with 95% limits of agreement between –2.1 and 5.1 l/minute (Figure 1). Conclusion Using the CardioQ™ as a reference, the ECOM™ system cannot be recommended as a clinical cardiac output measurement technique in abdominal aortic surgery, due to its poor correlation and wide limits of agreement. y Methods Twenty-two ALI patients were prospectively enrolled. Techniques to measure cardiac output: minimally invasive method versus thermodilution MostCare is resulted to be reliable and accurate even in hemodynamically unstable patients. It would be interesting to study the new device before and after having modifi ed the therapy, such as fl uid challenge or inotropic therapy or the use of vasopressors. References 1. Zangrillo A, et al.: J Cardiothorac Vasc Anesth 2010, 24:265-269. 2. Scolletta S, et al.: Br J Anaesth 2005, 95:159-165. 3. Romano SM, et al.: Crit Care Med 2002, 30:1834-1841. 1. Zangrillo A, et al.: J Cardiothorac Vasc Anesth 2010, 24:265-269. 2. Scolletta S, et al.: Br J Anaesth 2005, 95:159-165. 3. Romano SM, et al.: Crit Care Med 2002, 30:1834-1841. Techniques to measure cardiac output: minimally invasive method versus thermodilution versus thermodilution A Donati, S Tondi, A Carsetti, R Domizi, C Melia, D Kolgjini, C Munch, C Scorcella, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2012, 16(Suppl 1):P216 (doi: 10.1186/cc10823) Università Politecnica delle Marche, Ancona, Italy Critical Care 2012, 16(Suppl 1):P216 (doi: 10.1186/cc10823) Introduction Hemodynamic monitoring is important to manage critically ill patients. The thermodilution pulmonary catheter is considered the gold standard; however, it is invasive and associated with the onset of complications. Our study compared cardiac output (CO) obtained with the MostCare (COMC), which uses the pressure recording analytical method, to CO obtained with a Swan–Ganz (COSG) catheter in hemodynamically unstable patients. Introduction Hemodynamic monitoring is important to manage critically ill patients. The thermodilution pulmonary catheter is considered the gold standard; however, it is invasive and associated with the onset of complications. Our study compared cardiac output (CO) obtained with the MostCare (COMC), which uses the pressure recording analytical method, to CO obtained with a Swan–Ganz (COSG) catheter in hemodynamically unstable patients. Conclusion These results showed that frequent artefacts and distortions induced by the fl uid-fi lled tubing could modify the arterial waveform and could lead to inaccurate therapy [1]. More attention should be paid to the quality of the pressure signal. Reference Methods We conducted a prospective clinical study in our cardiosurgical ICU. Sixteen post-cardiosurgical adult patients were enrolled. They had a Swan–Ganz catheter and were mechanically ventilated. The Swan– Ganz catheter was connected to the monitor Vigilance Edwards®, while the MostCare was connected to the patient’s artery. For each patient three measurements of CO have been carried out and the mean was considered for statistical analysis. The correlation coeffi cient, Bland– Altman test and percentage of error were measured. Results The correlation coeffi cient between COSG and COMC was 0.824 (0.567 to 0.935, 95% CI; P <0.001) The Bland–Altman analysis showed S78 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P218). a mean diff erence between the two methods (bias) of 0.22 ± 0.55 l/ minute/m2 with lower and upper 95% limits of confi dence of –0.87 and 1.30 l/minute/m2 respectively. The percentage of error was of 25%. Figure 1 (abstract P218). p y p g Conclusion This study demonstrated a good correlation between the two methods. P217f Eff ects of cardiac output levels on the measurement of transpulmonary thermodilution cardiac output in patients with acute lung injury K Kao, H Hu, C Hung, C Chang, C Huang Chang Gung Memorial Hospital, Kwei-Shan,Taoyuan, Taiwan Critical Care 2012, 16(Suppl 1):P217 (doi: 10.1186/cc10824) p References References 1. Harvey S, et al.: Lancet 2005, 366:472-477. 2. Koo KKY, et al.: Crit Care Med 2011, 39:1613-1618. 1. Harvey S, et al.: Lancet 2005, 366:472-477. 2. Koo KKY, et al.: Crit Care Med 2011, 39:1613-1618. Methods Seven consecutive patients (seven male, zero female), mean age 56.5 (± 12) years, were enrolled into the study. ICO data were Eff ects of cardiac output levels on the measurement of transpulmonary thermodilution cardiac output in patients with acute lung injury Paired bolus transpulmonary thermodilution cardiac index (BCItp) and continuous PA thermodilution cardiac index (CCIpa) data were recorded at baseline and repeated immediately and at 2, 4, and 6 hours after volume expansion with a 500 ml infusion of 10% pentastarch (HES 200/0.5). Cardiac output monitoring in cirrhotic patients: EV1000 versus pulmonary artery catheter – preliminary data G Costa, T Cecconet, D Baron, G Serena, P Chiarandini, L Pompei, L Vetrugno, G Della Rocca University of Udine, Italy Critical Care 2012, 16(Suppl 1):P219 (doi: 10.1186/cc10826) Results One hundred and ten paired CI measurements were recorded and divided into four quartiles from the lowest to the highest CCIpa. The mean BCItp was higher than CCIpa, and the Bland–Altman analysis revealed a bias of 0.57 ± 0.75 l/minute/m2. The limits of agreement (2SD) were +2.07 to –0.93 l/minute/m2. BCItp correlated closely with CCIpa (R = 0.887). CCIpa negatively correlated with the diff erence between BCItp and CCIpa (R = –0.26). The bias of quartile 1 with the least CCIpa was signifi cantly greater than those of the three other quartiles. Introduction The EV1000 platform, a new calibrated device for intermittent and continuous cardiac output monitoring, has recently been introduced into clinical practice [1]. This study aims to assess the level of agreement between intermittent and continuous cardiac output obtained from VolumeView (ICOvv and CCOvv) connected to the EV1000 platform (Edwards Lifesciences, Irvine, CA, USA) and intermittent (ICOvig) and continuous cardiac output (CCOvig) obtained using an advanced pulmonary artery catheter (PAC) connected to the Vigilance System (Edwards Lifesciences) in cirrhotic patients undergoing liver transplantation. i Conclusion In ALI patients, transpulmonary thermodilution is a clinically acceptable and interchangeable alternative to PA thermodilution for CO measurement. Levels of CO weakly and negatively correlate with the diff erence between BCItp and CCIpa. There is greater overestimation of BCItp over CCIpa in low than in high CO states. Comparison of bioimpedance and oesophageal Doppler cardiac output monitoring during abdominal aortic surgery Agreement and precision between CO values were evaluated with Bland–Altman analysis. The percentage error (PE) was calculated as 2SD / mean CO [2]. Results Twenty-one ICO data pairs were compared. Two patients were excluded from CCO data analysis for technical reasons. A total 240 CCO data pairs from fi ve patients were analysed. Data yielded were analysed as total and for CO values lower and higher than 8 l/minute (Table 1). Conclusion These data, even if very preliminary, showed low agreement and high PE either for intermittent and continuous CO obtained from the VolumeView. However, for CO data lower than 8 l/minute the PE was improved. percentage error (PE) was calculated as 2SD / mean CO [2]. Results Twenty-one ICO data pairs were compared. Two patients were excluded from CCO data analysis for technical reasons. A total 240 CCO data pairs from fi ve patients were analysed. Data yielded were analysed as total and for CO values lower and higher than 8 l/minute (Table 1). Conclusion These data, even if very preliminary, showed low agreement and high PE either for intermittent and continuous CO obtained from the VolumeView. However, for CO data lower than 8 l/minute the PE was improved. Results Twenty-one ICO data pairs were compared. Two patients were excluded from CCO data analysis for technical reasons. A total 240 CCO data pairs from fi ve patients were analysed. Data yielded were analysed as total and for CO values lower and higher than 8 l/minute (Table 1). TPTD Conclusion The PCCO method cannot replace the transpulmonary thermodilution method in critically ill children. Conclusion These data, even if very preliminary, showed low agreement and high PE either for intermittent and continuous CO obtained from the VolumeView. However, for CO data lower than 8 l/minute the PE was improved. P221 References 1. Bendjelid et al.: Crit Care 2010, 14:R209. 2. Cecconi et al.: Crit Care 2009, 13:201. 1. Bendjelid et al.: Crit Care 2010, 14:R209. Introduction With respect to a radial arterial pressure measurement, a more central achievement of this pressure should improve the reliability of endotracheal bioimpedance cardiac output (CO) monitoring. We therefore compared prospectively the impact on accuracy of this device, in comparison with thermodilution (TD) CO. P221 Impact of arterial catheter location on the accuracy of cardiac output provided by an endotracheal bioimpedance device F Gennart, S Beckers, C Verborgh, A De baerdemaeker, J Poelaert UZ Brussels, Belgium Critical Care 2012, 16(Suppl 1):P221 (doi: 10.1186/cc10828) Comparison of bioimpedance and oesophageal Doppler cardiac output monitoring during abdominal aortic surgery Table 1 (abstract P219). Bias, 2SD and PE for all data pairs and for CO higher and lower than 8 l/minute Table 1 (abstract P219). Bias, 2SD and PE for all data pairs and for CO higher and lower than 8 l/minute Bias 2SD PE (%) ICO Total –0.89 3.6 49 <8 –1.37 2.4 40 >8 –0.05 4.8 52 CCO Total –0.83 4.88 63 <8 –1.8 2.4 37 >8 2.07 5.64 47 g g Littlebaelt Hospital Kolding, Denmark Introduction Abdominal aortic surgery is a high-risk procedure. Cardiac output monitoring allowing haemodynamic optimisation may reduce the complication rate. Minimally invasive, continuous techniques are preferable. Cardiac output using oesophageal Doppler has been validated in several studies, showing good agreement with the gold standard. The aim of this study was to assess the degree of correlation and agreement between cardiac output measured by oesophageal Doppler and bioimpedance obtained from an endotracheal tube. Methods Twelve patients scheduled for elective abdominal aortic surgery were included. Patients were intubated with an ECOM™ endotracheal tube (ConMed Corporation, NY, USA) which was connected to the ECOM™ monitoring system. An oesophageal Doppler S79 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 5 hours and 33 minutes (range 14 minutes to 15 hours). The correlation coeffi cient between the COTPTD and PCCO was 0.85 (P <0.0001). The Bland–Altman analysis showed a mean bias of 0.06 l/minute (limits of agreement (LoA) ± 2.22 l/minute) (Figure 1). The percentage error was 43%. The correlation coeffi cient between the recalibration interval and the bias between COTPTD and PCCO was –0.26 (P = 0.05). There was no correlation between COTPTD and PCCO (r = 0.09 (P = 0.57)). Conclusion The PCCO method cannot replace the transpulmonary thermodilution method in critically ill children. obtained from the two devices after ICU admission (T0) and after 12 (T12) and 24 hours (T24). CCOvig and CCOvv were recorded every hour from T0 up to 48 hours after ICU admission. Agreement and precision between CO values were evaluated with Bland–Altman analysis. The percentage error (PE) was calculated as 2SD / mean CO [2]. obtained from the two devices after ICU admission (T0) and after 12 (T12) and 24 hours (T24). CCOvig and CCOvv were recorded every hour from T0 up to 48 hours after ICU admission. Haemodynamic changes during the peri-extubation period using bioreactance fl ow monitoringi l g J Thirsk, D Magimairaj, A Douiri, D Hadfi eld, P Hopkins Ki ’ H lth P t L d UK King’s Health Partners, London, UK Results The sample was 19 males, nine females, age 60.2 ± 11.8 years; APACHE II score 23.3± 5.4. The 280 pairs of CIpc and CItd showed a signifi cant correlation (P <0.001; r = 0.907). There was no diff erence between CIpc versus CItd (4.15 ± 1.46 vs. 4.09 ± 1.41 l/minutesqm; P = 0.265). Bland–Altman analysis demonstrated a mean bias of –0.061 ± 0.603 l/minutesqm (lower and upper levels of agreement –1.24 and 1.12l/minutesqm; percentage error of 28.7%). In univariate analyses, the bias CItd2–CIpc was not correlated to the interval to the last calibration (P = 0.705; r = –0.023), but it was correlated to CIpc immediately before recalibration (r = –0.275; P <0.001) and to changes from CIpc versus the previous CItd1 (Delta-CIpc–CItd1; r = –0.504; P <0.001). These fi ndings were confi rmed in the validation collective (P <0.001). Multiple regression analysis demonstrated independent association of the bias to Delta-CIpc–CItd1. This association was best described by bias CItd2–CIpc = –0.014 – 0.372x + 0.145x2 – 1.260x3 with x = Delta-CIpc–CItd1. This formula as a potential calibration index provided ROC AUCs of 0.882 and 0. 751 (P <0.001) to predict a bias CItd2–CIpc >20% or <–20% in the evaluation collective. This formula was confi rmed with ROC AUCs of 0.809 and 0.714 (P <0,001) to predict a bias CItd2–CIpc >20% or <–20% in the independent validation collective. Conclusion The diff erence CIpc–CItd1 is an independent predictor of the bias CItd2–CIpc. A calibration index was developed and validated. It could be a useful decision support to initiate the next TD. King’s Health Partners, London, UK Critical Care 2012, 16(Suppl 1):P224 (doi: 10.1186/cc10831) g Critical Care 2012, 16(Suppl 1):P224 (doi: 10.1186/cc10831 Introduction Here we present a prospective, observational study examining the eff ect of extubation on cardiac index, measured by bioreactance (Nicom Cheetah), in critically ill patients with or without a history of left ventricular impairment [1]. A number of simple interventions are known to improve the process of weaning patients from mechanical ventilation. Despite this progress, the pathophysiology underlying failure to wean remains incompletely understood. In particular, the role of cardiac ventricular dysfunction may be underestimated [2]. P223 Methods Fourteen patients undergoing cardiac surgery with cardiac output monitoring by TD have been enrolled. A specially designed endotracheal tube (ECOM; ConMed) was placed in conjunction with a catheter located either in the brachial (18 G) or in the radial (20 G) artery in each group of seven patients. Six individual measurements have been carried out in each patient at fi xed period, resulting in a total of 42 measurements for each subset. The mean CO by TD was compared with CO by ECOM for each operative period and assessed for agreement by linear regression, Bland–Altmann analysis and percentage error methods. The measurement error should not exceed 30% to be considered as valid, according to Critchley and colleagues. P224 Haemodynamic changes during the peri-extubation period using bioreactance fl ow monitoring J Thirsk, D Magimairaj, A Douiri, D Hadfi eld, P Hopkins King’s Health Partners, London, UK Critical Care 2012, 16(Suppl 1):P224 (doi: 10.1186/cc10831) P224 Haemodynamic changes during the peri-extubation period using bioreactance fl ow monitoring J Thirsk, D Magimairaj, A Douiri, D Hadfi eld, P Hopkins King’s Health Partners, London, UK Critical Care 2012, 16(Suppl 1):P224 (doi: 10.1186/cc10831) g References 1. Critchley et al.: J Clin Monit 1999, 15:85-91. 2. Bland et al.: Lancet 1986, i:307-310. p y p p g Methods A prospective study in 51 patients undergoing open elective abdominal aortic surgery – 30 patients with AAD and 21 with AOD. CF values were obtained at baseline, before induction of anaesthesia (T1) and 30 minutes after reperfusion (T2). Cardiac output monitoring using the LiDCOplus™ monitor in abdominal aortic surgery: changes in calibration factor in aortic aneurysm disease versus aortic occlusive disease y HK Jørgensen, J Bisgaard, T Gilsaa Lillebaelt Hospital, Kolding, Denmark Critical Care 2012, 16(Suppl 1):P223 (doi: 10.1186/cc10830) y HK Jørgensen, J Bisgaard, T Gilsaa Lillebaelt Hospital, Kolding, Denmark Critical Care 2012, 16(Suppl 1):P223 (doi: 10.1186/cc10830) y HK Jørgensen, J Bisgaard, T Gilsaa Lillebaelt Hospital, Kolding, Denmark Critical Care 2012, 16(Suppl 1):P223 (doi: 10.1186/cc10830) Introduction Monitoring of cardiac output (with subsequent haemodynamic optimisation) may improve outcome after high-risk surgery. The pulmonary artery catheter is still considered the gold standard, but has potential serious complications. Much eff ort has been put into developing equally good, but less invasive techniques. One of these, the LiDCOplus™ system, uses pulse power analysis to calculate cardiac output and is calibrated by a lithium indicator dilution technique. Since cardiac output is aff ected by the compliance of the aorta, the LiDCO calculates a calibration factor (CF) each time it is calibrated. The purpose of this study was to investigate whether insertion of aortic prosthetic material would aff ect aortic compliance and thereby the CF. It was hypothesised that the change in CF would be larger in patients with aortic occlusive disease (AOD) than in patients with aortic aneurysm disease (AAD), since previous studies have shown that these two groups diff er considerably on both haemodynamic capacity and their response to aortic cross-clamping [1]. g y g Results Mean patient age was 71 years (56 to 89) (13 male, one female). R2 values of 0.47 (P <0.01) and 0.63 (P <0.01) in the linear regressions and errors of 41% and 50% were found for the radial and brachial catheter data, respectively. See Figures 1 and 2. y Conclusion Accuracy was considerably improved using a brachial artery catheter. Nevertheless, measurement errors between TD and ECOM using either a radial or brachial catheter both exceed 30%. Based on these results and under the current technical conditions, ECOM should not replace TD in CO monitoring for patients undergoing cardiac surgery. Pulse contour cardiac output monitoring is less reliable in critically ill children Pulse contour cardiac output monitoring is less reliable in critically ill children JC Verheul, A Nusmeier, J Lemson Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P220 (doi: 10.1186/cc10827) JC Verheul, A Nusmeier, J Lemson Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P220 (doi: 10.1186/cc10827) JC Verheul, A Nusmeier, J Lemson JC Verheul, A Nusmeier, J Lemson Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Critical Care 2012, 16(Suppl 1):P220 (doi: 10.1186/cc10827) Figure 2 (abstract P221). Figure 1 (abstract P221). Figure 1 (abstract P221). Introduction Intermittent cardiac output measurement using the transpulmonary thermodilution (TPTD) method is considered to be the gold standard in young children but a validated continuous cardiac output technique is not available in these patients. We compared the continuous pulse contour cardiac output (PCCO) measurements with the TPTD method in critically ill children. y Methods We compared PCCO, measured with the PiCCO device (Pulsion, Munich, Germany), with TPTD measurements (COTPTD) using the same device in a general pediatric intensive care (PICU) population. Because PCCO is calibrated with each TPTD measurement (COTPTD) we compared the mean PCCO value just before a new TPTD measurement was done. We approved only COTPTD measurement consisting of three consecutive TPTD measurements and we checked the thermodilution curve for a temperature diff erence of at least 0.2°C and a normal appearance. Only the intervals between two approved series of TPTD measurements were analysed. We calculated the correlation coeffi cient and used the Bland–Altman method for analysis. y Results Sixty-one measurements in 10 children were included. Mean age was 24.5 (range 5 to 123) months; mean weight was 11.2 (range 3.8 to 18) kg, mean heart rate was 131/minute (range 87 to 193) and the mean blood pressure was 73 (range 49 to 96) mmHg. The mean COTPTD was 2.60 (range 0.66 to 5.64) l/minute, mean cardiac index was 5.16 (range 2.76 to 10.83) l/minute/m2 and mean duration of the interval was Figure 1 (abstract P221). Figure 1 (abstract P221). Figure 1 (abstract P220). Bland–Altman analysis of COTPTD and PCCO. Figure 2 (abstract P221). Figure 1 (abstract P220). Bland–Altman analysis of COTPTD and PCCO. Figure 2 (abstract P221). Figure 2 (abstract P221). S80 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Reference 1. Shteinberg D, et al.: Eur J Vasc Endovasc Surg 2000, 20:462-465. Methods In 28 consecutive patients 56 datasets each including six TDs were recorded. In each triplicate TD measurement, CIpc was recorded immediately before recalibration by TD and compared to CItd. Results derived from this evaluation collective were validated in an independent second collective of 48 patients with 67 datasets. SPSS 19 software was used. 1. Shteinberg D, et al.: Eur J Vasc Endovasc Surg 2000, 20:462-465. Accuracy of the PiCCO2-derived pulse contour cardiac index (CIpc): development and validation of a calibration index in two independent collectives Results AAD patients were older (70 vs. 65 years, P <0.05), predominantly males (80% vs. 47%), weighed more (80 kg vs. 73 kg, P <0.1) and preoperative cardiac co-morbidity was more prevalent (43% vs. 14%). No diff erence was found in the use of epidural analgesia, vasopressors, or inotropes between the groups. At T1, CF was signifi cantly higher for AAD = 0.83 versus AOD = 0.68 (P = 0.01). After reperfusion, T2, there was no signifi cant diff erence in CF, AAD = 0.86 versus AOD = 0.81 (P = 0.53). The percentage change in CF from T1 to T2 was signifi cantly larger in AOD than in AAD (20% vs. 1.3%) (P <0.05). p W Huber, J Koenig, B Saugel, T Schuster, R Schmid, S Mair Klinikum Rechts der Isar, Technical University of Munich, Germany Critical Care 2012, 16(Suppl 1):P222 (doi: 10.1186/cc10829) Introduction After calibration by thermodilution (TD), the PiCCO device is able to assess CO using pulse contour (PC) analysis. Despite an overall good correlation of CItd and CIpc in several studies, the manufacturer suggests recalibration by TD after 8 hours. A calibration index derived from PC parameters indicating a certain probability of a relevant bias and triggering the next calibration would be of great practical use. Therefore, it was the aim of our study to prospectively evaluate predictors of the bias CItd–CIpc exactly 1 hour, 2 hours, 4 hours, 6 hours and 8 hours after the last calibration. Conclusion Operative insertion of an abdominal aortic prosthesis signifi cantly aff ects the calibration factor in patients with AOD, indicating an increase in aortic compliance and the need for recalibration of the LiDCOplus™. No signifi cant change was seen in patients with aortic aneurysm disease. Reference P222 Accuracy of the PiCCO2-derived pulse contour cardiac index (CIpc): development and validation of a calibration index in two independent collectives W Huber, J Koenig, B Saugel, T Schuster, R Schmid, S Mair Klinikum Rechts der Isar, Technical University of Munich, Germany Critical Care 2012, 16(Suppl 1):P222 (doi: 10.1186/cc10829) Accuracy of the PiCCO2-derived pulse contour cardiac index (CIpc): development and validation of a calibration index in two independent collectives Left ventricular stroke volume measurement by impedance cardiography correlates with echocardiography in neonates l h 1 h1 Ob h 1 ll 2 l2 S 1 , , , , , g 1University Medical Center Hamburg – Eppendorf, Hamburg, Germany; 2University Center for Cardiology and Cardiothoracic Surgery, Hamburg, Germany Introduction Thermodilution (TD) is a gold standard for cardiac index (CI) measurement. The aim of this study is to compare intermittent bolus TDCI with intermittent automatic calibration CI (AutoCI) and continuous CI (CCI) obtained by pulse contour analysis with PiCCO2 (PiCCI) and Pulsiofl ex (PuCCI). Critical Care 2012, 16(Suppl 1):P225 (doi: 10.1186/cc10832) Introduction The aim of this study was to validate impedance cardiography (electrical velocimetry (EV)) as a continuous noninvasive cardiac output monitoring in neonates and infants. As the reference method, discontinuous transthoracic echocardiography (TTE) was used. l Methods A prospective study in 20 patients (all mechanically ventilated, 14 male). Age 54.4 ± 16.7, BMI 28.1 ± 7.3, SAPS II 52.9 ± 13.4, APACHE II score 26.7 ± 7.8 and SOFA score 10 ± 3. All patients underwent PiCCO monitoring via a femoral line whilst the radial line was kept in place during four 8-hour time periods (in the fi rst two periods the Pulsiofl ex was connected to the radial line, in the last two it was connected to the femoral line). In the fi rst and third 8-hour periods the Pulsiofl ex was calibrated with the TDCI obtained at baseline, for the second and fourth 8-hour periods the Pulsiofl ex was calibrated with the AutoCI value. Simultaneous PiCCI and PuCCI measurements were obtained every 2 hours while simultaneous TDCI and AutoCI were obtained every 8 hours. The PiCCI and PuCCI values were recorded within 5 minutes before TDCI was determined. We also looked at the eff ects of 22 interventions: passive leg raise (n = 6), fl uid bolus (n = 5), change in vasopressor (n = 9) or dobutamine (n = 1), increase in sedation (n = 1). Statistical analysis was performed using Pearson correlation and Bland–Altman analysis. Methods In a prospective single-center observational study, simultaneous left ventricular stroke volume (LVSV) measurements by EV (using an Aesculon® Monitor) and by TTE were compared. LVSV measurement by TTE was based on the aortic valve velocity time integral multiplied by the area of the aortic valve outfl ow tract. Haemodynamic changes during the peri-extubation period using bioreactance fl ow monitoringi Methods Cardiac index was measured by bioreactance monitoring at 30-second to 60-second intervals for 1 hour pre and 1 hour post extubation. Individual data were presented by box plot, showing median and interquartile ranges (Figure 1). Combined results from multiple patients in each test group were analysed by covariance (Stata version 11.2). Results Group A (n = 5) had impaired left ventricular systolic function, documented on formal transthoracic echo, of which three had ejection fractions <25%. One patient in this group failed extubation due to cardiogenic pulmonary oedema. Group B (n = 6) had normal systolic function. Figure 1 shows representative absolute data obtained from Conclusion The diff erence CIpc–CItd1 is an independent predictor of the bias CItd2–CIpc. A calibration index was developed and validated. It could be a useful decision support to initiate the next TD. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 S81 Figure 1 (abstract P224). a patient in each group. There was a statistical diff erence between the two groups (P = 0.02). In the impaired LV group, the cardiac index fell from 3.2 l/minute/m2 (± 0.5) to 2.9 l/minute/m2 (± 2.5). higher LVSVs resulting in higher EV than TTE measurements. The bias defi ned by the diff erence of the means of the two methods was 9.65%, with a mean percentage error of the individual measurements of 55%. Based on the Bland–Altman analysis, a deduced approximated correction factor between TTE-LVSV and EV-LVSV was TTE-LVSV = EV- LVSV0.539×100.335  (EV-LVSV)×2.2. Conclusion In this small observational study we demonstrated a consistent fall in cardiac index post extubation in patients with known cardiac ventricular dysfunction when compared with patients with normal hearts. These data suggest that bioreactance monitoring may be valuable during spontaneous breathing trials and extubation. References Conclusion Correlation between EV and TTE in LVSV measurement was signifi cant. Bland–Altman analysis showed that – despite a large mean error of the individual measurements of 55% – the bias between the means of the two methods was only 9.65%. A correction factor between TTE and EV could be deduced. 1. Benomar B, Ouattara A, Estagnasie P, Brusset A, Squara P: Fluid responsiveness predicted by non-invasive bioreactance-based passive leg raise test. Intensive care Med 2010, 36:1875-1881. 2. Papanikolaou J, Makris D, Saranteas T, et al.: New insights into weaning from mechanical ventilation: left ventricular diastolic dysfunction is a key player. Validation of less-invasive hemodynamic monitoring with Pulsiofl ex in critically ill patients Validation of less-invasive hemodynamic monitoring with Pulsiofl e in critically ill patients M Peetermans, W Verlinden, J Jacobs, A Verrijcken, S Pilate, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, ML Malbrain ZNA Stuivenberg, Antwerp, Belgium Critical Care 2012, 16(Suppl 1):P226 (doi: 10.1186/cc10833) M Peetermans, W Verlinden, J Jacobs, A Verrijcken, S Pilate, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, ML Malbrain ZNA Stuivenberg, Antwerp, Belgium C iti l C 2012 16(S l 1) P226 (d i 10 1186/ 10833) Haemodynamic changes during the peri-extubation period using bioreactance fl ow monitoringi Intensive Care Med 2011, 37:1976-1985. P225 Left ventricular stroke volume measurement by impedance cardiography correlates with echocardiography in neonates ME Blohm1, J Hartwich1, D Obrecht1, G Müller2, J Weil2, D Singer1 1University Medical Center Hamburg – Eppendorf, Hamburg, Germany; 2University Center for Cardiology and Cardiothoracic Surgery, Hamburg, Germany Critical Care 2012, 16(Suppl 1):P225 (doi: 10.1186/cc10832) P227 P227 A preliminary study on the use of noninvasive hemodynamic monitoring with the Nexfi n monitor in critically ill patients M Peetermans, W Verlinden, J Jacobs, A Verrijcken, S Pilate, N Van Regenmortel, I De laet, K Schoonheydt, H Dits, ML Malbrain ZNA Stuivenberg, Antwerp, Belgium Critical Care 2012, 16(Suppl 1):P227 (doi: 10.1186/cc10834) Results The models accurately predicted maximum ventricular pressures and volumes, not used in the identifi cation process, to mean percentage errors of 7.1% and 6.7% (less than measurement error ~10%). Mean modelled pulmonary vascular resistances (PVR) compared well (R2 = 0.81 for APE and R2 = 0.95 for SS) to experimentally derived values. Importantly, in the APE study a 91% rise from baseline in the mean PVR was identifi ed with an 89% increase seen in the SS pigs. Contrasting behaviour between the two studies was observed for systemic vascular resistance (SVR) with a maximum drop of 40% from baseline recorded at T120 for SS, indicating a loss of vascular tone as expected, where at the same time in the APE study the average SVR had increased by 13%. An increase in the ratio of right to left ventricle end volume was identifi ed in all nine pigs, indicating right ventricular distension and a leftward shift in the intraventricular septum.i Introduction Noninvasive hemodynamic monitoring may become a new tool in the ICU armamentarium. The Nexfi n monitor (BMEYE, Amsterdam, the Netherlands) enables continuous noninvasive analysis of the fi nger blood pressure waveform using an infl atable fi nger cuff , a technology based on the volume-clamp principle of Penaz in combination with the physical criteria of Wesseling. The aim of the present study was to validate the Nexfi n in a mixed population of medical ICU patients and to look for a pattern recognition that may be linked with outcome. g y Methods A prospective study in 40 patients admitted to the medical ICU (17 patients mechanically ventilated, M/F ratio 1/1). Age 63.5 ± 16.7, BMI 26.4 ± 5.4, APACHE II score 20.8 ± 9.5, SAPS II 45.9 ± 18.9, SOFA score 7.2 ± 4.2. For all patients, simultaneous recording of arterial pressure by radial line (n = 46), by PiCCO monitor (n = 15) or by NIBP measurement with arm cuff (n = 17) was compared with noninvasive hemodynamic parameters obtained with the Nexfi n monitor. P227 Statistical analysis was performed with Student’s t test, Pearson correlation and Bland–Altman analysis. Conclusion These results indicate that subject-specifi c cardiovascular models are capable of tracking well-known global hemodynamic trends of two common forms of shock in the ICU. The method shows potential and could provide a means for continuous cardiovascular monitoring at little extra cost as no extra measurements or expensive devices are required. P229 y Results A total of 69 measurements in 40 patients were performed. In three patients measurement with the Nexfi n was not possible. For CO (26 paired measurements), values were 6.4 ± 2.1 l/minute (range 3.3 to 12). The Pearson correlation coeffi cient comparing Nexfi n-CO with reference CO showed a good correlation (R2 = 0.5). Bland–Altman analysis comparing both CO techniques revealed a mean bias ±2SD (LA) of 0.7 ± 3.9 l/minute (58.3% error). The MAP was 84.6 ± 17.7 mmHg (57.5 to 131.5) and values obtained with the Nexfi n correlated well with the reference method (PiCCO in eight; radial line in 43) with an R2 of 0.75. Bland–Altman analysis comparing both MAP techniques revealed a mean bias ±2SD (LA) of 0.2 ± 19.7 mmHg (23.3% error). However, Nexfi n-MAP did not correlate well with NIBP (R2 = 0.1). The nine patients that died in the ICU had higher APACHE II (P = 0.07), SAPS II (P = 0.07) and SOFA (P = 0.01) scores and signifi cantly lower MAP (P = 0.028) and lower dp/dtmax (P = 0.029), a marker for contractility. There were no outcome diff erences with regard to subgroup analysis in patients with either low or high CO or SVR. A Ercole, SM Bishop, SI Yarham, VU Navapurkar, DK Menon University of Cambridge, UK y g Critical Care 2012, 16(Suppl 1):P229 (doi: 10.1186/cc10836) y g Critical Care 2012, 16(Suppl 1):P229 (doi: 10.1186/cc10836) Introduction Physiological instability is a common clinical problem in the critically ill. Physiological adaptation can be regarded as a dynamic process, with stability being conferred by a number of apparently complex, fl uctuating homeokinetic processes [1]. Many natural systems are nonlinear, and seemingly random fl uctuations may result as a consequence of their underlying dynamics. Fractal geometry off ers a method to characterize the underlying nonlinear state, providing a technique for monitoring complex physiology in real time, which may be of clinical importance. Methods We employ the wavelet modulus maxima technique to characterize the multifractal properties of physiological time series such as heart rate (HR) and mean arterial pressure (MAP) under conditions of clinical physiological instability. We calculated point estimates for the dominant Hölder exponent (hm) and multifractal spectrum width-at- half-height (WHH). We investigated how these parameters changed with pharmacological interventions such as vasoconstriction. Conclusion The preliminary results of this ongoing prospective trial indicate that in unstable critically ill patients CO and MAP can be monitored noninvasively with the Nexfi n. The exact patient population for this technology has yet to be defi ned and more patients are probably needed for pattern recognition, although the results indicate that low MAP and dp/dtmax are associated with poor outcome. g Results Hypotensive patients showed lower values of hm for MAP, consistent with a more highly fl uctuating, antipersistent and complex behavior. Blood pressure support with pharmacological vasoconstriction led to a transient increase in hm for MAP (Figure 1) revealing the appearance of longer-range correlations, but did not aff ect hm as estimated for HR. On the other hand, supporting the heart rate with atropine had no eff ect on hm for MAP, but did tend to increase hm for HR. Left ventricular stroke volume measurement by impedance cardiography correlates with echocardiography in neonates l h 1 h1 Ob h 1 ll 2 l2 S 1 A total of 102 healthy neonates with normal biventricular cardiac morphology (including PDA or patent foramen ovale) were included – further patient details: 43 female, 59 male, median weight 3.32 kg, median length 51 cm, median age 49.24 hours, mean heart rate 133 ± 22/ minute. In total 328 simultaneous LVSV measurements in triplicate irrespective of respiratory cycle were analyzed. Results Signifi cant correlations (P <0.05) were noted between EV-LVSV and body weight, TTE-LVSV and body weight, EV-LVSV and age, TTE-LVSV and age. A signifi cant inverse correlation was seen between EV-LVSV and heart rate, and TTE-LVSV and heart rate. No signifi cant correlation was found for EV-LVSV and age (if age ≤120 hours). No signifi cant eff ect was seen for a small persistent foramen ovale (n = 66) and a small PDA (n = 26) on EV-LVSV and TTE-LVSV in the observed cohort. Bland–Altman analysis of logarithmic data showed a bias of the EV-LVSV measurements in comparison to the TTE-LVSV measurements with smaller LVSVs resulting in lower EV than TTE measurements and y Results In total, 305 paired PiCCI–PuCCI and 128 paired AutoCI–TDCI values were obtained. TDCI values ranged from 1.5 to 6.7 l/minute/m2 (mean 3.9 ± 1), AutoCI from 2.4 to 6.5 (3.8 ± 0.8), PiCCI from 1.5 to 7.1 (3.8 ± 1.2) and PuCCI from 2 to 7.6 (3.8 ± 1). The Pearson correlation coeffi cient comparing all and mean PuCCI and PiCCI values had an R2 of 0.77 and 0.86 respectively; for AutoCI and TDCI, R2 was 0.76. The above R2 values were 0.73, 0.84 and 0.71 respectively when the Pulsiofl ex was connected to a radial line. Changes in AutoCI correlated well with changes in TDCI (R2 = 0.68), as did changes in PuCCI versus changes S82 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 immunocompromised and immobile state of ICU patients. This research retrospectively tests the ability of a computer-based method to monitor acute hemodynamic changes in pigs. If proven, this method could assist ICU staff by providing a clear physiological, patient-specifi c picture of cardiovascular status for decision support. in PiCCI (R2 = 0.53). PPV obtained from Pulsiofl ex and PiCCO correlated better than SVV (R2 = 0.86 vs. 0.62). Left ventricular stroke volume measurement by impedance cardiography correlates with echocardiography in neonates l h 1 h1 Ob h 1 ll 2 l2 S 1 Changes in PiCCI and PuCCI induced by an intervention correlated well with each other (R2 = 0.94). Bland– Altman analysis comparing AutoCI with TDCI revealed a mean bias ±2SD (LA) of 0.05 ± 0.94 l/minute/m2 (with 27.3% error) while analysis of PuCCI versus PiCCI showed a bias ±LA of 0.01 ± 1.12 (29.1% error). Conclusion Although TDCI remains a gold standard, the preliminary results of an ongoing prospective study indicate that in unstable critically ill patients CI can be reliably monitored with Pulsiofl ex technology. Moreover, the Pulsiofl ex was also able to keep track of changes in CI. in PiCCI (R2 = 0.53). PPV obtained from Pulsiofl ex and PiCCO correlated better than SVV (R2 = 0.86 vs. 0.62). Changes in PiCCI and PuCCI induced by an intervention correlated well with each other (R2 = 0.94). Bland– Altman analysis comparing AutoCI with TDCI revealed a mean bias ±2SD (LA) of 0.05 ± 0.94 l/minute/m2 (with 27.3% error) while analysis of PuCCI versus PiCCI showed a bias ±LA of 0.01 ± 1.12 (29.1% error). in PiCCI (R2 = 0.53). PPV obtained from Pulsiofl ex and PiCCO correlated better than SVV (R2 = 0.86 vs. 0.62). Changes in PiCCI and PuCCI induced by an intervention correlated well with each other (R2 = 0.94). Bland– Altman analysis comparing AutoCI with TDCI revealed a mean bias ±2SD (LA) of 0.05 ± 0.94 l/minute/m2 (with 27.3% error) while analysis of PuCCI versus PiCCI showed a bias ±LA of 0.01 ± 1.12 (29.1% error). p pp Methods In two porcine studies, APE (n = 5) and SS (n = 4) were induced using autologous blood clots and endotoxin infusions. Hemodynamic measurements were recorded every 30 minutes for 4 hours (n = 80). Subject-specifi c cardiovascular models were identifi ed from typical ICU measurements obtained from each of these datasets, including aortic and pulmonary artery pressure, stroke volume, heart rate, global end- diastolic volume, and mitral and tricuspid valve closure times. Model outputs and identifi ed parameters were compared to experimentally derived indices, measurements not used in the identifi cation process, and known trends to validate the accuracy of the models. Conclusion Although TDCI remains a gold standard, the preliminary results of an ongoing prospective study indicate that in unstable critically ill patients CI can be reliably monitored with Pulsiofl ex technology. Moreover, the Pulsiofl ex was also able to keep track of changes in CI. Homeodynamic complexity: multifractal analysis of physiological instability Homeodynamic complexity: multifractal analysis of physiological instability A Ercole, SM Bishop, SI Yarham, VU Navapurkar, DK Menon University of Cambridge, UK P228 P228 Computer-based monitoring of global cardiovascular dynamics during acute pulmonary embolism and septic shock in swine JA Revie1, DJ Stevenson1, JG Chase1, BC Lambermont2, A Ghuysen2, P Kolh2, GM Shaw3, T Desaive2 1University of Canterbury, Christchurch, New Zealand; 2University of Liege, Belgium; 3Christchurch Hospital, Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P228 (doi: 10.1186/cc10835) P228 Computer-based monitoring of global cardiovascular dynamics during acute pulmonary embolism and septic shock in swine JA Revie1, DJ Stevenson1, JG Chase1, BC Lambermont2, A Ghuysen2, P Kolh2, GM Shaw3, T Desaive2 1University of Canterbury, Christchurch, New Zealand; 2University of Liege, Belgium; 3Christchurch Hospital, Christchurch, New Zealand Critical Care 2012, 16(Suppl 1):P228 (doi: 10.1186/cc10835) P230 Accuracy of conventional urinary output monitoring in the ICU E Bouwhuijsen, A Oude Lansink, MW Nijsten, W Dieperink University of Groningen, University Medical Center Groningen, the Netherlands Critical Care 2012, 16(Suppl 1):P230 (doi: 10.1186/cc10837) Introduction In patients who are treated in the ICU an accurate fl uid balance is an important tool to assess their hydration status. In most ICUs, intake of fl uid is monitored precisely by sophisticated volumetric infusion and feeding pumps. In contrast to fl uid intake, fl uid output – especially urine as its most important component – is usually monitored visually by hourly assessment of the amount of fl uid lost and urine production. Thus measurement of urinary output is a repetitive procedure 24 times a day which requires handling of the urinary collection system, visual assessment and manual data recording, actions that are easily aff ected by human errors. l g Results One patient was excluded due to missing data. There were signifi cant increases in mean arterial pressure (MAP), left ventricular dimensions at the end of diastole and systole, stroke volume (SV), cardiac output (CO) and BNP after the fl uid challenge, while the heart rate decreased. Impaired cardiac contractility was defi ned as an ejection fraction (EF) <50%. The left ventricular end-systolic dimension (LVESd) before (4 cm vs. 2.9 cm) and after the fl uid challenge (4.2 cm vs. 3.29 cm) was signifi cantly greater (statistically and beyond reference intervals) in the EF <50% group compared to the EF >50% group. In the group with EF <50, the median LVEDd2 and LVESd2 post fl uid challenge increased to values of 5.72 cm and 4.28 cm respectively (above the reference thresholds). For the group with EF >50, the median post-challenge LVEDd2 and LVESd2 increased signifi cantly (P = 0.01) to 5.38 cm and 3.39 cm but within the reference thresholds. The BNP increased by 53.6% in the EF <50 group in contrast to a decrease by 12.7% in the EF >50 group. The median EF in the EF <50 and EF >50 groups were signifi cantly diff erent (0.44 vs. 0.66 respectively). . Pirracchio R, et al.: Impaired plasma BNP clearance in human septic shock. Cri Care Med 2008, 36:2542-2546. Computer-based monitoring of global cardiovascular dynamics during acute pulmonary embolism and septic shock in swine m Conclusion We demonstrate increasing signal complexity under physiological challenge consistent with the activation of homeokinetic processes. Diff erential fractal behavior for HR and MAP suggests that the homeokinetic systems are recruited in a targeted way depending Introduction Acute pulmonary embolism (APE) and septic shock (SS) are highly prevalent dysfunctions in the ICU due to the Introduction Acute pulmonary embolism (APE) and septic shock (SS) are highly prevalent dysfunctions in the ICU due to the S83 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 pp http://ccforum.com/supplements/16/S1 Figure 1 (abstract P229). Figure 1 (abstract P229). on the physiological challenge. Pharmacological restoration of homeo- stasis leads to system decomplexifi cation suggesting that homeokinetic mechanisms are derecruited as physiology is restored. We suggest fractal geometry provides a method for characterizing physiological instability and measuring the homeokinetic stress response during physiological challenges. Reference 1. Goldberger AL, Amaral LAN, Hausdorff JM, Ivanov PC, Peng C-K, Stanley HE: Proc Natl Acad Sci U S A 2002, 99:2466-2472. Introduction The aim of the study is to describe the hemodynamic changes and relate them to the changes in B-type natriuretic peptide (BNP) following fl uid challenge in patients with severe sepsis and septic shock. Methods This prospective observational study enrolled 30 patients with severe sepsis or septic shock who required a fl uid challenge within 48 hours of admission to the ICU. All patients had a basic cardiac echocardiogram (echo) performed, blood for BNP collected and baseline hemodynamic measurements recorded. A 500 ml colloid challenge was administered within 30 minutes. The echo and hemodynamic measurements were repeated at this point. One hour after the fl uid challenge the BNP test was repeated. Changes in B-type natriuretic peptide and related hemodynamic parameters following a fl uid challenge in patients with severe sepsis or septic shock Changes in B-type natriuretic peptide and related hemodynamic parameters following a fl uid challenge in patients with severe sepsis or septic shock S Omar, LR Mathivha, A Ali Wits University, Johannesburg, South Africa Critical Care 2012, 16(Suppl 1):P231 (doi: 10.1186/cc10838) Reference Assessment fl uid responsiveness in septic shock patients: a comparison of automated pulse pressure variation and manually calculated pulse pressure variation Results At baseline, PP and EKG were both <12%. PP were signifi cantly correlated with EKG (r2 = 0.89, P <0.001). Volume loss induced by haemorrhage increased signifi cantly PP and EKG. Moreover, during this state, PP were signifi cantly correlated with EKG (r2 = 0.86, P <0.001). Retransfusion signifi cantly decreased both PP and EKG, and PP were signifi cantly correlated with EKG (r2 = 0.90, P <0.001). S Panyawatanaporn, B Khwannimit S Panyawatanaporn, B Khwannimit y p Prince of Songkla University, Hat Yai, Thailand Prince of Songkla University, Hat Yai, Thailand Critical Care 2012, 16(Suppl 1):P234 (doi: 10.1186/cc10841) g y Critical Care 2012, 16(Suppl 1):P234 (doi: 10.1186/cc10841) Introduction Pulse pressure variation (PPV) is an accurate predictor of fl uid responsiveness in mechanically ventilated patients. The aim of this study was the assessment and comparison of the ability of automated PPV, when measured by an IntelliVue MP 70 monitor, and manually calculated PPV to predict fl uid responsiveness in mechanically ventilated septic shock patients. gi y Conclusion Available correlations between PP and EKG at each time of the study were observed, meaning that EKG is a reliable parameter to estimate the changes in intravascular volume status and provide experimental confi rmation of the Brody eff ect [2]. References i yf References p p Methods We conducted a prospective study on 36 septic shock patients. Automated and manually calculated PPV and other hemodynamic data were recorded before and after fl uid administration of 500 ml of 6% hydroxyethyl starch (130/0.4) over 30 minutes. Responders were defi ned as patients with an increase in their cardiac index >15% after fl uid loading. 1. Cannesson M, Keller G, Desebbe O, Lehot JJ: Relations between respiratory changes in R-wave amplitude and arterial pulse pressure in mechanically ventilated patients. J Clin Monit Comput 2010, 24:203-207. p p 2. Brody DA: A theoretical analysis of intracavitary blood mass infl uence on the heart–lead relationship. Circ Res 1956, 4:731-738. 2. Brody DA: A theoretical analysis of intracavitary blood mass infl uence on the heart–lead relationship. Circ Res 1956, 4:731-738. Results The agreement (mean bias ± SD) between automated and manually calculated PPV was 4.03 ± 7.37%. The baseline automated PPV correlated with the baseline manually calculated PPV (r = 0.79, P <0.01). Twenty-three (63.9%) patients were classifi ed as fl uid responders. References 1. Intensive Care Med 2011, 37:233-240. 2. Crit Care Med 2088, 36:2810-2816. P232 The Brody eff ect to detect hypovolemia in clinical practice R Giraud, N Siegenthaler, DR Morel, K Bendjelid Hôpitaux Universitaires de Genève, Switzerland Critical Care 2012, 16(Suppl 1):P232 (doi: 10.1186/cc10839) P232 The Brody eff ect to detect hypovolemia in clinical practice R Giraud, N Siegenthaler, DR Morel, K Bendjelid Hôpitaux Universitaires de Genève, Switzerland Critical Care 2012, 16(Suppl 1):P232 (doi: 10.1186/cc10839) Table 1 (abstract P233). Hemodynamic variables at 0 and 24 hours Variable 0 hours 24 hours P value CVP 15.51 16.2 >0.1 MAP 73.6 76.4 >0.1 SVV% 13.61 10.8 >0.1 Arterial lactate 3.9 ± 2.6 2.3 ± 1.5 <0.001 PO2/FiO2 120.6 ± 42 193.4 ± 76 <0.001 Introduction The electrocardiogram (EKG) is a common monitoring method in intensive care medicine. Several studies suggest that changes in EKG morphology may refl ect changes in volume status. The Brody eff ect, a theoretical analysis of left ventricular chamber size infl uence on QRS-wave amplitude, is the key element of this phenomenon. It is characterized by an increase in QRS-wave amplitude induced by an increase in ventricular preload [1]. This study investigated the infl uence of changes in intravascular volume status on respiratory variations of QRS-wave amplitude (EKG) compared with respiratory pulse pressure variations (PP). study period were 5.1 ± 2.6 l. Arterial lactates reduced signifi cantly without worsening of hypoxia. The PO2/FiO2 ratio increased signifi cantly at 24 hours. Twenty-two out of 37 survived (59.45%) until hospital discharge. See Table 1.l g Conclusion SVV guided fl uid therapy in septic shock with ARDS may improve shock by optimizing preload in a targeted way without worsening oxygenation. Methods In 17 pigs, EKG and arterial pressure were recorded. QRS- wave amplitude was measured from the Biopac recording ensuring that in all animals EKG electrodes were always at the same location. Maximal QRS amplitude (EKGmax) and minimal QRS amplitude (EKGmin) were determined over one respiratory cycle. EKG was calculated as 100×((EKGmax – EKGmin) / (EKGmax + EKGmin) / 2). EKG and PP were simultaneously recorded. Measurements were performed during normovolaemic conditions, after haemorrhage and following retransfusion with constant tidal volume (10 ml/kg) and respiration rate (15/minute).i Stroke volume variation guided fl uid therapy in septic shock with ARDS S Jog, D Patel, M Patel, S Sable S Jog, D Patel, M Patel, S Sable Deenanath Mangeshkar Hospital and Research Centre, Pune, India Critical Care 2012, 16(Suppl 1):P233 (doi: 10.1186/cc10840) g, , , eenanath Mangeshkar Hospital and Research Centre, Pune, India Deenanath Mangeshkar Hospital and Research Centre, Pune, India Critical Care 2012, 16(Suppl 1):P233 (doi: 10.1186/cc10840) Introduction Optimal fl uid resuscitation guided by central venous pressure (CVP) in patients having septic shock with ARDS is a perplexed issue having risk of underfi lling and worsening of shock versus fl uid overload leading to pulmonary edema. Whether stroke volume variation (SVV) (Flotrac-Vigileo system) guided fl uid resuscitation has an impact on improvement of shock, oxygenation and mortality were tested in this single-center prospective study [1,2]. i Conclusion Our results indicate that the automated PPV, obtained by the IntelliVue MP 70 monitor, and manually calculated PPV, showed comparable performance for predicting fl uid responsiveness in passively ventilated septic shock patients. Methods Inclusion criteria were: (1) septic shock patients with dose of norepinephrine ≥0.1 μg/kg/minute or dopamine ≥10 μg/kg/ minute; (2) CVP ≥12 mmHg; (3) PO2/FiO2 ratio ≤200 with ARDSnet protocol ventilation under deep sedation. Exclusion criteria were atrial/ventricular arrhythmias, spontaneous triggering of inspiration, established renal failure needing continuous renal replacement therapy (CRRT). During the 24-hour study period, SVV was continuously monitored with the third-generation Flotrac-Vigileo system (version 3.02). Intravenous fl uids were given in the boluses of 250 to 500 cm3 to keep SVV <12% throughout the study period. Vasopressor infusion was titrated to keep MAP >70 mmHg. Assessment fl uid responsiveness in septic shock patients: a comparison of automated pulse pressure variation and manually calculated pulse pressure variation Automated PPV and manually calculated PPV were signifi cantly higher in responders than in nonresponders (16.0 ± 4.5% vs. 7.2 ± 2.0% and 11.1 ± 5.6 vs. 4.6 ± 2.8%, respectively; P <0.001 for both). The area under the receiver operating characteristic curves of automated PPV was signifi cantly greater than the manually calculated PPV (0.982 vs. 0.87, respectively; P = 0.04). The optimal threshold values for predicting fl uid responsiveness were 11% for automated PPV (sensitivity 91.3%, specifi city 92.3%) and 13% for manually calculated PPV (sensitivity 73.9%, specifi city 84.6%). P230 Multiple regression analysis found LV dimension at end diastole at baseline was one of four independent predictors of an increase in %BNP.il g yf y Methods In a bench test we investigated the accuracy and precision of conventional urinary output monitoring, by visual hourly readings and manual data recording, as performed by experienced intensive care nurses with the purpose to provide insight into potential errors in urinary output measurement as well as identifying systematic sources of error. Two diff erent types of ordinary 24-hour urine meters were used. The meters were fi lled with a predetermined amount (gold standard) of yellow lemonade. Both urine meters were fi lled with variable but identical volumes for a range of 8 to 325 ml, to a total amount of 3,600 ml. Hereafter the nursing staff manually recorded the reading of 48 prefi lled urine meters. p p Conclusion A signifi cant increase in %BNP after a fl uid challenge (irrespective of initial value) may indicate that cardiac contractility is impaired and the LV dilated, indicating a strategy away from fl uid resuscitation and towards inotrope use. i Results Forty-eight nurses performed 2,285 urine volume measure- ments in two diff erent types of ordinary urine meters (Bard Urine meter drainage bag; Bard Medical, Covington, Georgia, USA and Rüsh U-bag; Jiangsu, People’s Republic of China). The mean measured output for the Bard urine meter was 3,688 ml, SD ±45 and for the Rüsh urine meter 3,692 ml, SD ±55. The limits of agreement between both types of urine meters were 2.4% to 2.6% respectively. Compared with the gold standard, analysis demonstrated deviations of 2.6% for both types of urine meters. Reference Conclusion Conventional urinary output measurement with ordinary urine meters constitutes a simple and accurate method for measuring urine volume in the ICU. S84 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P233). Hemodynamic variables at 0 and 24 hours P237 Microcirculatory blood fl ow is related to clinical signs of impaired organ perfusion, and its dynamics to the macrohemodynamic concept of fl uid responsiveness A Pranskunas1, M Koopmans2, V Pilvinis3, P Koetsier2, EC Boerma2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Medical Centre Leeuwarden, the Netherlands; 3Hospital of Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2012, 16(Suppl 1):P237 (doi: 10.1186/cc10844) Microcirculatory blood fl ow is related to clinical signs of impaired organ perfusion, and its dynamics to the macrohemodynamic concept of fl uid responsiveness A Pranskunas1, M Koopmans2, V Pilvinis3, P Koetsier2, EC Boerma2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Medical Centre Leeuwarden, the Netherlands; 3Hospital of Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2012, 16(Suppl 1):P237 (doi: 10.1186/cc10844) Microcirculatory blood fl ow is related to clinical signs of impaired organ perfusion, and its dynamics to the macrohemodynamic concept of fl uid responsiveness Results Ninety volume expansions in 68 patients were performed during the study period. Central venous catheter was present 58.9% of the time. In 41.1% of the cases patients were in spontaneous ventilation. No patients used a pulmonary artery catheter. An echocardiography machine with an attending physician trained for critical care echocardiography was available in 8.9%. An arterial catheter was available in 21% of the volume expansions and mechanical ventilation was present in 31.1% of the cases (67.3% of ventilated patients were using controlled mode of ventilation). The association of mechanical ventilation in controlled mode with an arterial catheter in place and no restrictions for performing analysis of dynamic parameters was present in only 7.7% of patients. Considering all dynamic parameters described here, the use of any method for predicting fl uid responsiveness was possible in 15.6% of the volume expansions performed in our ICU.l Introduction Fluid responsiveness is not equal to a clinical need for fl uid therapy. The aim of our study was to assess the incidence of microcirculatory fl ow alterations, according to a predefi ned arbitrary cut-off value, in patients with clinical signs of impaired organ perfusion. The secondary endpoint was to establish the correlation between the microcirculatory and macrocirculatory response to a fl uid challenge. Methods We performed a prospective, single-centre, observational study. Included were ICU patients ≥18 years with invasive hemo- dynamic monitoring and clinical signs of impaired organ perfusion, as the principal reason for fl uid administration. P237 Fluid challenge was performed by the infusion of 500 ml crystalloid or a balanced colloid (Volulyt®) solution in 30 minutes. Before and after fl uid challenge, systemic hemodynamics and direct in vivo observation of the micro- circulation were obtained with sidestream dark-fi eld imaging. Assess- ment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI) and proportion of perfused vessels), and diff usion distance (perfused vessel density and total vessel density) was done using a semiquantitative method. p p p Conclusion The use of dynamic parameters for predicting fl uid responsiveness in the ICU may have restricted applicability since the necessary conditions are often not present. P236 Fluid responsiveness during weaning from mechanical ventilation M Geisen, UM Schmid, O Dzemali, A Zollinger, CK Hofer Triemli City Hospital, Zürich, Switzerland Critical Care 2012, 16(Suppl 1):P236 (doi: 10.1186/cc10843) g Results We enrolled 50 patients. MFI <2.6 was present in 66% of the patients. After fl uid challenge, signs of impaired organ perfusion reduced from 100% to 68% of the patients, P <0.001. The incidence of MFI <2.6 decreased to 46%, and was higher in patients with persistent signs of impaired organ perfusion: 56% versus 25%, P = 0.04. Median MFI increased from 2.5 (2.3 to 2.8) at baseline to 2.7 (2.4 to 2.8) after fl uid challenge, P = 0.003, but its change was only signifi cant in fl uid- responsive patients. Introduction To overcome the limited accuracy of functional hemodynamic parameters such as stroke volume and pulse pressure variation (SVV and PPV) during spontaneous breathing, a passive leg raising (PLR) maneuver has been suggested as a reliable predictor of fl uid responsiveness [1,2]. The aim of this study was to evaluate fl uid responsiveness using SVV, PPV and PLR during the transition from controlled to spontaneous breathing. Conclusion These data demonstrate a relationship between clinical signs of impaired organ perfusion and MFI <2.6. Fluid responsiveness did not discriminate between patients with and without clinical signs of impaired organ perfusion or MFI <2.6. However, signifi cant improvement of microvascular alterations and attenuation of clinical signs of impaired organ perfusion was restricted to patients who were fl uid responsive. Noninvasive assessment of microvascular perfusion may help to defi ne patients with potential need for fl uid therapy, and to evaluate its eff ect. Methods Thirty-four patients after off -pump CABG were enrolled. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Prediction of a fl uid response with SVV/PPV was less reliable in spontaneous breathing. PLR predicted fl uid responsiveness, but was less accurate than previously reported. Conclusion Prediction of a fl uid response with SVV/PPV was less reliable in spontaneous breathing. PLR predicted fl uid responsiveness, but was less accurate than previously reported. To ensure appropriate indication of fl uid administration, evaluation of fl uid responsiveness by dynamic parameters is suggested, although it requires specifi c conditions not always present in ICU patients. The aim of this study was to analyze the applicability of parameters for evaluation of fl uid responsiveness in the ICU. y References 1. Marik et al.: Crit Care Med 2009, 37:2642-2647. 2. Monnet et al.: Crit Care Med 2006, 34:1402-1407. References 1. Marik et al.: Crit Care Med 2009, 37:2642-2647. 2. Monnet et al.: Crit Care Med 2006, 34:1402-1407. l Methods We conducted a prospective observational study in two ICUs. Volume expansions performed in ICU patients at the discretion of the physician in charge were analyzed for the presence of conditions that allowed adequate fl uid responsiveness evaluation. The presence of central venous, pulmonary arterial or peripheral arterial catheters, invasive mechanical ventilation and ventilator settings, echocardiography availability, presence of arrhythmias, use of sedation and vasoactive drugs were registered. Percentages of patients who fulfi lled conditions for dynamic parameters (such as pulse pressure variation, stroke volume variation and echocardiographic analysis) were recorded. P237 Measurements were performed in the ICU using a PiCCO2 system. Fluid (500 ml) was given: (A) during controlled mechanical ventilation, (B) during pressure support ventilation with spontaneous breathing and (C) after extubation. The stroke volume (SV), SVV and PPV as well as the mean arterial pressure and heart rate were assessed. A PLR was performed before fl uid administration at all three time points. Fluid response was defi ned as an increase in SV >15%. Prediction of fl uid responsiveness was tested using ROC analysis.i P238 P238 Frank–Starling and Guyton together at bedside during a fl uid challenge H Aya, M Cecconi, M Geisen, C Ebm, M Grounds, N Fletcher, A Rhodes St George’s Hospital, London, UK Critical Care 2012, 16(Suppl 1):P238 (doi: 10.1186/cc10845) P235 P235 Applicability of methods for fl uid responsiveness prediction in the ICU P Mendes1, L Miranda1, M Park1, L Azevedo1, B Rodrigues2, E Queiroz2, G Schettino2, L Taniguchi1 1Hospital das Clinicas, São Paulo, Brazil; 2Hospital Sirio Libanes, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P235 (doi: 10.1186/cc10842) Critical Care 2012, 16(Suppl 1):P235 (doi: 10.1186/cc10842 Results Thirty-seven patients with severe sepsis-induced multiorgan dysfunction syndrome with average APACHE II score of 24.6 and PEEP of 8.2 cm were enrolled. SVV guided fl uids received during the 24-hour Introduction Volume expansion is a frequent and widely used therapy in hemodynamically unstable patients but may lead to complications. Introduction Volume expansion is a frequent and widely used therapy in hemodynamically unstable patients but may lead to complications. S85 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Prediction of fl uid responsiveness in intensive care (PREFERENCE study): fl uid challenge versus passive leg raising in high-risk surgical patients Prediction of fl uid responsiveness in intensive care (PREFERENCE study): fl uid challenge versus passive leg raising in high-risk surgical patients Introduction Massive fl uid resuscitation followed by hypoperfusion abnormalities in the ICU is a risk factor for development of intra- abdominal hypertension (IAH). The aim of our study was to determine what infl uence would have the control of daily fl uid balance on the incidence of IAH in patients after extensive abdominal surgery or after abdominal trauma. g p M Cecconi, F Caliandro, J Mellinghoff , D Dawson, S Ranjan, M Hamilton, M Grounds, A Rhodes St Georges Hospital, London, UK Critical Care 2012 16(Suppl 1):P239 (doi: 10 1186/cc10846) Introduction The aim of this study is to evaluate whether the passive leg raising (PLR) maneuver could be used to predict fl uid responsiveness in awake postoperative patients admitted to the ICU. PLR has been demonstrated to be a good indicator of fl uid responsiveness even in spontaneously breathing patients, but few data are available in the immediate postoperative period. Nexfi n is a new cardiac output monitor that measures and tracks stroke volume (SV) by analyzing the arterial pressure pulse contour noninvasively from a fi nger probe. Methods A prospective observational study included a total of 82 adult patients (age: 59 ± 10 years, APACHE II score at admission: 18 ± 11, predicted mortality according to APACHE II score at admission: 34%, observed in-hospital mortality: 20%), number of patients with intra- abdominal pressure (IAP) above 12 at admission: 23 (28%), admitted to a single ICU with the diagnosis of abdominal trauma (n = 22) or after abdominal surgery (n = 60). During fi rst 7 days the fl uid intake and balance was monitored and corrected at 6-hour intervals not to exceed 1,500 ml positivity over 24 hours (oncodiuretic therapy was administered – repeated boluses of starch solutions and/or albumin followed by furosemide). IAP was measured from admission twice daily (standardized measurement by instillation of 25 ml normal saline into the bladder). A sustained elevation of the IAP above 12 mmHg in two consecutive measurements was considered as IAH.l p p yi g p Methods We enrolled self-ventilating patients admitted to the ICU postoperatively. A PLR maneuver (45° bed tilt from the 30 to 45° head up) was performed and followed by a fl uid challenge (FC, 250 ml fl uid bolus over 5 minutes). Frank–Starling and Guyton together at bedside during a fl uid challenge Results In 34 patients signifi cant hemodynamic changes were observed, with 19 (55.9%), 22 (64.7%), and 13 (40.6%) responders at time points A, B and C, respectively. Prediction of fl uid responsiveness is depicted in Table 1. H Aya, M Cecconi, M Geisen, C Ebm, M Grounds, N Fletcher, A Rhodes St George’s Hospital, London, UK Critical Care 2012, 16(Suppl 1):P238 (doi: 10.1186/cc10845) Table 1 (abstract P236). Prediction of fl uid responsiveness A B C AUC P value TS% AUC P value TS% AUC P value TS% SVV 0.88 0.0001 15.5 0.70 0.056 12.5 0.56 0.604 13.5 PPV 0.83 0.001 14.5 0.69 0.063 11.0 0.48 0.863 11.5 PLR SV% 0.72 0.028 8.0 0.74 0.021 10.0 0.70 0.058 8.0 TS, threshold. Table 1 (abstract P236). Prediction of fl uid responsiveness Introduction According to Guyton, the diff erence between mean systemic fi lling pressure (Pms) and right atrial pressure (RAP) is the venous pressure gradient (VP). This is proportional to venous return and cardiac output (CO). According to the Frank–Starling law a fl uid challenge successfully increases the stroke volume if the preload increases in the ascending part of the curve. The aim of this study was to assess the signifi cance of the analogue of the Pms (Pmsa) measured with the Navigator™ (Applied Physiology, St Leonards, Australia), the central venous pressure (CVP) (as a surrogate of RAP) and the VP during a fl uid challenge. Introduction According to Guyton, the diff erence between mean systemic fi lling pressure (Pms) and right atrial pressure (RAP) is the venous pressure gradient (VP). This is proportional to venous return and cardiac output (CO). According to the Frank–Starling law a fl uid challenge successfully increases the stroke volume if the preload increases in the ascending part of the curve. The aim of this study was to assess the signifi cance of the analogue of the Pms (Pmsa) measured with the Navigator™ (Applied Physiology, St Leonards, Australia), the central venous pressure (CVP) (as a surrogate of RAP) and the VP during a fl uid challenge. S86 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods A prospective observational study was performed in postsurgical patients. Patients were monitored with a central venous catheter, an arterial line, a calibrated LiDCO™plus (LiDCO, Cambridge, UK) and the Navigator™. P240 P240 Tight control of fl uid balance may reduce incidence of intra- abdominal hypertension in patients after major abdominal surgery and trauma: a pilot study P Szturz1, J Maca1, J Neiser1, J Jahoda1, R Kula1, JT Tichy2 1Faculty Hospital Ostrava, Czech Republic; 2Yeovil District Hospital, Yeovil, UK Critical Care 2012, 16(Suppl 1):P240 (doi: 10.1186/cc10847) P240 Tight control of fl uid balance may reduce incidence of intra- abdominal hypertension in patients after major abdominal surgery and trauma: a pilot study Conclusion Our study demonstrates that the Navigator™ may be used to monitor the eff ect of fl uid challenges by assessing the change in VP to the challenge. Frank–Starling and Guyton together at bedside during a fl uid challenge A 250 ml fl uid challenge was used to maximise the stroke volume (SV). Data were recorded before and after the fl uid challenge which was given over 5 minutes. A positive response to the fl uid challenge was defi ned as either a stroke volume or CO increase of 10% or more. P240 Tight control of fl uid balance may reduce incidence of intra- abdominal hypertension in patients after major abdominal surgery and trauma: a pilot study P Szturz1, J Maca1, J Neiser1, J Jahoda1, R Kula1, JT Tichy2 1Faculty Hospital Ostrava, Czech Republic; 2Yeovil District Hospital, Yeovil, UK Critical Care 2012, 16(Suppl 1):P240 (doi: 10.1186/cc10847) Figure 1 (abstract P239). Figure 1 (abstract P239). UK) and the Navigator™. A 250 ml fl uid challenge was used to maximise the stroke volume (SV). Data were recorded before and after the fl uid challenge which was given over 5 minutes. A positive response to the fl uid challenge was defi ned as either a stroke volume or CO increase of 10% or more. Results Twenty-fi ve fl uid challenges in 14 patients were observed. In seven cases (28%), the fl uid challenge increased SV (and CO) by ≥10% (Table 1). At baseline there were no diff erences between HR, Pmsa, CVP or ΔVP for responders or nonresponders. The responders had greater changes in ΔVP in response to the challenge. Table 1 (abstract P238). Haemodynamic parameters in responders and nonresponders Nonresponders Responders P value MAP 78.4 71.4 0.07 Pmsa 17 15 0.3 CVP 9.7 9 0.7 HR 89 91 0.7 ΔVP 7.2 6 0.09 ΔPmsa 19.3 22.1 0.6 ΔCVP 2.9 1.6 0.1 Δ(ΔVP) 1.1 24.8 <0.01 Conclusion Our study demonstrates that the Navigator™ may be used to monitor the eff ect of fl uid challenges by assessing the change in VP to the challenge Table 1 (abstract P238). Haemodynamic parameters in responders and nonresponders P239 P239 Prediction of fl uid responsiveness in intensive care (PREFERENCE study): fl uid challenge versus passive leg raising in high-risk surgical patients M Cecconi, F Caliandro, J Mellinghoff , D Dawson, S Ranjan, M Hamilton, M Grounds, A Rhodes St Georges Hospital, London, UK Critical Care 2012, 16(Suppl 1):P239 (doi: 10.1186/cc10846) P241 P241 Negative fl uid balance 48 hours after admission improves survival at 28 days in critically ill patients M Cuartero, AJ Betbese, K Nuñez, J Baldira, L Zapata Hospital De Sant Pau, Barcelona, Spain Critical Care 2012, 16(Suppl 1):P241 (doi: 10.1186/cc10848) yf Results Data of 58 patients were collected. The mean length of hospital stay was 9 ± 3 days. Postoperative complications included suture leak (3.4%), surgical site infection (6.9%), and heart failure (3.4%) with no other medical or surgical complications. Total fl uids during surgery were 2,775 ± 934 ml (1,259 ± 498 ml crystalloids, 1,302 ± 622 ml colloids, 214 ± 330 ml RBC). Fluid infusion and urine output were 10.6 ± 3.2 and 1.8 ± 1.2 ml/kg hour respectively. Hemodynamic data are shown in Table 1. Introduction Fluid infusion may be lifesaving in critically ill patients, but following initial resuscitation a positive fl uid balance is associated with increased mortality. This study aimed to determine whether a negative fl uid balance (≤–500 ml) within the fi rst 48 hours of admission in the ICU is associated with improved survival at 28 days in a heterogeneous cohort of critically ill patients. Table 1 (abstract P242). Hemodynamic variables Start SG End SG P value CI 2.85 ± 0.9 3.16 ± 0.4 0.020 SVI 40 ± 10 41 ± 8 0.59 HR 70 ± 12 77 ± 12 <0.001 PPV 7 ± 5 4 ± 2 0.001 MAP 76 ± 14 83 ± 14 0.002 SVRI 2051 ± 588 2026 ± 551 0.74 Conclusion GDT with MostCare® resulted in a low incidence of postoperative complications and provided an optimal hemodynamic management during major colon surgery. References 1. Gan TJ, et al.: Anesthesiology 2002, 97:820-826. 2. Vincent JL, et al.: Crit Care 2011, 15:229. Table 1 (abstract P242). Hemodynamic variables Table 1 (abstract P242). Hemodynamic variables y p Methods We conducted a retrospective study in a 20-bed ICU at a university-affi liated teaching hospital. Patients admitted for acute heart failure, those who required dialysis before admission and those who died within 24 hours after admission were excluded. Demographic data, SAPS II and APACHE II scores were recorded at admission and SOFA, fl uid balance, hemodynamic, respiratory and renal variables once per day. Variables were compared between survivors and nonsurvivors and between patients who did and those who did not achieve negative fl uid balance by day 2 of admission. Prediction of fl uid responsiveness in intensive care (PREFERENCE study): fl uid challenge versus passive leg raising in high-risk surgical patients Changes in SV during PLR and after administration of FC were monitored with the Nexfi n monitor. Receiver operator characteristic (ROC) analysis was performed.i y Results Forty-fi ve patients were enrolled. Twenty-three patients responded to the FC with an increase of SV >5%. Twenty-eight patients (62%) were excluded from the PLR analysis as a result of haemodynamic instability (diff erence in heart rate, mean arterial pressure or SV baselines pre PLR and pre FC >5%). Seventeen patients were analyzed. The area under the curve for the ROC analysis was 0.93 (SE = 0.06; P = 0.003) (Figure 1). A SV increase >1% during a PLR test predicts a SV increase >5% after FC with a sensitivity of 75% and a specifi city of 78%. Conclusion In 62% of patients a PLR test could not be performed due to haemodynamic instability. In these patients, FC is the best way to assess fl uid responsiveness. During haemodynamic stability PLR shows great sensitivity and specifi city to predict fl uid responsiveness. The Nexfi n monitor can be used to track SV changes both during FC and during a PLR test. Results The incidence of IAH in relation to daily fl uid intake and daily fl uid balance is shown in Figure 1. Conclusion The incidence of IAH in patients after abdominal surgery or abdominal trauma may exceed the value of 40%, especially in situations associated with massive fl uid resuscitation [1]. We have identifi ed a close relationship of the daily dynamics of changes in IAP and fl uid balance. When we maintained the daily fl uid balance not to exceed the positivity of 1,500 ml/24 hours, the incidence of IAH in our study dropped from 28% to less than 20%, despite high daily fl uid intake (about 5,000 to 8,000 ml/day). Tight control of the fl uid balance seems an eff ective method to prevent the development of IAH. Reference Ball ChG, et al.: The secondary abdominal compartment syndrome: not just another post-traumatic complication. Can J Surg 2008, 51:399-405. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S87 Figure 1 (abstract P240). Fluid intake, fl uid balance and incidence of IAH. 1. Boyd JH, Forbes J, Nakada T, Walley KR, Russell JA: Fluid resuscitation in septic shock: a positive fl uid balance and elevated central venous pressure are associated with increased mortality. Crit Care Med 2011, 39:259-265. P241 Multiple logistic regression was used to identify variables signifi cantly associated with ICU mortality in the univariate analysis. Survival was assessed using Kaplan–Meier analysis. y Results We studied 87 patients: 53 males, mean age 58 ± 18 years, SAPS II 39.3 ± 15.8, APACHE II score 15.9 ± 7.5, SOFA score 5.0 ± 3.4, and ICU stay 10.3 ± 9.8 days. The main syndrome diagnosis at admission was septic shock (n = 26), acute respiratory failure (n = 19), trauma (n = 13), neurocritical illness (n = 14) and others (n = 15). Overall mortality in the ICU reached 20.7% and survival at 28 days was 73.6%. When patients were classifi ed according to 28-day outcome, we observed statistically signifi cant diff erences in negative fl uid balance at 48 hours (P <0.001), SAPS II (P <0.001), APACHE II score (P = 0.007), age (P = 0.046) and incidence of acute kidney injury at admission (P = 0.02; defi ned as at least Risk in RIFLE criteria), but urinary output, hemodynamic and respiratory parameters did not diff er. Multivariate analysis showed that negative fl uid balance at 48 hours was independently associated with improved survival: odds ratio = 7.9 (P = 0.013). Kaplan–Meier analysis showed that survival was signifi cantly lower in patients without negative fl uid balance at 48 hours (P = 0.015).il P243 P243 Implementation of an optimal fl uid management protocol using the PiCCO system delays development of ARDS secondary to severe sepsis N Saito, T Yagi, Y Hara, H Matsumoto, K Mashiko Chiba-Hokusoh Hospital, Nippon Medical School, Chiba, Japan Critical Care 2012, 16(Suppl 1):P243 (doi: 10.1186/cc10850) P243 Implementation of an optimal fl uid management protocol using the PiCCO system delays development of ARDS secondary to severe sepsis Introduction Acute respiratory distress syndrome (ARDS) is often associated with sepsis, and one recommended approach in the fl uid management of ARDS is to keep sepses dry. On the other hand, optimal fl uid management after the early phase of septic shock remains unknown. An excessive positive fl uid balance in patients with septic shock is associated with increased mortality and morbidity. We implemented an optimal fl uid management (OFM) protocol using the PiCCO system from April 2009. The purpose of this study was to Introduction Acute respiratory distress syndrome (ARDS) is often associated with sepsis, and one recommended approach in the fl uid management of ARDS is to keep sepses dry. On the other hand, optimal fl uid management after the early phase of septic shock remains unknown. An excessive positive fl uid balance in patients with septic shock is associated with increased mortality and morbidity. We implemented an optimal fl uid management (OFM) protocol using the PiCCO system from April 2009. The purpose of this study was to Conclusion Our fi ndings show that negative fl uid balance 48 hours after admission may correlate with better outcome in a heterogeneous population of critically ill patients. Goal-directed fl uid and hemodynamic therapy in major colon surgery with the pressure recording analytical method cardiac output monitor (MostCare®-PRAM®): prospective analysis of 58 patients Goal-directed fl uid and hemodynamic therapy in major colon surgery with the pressure recording analytical method cardiac output monitor (MostCare®-PRAM®): prospective analysis of 58 patients JM Alonso-Iñigo, MJ Fas, V Osca, A Nacher, JE Llopis Hospital Universitario de la Ribera, Alzira, Spain Critical Care 2012, 16(Suppl 1):P242 (doi: 10.1186/cc10849) Introduction Optimal fl uid therapy in colon surgery is controversial. The aim of this study is to assess the impact on postoperatory complications, fl uid administration, and length of hospital stay, of GD fl uid and hemodynamic therapy protocol based on PRAM®-MostCare® hemodynamic variables. y Methods Patients scheduled for elective colorectal operations were included. The MostCare® was connected to a radial artery catheter for hemodynamic monitoring. Hemodynamic variables including the stroke volume index (SVI), cardiac index (CI), and pulse pressure variation (PPV) were measured. Fluids and hemodynamic drugs were administered to achieve the primary endpoints: CI 2.5 to 4.5, PPV <13%, SVRI >1,500, MAP similar to basal values. Hemodynamic data were noted at induction and at the end of surgery. Data of fl uids, urine output, postoperative complications and length of stay were assessed. Descriptive statistics were used. A paired-simple t test was used for the analysis of the diff erences between variables. Indexation of extravascular lung water in unselected adult patients with and without mechanical ventilation: a prospective study in 50 patients with 843 transpulmonary thermodilutions W Huber, B Saugel, D Paradellis, J Hoellthaler, V Phillip, C Schultheiss, P Thies, U Mayr, A Herrmann, RM Schmid Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P245 (doi: 10.1186/cc10852) Indexation of extravascular lung water in unselected adult patients with and without mechanical ventilation: a prospective study in 50 patients with 843 transpulmonary thermodilutions W Huber, B Saugel, D Paradellis, J Hoellthaler, V Phillip, C Schultheiss, P Thies, U Mayr, A Herrmann, RM Schmid Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P245 (doi: 10.1186/cc10852) Indexation of extravascular lung water in unselected adult patients with and without mechanical ventilation: a prospective study in 50 patients with 843 transpulmonary thermodilutions W Huber, B Saugel, D Paradellis, J Hoellthaler, V Phillip, C Schultheiss, P Thies, U Mayr, A Herrmann, RM Schmid Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P245 (doi: 10.1186/cc10852) Methods A retrospective study was conducted in a Japanese mixed ICU of a tertiary-care teaching hospital from July 2007 to March 2011. Our protocol includes daily volume assessment using the PiCCO system after ICU admission and a change of fl uid therapy after evaluation; for example, additional diuretic use or fl uid restriction. We retrospectively analyzed 96 consecutive patients with severe sepsis or septic shock who required mechanical ventilation between July 2007 and December 2010. We divided patients into the OFM protocol group (P; n = 49; April 2009 to December 2010) and the control group (C; n = 47; July 2007 to March 2009) and compared their clinical and laboratory data. Introduction Extravascular lung water (EVLW) has been indexed to actual BW (BW-act), termed the EVLW index (ELWI). Since in obese patients indexation to BW-act might inappropriately diminish the indexed ELWI-act, ELWI indexed to predicted BW (ELWI-pred) has been introduced. Indexation of EVLW to height might be superior to ELWI- pred/-act. Recent data in a selected collective of ARDS patients suggest that indexation to height might improve the predictive capabilities of ELWI regarding pO2/FiO2. We aimed to investigate which indexation of EVLW provides the best association of ELWI and pO2/FiO2 in patients without pulmonary impairment or without ventilation. Results Median (IQR) age was 69.5 (55.5 to 78.5) years, and the median APACHE II score and SOFA score were 23.0 (19.0 to 27.0) and 10.0 (7.0 to 12.0), respectively. The proportion of patients with septic shock was 75%. There was no diff erence in patient characteristics between the two groups. At 28 days, the mortality rate was similar in both groups (P: 14.3%; C: 17.0%; P = 0.78). P246 Methods We studied 20 septic poytraumatized patients (mean ISS score 35) with ARDS syndrome and ELWI >10, with good renal function and on medical treatment with levophed. We administered furosemide 10 mg/hour, for 24 hours, while at the same time we confronted the septic source. During all these we noted PO2/FiO2, MAP, CVP and ELWI every 8 hours. Moreover, we noted the changes in the levophed dose and the total balance of fl uid at the end of the 24-hour interval. How to perform indexing of extravascular lung water data S Wolf 1, A Riess2, J Landscheidt2, C Lumenta2, L Schuerer2, P Friederich2 1Charite Berlin, Germany; 2Klinikum Bogenhausen, Munich, Germany Critical Care 2012, 16(Suppl 1):P246 (doi: 10.1186/cc10853) Introduction Extravascular lung water (EVLW) is a marker for the severity of acute lung injury. To allow assessment of normal and pathologic states, traditionally EVLW data are either indexed to real or predicted body weight. Surprisingly and despite widespread use, this has so far not been validated in a larger cohort of subjects without cardiopulmonary compromise. The aim of the study was to prospectively evaluate a diff erent ways of indexing EVLW data. l Results The ELWI rate was up 12 to 16 before the administration of furosemide. We marked that after the administration of furosemide and at the end of the fi rst 8-hour interval, the ELWI rate decreased about 2 to 3 units but we had to increase the dose of vasoconstriction, until the end of the 24-hour interval the ELWI rate restored to the initial high level and could not manage to decrease the dose of vasoconstriction to have a negative balance of fl uids. The improvement of PO2/FiO2 was insignifi cant statistically and we confronted operatively the septic source for fi ve to 20 patients at the end of the 24-hour interval. yf y g Methods EVLW was measured using single indicator transpulmonary thermodilution at predefi ned time points in 101 patients requiring elective brain tumor surgery. This database was used to investigate the properties of indexing EVLW data to real and predicted body weight, body surface area and body height. Conclusion We managed to decrease very little the ELWI rate, only temporarily after the administration of furosamide about a 24-hour interval. P245 examine the eff ect of the OFM protocol in comparison with historical controls. Reference 1. Boyd JH, Forbes J, Nakada T, Walley KR, Russell JA: Fluid resuscitation in septic shock: a positive fl uid balance and elevated central venous pressure are associated with increased mortality. Crit Care Med 2011, 39:259-265. S88 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Indexation of extravascular lung water in unselected adult patients with and without mechanical ventilation: a prospective study in 50 patients with 843 transpulmonary thermodilutions W Huber, B Saugel, D Paradellis, J Hoellthaler, V Phillip, C Schultheiss, P Thies, U Mayr, A Herrmann, RM Schmid Klinikum Rechts der Isar, Technischen Universität München, Munich, Germany Critical Care 2012, 16(Suppl 1):P245 (doi: 10.1186/cc10852) The incidence of ARDS after ICU admission in the P group was signifi cantly lower than that in the C group (P: 20.4%; C: 57.4%; P = 0.02). In addition, the onset of ARDS in the P group occurred later than that in the C group (P <0.01). Achievement of a negative water balance in the P group occurred earlier than in the C group. The incidence of AKI (RIFLE criteria: failure) and another organ failure was similar in both groups. Multivariate regression analysis revealed that the OFM protocol independently suppressed the onset of ARDS (OR 0.17 (P = 0.001; 95% CI: 0.06 to 0.51)). p y p Methods In 50 consecutive ICU patients with PiCCO monitoring, 843 triplicate measurements of EVLW and simultaneous blood gas analysis were performed. The endpoint was prediction of pO2/FiO2 <200 mmHg and other critical thresholds provided by unindexed EVLW as well as ELWI indexed to ideal BW, adjusted BW, BMI, body surface area, height and total lung capacity. Results Measurements in patients without pulmonary impairment 463/843 (54.9%); acute 188/843 (22.3%), chronic 106/843 (12.6%), and both acute and chronic pulmonary disease 86/843 (10.2%). Mechanical ventilation in 458/843 (54.3%) measurements. The largest ROC AUCs regarding pO2/FiO2 <200 mmHg were found for ELWI-height (AUC 0.658; 95% CI 0.554 to 0.735) and EVLW (0.655; 95% CI 0.544 to 0.732), the lowest AUC for ELWI-act (0.629; 95% CI 0.514 to 0.742). Similarly ELWI-height and unindexed EVLW provided the largest ROC AUCs regarding pO2/FiO2 >300 mmHg (0.659 and 0.657), normal pO2/FiO2 (>381 mmHg; 0.665 and 0.657) and acute and/or chronic pulmonary impairment (0.622 and 0.625). All these associations were signifi cant with P <0.001. Among patients with pulmonary impairment, fi rst values of ELWI-height and EVLW provided the largest ROC AUCs regarding mortality (0.815 and 0.815; P = 0.016) compared to ELWI-act (0.694; P = 0.136) and APACHE II score (0.792; P = 0.025). Conclusion Implementation of an OFM protocol using the PiCCO system signifi cantly decreased the development of ARDS secondary to severe sepsis with no other complications. P244 Confrontation of the increase in ELWI rate regarding the septic polytraumatised patient administering furosemide: is it eff ective? P Sarafi dou, E Pappa, D Dimitriadou, D Litis, I Pavlou KAT General Hospital Kifi sia, Athens, Greece Critical Care 2012, 16(Suppl 1):P244 (doi: 10.1186/cc10851) Conclusion Indexation to BW-act results in reduced predictive capabilities compared to no indexation at all. ELWI-pred performs slightly better than ELWI-act, but our data do not support that ELWI-pred is superior to no indexation at all in adult ICU patients. In this unselected and prospectively evaluated collective, the highest predictive capabilities regarding several predefi ned thresholds were found for ELWI-height. Introduction The ELWI rate (measurable with the PICCO catheter) conveys the extravascular lung water. The increase of the ELWI rate is a frequent fi nding in heavily septic patients and it is connected with very high mortality. The purpose of this study is to fi nd out if the administration of furosemide is possible to reduce eff ectively the ELWI rate and if this decrease can be maintained regardless of the confrontation of the sepsis. P247 P247 Near-normal values of extravascular lung water in children J Lemson1, C Cecchetti2, A Nusmeier1 1Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 2IRCS Bambino Gesù Roma, Rome, Italy Critical Care 2012, 16(Suppl 1):P247 (doi: 10.1186/cc10854) Introduction Extravascular lung water (EVLW) refl ects the amount of pulmonary edema and can be measured at the bedside using the transpulmonary thermodilution method (TPTD) incorporated in the PiCCO device (Pulsion, Germany). Currently, normal values of EVLW for the use in children are unavailable. This study was designed to collect near-normal values of EVLW in children after recovery from critical illness. g Results Twenty patients were enrolled in this study. All patients had septic shock. Six of all had lung involvement. Twelve patients received mechanical ventilation. The mean of net fl uid balance was +2,228 ± 1,982 ml and the mean of duration between two ultrasound measurements was 31 ± 13 hours. The means of TBS at pre and post fl uid therapy were 37 ± 26 and 64 ± 29 respectively (P <0.0001, 95% CI 13.47 to 33.67). This increase was found in all areas of measurement. In particular, the number of B-lines measured at the anterior axillary line area very well correlated to the TBS (r = 0.90, P <0.01) and its increment had reverse correlation to the PaO2/FiO2 ratio (r = 0.704, P <0.05). The volume of fl uid per one B-line increasing was 119 ± 134 ml. The interobserver reliability between two ultrasound readers was very high (r = 0.92, P <0.01). The changing of TBS did not correlated to that of the chest radiologic score for EVLW assessment (r = 0.002, P >0.05). There was no linear correlation observed between net fl uid balance and total number of increasing B-lines.il Methods In this prospective observational multicenter study (fi ve sites), pediatric TPTD measurements were collected from children admitted to a pediatric ICU without or after resolution of pulmonary abnormalities. Inclusion criteria were minimal or no respiratory support and stable hemodynamics. We searched typically for the last lung water measurement prior to removal of the PiCCO system. EVLW was indexed using predicted body weight (EVLWI) calculated using height, based upon WHO data. p Results Fifty-fi ve children aged from 0 to 16 years were included. P247 Mean values (range) were: age 6.5 (0.04 to 16) years, weight 25.8 (3.7 to 80) kg, mean arterial blood pressure 79 (48 to 131) mmHg, PaO2/FiO2 ratio 388 (171 to 662) mmHg, cardiac index (CI) 4.5 (2.2 to 6.7) l/minute/m2, global end-diastolic volume (GEDVI) 490 (211 to 718) ml/m2, EVLWI 12.7 (4.7 to 34.6) ml/kg. Figure 1 shows the logarithmic relation between EVLWI and age with an r2 of 0.7. There was no signifi cant correlation between GEDVI or CI and age. g Conclusion The number of B-lines defi nitely increased after fl uid resuscitation in shock and correlated to the deterioration of pulmonary gas exchange. These data support the benefi t of transthoracic portable ultrasound for assessment of the increment of EVLW in shock patients receiving fl uid resuscitation. P248 indexing to body height presents an alternative method without dependence on physical properties or gender of a patient, yielding a uniform 95% confi dence interval of normal values from 0.22 to 0.43 l/m. Conclusion Traditional ways of indexing EVLW data do not resolve value dependence on physical properties or gender. Therefore, the currently used defi nition of a normal range from 3 to 8 ml/kg seems to be invalid. Our data suggest indexing EVLW to plain body height instead of weight-based methods. As we are not aware of any abnormal hemodynamic profi le for brain tumor patients, we propose our fi ndings as a close approximation to normal values. This will require further validation in critically ill patients. indexing to body height presents an alternative method without dependence on physical properties or gender of a patient, yielding a uniform 95% confi dence interval of normal values from 0.22 to 0.43 l/m. Conclusion Traditional ways of indexing EVLW data do not resolve value dependence on physical properties or gender. Therefore, the currently used defi nition of a normal range from 3 to 8 ml/kg seems to be invalid. Our data suggest indexing EVLW to plain body height instead of weight-based methods. As we are not aware of any abnormal hemodynamic profi le for brain tumor patients, we propose our fi ndings as a close approximation to normal values. This will require further validation in critically ill patients. P246 The small improvement of the PO2/FiO2 fi nally leads to a decrease of the ELWI rate but it is not important statistically and on the other side leads to an increase of vasoconstriction. Therefore, this method is not eff ective. We gain only a short time for the safer surgical treatment if it is needed Results EVLW indexed to predicted body weight was inversely correlated with a patient’s body height (P <0.001), while values indexed to real body weight remained inversely dependent on weight (P <0.001). Indexing to estimated body surface area, again based on real or predicted body weight, provided no advantage. In contrast, S89 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 gl References l References 1. Bouhemad B, Zhang M, Lu Q, et al.: Clinical review: Bedside lung ultrasound in critical care practice. Crit Care 2007, 11:205. 1. Bouhemad B, Zhang M, Lu Q, et al.: Clinical review: Bedside lung ultrasound in critical care practice. Crit Care 2007, 11:205. Conclusion Near-normal values of EVLW in children are strongly correlated with age. Based upon these data, normal values can be constructed for future clinical use. 2. Soldati G, Copetti R, Sher S: Sonographic interstitial syndrome: the sound of lung water. J Ultrasound Med 2009, 28:163-174. 2. Soldati G, Copetti R, Sher S: Sonographic interstitial syndrome: the sound of lung water. J Ultrasound Med 2009, 28:163-174. Figure 1 (abstract P247). Transthoracic ultrasound assessment of B-lines for identifying the increment of extravascular lung water in shock patients requiring fl uid resuscitation Transthoracic ultrasound assessment of B-lines for identifying the increment of extravascular lung water in shock patients requiring fl uid resuscitation l P Theerawit, N Tomuan, Y Sutherasan, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P248 (doi: 10.1186/cc10855) l P Theerawit, N Tomuan, Y Sutherasan, S Kiatboonsri Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P248 (doi: 10.1186/cc10855) Introduction Sonographic B-lines are commonly observed in cases of increasing extravascular lung water (EVLW). These fi ndings became prominent when interstitial and alveolar tissues were fi lled with fl uid [1,2]. Thus, we hypothesized that the increment of sonographic B-lines would be observed when the EVLW increased after fl uid resuscitation in shock patients and be associated with the impaired gas exchange. Introduction Sonographic B-lines are commonly observed in cases of increasing extravascular lung water (EVLW). These fi ndings became prominent when interstitial and alveolar tissues were fi lled with fl uid [1,2]. Thus, we hypothesized that the increment of sonographic B-lines would be observed when the EVLW increased after fl uid resuscitation in shock patients and be associated with the impaired gas exchange. Methods Transthoracic portable ultrasound before and after fl uid resuscitation was performed. Patients with pleural disease were excluded. The B-lines were measured in 23 lung zones. The total numbers of B-lines seen in each patient were counted as the total B-line score (TBS). The primary outcome was to demonstrate the increase of TBS after fl uid resuscitation. The secondary outcome was to examine the magnitude of the incremental number of TBS. p p g g Methods Transthoracic portable ultrasound before and after fl uid resuscitation was performed. Patients with pleural disease were excluded. The B-lines were measured in 23 lung zones. The total numbers of B-lines seen in each patient were counted as the total B-line score (TBS). The primary outcome was to demonstrate the increase of TBS after fl uid resuscitation. The secondary outcome was to examine the magnitude of the incremental number of TBS. P252 Evaluation of eff ectiveness and safety of hydroxyethyl starch (HES 130 kDa/0.4) in burn resuscitation A Mokline, I Rahmani, L Gharsallah, H Oueslati, B Gasri, I Harzallah, A Ksontini, A Messadi Burn and Trauma Center, Tunis, Tunisia Critical Care 2012, 16(Suppl 1):P252 (doi: 10.1186/cc10859) Methods This study was a prospective, randomized, active-controlled study comparing the hemodynamics, effi cacy, and safety of HES 130/0.4 to that of albumin in patients undergoing pancreaticoduodenectomy. Eligible adult patients of both sexes were assigned following the surgery into either the HES group or the albumin group at a ratio of 1:1. Crystalloids for hydration and colloid therapy for volume support were administered. The primary endpoint of this study was the hemodynamic evaluation. Secondary endpoints were measurement of the input– output, ICU stay, ventilation time, length of hospital stay, time to liquid mealtime and the use of blood products. Safety assessment was carried out by performing physical examination, laboratory examination, and assessment of any adverse events during the study period. Introduction Excessive fl uid resuscitation of large burn injuries has been associated with adverse outcomes including worsening of burn oedema, conversion of superfi cial into deep burns, and compartment syndromes. So, there have been eff orts recently to address these concerns, particularly with the use of physiologically balanced fl uids. Starches, as eff ective plasma expanders, may limit resuscitation requirements and burn oedema. This study aims to evaluate clinical results of HES in early burn resuscitation of major burn-injured patients. Methods A case–control study conducted in a burn care center in Tunis. Adult burned patients admitted within the fi rst 24 hours post burn, with a burn injury exceeding 30% of total body surface area, from 1 January to 31 December 2010 were included. Exclusion criteria were pregnancy, history or biochemical evidence of renal impairment on admission (serum creatinine >130 μmol/l), history or hematological evidence of disorders of hemostasis. Fluid volume resuscitation was evaluated according to the Parkland formula. HES supplementation was limited to 33 ml/kg/24 hours. The HES supplementation group was compared with a group of patients from the same center matched in age, sex and severity of burns at baseline. y g y p Results A total of 50 patients were randomized to study groups (25 each). Eff ect of balanced versus unbalanced HES solution on cytokine response in a rat model of peritonitis Eff ect of balanced versus unbalanced HES solution on cytokine response in a rat model of peritonitis M Schläpfer1, M Urner1, S Voigtsberger1, R Schimmer2, B Beck-Schimmer1 1University Hospital Zurich, Switzerland; 2University of Zurich, Switzerland Critical Care 2012, 16(Suppl 1):P251 (doi: 10.1186/cc10858) M Schläpfer1, M Urner1, S Voigtsberger1, R Schimmer2, B Beck-Schimmer1 1University Hospital Zurich, Switzerland; 2University of Zurich, Switzerland Critical Care 2012, 16(Suppl 1):P251 (doi: 10.1186/cc10858) Introduction Sepsis with multiple organ failure remains a leading cause of death in ICUs. Acute renal failure is a common complication of severe sepsis and septic shock. The eff ect of hydroxyethyl starch (HES) on the kidney as well as on liver tissue remains controversial and has never been tested in detail. We investigated in a model of fecal peritonitis the infl uence of fl uid resuscitation with HES 6% in unbalanced versus balanced solutions on infl ammatory mediator expression in renal and hepatic tissue. y Results The EG patients with degree I shock had higher hemodynamics parameters (BPsys, BPdias, MAP) and less expressed tachycardia as compared to the CG patients with degree I shock (P <0.05). The EG patients with degree II shock had higher hemodynamics parameters (BPsys, MAP, ESV, CI, SVR) as compared to the CG patients with degree II shock (P <0.05). The change of the fl uid therapy tactics in the EG resulted in the normalization of the HR, SVR and in the increase of the BPsys, MAP, ESV and CI in patients of both degrees of shock during transportation. The values of the EG were higher than in the CG during all periods of the transportation (P <0.05).l Methods Cecal ligation and puncture was performed in anesthetized Wistar rats (CLP group). Sham group animals were treated in the same manner but without CLP. One hour after this procedure, Ringer lactate (RL) was intravenously infused to all animals at a volume of 30 ml/kg. Two hours after initiation of injury rats received RL (control, 75 ml/kg), unbalanced HES 130/0.42 (HES, 25 ml/kg) or balanced HES 130/0.42 (Tetraspan, 25 ml/kg). Animals were euthanized 4 hours after induction of peritonitis. Monocyte chemotactic protein-1, intercellular adhesion molecule-1, and TNFα mRNA expression were assessed in the kidneys and liver. Linear regression was used to evaluate infl uence of the diff erent fl uid resuscitation procedures on infl ammatory mediator expression. Eff ect of balanced versus unbalanced HES solution on cytokine response in a rat model of peritonitis p p Conclusion Inclusion of the HES 130/04 starch in the fl uid therapy complex of the patients with traumatic shock in polytrauma allows one to normalize hemodynamics values at short notice and to support them adequately during all periods of transportation. A prospective, randomized, clinical trial comparing the hemodynamics, effi cacy, and safety of 6% hydroxyethyl starch 130/0.4 compared to albumin in postoperative patients undergoing pancreaticoduodenectomy Results CLP had a signifi cant eff ect on production of infl ammatory mediators in the kidneys (P ≤0.03) and liver (P ≤0.02). While HES did not alter expression of infl ammatory mediators compared to RL, fl uid resuscitation with Tetraspan provoked a burst in infl ammatory mediator expression, which was at least threefold higher in the kidneys (P <0.001) and eightfold in the liver (P = 0.001) compared to the RL group.l p y SK Hong1, K Kyoung2, Y Kim1, S Kim1 1Ulsan University College of Medicine, Asan Medical Center, Seoul, South Korea; 2Inje University College of Medicine, Harundae Paik Hospital, Busan, South Korea Critical Care 2012, 16(Suppl 1):P250 (doi: 10.1186/cc10857) Conclusion While unbalanced HES did not show a proinfl ammatory eff ect on renal and hepatic tissue in early sepsis, the balanced HES solution upregulated infl ammatory mediators. Further studies have to be performed to elucidate this phenomenon in detail and to assess the functional implication of these results. Critical Care 2012, 16(Suppl 1):P250 (doi: 10.1186/cc10857) Introduction Hypovolemia is often present in patients undergoing extensive abdominal surgery. As the fi rst colloid used in the clinical setting, albumin is still widely employed during perioperative periods. We hypothesized that 6% hydroxyethyl starch (HES) 130/0.4 is equally effi cacious and has the added advantages of its low cost and convenience of use. This study’s objective is to compare the hemodynamics, effi cacy, and safety of HES 130/0.4 compared with that of albumin. P252 P250 P250 A prospective, randomized, clinical trial comparing the hemodynamics, effi cacy, and safety of 6% hydroxyethyl starch 130/0.4 compared to albumin in postoperative patients undergoing pancreaticoduodenectomy SK Hong1, K Kyoung2, Y Kim1, S Kim1 1Ulsan University College of Medicine, Asan Medical Center, Seoul, South Korea; 2Inje University College of Medicine, Harundae Paik Hospital, Busan, South Korea Critical Care 2012, 16(Suppl 1):P250 (doi: 10.1186/cc10857) P251 24 hours after trauma. The distance was 177 ± 9 km. The components of the fl uid therapy in the CG were crystalloids and dextrans. The latter were not used in degree I shock. Crystalloid infusion was carried out on the basis of 3 ml crystalloids per 1 ml blood loss. The crystalloids and HES 130/04 starch were used in the EG. The dose of HES 130/04 starch comprised 10 to 25 ml/kg of the body mass and depended on the shock severity state. Statistical analysis was performed using Statistica 6.1. We used the Mann–Whitney criterion. Fluid therapy tactics in patients with polytrauma during interhospital transportation D Skopintsev, S Kravtsov, A Shatalin, V Agadzhanyan Federal State Budgetary Medical Prophylactic Institution, Scientifi c Clinical Center of Miners’ Health Protection, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P249 (doi: 10.1186/cc10856) Introduction The study’s aim was to carry out a comparative evaluation of fl uid therapy’s infl uence using 130/04 hydroxyethyl (HES) starch and dextrans on hemodynamics values in patients with traumatic shock in polytrauma during interhospital transportation. Introduction The study’s aim was to carry out a comparative evaluation of fl uid therapy’s infl uence using 130/04 hydroxyethyl (HES) starch and dextrans on hemodynamics values in patients with traumatic shock in polytrauma during interhospital transportation. Figure 1 (abstract P247). p y g p p Methods Eighty patients with polytrauma were included in the study. Mean age was 35 ± 1 years. All patients were divided into two similar groups: experimental (EG) and control (CG). Each group was apportioned by two subgroups depending on the shock severity. Subgroup 1 consisted of patients with degree I shock, subgroup 2 comprised patients with degree II shock. The Algover–Burry index was used to evaluate the shock severity. ISS was applied to determine the injuries’ severity. The injuries’ severity values of the EG were the following: in subgroup 1, 25 ± 1 points; in subgroup 2, 46 ± 2 points. The values of the CG were 26 ± 1 in the fi rst subgroup and 44 ± 2 points in the second subgroup. All patients were transported during the fi rst S90 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P252 The volume of the crystalloid was the same in both groups; however, signifi cantly more colloids were infused after 24 hours post surgery in the HES group than in the albumin group, the voluven patient group had lower heart rates, and the diff erence in the lowest MAP value was –1.64 mmHg (lower limit of confi dence interval, –8.228 mmHg) than in the albumin group. Routine hematology and biochemical profi les, including blood coagulation test and renal function assessment, were comparable in the two groups. The mean duration of the ICU stay, ventilation, hospital stay, and tolerance of a liquid meal were similar. The mean cost of the colloid was signifi cantly lower in the HES 130/0.4 group than in the albumin group (P <0.001). Conclusion This study demonstrated that 6% HES 130/0.4 may be used as a valuable alternative to 5% albumin in patients undergoing extensive abdominal surgery, as its low cost is also of value. g y Results Patients were assigned to two groups: G1 (n = 15): HES supplemented, and G2 (n = 15): crystalloids only. The mean age was 44 ± 18 years old for G1 and 43 ± 17 years old for G2. The average TBSA was 51.8% ± 19 for G1 versus 43.6 ± 7 for G2. The addition of S91 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 biomarkers in a pilot study of early septic shock patients resuscitated with either 5% albumin or normal saline. HES 130 kDa/0.4 reduces signifi cantly body weight gain within the fi rst 72 hours after injury: 8 kg for G1 versus 13.6 kg for G2 (P = 0.002), occurrence of ALI (35% for G1 versus 65% for G2) (P = 0.01), and length of ICU stay (19 days ± 13 for G1 vs. 30 days ± 15 for G2). There was no evidence of renal dysfunction with the use of HES in burns patients comparative to the crystalloids group. Methods Patients presenting in early septic shock received albumin or saline in a randomized, double-blind pilot study. Blood and urine was collected at enrolment and 6, 12, 24, 72 hours and 7 days later and processed using standard operating procedures. A panel of 27 cytokines, chemokines and growth factors was measured by multiplex technology. f References 1. Maitland K, et al.: N Engl J Med 2011, 364:2483-2495. 2. Prange HD, et al.: J Appl Physiol 2001, 91:33-38. Methods This was an observational study performed during 4 July 2007 to 31 March 2009 in the 10-bed ICU of Pramongkutklao Hospital in severe sepsis or septic shock patients who already had a central venous catheter inserted. Two blood samples were collected and sent to the laboratories for blood gas analysis. We then calculated for the correlation using correlation and linear regression analysis. Study of the correlation between central venous oxygen saturation and venous saturation from the antecubital vein in severe sepsis/ septic shock patients K Piyavechviratana, W Tangpradubkiet Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P255 (doi: 10.1186/cc10862) Study of the correlation between central venous oxygen saturation and venous saturation from the antecubital vein in severe sepsis/ septic shock patients K Piyavechviratana, W Tangpradubkiet Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P255 (doi: 10.1186/cc10862) Introduction Early goal-directed therapy has been used for severe sepsis and septic shock in the ICU to achieve a balance between systemic oxygen delivery and oxygen demand before global tissue hypoxia develops and proceeds to multiorgan failure. One of the resuscitation end points includes normalized values for central venous oxygen saturation (ScvO2) that needs insertion of a central venous catheterization, which is still impractical in small-to-medium-sized hospitals in Thailand. The purpose of this study was to examine whether the venous oxygen saturation from the antecubital vein has correlation with the central venous oxygen saturation or can be applied instead of the central venous oxygen saturation. f Conclusion Saline infusion worsens lactic acidosis, despite similar blood pressure, when compared to plasmalyte. The mechanisms responsible for this eff ect are unclear. However, deoxygenated hemoglobin readily binds hydrogen ions, forming HbH+, which is stabilized in the presence of chloride [2]. Consequently, the oxygen affi nity for hemoglobin is reduced, which could impair oxygen delivery, perpetuating the lactic acidosis. Further study is needed to better understand the mechanisms of this eff ect and their clinical relevance. Normal saline resuscitation worsens lactic acidosis in experimental sepsis h h b l ll p F Zhou, ME Cove, ZY Peng, J Bishop, K Singbartl, JA Kellum University of Pittsburgh Medical School, Pittsburgh, PA, USA Critical Care 2012, 16(Suppl 1):P253 (doi: 10.1186/cc10860) Introduction Infusing large volumes of 0.9% sodium chloride (saline) causes hyperchloremic acidosis. The clinical relevance of this eff ect remains contentious and saline is still the most commonly used resuscitation fl uid in the US. However, a recent trial showed that saline or albumin in saline increased mortality in children with malarial sepsis, compared to no fl uid [1]. Infusion of these fl uids may have perpetuated the underlying metabolic acidosis sepsis, causing cardiovascular collapse and death. In this study, we investigated the eff ect of saline versus a balanced crystalloid (plasmalyte) in a cecal ligation and puncture (CLP) model of sepsis. We hypothesized that saline resuscitation would increase acidosis and worsen hemodynamics, compared to resuscitation using a balanced crystalloid. Conclusion In this cohort of patients treated with albumin or saline in early septic shock, there appeared to be a marked increase in the clustering of early T-cell-mediated immune responses. Also striking was the blunted rise in urine NGAL over time for patients in the albumin fl uid group. These results should be considered hypothesis generating and prompt further studies to explore possible biological mechanisms for albumin resuscitation in sepsis. y Methods Fifty adult male Sprague–Dawley rats were subjected to CLP (25% cecum length, two punctures with a 25-gauge needle). Eighteen hours later, they were randomly assigned to receive either 30 ml/kg saline (n = 25) or plasmalyte (n = 25) over 4 hours. Arterial blood gases, serum creatinine, urea, and lactate were measured at baseline, 18 hours after CLP (before resuscitation), after resuscitation, and 24 hours after resuscitation. Blood pressure and pulse rate were measured during fl uid infusion.i P255 l Results Saline-treated animals developed signifi cantly higher levels of serum chloride (111 mmol/l vs. 102 mmol/l, P <0.0001) and lower pH (7.35 v. 7.44, P <0.01) compared to plasmalyte. In addition, lactate was signifi cantly higher after fl uid infusion in the saline group (4.8 mmol/l vs. 2.5 mmol/l, P <0.001) compared to plasmalyte, despite being similar before infusion (2.61 vs. 2.39, P >0.05). However, neither mean arterial blood pressure (83 mmHg vs. 91 mmHg, P >0.10) nor heart rate (310 vs. 299, P >0.10) diff ered between the two groups. P252 Mean values were separated by treatment and analyzed using R to generate heat maps, by principal component analysis (PCA) and hierarchal clustering. Urinary neutrophil gelatinase-associated lipocalin (NGAL) was measured by ELISA.i Conclusion HES supplementation in early burn resuscitation allows, for smaller fl uid volume requirement, less tissue oedema. This along with a signifi cantly lower in ALI occurrence and length of ICU stay. P253 p y Results Twenty-fi ve patients (median age 66 years, median APACHE II score 26) received albumin (median amount 3 l) and 21 (median age 62 years, median APACHE II score 22) received normal saline (median amount 3.5 l) as study fl uid over 7 days. PCA revealed that 60% of the variance in the chemokines was accounted for with the fi rst two components. Analyzing the fi rst component using a threshold of greater than 0.5 or –0.5 we saw a clustering of IL-17, IL-12p70, IL-9 and IL-5. Heat map analysis suggests that by 72 hours albumin-resuscitated patients are distinguished by the cluster of IL-17, IL-9 and Il-12p70 and VEGF when compared to saline. Hierarchal clustering also separates IL-17, IL-19, IL-12p70 and IL-2 in the albumin-treated patients but not the saline-treated patients at 72 hours. At enrolment, mean urine NGAL levels were greater than 1,000 ng/ml (albumin 1,121 ± 2,172 (n = 21), saline 1,375 ± 3,197 (n = 17)). Over the next 24 hours there was a marked increase in urine NGAL in the saline-resuscitated patients, peaking at 5,793 ± 15,948 ng/ml, whereas levels remain blunted over the fi rst 12 hours, peaking at 2,216 ± 3,177 ng/ml at 24 hours in the albumin group. Normal saline resuscitation worsens lactic acidosis in experimental sepsis F Zhou, ME Cove, ZY Peng, J Bishop, K Singbartl, JA Kellum University of Pittsburgh Medical School, Pittsburgh, PA, USA Critical Care 2012, 16(Suppl 1):P253 (doi: 10.1186/cc10860) Curve analysis of tissue oxygen desaturation after a venous occlusion test does not identify the central venous hemoglobin oxygen saturation Curve analysis of tissue oxygen desaturation after a venous occlusion test does not identify the central venous hemoglobin oxygen saturation yg G Friedman1, C Alan2, A Meregalli2, A Lima3, J Bakker3 1UFRGS, Porto Alegre, Brazil; 2Complexo Hospitalar Santa Casa, Porto Alegre, Brazil; 3University Medical Center Erasmus, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P257 (doi: 10.1186/cc10864) y G Friedman1, C Alan2, A Meregalli2, A Lima3, J Bakker3 1UFRGS, Porto Alegre, Brazil; 2Complexo Hospitalar Santa Casa, Porto Alegre, Brazil; 3University Medical Center Erasmus, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P257 (doi: 10.1186/cc10864) Introduction We aim to compare the time for the equivalence of tissue oxygen saturation (StO2) with central venous hemoglobin oxygen saturation (ScvO2) measured at depths of 15 and 25 mm. Methods Twenty-one critically ill patients were included. The ScvO2 was measured by blood gas analysis. Thenar StO2 was continuously monitored (Model 650 InSpectra Tissue Spectrometer; Hutchinson Technology Inc., MN, USA) in 15 mm (StO2_15) and 25 mm (StO2_25) depths. The venous occlusion was performed using an automatic pneumatic device maintaining infl ation pressure 10 mmHg above the diastolic pressure. A StO2 desaturation curve was plotted to identify the time for equivalence to ScvO2. Introduction We aim to compare the time for the equivalence of tissue oxygen saturation (StO2) with central venous hemoglobin oxygen saturation (ScvO2) measured at depths of 15 and 25 mm. Introduction We aim to compare the time for the equivalence of tissue oxygen saturation (StO2) with central venous hemoglobin oxygen saturation (ScvO2) measured at depths of 15 and 25 mm. 2 Methods Twenty-one critically ill patients were included. The ScvO2 was measured by blood gas analysis. Thenar StO2 was continuously monitored (Model 650 InSpectra Tissue Spectrometer; Hutchinson Technology Inc., MN, USA) in 15 mm (StO2_15) and 25 mm (StO2_25) depths. The venous occlusion was performed using an automatic pneumatic device maintaining infl ation pressure 10 mmHg above the diastolic pressure. A StO2 desaturation curve was plotted to identify the time for equivalence to ScvO2. the predicting equation were 69.23%, 77.41%, 85.71% and 56.25% respectively. The accuracy was 75%. See Figure 1. q 2 Results Age: 59 ± 17 years, APACHE II score: 21 ± 7, SOFA score: 7 ± 4, ScvO2: 75 ± 6%, blood lactate: 1.6 ± 1.2 mmol/l, capillary refi ll time: 9.1 ± 8.1 seconds, body temperature: 36.7 ± 1.1°C. Measurements were performed for up to three consecutive days (total measurements: 43). Albumin in early septic shock resuscitation: examination of plasma and urine infl ammatory markers Albumin in early septic shock resuscitation: examination of plasma and urine infl ammatory markers A Fox-Robichaud1, C Leger2, KD Burns3, E Sabri3, B Lo1, P Kubes2, LA McIntyre3 1McMaster University, Hamilton, Canada; 2University of Calgary, Canada; 3Ottawa Hospital Research Institute, Ottawa, Canada Critical Care 2012, 16(Suppl 1):P254 (doi: 10.1186/cc10861) A Fox-Robichaud1, C Leger2, KD Burns3, E Sabri3, B Lo1, P Kubes2, LA McIntyre3 1M M U i i H il C d 2U i i f C l C Results Of the 44 enrolled patients, 24 were males (54.54%). Mean age was around 69.86 ± 16.819 years. A total of 84.1% was in septic shock. The most common source of infection was pneumonia (38.6%). The central ScvO2 and peripheral venous oxygen saturation ranges and means were 46.0 to 93.2%, 31.5 to 99.0% and 71.66 ± 10.39%, 71.18 ± 19.79% respectively. The correlation between ScvO2 and antecubital venous oxygen saturation signifi cant P value was 0.000: calculated ScvO2 = 52.386 + 0.271(peripheral), R2 = 0.266. The specifi city, sensitivity, positive predictive value and negative predictive value of y 1McMaster University, Hamilton, Canada; 2University of Calgary, Canada; 3Ottawa Hospital Research Institute, Ottawa, Canada Critical Care 2012, 16(Suppl 1):P254 (doi: 10.1186/cc10861) Introduction A recent meta-analysis has suggested that albumin may be benefi cial in sepsis; however, there is no clear biological rationale for the pharmacological use of this negative acute-phase protein. Our objective was to describe the temporal production of plasma and urine Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 S92 Figure 1 (abstract P255). Central and peripheral oxygen saturation range. P257 5 Curve analysis of tissue oxygen desaturation after a venous occlusion test does not identify the central venous hemoglobin oxygen saturation G Friedman1, C Alan2, A Meregalli2, A Lima3, J Bakker3 1UFRGS, Porto Alegre, Brazil; 2Complexo Hospitalar Santa Casa, Porto Alegre, Brazil; 3University Medical Center Erasmus, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P257 (doi: 10.1186/cc10864) Curve analysis of tissue oxygen desaturation after a venous occlusion test does not identify the central venous hemoglobin oxygen saturation Curve analysis of tissue oxygen desaturation after a venous occlusion test does not identify the central venous hemoglobin oxygen saturation In four patients the equivalence was not identifi ed. The curve analysis showed that StO2 desaturation time equivalency for ScvO2 was greater for StO2_15 than for StO2_25 (88 ± 54 seconds vs. 79 ± 56 seconds, P <0.01). The Pearson correlation index for equivalence times for StO2_15 and StO2_25 was 0.92 (P <0.001), but Bland–Altman analysis showed a signifi cant diff erence between the times (mean diff erence: StO2_25 – StO2_25: –7.9 ± 37.8 seconds). An arbitrary time of 80 seconds identifi es the ScvO2 in 58% of cases. Conclusion Venous oxygen saturation from the antecubital vein was not the exact value of central venous oxygen saturation but there were signifi cant correlations. Central venous hyperoxia is related to changes in tissue perfusion and morbi-mortality of patients in shock G Friedman1, A Do Canto2, D Araujo2 1UFRGS, Porto Alegre, Brazil; 2Complexo Hospitalar Santa Casa, Porto Alegre, Brazil Critical Care 2012, 16(Suppl 1):P256 (doi: 10.1186/cc10863) Central venous hyperoxia is related to changes in tissue perfusion and morbi-mortality of patients in shock G Friedman1, A Do Canto2, D Araujo2 1UFRGS, Porto Alegre, Brazil; 2Complexo Hospitalar Santa Casa, Porto Alegre, Brazil Critical Care 2012, 16(Suppl 1):P256 (doi: 10.1186/cc10863) i 2 Conclusion The analysis of the StO2 desaturation curve does not adequately identify the hemoglobin central venous oxygen saturation. References Introduction Mixed or central venous hyperoxia is associated with organ dysfunction and worse mortality. Venous hyperoxia may refl ect altered tissue oxygen extraction. We aim to assess the relationship between central venous hyperoxia (ScvO2) and markers of tissue perfusion; and to evaluate the relationship between central venous hyperoxia and morbidity. 1. Lima A, et al.: The relation of near-infrared spectroscopy with changes in peripheral circulation in critically ill patients. Crit Care Med 2011, 39:1649. 2. Bezemer R, et al.: Assessment of tissue oxygen saturation during a vascular occlusion test using near-infrared spectroscopy: the role of probe spacing and measurement site studied in healthy volunteers. Crit Care 2009, 13(Suppl 5):S4. y y Methods The setting was a university general ICU with 18 beds. The population was adult patients (age >18 years) in circulatory shock. Blood lactate, arterial and central venous blood gases were collected on admission to the study and after 6, 12, 18 and 24 hours of shock. Venous hyperoxia was defi ned as a ScvO2 ≥85%. The severity of the patients was assessed using the APACHE II score on admission to the study. Mortality was evaluated in the ICU and after 28 days. 3. Lima A, et al.: Low tissue oxygen saturation at the end of early goal- directed therapy is associated with worse outcome in critically ill patients. Crit Care 2009, 13(Suppl 5):S13. 3. Lima A, et al.: Low tissue oxygen saturation at the end of early goal- directed therapy is associated with worse outcome in critically ill patients. Crit Care 2009, 13(Suppl 5):S13. 1. Pope JV, et al.: Multicenter study of central venous oxygen saturation (ScvO2) as a predictor of mortality in patients with sepsis. Ann Emerg Med 2010, 55:40-46. Lactate in burn patients: biomarker of sepsis and mortality y y y Results Preliminary data from 40 patients (205 measurements) are presented. Mean blood lactate levels were higher (3.2 vs. 2.3 mmol/l), the mean venoarterial CO2 diff erence was lower (4.7 vs. 5.8 mmHg), and the mean base defi cit was greater (11 vs. 8 mEq/l) for patients with venous hyperoxia at any time. Mean APACHE II score was higher (28 vs. 24) for patients with venous hyperoxia. The ICU mortality was higher (4/5 (80%) vs. 17/35 (46%)) among patients who already had venous hyperoxia at time 0. The proportion of death remained the same in the next day among patients that persisted or developed venous hyperoxia in the following 18 hours. A Mokline, L Gharsallah, A Abdenneji, H Oueslati, I Rahmani, B Gasri, I Jami, A Ghanem, A Messadi A Mokline, L Gharsallah, A Abdenneji, H Oueslati, I Rahmani, B Gasri, I Jami, A Ghanem, A Messadi Can we predict arterial lactate from venous lactate in the emergency department? A Mikami1, S Ohde2, G Deshpande2, T Mochizuki1, N Otani1, S Ishimatsu1 1St Luke’s International Hospital, Tokyo, Japan; 2St Luke’s Life Science Institute, Tokyo, Japan Results BL and TL were higher in septic shock patients compared to nonseptic shock patients (AUCs of 276 vs. 176 and 355 vs. 273 mmol/ lhours, respectively; Welch’s t test: P <0.0001). X-ApEn for MDL/BL was lower in septic shock patients compared to those without septic shock (mean ± SD: 0.79 ± 0.12 vs. 1.14 ± 0.13, respectively; t test: P <0.0001). Cross-correlation of TL versus BL was stronger in septic shock patients, with TL leading BL by 4 hours compared to TL versus BL with no lag time (r = +0.85, P <0.0001 and r = +0.66, P <0.0001, respectively) than in nonseptic shock patients (r = +0.58, P = 0.0003 with TL leading BL by 4 hours and r = +0.66, P <0.0001 with no lag time; z statistic = 2.41 and P = 0.016 for leading BL compared to z statistic=0.036, P = 0.971 for no lag time). Introduction Analysis of arterial blood has an important role in the clinical assessment of critically ill patients. Particularly, measured arterial lactate (a-Lac) provides valuable information on peripheral circulatory failure, although it is invasive and frequent measurement is often impractical. The aim of this study is to clarify the relationship between a-Lac and the more easily accessed venous lactate (v-Lac) and to generate a formula to predict a-Lac using v-Lac and other laboratory data. Conclusion In septic shock patients, tissue lactate levels – measured by MD – are higher compared to nonseptic shock patients. Furthermore, TL is better correlated with and precedes – within 4 hours – BL in septic shock patients compared to nonseptic shock patients. Further studies are warranted to assess the clinical value of serial TL monitoring. Methods A prospective cohort study was conducted from June to November 2011 in the emergency department at a tertiary-level community hospital in Tokyo, Japan. Patients were eligible for entry into the study if an arterial blood gas (ABG) analysis was required for appropriate diagnostic care by the treating physician. Arterial and venous samples were taken within 5 minutes of each other from the ipsilateral radial artery and cephalic vein. Samples were analyzed as soon as possible after collection on the same blood gas analyzer. P261 d Admission lactate and outcome after high-risk surgery M Geisen, HD Aya, C Ebm, N Arulkumaran, MA Hamilton, M Grounds, A Rhodes, M Cecconi St George’s Hospital NHS Trust, London, UK Critical Care 2012, 16(Suppl 1):P261 (doi: 10.1186/cc10868) Introduction The aim of this study was to assess the ability of serum lactate level in patients admitted to the ICU after surgery to predict outcome. Introduction The aim of this study was to assess the ability of serum lactate level in patients admitted to the ICU after surgery to predict outcome. Methods A retrospective, clinical observational study in patients undergoing high-risk surgery admitted to a 17-bed ICU of a large teaching hospital. Data were obtained during haemodynamic optimization using an established GDT protocol in the fi rst 8 hours after admission and included demographic data as well as haemodynamic and laboratory parameters. Outcome data included morbidity (defi ned as >1 complications on the postoperative morbidity survey) and clinical outcome (hospital mortality, length of ICU stay, length of hospital stay, readmission to the ICU). i Results Seventy-two arterial samples from 72 patients (61% male; mean age, 58.2 years) were included in the study. Indications for ABG included respiratory failure (16%), assessment of shock (21%), altered mental status (26%), and others (36%). An initial regression equation was derived from univariate linear regression analysis: (a-Lac) = –0.259 + (v-Lac)×0.996. Subsequent multivariate forward stepwise logistic regression analysis, incorporating venous lactate and venous pO2 (v-pO2), generated the following equation: (a-Lac) = –0.469 + (v- pO2)×0.005 + (v-Lac)×0.997. Calculated R-squared values by single and multiple regression were 0.94 and 0.96, respectively. Results Sixty-seven patients were included. Lactate clearance (decrease of lactate >10% in 2 hours) occurred in 64 patients (96%). Sixty patients developed at least one surgical complication. There were no signifi cant correlation between lactate levels on admission and development of Conclusion Venous lactate estimates showed a high correlation with arterial values and our data provide two clinically useful equations to calculate a-Lac from v-Lac data. Considering clinical fl exibility, Lac = –0.259 + VLac×0.996 might be more useful, while avoiding a time-consuming and invasive procedure. Table 1 (abstract P261). A Mokline, L Gharsallah, A Abdenneji, H Oueslati, I Rahmani, B Gasri, I Jami, A Ghanem, A Messadi Burn and Trauma Center, Tunis, Tunisia Critical Care 2012, 16(Suppl 1):P258 (doi: 10.1186/cc10865) Introduction In this study, we attempted to assess whether the early plasma lactate (PL) level is a useful biomarker to predict septic complications and outcome in burn patients. Methods A retrospective study was conducted in the burn care center in Tunis. Patients admitted within 24 hours from the thermal injury, from 1 January 2009 to 30 June 2010, were included. PL was measured early in the fi rst 24 hours and controlled more than twice. For each measurement, 5 ml venous blood was drawn into a heparin-coated syringe. The normal lactate value was defi ned as 1 ± 0.5 mmol/l. Conclusion These preliminary data suggest that the presence of central venous hyperoxia is associated with persistent changes in perfusion. The presence of venous hyperoxia at both the onset of shock and in the following hours is associated with a worse clinical outcome. Reference i Results Over an 18-month period of study, 80 patients were enrolled. There were 60 males and 20 females. The mean age was 40.7 ± 19.5 and the average TBSA was 32 ± 21%. Upon admission, patients with an initial lactate value of more than 2 mmol/l were 86.7%. Fifty-eight percent of them have a lactate initial value of more than 4 mmol/l. In order to evaluate the potential impact of using early lactate measurements (H24 post burn injury) as predictor biomarker of sepsis in burn patients, a linear discrimination function was performed, by measuring the area under the ROC curve, and found that initial lactate value of more than 4 mmol/l provides the best sensitivity and specifi city: 88% and 79% 1. Pope JV, et al.: Multicenter study of central venous oxygen saturation (ScvO2) as a predictor of mortality in patients with sepsis. Ann Emerg Med 2010, 55:40-46. S93 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 is a good prognostic fi nding, whereas persistent BL elevation portends poor outcome. Microdialysis (MD) enables direct monitoring of tissue metabolic changes. This study aimed to describe the dynamics of MD-assessed tissue lactate (TL) vis-à-vis BL in septic patients with and without shock. respectively. Also, the PL cut-off value for prediction of mortality was 4 mmol/l with a good sensitivity (86%) and specifi city (92%). P261 d Lactate on admission, complications and clinical outcome Lactate Lactate <1.7 mmol >1.7 mmol (n = 46) (n = 21) P value Complications 41 (89%) 19 (90.5%) NS Complications >1 36 (78.3%) 16 (76.2%) NS Total complications per patient 3 (2 to 7) 4 (2 to 7) NS Hospital stay 14 (8 to 39) 13 (8 to 24) NS ICU stay 1 (1 to 2) 2 (1 to 10) 0.045 Readmission to ICU 3 (6.5%) 6 (28.6%) 0.022 Mortality 2 (4.3%) 3 (14.3%) NS Table 1 (abstract P261). Lactate on admission, complications and clinical outcome P259 Can we predict arterial lactate from venous lactate in the emergency department? A Mikami1, S Ohde2, G Deshpande2, T Mochizuki1, N Otani1, S Ishimatsu1 1St Luke’s International Hospital, Tokyo, Japan; 2St Luke’s Life Science Institute, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P259 (doi: 10.1186/cc10866) A Mokline, L Gharsallah, A Abdenneji, H Oueslati, I Rahmani, B Gasri, I Jami, A Ghanem, A Messadi The area under the ROC curve was 0.96. Conclusion Lactate appears to be a powerful predictor biomarker of sepsis and mortality in burn patients. A serum lactate of 4 mmol/l provides the best sensitivity and specifi city. Methods We measured BL and thigh adipose tissue TL serially every 4 hours for 6 days in 88 patients with septic shock and 45 patients at various sepsis stages hospitalized in a tertiary-care hospital ICU. Analysis was done with measurement of the area under the curve (AUC) of lactatehours, cross-approximate entropy (X-ApEn) and cross-correlation. Comparisons of septic shock versus nonseptic shock patients’ results were done with t tests and z statistics. Can we predict arterial lactate from venous lactate in the emergency department? Univariate linear regression analysis was conducted to generate an equation to calculate a-Lac incorporating only v-Lac. Then, a multivariate forward stepwise logistic regression model (P value of 0.05 for entry, 0.1 for removal) was used to generate an equation including v-Lac and other potentially relevant variables including age, sex, systolic blood pressure, heart rate, and venous blood parameters (pH, pO2, pCO2, hemoglobin, creatine kinase, potassium). A Bland–Altman plot was drawn and the two equations were compared for model fi tting using R-squared. P260 Cross-correlation analysis of blood and microdialysis-assessed tissue lactate monitoring: a study in critically ill septic patients I Ilias1, P Kopterides2, N Nikitas2, D Vassiliadi2, M Theodorakopoulou2, E Papadomichelakis2, M Lygnos2, A Flevari2, M Rizos2, F Frantzeskaki2, C Diakaki2, E Paramythiotou2, E Dimitriadou2, S Orfanos2, A Armaganidis2, I Dimopoulou2 1‘Elena’ Hospital, Athens, Greece; 2‘Attiko’ University Hospital, Haidari, Athens, Greece Critical Care 2012, 16(Suppl 1):P260 (doi: 10.1186/cc10867) Cross-correlation analysis of blood and microdialysis-assessed tissue lactate monitoring: a study in critically ill septic patients I Ilias1, P Kopterides2, N Nikitas2, D Vassiliadi2, M Theodorakopoulou2, E Papadomichelakis2, M Lygnos2, A Flevari2, M Rizos2, F Frantzeskaki2, C Diakaki2, E Paramythiotou2, E Dimitriadou2, S Orfanos2, A Armaganidis2, I Dimopoulou2 1‘Elena’ Hospital, Athens, Greece; 2‘Attiko’ University Hospital, Haidari, Athens, Greece Critical Care 2012, 16(Suppl 1):P260 (doi: 10.1186/cc10867) Critical Care 2012, 16(Suppl 1):P260 (doi: 10.1186/cc10867) Introduction In the critical care setting, blood lactate (BL) concentration is measured to assess – albeit indirectly – tissue oxygenation. In addition, serial BL measurements are clinically useful since a drop in BL S94 Critical Care 2012, Volume 16 Suppl 1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P262). Figure 1 (abstract P261). Prediction of ICU readmission according to initial lactate concentration. Figure 1 (abstract P262). Figure 1 (abstract P262). consecutive hours on the fi rst, third, and fi fth days postoperatively. The second group (G2; n = 26) in addition to oxygen received NAC 200 mg/day for 5 days. The third group (G3; n = 26) was the control group which did not receive any oxygen variation. On postoperative day 6, hemoglobin, hematocrit and reticulocytes were measured and compared to the baseline values. A total of fi ve patients (three in G1 and two in G2) were excluded for discontinuing oxygen and/or early discharge from hospital. Figure 1 (abstract P261). Prediction of ICU readmission according to initial lactate concentration. g Results The reticulocyte count in G1 showed statistically diff erent values compared to G2 and G3. These fi ndings correlate with other clinical trials [2]. The fact that no statistical diff erence of hemoglobin level was recorded could be attributed to the lack of follow-up after patient discharge (postoperative day 6). See Figure 1.f complications and length of hospital stay. Nine patients (13%) were readmitted to the ICU. P260 A receiving operator characteristic analysis for readmission to the ICU showed an area under the curve of 0.79. A lactate higher than 1.7 mmol/l on admission had a sensitivity of 75% and a specifi city of 74% to predict ICU readmission (Figure 1). Patients with a lactate on admission >1.7 mmol/l also had a longer length of ICU stay (Table 1). Conclusion Induced relative hypoxia seems to be an eff ective stimulus for reticulocyte synthesis. However, further investigations are needed to confi rm these fi ndings and their impact on hemoglobin. References Conclusion Lactate on admission correlates with length of ICU stay and readmission to the ICU. 1. Balestra C, et al.: J Appl Physiol 2006, 100:512-518. 2. Theunissen et al.: Crit Care 2011, 15(Suppl 1):P422. References 2. Jansen TC, et al.: Am J Respir Crit Care Med 2010, 182:752-761. 1. Balestra C, et al.: J Appl Physiol 2006, 100:512-518. 2. Theunissen et al.: Crit Care 2011, 15(Suppl 1):P422. P263 P262 Eff ects of induced relative hypoxia during the postoperative period of abdominal oncologic surgery, on hemoglobin and reticulocyte levels: a prospective, randomized controlled clinical trial M Khalife1, K Wiams1, M Ben Aziz1, M Paesmans1, C Balestra2, M Sosnowski1 1Institut Jules Bordet, Brussels, Belgium; 2Divert Alert Network Europe Research Division, Brussels, Belgium Critical Care 2012, 16(Suppl 1):P262 (doi: 10.1186/cc10869) P263 Pre-emptive hypothermia during resuscitated porcine hemorrhagic shock J Matallo1, W Stahl1, M Gröger1, A Seifritz1, O Mccook1, M Georgieff 1, P Asfar2, M Matejovic3, E Calzia1, P Radermacher1, F Simon1 1University Medical School, UIm, Germany; 2University Hospital, Angers, France; 3Charles University, Plzeñ, Czech Republic Critical Care 2012, 16(Suppl 1):P263 (doi: 10.1186/cc10870) Carbon monoxide therapy protects against hepatic microvascular injury in a mouse model of murine hemorrhagic shock and resuscitationi Carbon monoxide therapy protects against hepatic microvascular injury in a mouse model of murine hemorrhagic shock and resuscitationi H Gomez, I Nassour, P Loughran, J Brumfi eld, L Otterbein, B Zuckerbraun University of Pittsburgh, PA, USA g p g Results All models presented good fi t (P >0.05) and discrimination. An AUC of 0.83 and 0.86 was obtained for the pneumonia and pancreatitis subgroups, respectively, compared to an AUC of 0.81 obtained for the general population of patients. A set of common predictive variables was found for the general population of patients: arterial base excess, noninvasive blood pressure and lactic acid. Additionally, group- specifi c predictive variables were found for each of the two subgroups of patients: white blood cell count for pneumonia patients, and temperature for pancreatitis patients. y g Critical Care 2012, 16(Suppl 1):P264 (doi: 10.1186/cc10871) Introduction The purpose of this study is to evaluate the eff ects of inhaled carbon monoxide (CO) as an adjunct to resuscitation on hepatic microvascular and endothelial integrity in a murine model of hemorrhagic shock and resuscitation (HSR). Others and ourselves have previously demonstrated that CO can protect against organ injury in experimental models of HSR [1]. Additionally, CO can prevent tissue hypoxia during hemorrhage. Based upon this we hypothesized that CO prevents hepatic injury and prevents hepatic hypoxia by maintaining endothelial integrity and the hepatic microvascular circulation. Conclusion Generally, accurate and well-calibrated predictive risk models were obtained for the impending use of vasopressors in an ICU. However, signifi cantly more accurate and well-calibrated models were developed for the two subpopulations – pneumonia and pancreatitis – than for the general population of ICU patients. This fi nding challenges one-model-fi ts-all approaches to overall predictive risk modeling and instead supports tailored modeling that is at least stratifi ed at a disease level. Methods Male C57BL/6 mice underwent sham operation or hemorrhage to a target MAP of 25 mmHg. Mice were maintained at this pressure for 120 minutes and then resuscitated with Ringer’s lactate at two times the volume of total shed blood. Mice were sacrifi ced 4 hours after resuscitation. Mice were randomized to receive room air or inhaled CO (250 ppm) for 30 minutes starting 90 minutes into the shock period (n = 6 to 8 per group). Relative hepatic hypoxia was determined using EF5 immunofl uorescence. Pre-emptive hypothermia during resuscitated porcine hemorrhagic shockf J Matallo1, W Stahl1, M Gröger1, A Seifritz1, O Mccook1, M Georgieff 1, P Asfar2, M Matejovic3, E Calzia1, P Radermacher1, F Simon1 1University Medical School, UIm, Germany; 2University Hospital, Angers, France; 3Charles University, Plzeñ, Czech Republic Critical Care 2012, 16(Suppl 1):P263 (doi: 10.1186/cc10870) Critical Care 2012, 16(Suppl 1):P262 (doi: 10.1186/cc10869) Introduction Anemia is a frequent complication in oncologic patients. Erythropoietin (EPO) stimulating agents are known as alternatives to transfusion. However, they expose patients to thrombosis and are expensive. Recently, a new phenomenon, the normobaric oxygen paradox (NOP), has been described. In brief, transient hyperoxia followed by a prolonged return to normoxia acts as an eff ective trigger for EPO production. The mechanism depends on free oxygen radicals and on reduced glutathione (GSH) availabilities. Also, N-acetylcystein (NAC) is known to regenerate the stock of GSH. Very few clinical trials have investigated this phenomenon [1]. The goal of this study was to test the NOP theory on the evolution of hemoglobin and reticulocytes in patients receiving intermittent oxygen with or without NAC compared to a control group. Introduction The role of hypothermia in hemorrhagic shock is still a matter of debate [1]. Therefore, we studied the eff ects of deliberate, pre-emptive hypothermia on hemodynamics and organ function during long-term porcine hemorrhage and resuscitation. Methods Anesthetized and instrumented pigs were randomly assigned to 32°C (n = 7), 35°C (n = 7), and 38°C (n = 6) of core temperature and subjected to 4 hours of hemorrhage (removal of 40% of the calculated blood volume, additional removal/retransfusion of blood to maintain mean arterial pressure (MAP) = 30 mmHg). After 12 hours of reperfusion comprising retransfusion of shed blood, colloid fl uid resuscitation and noradrenaline to keep MAP at pre-shock levels, animals were rewarmed to 38°C. Data (median, quartiles) were obtained before and at the end of the shock phase as well as at 12 and 22 hours of resuscitation, intergroup diff erences were analyzed using a Kruskal–Wallis ANOVA on ranks. Methods This prospective, randomized study included 78 patients (three groups). The fi rst group (G1; n = 26) received 60% FiO2 for 2 S95 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Fluid balance and noradrenaline requirements did diff er between groups. 1. Kim HS, Loughran PA, Rao J, Billiar TR, Zuckerbraun BS: Am J Physiol 2008, 295:G146-G152. P265 Two modeling approaches were used – fuzzy modeling (FM) and logistic regression (LR) – combined with a sequential forward feature selection process. For each group of patients, the selected dataset was divided into two parts: one for feature selection and the other for 10-fold cross-validation. The models’ calibration was assessed using the Hosmer–Lemeshow goodness-of-fi t test, and discrimination using the area under the receiver-operating curve (AUC).i P264 Carbon monoxide therapy protects against hepatic microvascular injury in a mouse model of murine hemorrhagic shock and resuscitation H Gomez, I Nassour, P Loughran, J Brumfi eld, L Otterbein, B Zuckerbraun University of Pittsburgh, PA, USA Critical Care 2012, 16(Suppl 1):P264 (doi: 10.1186/cc10871) P265 Customized modeling to predict the use of vasopressors in ICUs A Fialho1, F Cismondi1, S Vieira2, S Reti3, L Celi3, M Howell3, J Sousa3, S Finkelstein1 1Massachusetts Institute of Technology, Cambridge, MA, USA; 2Technical University of Lisbon, Instituto Superior Técnico, Lisbon, Portugal; 3Beth Israel Deaconess Medical Centre, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P265 (doi: 10.1186/cc10872) Customized modeling to predict the use of vasopressors in ICUs A Fialho1, F Cismondi1, S Vieira2, S Reti3, L Celi3, M Howell3, J Sousa3, S Finkelstein1 Introduction Vasopressors belong to a powerful class of drugs extremely useful for managing hypotension in patients with systemic shock. Being able to predict a patient’s impending use of vasopressors could be benefi cial as the central line insertion protocol could be initiated in a safe and timely fashion and, a central line would only be inserted if the patient has a likely future vasopressor need. Our goal in this work was to develop predictive risk models for the impending use of vasopressors in an ICU, and to make model comparisons between the general population and patients with pneumonia and pancreatitis. Methods We performed a retrospective cohort study using data from four diff erent adult ICUs at a tertiary-care hospital. Data contained 1,484 adult ICU patients, including a subgroup of 475 patients with an ICD9 diagnosis of pneumonia and 104 with an ICD9 diagnosis of pancreatitis. Two modeling approaches were used – fuzzy modeling (FM) and logistic regression (LR) – combined with a sequential forward feature selection process. For each group of patients, the selected dataset was divided into two parts: one for feature selection and the other for 10-fold cross-validation. The models’ calibration was assessed using the Hosmer–Lemeshow goodness-of-fi t test, and discrimination using the area under the receiver-operating curve (AUC).i Conclusion Deliberate, pre-emptive moderate hypothermia slowed but did not protect against hemorrhagic shock and resuscitation- induced organ dysfunction, possibly due to a delayed but not attenuated infl ammatory response. Acknowledgements Supported by the Bundesministerium der Verteidigung (M/SABX/8A004). Reference 1. Tisherman S: J Intensive Care Med 2010, 25:240-242. Methods We performed a retrospective cohort study using data from four diff erent adult ICUs at a tertiary-care hospital. Data contained 1,484 adult ICU patients, including a subgroup of 475 patients with an ICD9 diagnosis of pneumonia and 104 with an ICD9 diagnosis of pancreatitis. Carbon monoxide therapy protects against hepatic microvascular injury in a mouse model of murine hemorrhagic shock and resuscitationi Sinusoidal integrity was determined by scanning electron microscopy of the hepatic sinusoidal endothelium and Evan’s blue tissue levels. Leukocyte stasis, rolling, and adhesion were determined using intravital microscopy of post-sinusoidal hepatic venules. Statistical analysis was determined by ANOVA. Pre-emptive hypothermia during resuscitated porcine hemorrhagic shockf At 12 hours of reperfusion – that is, immediately before rewarming – the 32°C group showed the lowest blood levels of creatinine (P = 0.026), troponin I (P = 0.053), the thrombin–antithrombin complexes (P = 0.012), and von Willebrand factor (P = 0.012). At the end of the experiment – that is, after rewarming – all these intergroup diff erences had disappeared, but the 32°C group presented with arterial hypotension (P = 0.039), the most severe visceral organ acidosis (portal and hepatic venous base excess: P = 0.044, P = 0.022, respectively), and the highest TNFα blood levels (P = 0.030). P267 Examination of out-of-hospital cardiac arrest patients with the Utstein style in Saga prefecture, Japan T Iwamura, Y Sakamoto, N Kutsukata, T Hitomi, K Seki, M Koga, T Yamashita, A Nakashima, Y Nishimura, M Yahata, K Yamada Saga University, Saga, Japan Critical Care 2012, 16(Suppl 1):P267 (doi: 10.1186/cc10874) Results Cardiac massage was not performed adequately in 48% of laypersons. This was statistically signifi cantly more than among lifeguards (16%) and instructors (23%). The median response times of laypersons and lifeguards were 15 seconds and 16 seconds, respectively; this was statistically (P <0.05) longer than instructors (12 seconds). The median percentage of the time of massaging in group of laypersons was 51% (56 to 58%, 25th to 75th percentiles), which was statistically signifi cantly smaller than in the group of lifeguards (64%, 62 to 66%) and in the control group (67%, 62 to 69%). Introduction Saga Prefecture is a small prefecture with an area of 2,439 km2 (place-of-residence 1,339 km2), a population of 849,709, and is located in northwestern Kyushu in the western part of Japan. Saga University has the only medical department in Saga Prefecture, Japan, and it is in charge of both the online and offl ine medical control of Saga. This report examined the present status of OHCA in Saga, which should be improved, and it aimed at exploring policies that can contribute to the improvement in a ROSC rate. g p Conclusion The majority of all laypersons approach CPR in about 15 seconds from identifi cation of unconsciousness. However, only about one-half of laypersons after the mandatory CPR course perform qualitative cardiac massage, which is signifi cantly less than among motivated laypersons. The latter perform qualitative massage and achieve the same percentage of the massaging time as instructors. Results suggest that widespread promotion of the CPR protocol with an AED among laypersons has limitations. Therefore, education of laypersons should particularly focus on groups that have intrinsic motivation. p Methods The study examined 785 OHCA cases using the emergency conveyance record (the Utstein style) submitted for the purpose of MC verifi cation by the fi re-fi ghting organization in Saga from 1 July 2010 to 31 June 2011. The fi re-fi ghting organization was classifi ed into fi ve areas (A to E) for every near medical classifi cation. Survival after out-of-hospital cardiac arrest during nights and weekends Survival after out-of-hospital cardiac arrest during nights and weekends Results Age, gender, cardiac arrest cause, initial waveform, witness, shock and drug use pre-hospital did not diff er signifi cantly between the fi ve regions. The ROSC rate was signifi cantly higher in A and C areas than in D and E areas (A: 40.1% to D: 24.4% P <0.01, A: 40.1% to E: 26.8% P <0.05, C: 39.9% to D: 24.4% P <0.05), and the ROSC rate of a hospital waveform of asystole was signifi cantly higher in A and C areas than in the other areas (A: 32.0% to B: 15.3%, D: 13.2%, E: 12.2% P <0.01, C: 27.8% to B: 15.3%, E: 12.2% P <0.05). There were signifi cantly fewer examples of oral instruction enforcement in the E area in comparison to the other areas (E: 39.7% to A: 62.5%, B: 65.7%, C: 65.9%, D: 62.0% P <0.01), and there were fewer examples of CPR enforcement in the D and E areas in comparison to the B and C areas (D: 50.8% to B: 63.9% P <0.05, E: 42.3% to B: 63.9% P <0.01, E: 42.3% to C: 59.7% P <0.05). CPR was not always delivered without oral instruction because the bystander CPR-less rate of the oral-instruction-less example to citizens was not less than 80% in all the areas. K Maekawa, K Tanno, M Hase, K Mori, Y Asai Sapporo Medical University, Sapporo, Japan Critical Care 2012, 16(Suppl 1):P269 (doi: 10.1186/cc10876) Introduction Out-of-hospital cardiac arrest (OOHCA) still has a low survival rate, despite considerable eff orts including early applications of basic life support and defi brillation in the pre-hospital setting. Post- resuscitation care after hospitalization, infl uencing the fi nal outcome, may be less available during nights and weekends because of hospital, staffi ng, and response factors. We sought to determine whether outcomes after OOHCA diff er during nights and weekends (off -hours) compared with daytimes of weekdays (on-hours). Methods We performed a retrospective analysis of 4-year data collected prospectively in a single institute. Adults with witnessed OOHCA of cardiac origin were recruited. The therapeutic strategy after hospitalization, including extracorporeal cardiopulmonary resuscitation (ECPR), therapeutic hypothermia (TH) and primary percutaneous coronary intervention (PCI), was dependent on the critical care physicians in charge. We used a propensity-score matching Conclusion An improvement of the quality of oral instruction could improve the ROSC rate. P268f all post-cardiac arrest syndrome patients were concentrated in a hospital having facilities for post-resuscitation management and provided intensive care, including appropriate hemodynamic and pulmonary management, therapeutic hypothermia, and percutaneous coronary intervention. The primary outcome measure was patient survival at 1 month with a favorable neurological outcome.i P267 Comparative examinations were conducted between the background (age, gender, cardiac arrest cause, initial waveform, and hospital waveform, witness, bystander CPR, oral instruction, and pre-hospital medical examination (shock, advanced airway management, and drug use)) and the ROSC rate between the fi ve areas. Statistical analyses included the chi-square test and Fisher’s test. Implementation of the fi fth link of the Chain of Survival concept for out-of-hospital cardiac arrest Implementation of the fi fth link of the Chain of Survival concept for out-of-hospital cardiac arrest Implementation of the fi fth link of the Chain of Survival concept for out-of-hospital cardiac arrest T Tagami1, R Tosa2, M Omura2, H Yokota1, H Hirama1 1Nippon Medical School, Tokyo, Japan; 2Aizu Chuo Hospital, Fukushima, Japan Critical Care 2012, 16(Suppl 1):P266 (doi: 10.1186/cc10873) y y Results EF5 staining demonstrated that hemorrhagic shock induced liver hypoxia, which was prevented by CO treatment. Scanning EM imaging of hepatic sinusoids demonstrated that HSR results in loss of normal endothelium, with loss of fenestrations, rounding of cells, and adherent circulating cells. CO therapy prevented these changes. Relative hepatic levels of Evans blue, suggesting endothelial leak, were increased 1.7 ± 0.23-fold in HSR compared to sham-operated mice (P <0.05). CO treatment minimized endothelial leak, resulting in a 1.23 ± 0.21-fold increase compared to sham (P <0.05 compared to air-treated HSR). In addition, leukocyte rolling and adhesion were signifi cantly diminished by CO as compared to the air-treated group in HSR. T Tagami1, R Tosa2, M Omura2, H Yokota1, H Hirama1 1Nippon Medical School, Tokyo, Japan; 2Aizu Chuo Hospital, Fukushima, Japan p Critical Care 2012, 16(Suppl 1):P266 (doi: 10.1186/cc10873) Introduction The 2010 resuscitation guidelines of the American Heart Association–International Liaison Committee on Resuscitation recommend an additional fi fth link (post-resuscitation care in a regional center) in the Chain of Survival concept for out-of-hospital cardiac arrest (OHCA) in addition to early access to emergency medical care (fi rst link), early cardiopulmonary resuscitation (second link), early defi brillation (third link), and early advanced cardiac life support (fourth link). However, no direct evidence supports its implementation. Our study aimed to determine the eff ectiveness of this fi fth link. Conclusion CO protected the hepatic sinusoidal endothelium from HSR-induced injury. Further investigations into the mechanisms of action are necessary. CO therapy may prove to be a useful resuscitative adjunct in the treatment of HSR. Methods This multicenter, region-wide, prospective clinical study involved all eligible OHCA patients in the Aizu region (n = 1,482, suburban/rural, Fukushima, Japan). Primary outcomes before (January 2006 to April 2008) and after (January 2009 to December 2010) the implementation of the fi fth link were evaluated. After implementation, S96 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Eff ectiveness and limitations of learning cardiopulmonary resuscitation with an automated external defi brillator in the curriculum of First Aid courses among lay people U Kovačič1, L Kosec2 1Faculty of Medicine, University of Ljubljana, Slovenia; 2General Hospital in Novo Mesto, Slovenia Critical Care 2012, 16(Suppl 1):P268 (doi: 10.1186/cc10875) g Results The primary outcome improved signifi cantly from 0.5% (before, 4/770) to 3.0% (after, 21/712) (P <0.0001). The multivariate odds ratio for the primary outcome was 8.3 (95% CI, 2.6 to 26.6) after the implementation of the fi fth link, 7.1 (CI, 2.0 to 25.1) for a bystander- witnessed arrest, and 5.0 (CI, 2.6 to 26.6) for early defi brillation. Introduction The eff ectiveness and limitations of widespread promotion of cardiopulmonary resuscitation (CPR) with an automated external defi brillator (AED) among the laity was investigated. Early, qualitative and continuous cardiac massage has been stressed in the 2010 ERC guidelines. Since 2009 about 45,000 laypersons attended the mandatory First Aid courses for drivers (organised by Slovenian Red Cross), which include learning CPR with an AED. i Conclusion The proportion of OHCA patients with a favorable neurological outcome improved signifi cantly after the implementation of the fi fth link of the Chain of Survival. This fi nding may require confi rmation in an urban setting and/or with randomized trials. Trial registration University Hospital Medical Information Network Clinical Trials Registry: UMIN000001607 [http://apps.who.int/ trialsearch/trial.aspx?trialid=JPRN-UMIN000001607] Methods One hundred laypersons who attended 4-hour classes in CPR before the driving lessons were compared to 60 motivated laypersons who attended 6-hour classes in CPR before starting to work as lifeguards in pools. Sixty instructors served as the control group. All participants (randomly assigned in pairs) got the same 6-minute case-based scenario on a manikin. Rescuers were changing every 2 minutes. Basic skills in CPR were determined by the two instructors and by a sensored manikin. Massage was assessed as qualitative if at least 90% of massages were provided with proper hand placement, adequate compression depth and adequate frequency. We measured the response time from the call for help to the start of heart massaging and the percentage of the time of massaging regarding the total time from start of massaging to the end of the scenario. P270 CPR initiated after telephone-assisted instruction produces a better outcome of bystander-witnessed out-of-hospital cardiac arrests than no bystander CPR but is less eff ective than CPR on the bystander’s own initiative H Inaba1, T Kamikura1, K Takase1, W Omi2, S Sakagami2, Y Myojo3, J Taniguchi3 1Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Kanazawa Medical Center, Kanazawa, Japan; 3Ishikawa Prefectural Central Hospital, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P270 (doi: 10.1186/cc10877) CPR initiated after telephone-assisted instruction produces a better outcome of bystander-witnessed out-of-hospital cardiac arrests than no bystander CPR but is less eff ective than CPR on the bystander’s own initiative Methods From the Japanese nationwide database for 431,968 OHCAs that occurred from January 2005 to December 2008, we extracted and analyzed 112,144 bystander-witnessed OHCAs without any involvement of physicians, using multiple logistic regression analysis. Results The analysis for all bystander-witnessed OHCAs revealed that both CC-only and conventional CPR following telephone CPR produce better outcomes than no bystander CPR (Table 1). The analysis for bystander-witnessed OHCAs with bystander CPR disclosed that CPR on the bystander’s own initiative produces a better outcome than CPR following telephone CPR (Table 2). Methods From the Japanese nationwide database for 431,968 OHCAs that occurred from January 2005 to December 2008, we extracted and analyzed 112,144 bystander-witnessed OHCAs without any involvement of physicians, using multiple logistic regression analysis. Results The analysis for all bystander-witnessed OHCAs revealed that both CC-only and conventional CPR following telephone CPR produce better outcomes than no bystander CPR (Table 1). The analysis for bystander-witnessed OHCAs with bystander CPR disclosed that CPR on the bystander’s own initiative produces a better outcome than CPR following telephone CPR (Table 2). g 1Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Kanazawa Medical Center, Kanazawa, Japan; 3Ishikawa Prefectural Central Hospital, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P270 (doi: 10.1186/cc10877) g 1Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Kanazawa Medical Center, Kanazawa, Japan; 3Ishikawa Prefectural Central Hospital, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P270 (doi: 10.1186/cc10877) Introduction Telephone CPR has been shown to increase the incidence of bystander CPR and is expected to improve the outcomes of out-of- hospital cardiac arrests (OHCAs). Critical times in pediatric out-of-hospital cardiac arrest J Tij 1 C Zh 2 C P h 1 L M i 2 J H hi 1 Table 1 (abstract P270). Comparison of survival between OHCAs without bystander CPR and bystander CPR in bystander-witnessed OHCAs Factor Adjusted odds ratio 95% CI Type of CPR No bystander CPR Reference CC-only CPR following telephone-CPR 1.66 1.49 to 1.84 Conventional CPR following telephone-CPR 1.67 1.48 to 1.89 CC-only CPR on bystander’s own initiative 2.22 1.99 to 2.49 Conventional CPR on bystander’s own initiative 2.36 2.10 to 2.66 Aetiology Presumed cardiac 2.44 2.27 to 2.63 Noncardiac Reference Time intervals Witness-call 0.98 0.97 to 0.98 Witness-fi rst CPR performed either by 0.97 0.96 to 0.98 citizens or by EMTs Call-arrival at patients 0.93 0.92 to 0.94 Comparisons of 1-month survival with favourable neurological outcomes between OHCAs without bystander CPR and with four types of bystander CPR in bystander-witnessed OHCAs (multiple logistic regression analysis). Table 1 (abstract P270). Comparison of survival between OHCAs without bystander CPR and bystander CPR in bystander-witnessed OHCAs Introduction Pediatric out-of-hospital cardiac arrest (OHCA) has a less than 10% survival. Studies of the scene time and level of emergency medical services (EMS) training in pediatric OHCA are lacking. The objectives of this study are to describe the scene time, level of training and the order and timing of arrival of fi rst responders to pediatric OHCA in a large, densely populated area, the Toronto region. Methods The Resuscitation Outcomes Consortium (ROC) Epistry- Cardiac Arrest database was queried for all patients <19 years old from December 2005 to November 2011 in the Toronto region for age, sex, event characteristics, underlying conditions, cause of the cardiac arrest, level of EMS care, time to EMS arrival, scene time, return of spontaneous circulation (ROSC) and survival to hospital discharge. Patients were excluded if they were declared dead at the scene. y Results Four hundred and fi fty-two patients with OHCA were included. Thirty-one percent were infants, 29.4% age 1 to 11 years (child), and 37.4% age 1 to 18 (adolescent) years with 62.8% of cases male. Thirty percent had a signifi cant past medical history. The causes of the cardiac arrest were trauma (14.4%), drowning (6.2%), sudden infant death syndrome (4.0%), and unknown in 63%. The fi rst EMS responders were fi re in 52.2%, advanced care paramedics in 25%, and primary care paramedics in 22.3%. Survival was increased the earlier the EMS arrived (P = 0.015). Survival after out-of-hospital cardiac arrest during nights and weekends BLS education to the area, a re-examination of the oral instruction manual in the applicable areas, and the suitable evaluation of various examples of agonal respiration are together expected to improve the ROSC rate. S97 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 2 (abstract P270). Eff ects of type and origin by bystander CPR on survival of bystander-witnessed OHCAs having bystander CPR Table 2 (abstract P270). Eff ects of type and origin by bystander CPR on survival of bystander-witnessed OHCAs having bystander CPR Factor Adjusted odds ratio 95% CI Type of CPR CC-only CPR 0.96 0.88 to 1.04 Conventional CPR Reference Origin of CPR Following telephone-CPR 0.73 0.67 to 0.80 On bystander’s own initiative Reference Aetiology Presumed cardiac 2.27 2.05 to 2.51 Noncardiac Reference Time intervals Witness-call 0.99 0.98 to 0.99 Witness-fi rst CPR performed either by 0.98 0.97 to 0.99 citizens or by EMTs Call-arrival at patients 0.88 0.87 to 0.90 Eff ects of type and origin by bystander CPR on 1-month survival with favourable neurological outcomes of bystander-witnessed OHCAs having bystander CPR (multiple logistic regression analysis). to reduce the diff erences of pre-hospital variables between patients arriving during off -hours and on-hours. Primary endpoint was 90-day survival after cardiac arrest. We evaluated the survival diff erence using the log-rank test and identifi ed the signifi cant interventions aff ecting outcome using the Cox regression model. Adjusted odds ratio 95% CI g g Results Of 185 patients, 131 arrived during off -hours (the off -hours group) and 54 arrived during on-hours (the on-hours group). The matching process selected 37 patients each from both groups. The matched off -hours group had a lower survival rate than the matched on-hours group (10.8% vs. 37.8%; log-rank P = 0.025). Multivariate Cox regression analysis showed that TH was associated with 90-day survival after cardiac arrest (adjusted hazard ratio (HR), 0.43; 95% CI, 0.23 to 0.79), but there were no signifi cant associations of ECPR (adjusted HR, 0.83; 95% CI, 0.50 to 1.37) and primary PCI (adjusted HR, 0.76; 95% CI, 0.42 to 1.38). Conclusion Lower survival rates after OOHCA during nights and weekends were seen at our institute. TH was more likely to be induced in patients arrived during daytimes of weekdays, and independently associated with survival benefi t. P270 The aim of present study was to clarify if the outcomes of bystander-witnessed OHCAs having CC-only and conventional CPR following telephone CPR may be better than those having no bystander CPR and if the type (CC-only and conventional) and origin (following telephone CPR and on bystander’s own initiative) may aff ect the outcomes of bystander-witnessed OHCAs with bystander CPR. g p Conclusion Telephone CPR improves the outcomes of bystander- witnessed OHCAS. However, eff orts to increase the incidence of early CPR on the bystander’s own initiative would be necessary to obtain a higher incidence of survival in bystander-witnessed OHCAs. Reference 1. Peberdy MA, Ornato JP, Larkin GL, et al.: Survival from in-hospital cardiac arrest during nights and weekends. JAMA 2008, 299:785-792. Eff ects of type and origin by bystander CPR on 1-month survival with favourable neurological outcomes of bystander-witnessed OHCAs having bystander CPR (multiple logistic regression analysis). P271 P271 Critical times in pediatric out-of-hospital cardiac arrest J Tijssen1, C Zhan2, C Parshuram1, L Morrison2, J Hutchison1 1Hospital for Sick Children, Toronto, Canada; 2University of Toronto, Canada Critical Care 2012, 16(Suppl 1):P271 (doi: 10.1186/cc10878) P272 Don’t stop your heart in front of your family: family as a bystander is associated with poor outcome of bystander-witnessed, bystander- CPR-performed out-of-hospital cardiac arrest Conclusion Despite educational eff orts, most family members do not appear to be good CPR performers. The fi rst responder system that enables a good CPR performer to reach the scene quickly may be needed for OHCAs witnessed by the family. H Inaba1, K Takase1, T Nishi1, T Kamikura1, Y Wato2, H Hamada3 1Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Kanazawa Medical University, Uchinada, Japan; 3Suzu General Hospital, Suzu, Japan H Inaba1, K Takase1, T Nishi1, T Kamikura1, Y Wato2, H Hamada3 1Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Kanazawa Medical University, Uchinada, Japan; 3Suzu General Hospital, Suzu, Japan p Critical Care 2012, 16(Suppl 1):P272 (doi: 10.1186/cc10879) Critical Care 2012, 16(Suppl 1):P272 (doi: 10.1186/cc10879) Introduction Early CPR with a considerable quality is essential for survival from out-of-hospital cardiac arrest (OHCA). This study was Table 1 (abstract P272). Backgrounds, characteristics and outcomes of OHCAs with reference to relation of bystander to victim Relation of bystander to victim Family Friends, colleagues and Others Background, characteristics and outcome (n = 25,119) passers-by (n = 5,191) (n = 14,938) P value Patient’s age, median (25 to 75%) 77 (66 to 84) 61 (50 to 73) 84 (75 to 90) <0.001 Sex – male (%) 61.6 76.8 44.7 <0.001 CPR following telephone CPR (%) 75.1 42.7 36.1 <0.001 Initial rhythm shockable (%) 16.3 33.4 9.8 <0.001 Tune intervals, minutes, median (25 to 75%) Collapse-call 2 (0 to 5) 2 (0 to 4) 2 (0 to 5) <0.001 Collapse-bystander CPR 3 (1 to 6) 1 (0 to 4) 0 (0 to 2) <0.001 Call arrival at patient 8 (6 to 11) 8 (6 to 11) 8 (6 to 10) <0.001 Outcomes 1-month survival (%) 8.1 17.2 9.2 <0.001 1-month survival with favorable 4.0 11.9 4.8 <0.001 neurological outcomes (%) Table 1 (abstract P272). Backgrounds, characteristics and outcomes of OHCAs with reference to relation of b Table 1 (abstract P272). Backgrounds, characteristics and outcomes of OHCAs with reference to relation of bystander to victim Relation of bystander to victim tract P272). Backgrounds, characteristics and outcomes of OHCAs with reference to relation of bystander to victim Table 2 (abstract P272). Critical times in pediatric out-of-hospital cardiac arrest J Tij 1 C Zh 2 C P h 1 L M i 2 J H hi 1 The timing of arrival of advanced paramedics at the scene appeared to be associated with survival although this was not statistically signifi cant (P = 0.22). Infants had a shorter scene time (P <0.001) and an earlier arrival of advanced care paramedics at the scene p g between OHCAs without bystander CPR and with four types of bystander CPR in bystander-witnessed OHCAs (multiple logistic regression analysis). between OHCAs without bystander CPR and with four types of bystander CPR in b t d it d OHCA ( lti l l i ti i l i ) S98 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 conducted to test our hypothesis that the relation of the bystander to the victim may aff ect the outcomes of OHCAs. (P = 0.04). A shorter scene time was associated with ROSC on arrival at the emergency department (P <0.001) and a nonsignifi cant trend for improved survival (P = 0.13). Adolescents were more likely to have ROSC on arrival at the emergency department (P <0.001) and more likely to survive (P <0.05) compared to children or infants. the victim may aff ect the outcomes of OHCAs. Methods From a Japanese nationwide database for 431,968 OHCAs that occurred from January 2005 to December 2008, we extracted and then analyzed 45,248 bystander-witnessed, bystander-CPR-performed OHCAs without any involvement of physicians. Backgrounds, characteristics and outcomes were compared among the three groups of OHCAs categorized by the bystander’s relation to victims. Multiple logistic regression analysis was applied to clarify if the relation may aff ect the 1-month survival with favourable neurological outcomes. Results When the bystander was family, CPR was more frequently initiated following telephone-assisted instruction and the interval between collapse and bystander CPR was signifi cantly prolonged. Univariate analysis followed by multiple logistic regression analysis revealed that family as a CPR performer signifi cantly decreases the 1-month survival with favourable neurological outcomes. See Tables 1 and 2.f yf Methods From a Japanese nationwide database for 431,968 OHCAs that occurred from January 2005 to December 2008, we extracted and then analyzed 45,248 bystander-witnessed, bystander-CPR-performed OHCAs without any involvement of physicians. Backgrounds, characteristics and outcomes were compared among the three groups of OHCAs categorized by the bystander’s relation to victims. Critical times in pediatric out-of-hospital cardiac arrest J Tij 1 C Zh 2 C P h 1 L M i 2 J H hi 1 Multiple logistic regression analysis was applied to clarify if the relation may aff ect the 1-month survival with favourable neurological outcomes. Conclusion The timing of arrival of advanced paramedics at the scene may have been associated with survival and a larger study is needed to confi rm this trend. A shorter scene time was associated with ROSC and a trend for increased survival. However, infants have shorter scene times but worse outcomes. To provide increased power and scope for this study we will expand it to include all 10 Regional Clinical ROC Centers and future analyses will include the remaining Utstein data fi elds and compare the eff ects of advanced versus basic life support interventions during resuscitation. f g Results When the bystander was family, CPR was more frequently initiated following telephone-assisted instruction and the interval between collapse and bystander CPR was signifi cantly prolonged. Univariate analysis followed by multiple logistic regression analysis revealed that family as a CPR performer signifi cantly decreases the 1-month survival with favourable neurological outcomes. See Tables 1 and 2.f P272 End-tidal carbon dioxide levels should be monitored during CPR and considered a useful prognostic value for determining the outcome of resuscitative eff orts and when to cease CPR in the fi eld. y y y Methods A total of 11 female pigs, body weights 50.3 ± 3.4 kg, were enrolled into a protocol of prolonged cardiac arrest treated by FF or FS ECMO ± IABP in a randomized fashion. Animals under general anesthesia had undergone 15 minutes of ventricle fi brillation (VF) with basal ECMO fl ow of 5 to 10 ml/kg/minute simulating low-fl ow CA followed by continued VF with ECMO fl ow of 100 ml/kg/minute. CPP, myocardial lactate metabolism and myocardial oxygen extraction were determined. P275i P275 Modifi ed clinical decision rule for termination-of-resuscitation in cases of refractory out-of-hospital cardiac arrest Y Goto1, T Maeda1, Y Goto2, H Inaba3 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan; 3Kanazawa University Graduate School of Medicine, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P275 (doi: 10.1186/cc10882) Results CPP decreased from baseline of 85 mmHg (72, 94.5) to 15 mmHg (10, 20.5) during CA. The fi rst CPP value on ECMO increased to 34 mmHg (26.5, 44) and during the further protocol gradually rose to signifi cantly higher CPP of 68 mmHg (45.5, 82) before CPR (P = 0.003). This phenomenon of gradual rise was even more pronounced in FF ECMO, animals started on FF ECMO completed the protocol with identical CPP values as at baseline (85 mmHg (80, 99) vs. 86 mmHg (78, 86), P = 0.55). Following CA, signifi cantly higher lactate levels were detected in animals started on FS ECMO in all post-arrest periods (P = 0.016 and P = 0.035 for diff erence in arterial and coronary sinus lactate levels, respectively). Oxygen extractions after a steep increase during CA declined immediately after ECMO initiation and remained further with no statistically signifi cant diff erences between respective ECMO arms (P for diff erence = 0.547). Resuscitability was high, we gained 5 minutes return of spontaneous circulation (ROSC) in eight animals (73%) and 60 minutes ROSC was present still in eight animals (73%).i Introduction Two international termination-of-resuscitation (TOR) rules for the emergency medical services (EMS) personnel have been proposed to identify nonsurvivors after out-of-hospital cardiac arrest (OHCA). P272 P273 Coronary perfusion pressure in a pig model of prolonged cardiac arrest treated by diff erent modes of venoarterial extracorporeal membrane oxygenation and intraaortic balloon counterpulsation J Bělohlávek1, M Mlcek2, M Huptych3, S Havranek1, P Ostadal4, A Linhart1, O Kittnar2 1General Teaching Hospital Prague, Czech Republic; 21st Medical School, Charles University, Prague, Czech Republic; 3Technical Institute Prague, Czech Republic; 4Homolka Hospital, Prague, Czech Republic Critical Care 2012, 16(Suppl 1):P273 (doi: 10.1186/cc10880) Coronary perfusion pressure in a pig model of prolonged cardiac arrest treated by diff erent modes of venoarterial extracorporeal membrane oxygenation and intraaortic balloon counterpulsation J Bělohlávek1, M Mlcek2, M Huptych3, S Havranek1, P Ostadal4, A Linhart1, O Kittnar2 1General Teaching Hospital Prague, Czech Republic; 21st Medical School, Charles University, Prague, Czech Republic; 3Technical Institute Prague, Czech Republic; 4Homolka Hospital, Prague, Czech Republic Critical Care 2012, 16(Suppl 1):P273 (doi: 10.1186/cc10880) p Results PetCO2 after 20 minutes of advanced life support averaged 0.97 ± 0.33 kPa in patients who did not have ROSC and 4.85 ± 1.74 kPa in those who did (P <0.001). PetCO2 after 15 minutes of advanced life support averaged 1.11 ± 0.39 kPa in patients who did not have ROSC and 3.65 ± 0.98 kPa in those who did (P <0.001). End-tidal carbon dioxide values of 1.9 kPa (14.3 mmHg) or less discriminated between the 578 patients with ROSC and 502 patients without. When a 20-minute end- tidal carbon dioxide value of 1.9 kPa (14.3 mmHg) or less was used as a screening test to predict ROSC, the sensitivity, specifi city, positive predictive value, and negative predictive value were all 100%. The 15-minute petCO2 value of 1.8 kPa had a sensitivity and NPV of 100% with high specifi city PPV value (98%). Introduction An extracorporeal membrane oxygenation (ECMO)- based approach is increasingly used in cardiac arrest (CA). However, little is known about coronary perfusion pressure progress over time in CA managed by ECMO. The aim of this study was to assess femoro- femoral (FF) compared to femoro-subclavian (FS) venoarterial ECMO in a pig model of prolonged CA on coronary perfusion pressure (CPP), myocardial metabolic recovery and resuscitability. g pi y Conclusion End-tidal carbon dioxide levels of more than 1.9 kPa (14.3 mmHg) after 20 minutes may be used to predict ROSC with accuracy. P274 p g y Methods We analysed 289,769 OHCA adult patients with presumed cardiac causes, using a prospectively recorded nationwide Utstein-style database in Japan over 5 years (2005 to 2009). The primary endpoint was 1-month survival with unfavourable neurological outcome, or Glasgow–Pittsburgh cerebral performance category (CPC) scale = 3 to 5. Results The overall rates of 1-month survival with CPC = 1 or 2 and collective 1-month survival were 2.55% and 5.22%, respectively. The incidences of misclassifi cation in the BLS, ALS and modifi ed BLS TOR rules for 1-month survival with CPC = 3 to 5 were 0.20%, 0.15% and 0.13%, respectively. The specifi city (95% CI) in the BLS, ALS and modifi ed BLS TOR rules for 1-month survival with CPC = 3 to 5 were 0.941 (0.935 to 0.946), 0.981 (0.978 to 0.984) and 0.972 (0.968 to 0.975), respectively. The area under the receiver operating characteristic curve in the BLS, ALS and modifi ed BLS TOR rules for 1-month survival with CPC = 3 to 5 were 0.865, 0.654 and 0.765, respectively.i P274 Capnometry successfully predicts outcome and determination of the cessation of cardiopulmonary resuscitation eff orts EH Hajdinjak1, ŠG Grmec2, MK Križmarić3, ET Torkar2, MS Skufca2, DB Buić-Rerečić2, MK Kovač2, MZ Zelinka2 1Center for Emergency Medicine Maribor, University of Maribor, University of Ljubljana, Slovenia, Maribor, Slovenia; 2Community Health Centre Ljubljana, University of Ljubljana, University of Maribor, Ljubljana, Slovenia; 3Faculty of Medicine, University of Maribor, Slovenia Critical Care 2012, 16(Suppl 1):P274 (doi: 10.1186/cc10881) P272 The fi rst is for use by responders providing basic life support (BLS) which includes three criteria: not witnessed by EMS personnel, no shocks are administered and no return of spontaneous circulation (ROSC). The other is for use by responders providing advanced life support (ALS) which adds two criteria: unwitnessed by a bystander and no bystander cardiopulmonary resuscitation. Simpler criteria as a universal TOR rule may be desirable for any level of EMS personnel. We performed this study to validate two TOR rules and a modifi ed BLS TOR rule which includes three criteria: unwitnessed arrest, no shocks administered and no ROSC achieved before arrival at hospital for predicting refractory OHCAs. Conclusion Our experimental study confi rmed that, in a pig model of prolonged cardiac arrest, VA ECMO, mainly the FF approach, increases signifi cantly the CPP over time, assures good metabolic recovery and off ers sustained reasonable resuscitability. Morrison LJ, et al.: N Engl J Med 2006, 355:478-487. P272 Relation of bystander to victim as a factor associated with 1-month survival of bystander-witnessed OHCAs having bystander CPRi Table 2 (abstract P272). Relation of bystander to victim as a factor associated with 1-month survival of bystander-witnessed OHCAs having bystander CPR Adjusted odds ratio (95% confi dence interval) Bystander-witnessed OHCAs Of presumed Of presumed cardiac etiology Factor with bystander CPR cardiac etiology with shockable initial rhythm Etiology of arrest Presumed cardiac 1.39 (1.24 to 1.55) Undefi ned Undefi ned Noncardiac Reference Initial rhythm Shockable 4.38 (3.95 to 4.85) 4.82 (4.29 to 5.42) Undefi ned Nonshockable Reference Reference Reference Patient’s age 0.97 (0.97 to 0.98) 0.97 (0.97 to 0.97) 0.98 (0.97 to 0.98) Sex Male 1.14 (1.02 to 1.26) 1.16 (1.02 to 1.32) 1.07 (0.90 to 1.26) Female Reference Reference Reference Relation of bystander to victim Family Reference Reference Reference Friend, colleague and passers-by 1.70 (1.49 to 1.95) 1.40 (1.19 to 1.64) 1.61 (1.42 to 1.81) Others 1.59 (1.42 to 1.78) 1.46 (1.27 to 1.68) 1.32 (1.10 to 1.59) elation of bystander to victim as a factor associated with 1-month survival of bystander-witnessed OHCAs having bystander CPR Table 2 (abstract P272). Relation of bystander to victim as a factor associated with 1-month survival of bystander-witnessed O y y Adjusted odds ratio (95% confi dence interval) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S99 P273 and partial pressure of end-tidal carbon dioxide (PetCO2) values were collected for each patient in cardiac arrest by the emergency physician. We hypothesized that an end-tidal carbon dioxide level of 1.9 kPa (14.3 mmHg) or more after 20 minutes and 1.8 kPa or more after 15 minutes of standard advanced cardiac life support would predict restoration of spontaneous circulation (ROSC). P276 Survival benefi t for patients receiving antibiotics following out-of- hospital cardiac arrest KJ Davies, ID Kerslake, J Walters, MJ Thomas Bristol Royal Infi rmary, Bristol, UK Critical Care 2012, 16(Suppl 1):P276 (doi: 10.1186/cc10883) Introduction Therapeutic hypothermia (TH) has become standard management following out-of-hospital cardiac arrest (OHCA). Recent evidence suggests TH increases the risk of pneumonia. We retrospectively assessed infective indicators after OHCA and evaluated the eff ect of antibiotics on survival. Results One-hundred and four patients survived more than 24 hours out of all 1,026 patients analyzed. Mean IL-6, S-100B and NSE levels in nonsurvivors (n = 51) were signifi cantly higher than those in survivors (n = 53) at all timepoints (P <0.01). Those in poor neurological outcome (n = 74) were signifi cantly higher than those in favorable neurological outcome (n = 29) at all timepoints (P <0.01). From the results of ROC analysis and multivariate analysis, IL-6 >240 pg/ml at 6 hours and S-100B >0.37 ng/ml on admission were chosen as an independent predictors of nonsurvival, and S-100B >0.07 ng/ml at 24 hours was chosen as that of poor neurological outcome. Subgroup analysis of 56 patients showed that mean levels of IL-6 at 6 hours, S-100B at 6 hours and S-100B at 24 hours in the maintained (n = 29) group were signifi cantly lower than those in the not-maintained group (n = 27) (P <0.05). f Methods We identifi ed all patients admitted to the ICU of a regional primary angioplasty hospital following OHCA from May 2007 to December 2010. We recorded ICNARC predicted mortality scores, blood and respiratory (protected catheter aspiration) culture results, white blood cell count (WBC) and C-reactive protein (CRP), hospital outcome and ICU length of stay. All chest radiographs (CXRs) were reviewed by a respiratory consultant (JW). Any antibacterial therapy was recorded. Results A total of 144 patients were admitted to the ICU following OHCA. Mean age was 61.7 years (95% CI 59.0 to 64.4). The mortality rate was 66.67% (58.62 to 73.84) with mean ICNARC predicted mortality of 77.11% (73.84 to 80.39). Of 144 patients, 138 (95.8%; 91.1 to 98.1) had at least one positive marker of infection within 72 hours. Sixty-four had microbiology samples analysed, 34 of which were positive (53.1%; 41.1 to 64.8%). Of 88 patients who had a CXR, 26 (29.6%; 21.0 to 39.8) had consolidation. P277 P277 Correlation between IL-6 and S-100B blood levels and outcome of post-cardiac arrest syndrome and infl uence of therapeutic hypothermia on these mediator blood levels K Shinozaki 1, S Oda2, T Sadahiro2, M Nakamura2, E Watanabe2, R Abe2, T Nakada2, Y Morita2, K Nakanishi3, N Kitamura4, H Hirasawa2 1Chiba Aoba Municipal Hospital, Chiba City, Japan; 2Chiba University, Chiba City, Japan; 3Narita Red Cross Hospital, Narita City, Japan; 4Kimitsu Chuo Hospital, Kisarazu City, Japan Critical Care 2012, 16(Suppl 1):P277 (doi: 10.1186/cc10884) Correlation between IL-6 and S-100B blood levels and outcome of post-cardiac arrest syndrome and infl uence of therapeutic hypothermia on these mediator blood levels K Shinozaki 1, S Oda2, T Sadahiro2, M Nakamura2, E Watanabe2, R Abe2, T Nakada2, Y Morita2, K Nakanishi3, N Kitamura4, H Hirasawa2 1Chiba Aoba Municipal Hospital, Chiba City, Japan; 2Chiba University, Chiba City, Japan; 3Narita Red Cross Hospital, Narita City, Japan; 4Kimitsu Chuo Hospital, Kisarazu City, Japan Critical Care 2012, 16(Suppl 1):P277 (doi: 10.1186/cc10884) Results Overall rates of 1-month survival and that with favorable neurological outcome were 56.7% (n = 5,604) and 40.6% (n = 4,013), respectively. Multivariate logistic regression analysis revealed that the odds ratio for age, shockable initial rhythm and collapse–ROSC time interval were 0.964 (95% CI 0.961 to 0.967), 3.564 (95% CI 3.232 to 3.934) and 0.967 (95% CI 0.963 to 0.970), respectively, and that these variables were identifi ed as the best variables for developing a prediction model. A statistical outcome prediction model using these three variables was as follows: Pf = exp(B) / [1 + exp(B)], where Pf is the probability of a favorable outcome and exp(B) is the exponential function of B: B = –0.037×age (years) + 0.635×(shockable rhythm (1 or 0)) – 0.034×(collapse–ROSC time interval (minutes)) + 2.540. The area under the receiver operating characteristic curve of this model for predicting 1-month favourable neurological outcome was 0.764. Introduction To elucidate the signifi cance of IL-6, S-100B and NSE in pathophysiology of post-cardiac arrest syndrome (PCAS), blood levels of those mediators sampled within the fi rst 24 hours after cardiac arrest (CA) were compared between groups classifi ed according to survival and neurological outcomes. Furthermore, infl uence of stability of core temperature with therapeutic hypothermia (TH) on these mediator blood levels was also investigated. Methods Nontraumatic out-of-hospital CA patients were included. Blood was sampled on admission, at 6 hours and 24 hours after CA, respectively. Capnometry successfully predicts outcome and determination of the cessation of cardiopulmonary resuscitation eff orts Capnometry successfully predicts outcome and determination of the cessation of cardiopulmonary resuscitation eff orts Š j j DB Buić-Rerečić2, MK Kovač2, MZ Zelinka2 1Center for Emergency Medicine Maribor, University of Maribor, University of Ljubljana, Slovenia, Maribor, Slovenia; 2Community Health Centre Ljubljana, University of Ljubljana, University of Maribor, Ljubljana, Slovenia; 3Faculty of Medicine, University of Maribor, Slovenia Critical Care 2012, 16(Suppl 1):P274 (doi: 10.1186/cc10881) Introduction Prognosis in patients suff ering out-of-hospital cardiac arrest is poor. Higher survival rates have been observed only in patients with ventricular fi brillation who were fortunate enough to have basic and advanced life support initiated soon after cardiac arrest. An ability to predict cardiac arrest outcomes would be useful for resuscitation. Changes in expired end-tidal carbon dioxide levels during cardio- pulmonary resuscitation (CPR) may be a useful, noninvasive predictor of successful resuscitation and survival from cardiac arrest, and could help in determining when to cease CPR eff orts. Conclusion We found that each TOR rule had high specifi city (ability to predict survivors with favourable neurological outcome) and low misclassifi cation rate as a universal TOR rule. The modifi ed BLS TOR rule is simpler and as reliable as the other two rules. In Japan, as EMS providers are legally prohibited from terminating resuscitation in the fi eld, the amendment of related laws and the establishment of national consensus would be necessary to apply these rules in the Japanese EMS system. Reference f Methods This is a prospective, observational study of 1,080 cases of out-of-hospital cardiac arrest. The patients were intubated and measurements of end-tidal carbon dioxide taken. Data according to the Utstein criteria, demographic information, medical data, Morrison LJ, et al.: N Engl J Med 2006, 355:478-487. S100 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P276 poor neurological outcome (CPC 3 to 5) and favorable neurological outcome (CPC 1 to 2), respectively. Factors signifi cantly correlated with survival and neurological outcomes were investigated by comparing baseline characteristics and mediator blood levels. Patients receiving TH were also included into subgroup analysis. If the core temperature was maintained at 33 ± 1°C for more than 18 hours within the fi rst 24 hours, the patient was classifi ed into maintained, and if not into not- maintained, and mediator blood levels were compared between the subgroups. P276 Survival benefi t for patients receiving antibiotics following out-of- hospital cardiac arrest Ninety-six of 115 patients (83.5%; 75.6 to 89.1) had a CRP >100 mg/l (normal value <10 mg/l) within 72 hours and 82 of 115 (71.3%; 62.5 to 78.8) had an abnormal WBC (<4.0 or >11.0×109/l). Fifty- six of 144 patients (38.9%; 31.3 to 47.0) received antibiotics during the fi rst 7 days of their ICU stay (mean time to fi rst dose 2.17 days; 1.69 to 2.66). The hospital mortality rate for these patients 53.6% (40.7 to 66.0) was signifi cantly less than those not receiving antibiotics 75.0% (65.0 to 82.9) (χ2 6.14, P = 0.01) with absolute risk reduction of 0.214 (0.055 to 0.365) and NNT of 5 (3 to 18). There was no diff erence in age (59.9 ± 4.2 vs. 62.9 ± 3.5) or ICNARC predicted mortality (75.1 ± 5.2 vs. 78.4 ± 4.2) between the groups. Conclusion IL-6 and S-100B levels within 24 hours after CA, but not NSE, are related to survival and neurological outcome. IL-6 and S-100B are considered to be important mediators for the pathophysiology of PCAS and TH may infl uence blood levels of these mediators. Early neurological outcome prediction model after bystander-witnessed out-of-hospital cardiac arrest: a nationwide population-based study Early neurological outcome prediction model after bystander-witnessed out-of-hospital cardiac arrest: a nationwide population-based study Y Goto1, T Maeda1, Y Goto2, H Inaba3 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan; 3Kanazawa University Graduate School of Medicine, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P278 (doi: 10.1186/cc10885) Y Goto1, T Maeda1, Y Goto2, H Inaba3 1Kanazawa University Hospital, Kanazawa, Japan; 2Yawata Medical Center, Komatsu, Japan; 3Kanazawa University Graduate School of Medicine, Kanazawa, Japan Critical Care 2012, 16(Suppl 1):P278 (doi: 10.1186/cc10885) g Conclusion The post-arrest management of OHCA is commonly complicated by infections, the diagnosis of which is delayed by a universal increase in infl ammatory markers, body temperature control, delay in the processing of samples and poor quality radiography. We have shown a signifi cant reduction in mortality in patients receiving antibiotics compared with patients who do not, despite there being no diff erence in age or predicted mortality between the groups. This could be due to treatment of an aspiration pneumonia, an anti-infl ammatory eff ect or that some patients did not survive long enough to receive antibiotics. It suggests that a formal clinical trial is warranted. Critical Care 2012, 16(Suppl 1):P278 (doi: 10.1186/cc10885 Introduction Identifi cation of prehospital prognostic factors in out-of- hospital cardiac arrests (OHCAs) with prehospital return of spontaneous circulation (ROSC) and establishment of a prediction model for survival with favorable neurological outcome may minimize the costs and save the medical resources. In this study, we developed a best model for predicting 1-month survival with favorable neurological outcome (defi ned as Glasgow–Pittsburgh cerebral performance category (CPC) scale = 1 or 2), using a logistic regression analysis. g g g y Methods Of 522,801 resuscitation-attempted adult patients after OHCAs, 9,876 bystander-witnessed arrests of presumed cardiac origin with prehospital ROSC were analyzed in a prospectively recorded nationwide Utstein-style database in Japan over 5 years (2005 to 2009). The endpoint was 1-month survival with favorable neurological outcome. We performed multivariate logistic regression analysis to develop a prediction model using the prehospital factors. P277 Population cooled post cardiac arrest D/C (n = 12) Died (n = 26) VF arrest 10 (83%) 18 (75%) P = 0.69 DT >30 minutes 1 (8%) 14 (58%) P = 0.005 First CPR <5 minutes 11 (92%) 17 (71%) P = 0.22 Cardiac aetiology 10 (83%) 16 (67%) P = 0.44 Conclusion Survival is improved in patients cooled post-out-of- hospital cardiac arrest [1,2]. Downtime is statistically signifi cant in the survival of cooled patients. Achieving optimal timing of cooling was no better in surviving versus dying populations. Cooling post-out-of- hospital cardiac arrest is expensive and time-consuming; selection criteria need to be evaluated to concentrate this resource on patients where there is a higher prospect of a positive outcome [2] Table 1 (abstract P280). Population cooled post cardiac arrest Table 1 (abstract P280). Population cooled post cardiac arrest Introduction Most patients admitted to the ICU after cardiac arrest die or have an unfavourable neurological outcome due to brain damage. Currently, the only treatment to reduce brain injury after cardiac arrest is mild hypothermia. Helium inhalation has shown promising results as a neuroprotective agent in animal models of cerebral infarction. If helium inhalation ameliorates neurological damage by reducing reperfusion injury in humans as well, this could be of great benefi t to patients. As no studies exist that investigate the use of helium ventilation in patients after cardiac arrest we investigated whether this treatment is safe and feasible. Conclusion Survival is improved in patients cooled post-out-of- hospital cardiac arrest [1,2]. Downtime is statistically signifi cant in the survival of cooled patients. Achieving optimal timing of cooling was no better in surviving versus dying populations. Cooling post-out-of- hospital cardiac arrest is expensive and time-consuming; selection criteria need to be evaluated to concentrate this resource on patients where there is a higher prospect of a positive outcome [2]. Methods A single-centre open-label intervention study was performed in a mixed 30-bed academic ICU, approved by the local medical ethics committee. Inclusion criteria: admission after a witnessed cardiac arrest, presenting with ventricular fi brillation or tachycardia, return of spontaneous circulation within 30 minutes, treatment with hypothermia. Exclusion criteria: pre-existing neurological disorders or the need for a FiO2 >50% or >10 mmHg PEEP on ICU admission. Helium was administered during 3 hours as a 1:1 mixture with oxygen, using a Servo-i ventilator. P281 P281 Therapeutic hypothermia for nonventricular fi brillation/ventricular tachycardia cardiac arrest S Jog1, D Patel1, M Patel1, R Kulkarni1, N Chouthai2 1Deenanath Mangeshkar Hospital and Research Centre, Pune, India; 2Wayne State University, Detroit, MI, USA Critical Care 2012, 16(Suppl 1):P281 (doi: 10.1186/cc10888) Therapeutic hypothermia for nonventricular fi brillation/ventricular tachycardia cardiac arrest p p p Results In total 25 patients were included, 20 (80%) male, age 64.8 ± 12.1 years, APACHE II score 20.0 ± 8.6, SAPS II 53.6 ± 18.6. Helium treatment was started 4:57 ± 0:54 hours after arrest. In one patient the treatment was stopped due to inadequate ventilation using the preset limits. This was not due to the helium ventilation and no adverse events due to helium ventilation were noted. Overall, nine (36%) patients had a poor outcome. S Jog1, D Patel1, M Patel1, R Kulkarni1, N Chouthai2 1Deenanath Mangeshkar Hospital and Research Centre, Pune, India; 2Wayne State University, Detroit, MI, USA Critical Care 2012, 16(Suppl 1):P281 (doi: 10.1186/cc10888) Introduction Although effi cacy of therapeutic hypothermia (TH) for cardiac arrest following ventricular tachycardia (VT)/ventricular fi brillation (VF) is a recommended therapy, the effi cacy of TH for non- VF/VT cardiac arrest is still not well studied. We conducted a study to evaluate effi cacy and outcomes of TH in non-VF/VT cardiac arrest patients in terms of survival and neurological outcome. Conclusion In this small study, we encountered no problems associated with helium treatment in patients admitted to the ICU after cardiac arrest. This opens the way for studies investigating the hypothesis that helium treatment reduces neurological injury in these patients. Methods TH was initiated with intravenous ice-cold saline and maintained with an external servo controlled cooling system (ESCCS); by Blanketrol II Hypo-Hyperthermia system (Cincinnati Sub-Zero Inc.) between 34 and 32°C for 24 hours. Gradual rewarming was also done with ESCCS. Non-VF/VT cardiac arrest patients with GCS ≤7 at 60 minutes of return of spontaneous circulation (ROSC) were enrolled. Standard hemodynamic monitoring and management was continued in all patients. P277 Then, patients that died within 24 hours after CA were excluded. Patients were classifi ed into nonsurvivors (died within 28 days) and survivors (survived for 28 days or longer), and classifi ed into Conclusion Three prehospital prognostic factors (age, shockable initial rhythm and collapse–ROSC time interval) were identifi ed as the best variables in predicting favorable neurological outcomes in OHCAs with prehospital ROSC. A model using these prehospital prognostic factors S101 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Seventy patients had a hospital admission of post-cardiac arrest. Five failed the inclusion criteria and six fulfi lled exclusion criteria. A total of 36 (51%) were cooled (Table 1). Twelve (33%) of the cooled population survived to hospital discharge (D/C), one (8%) cooled within 4 hours, three (25%) cooled for over 12 hours. Ten (28%) patients were cooled despite not having a cardiac cause. One (4%) of the 23 noncooled patients survived to hospital discharge, four (17%) had a cardiac cause. The median age of cooled population was 66 years (quartile range 53.5 to 74 years) and 44 years (quartile range 41 to 52 years) of the noncooled. Results Seventy patients had a hospital admission of post-cardiac arrest. Five failed the inclusion criteria and six fulfi lled exclusion criteria. A total of 36 (51%) were cooled (Table 1). Twelve (33%) of the cooled population survived to hospital discharge (D/C), one (8%) cooled within 4 hours, three (25%) cooled for over 12 hours. Ten (28%) patients were cooled despite not having a cardiac cause. One (4%) of the 23 noncooled patients survived to hospital discharge, four (17%) had a cardiac cause. The median age of cooled population was 66 years (quartile range 53.5 to 74 years) and 44 years (quartile range 41 to 52 years) of the noncooled. has shown a good predictive value for estimating 1-month survival with favorable neurological outcome in OHCA patients. Although this novel model needs to be validated using another external dataset, this model may help to minimize the cost and save medical resources. P279 P279 Helium ventilation is safe and feasible in ICU patients admitted after cardiac arrest D Brevoord, C Beurskens, N Juff ermans, W Van den Bergh, W Lagrand, B Preckel, J Horn Academic Medical Centre, University of Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P279 (doi: 10.1186/cc10886) Table 1 (abstract P280). P277 An independent data safety monitoring board reviewed all problems arising from the helium ventilation itself and all fatalities. Poor outcome was assessed with the Glasgow Outcome Score at 30 days: death and vegetative state were defi ned as poor outcome. Data are presented as mean ± SD or numbers and proportions. References 1. Holzer M, et al.: Crit Care Med 2005, 33:414-418. 2. Hay A, et al.: Anaesthesia 2008, 63:15-19. References 1. Holzer M, et al.: Crit Care Med 2005, 33:414-418. 2. Hay A, et al.: Anaesthesia 2008, 63:15-19. Therapeutic hypothermia in an out-of-hospital arrest population: are we selecting appropriately? The decrease in cerebral oxygenation during induction of TH was not associated with a major change in hemodynamic parameters (MAP before induction of TH: 79 mmHg ± 19; at 33°C: 82 mmHg ± 9), nor with a major change in systemic oxygenation (SpO2 before TH: 99% ± 1; at 33°C: 97% ± 3). In patients who survived until hospital discharge, SctO2 returned to baseline values, 3.5 hours after induction of TH, before the target temperature of 33°C was reached. In patients who did not survive the hospital stay, SctO2 remained lower than baseline values until the target temperature was reached. In these nonsurvivors, SctO2 values did only return to baseline values during maintenance of TH (10 hours after induction of TH). During maintenance of TH and rewarming (0.3°C), no further signifi cant changes in SctO2 values were observed. P282 Comparison of cold crystalloid and colloid infusions for induction of therapeutic hypothermia RS Skulec, AT Truhlar, ZT Turek, RP Parizkova, VC Cerny Charles University in Prague, University Hospital Hradec Kralove, Czech Republic Critical Care 2012, 16(Suppl 1):P282 (doi: 10.1186/cc10889) p Critical Care 2012, 16(Suppl 1):P282 (doi: 10.1186/cc10889) Introduction While cold crystalloids have been used for induction of therapeutic hypothermia after cardiac arrest [1,2], the eff ectiveness of cold colloids has not so far been evaluated. Therefore, we investigated the cooling eff ect of rapid intravenous infusion of cold crystalloid compared to colloid in a porcine model of ventricular fi brillation (VF). Methods VF was electrically induced in 22 anesthetized domestic pigs (33 ± 2 kg). Defi brillation was attempted after 15 minutes CPR using the AutoPulse (Zoll Medical, USA) and artifi cial ventilation. After spontaneous circulation was restored, the animals were randomized to receive either 1,500 ml of 1°C cold normal saline (group A; n = 9) within 20 minutes using a Zoll Power Infuser, or 1,500 ml of 1°C cold Voluven (6% hydroxyethyl starch 130/0.4 in 0.9% NaCl) (group B; n = 9), or no infusion (group C; n = 4). The animals were observed for 90 minutes following infusion. Cerebral, rectal, intramuscular, pulmonary artery, and subcutaneous fat body temperatures (BT) were continuously recorded using GES 130 temperature probes and GMH 3250 digital thermometers (Greisinger Electronic, Germany). Data were analyzed with JMP 3.2 software (SAS Institute, USA) and are expressed as a mean ± SD. P <0.05 was considered statistically signifi cant. Survey on the management of patients treated with therapeutic hypothermia post cardiac arrest in London hospitals A Walecka, SC Robert, A Prasad The Royal Free Hospital, London, UK Critical Care 2012, 16(Suppl 1):P284 (doi: 10.1186/cc10891) y gi Results In total, 46.6 ± 3.2 ml/kg cold normal saline was infused in group A, and 45.7 ± 2.7 ml/kg cold colloid in group B. The animals treated with cold fl uids achieved a signifi cant decrease of BT in all measurement sites while there was a spontaneous increase in group C (P <0.05). At the time of fi nishing infusion there was a greater decrease in cerebral and pulmonary artery BT in group A compared to group B (–1.7 ± 0.4 vs. –1.1 ± 0.3 °C, P = 0.002; and –2.1 ± 0.3 vs. –1.6 ± 0.2 °C, P <0.001 respectively). Area under the curve analysis of the decrease in intracerebral BT revealed a more vigorous cooling eff ect in group A compared to B (–91 ± 30 vs. –62 ± 27 °C/minute, P = 0.047). There was also a higher calculated enthalpy for crystalloid solution compared to colloid in the time-point of maximal BT decrease (33.9 ± 5.7 vs. 26.6 ± 3.4 kJ/kg, P <0.05). A Walecka, SC Robert, A Prasad The Royal Free Hospital, London, UK Introduction The second UK national survey on therapeutic hypothermia (TH) post cardiac arrest demonstrated an impressive increase in its implementation across the UK (from 28% to 85.6%) [1]. Therapeutic hypothermia, however, induces numerous physiological and pathophysiological changes and therefore should be performed in a standardised and controlled manner in order to be safe and eff ective. We carried out a telephone survey to determine the current TH practice in London hospitals. p Methods Thirty-two London intensive care units (ITUs) were contacted by telephone. The data were analysed using Excel spreadsheets. Conclusion Cold crystalloid infusions resulted in a more intense cooling eff ect than colloid infusions of the same temperature and infusion rate in this porcine model of cardiac arrest. by telephone. The data were analysed using Excel spreadsheets. Results Of the 32 ITUs contacted, 30 (93.7%) had been using therapeutic hypothermia following cardiac arrest. Fifteen (50%) of them were teaching hospitals and the remaining 15 (50%) were district general hospitals. Twenty-two (73.3%) hospitals had a protocol in place. External cooling was the preferred method used by 28 (93.3%) hospitals. Therapeutic hypothermia in an out-of-hospital arrest population: are we selecting appropriately? A Short, M Brett, L Donaldsoni Glasgow Royal Infi rmary, Glasgow, UK p Results A total of 13 patients with average GCS of 3.4 at 1 hour after ROSC were enrolled in the study. Average time for ROSC was 16.5 minutes. Demographic and baseline variables were comparable amongst survivors and nonsurvivors except age (survivors 43 years and nonsurvivors 65 years). Average duration to achieve target temperature was 4.9 hours. Five out of 13 (38.46%) patients survived without any neurological defi cit or cognitive dysfunction (Cerebral Performance Category – 1). Out of eight nonsurvivors, six died due to cardiogenic shock, one died due to refractory hypoxia and in one case relatives opted for withhold of aggressive care. Cardiac arrest was out of hospital in eight patients (three survivors and fi ve nonsurvivors) and intra-hospital in fi ve (two survivors and three nonsurvivors). Introduction We question how appropriately we select patients to undergo therapeutic hypothermia following out-of-hospital cardiac arrest. Methods The population was identifi ed through searching Wardwatcher between August 2006 and February 2011. Inclusion criteria were all patients with an ICU admission of out-of-hospital cardiac arrest. Exclusion criteria were: no CPR within the preceding 24 hours; admission from theatre; insuffi cient data. Data were gathered from Wardwatcher, Careview and patients’ case notes for age, arrest rhythm, downtime (DT) – time from arrest to return of spontaneous circulation, time to initiation of CPR, temperatures at various time points, cause of arrest and outcome. Statistical analysis was performed with Fisher’s exact test, signifi cance level of P <0.05. Permission for use of patient notes was granted from the consultant group of the ICU audited. i Conclusion TH may have benefi cial eff ects in the neurological outcome of patients having non-VT/VF cardiac arrest. Additional controlled studies are warranted to establish effi cacy of TH as a treatment for non- VT/VF cardiac arrest. S102 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P282 impairment. Temperature at admission was 34.6°C (±0.5°C). Patients reached the target temperature of 33°C, 4 hours after induction of TH. Two patients died during maintenance of TH due to refractory hemodynamic shock. In all patients, SctO2 values started above 65%. Two and a half hours after induction of TH, SctO2 values decreased with 9% (±3%). P284 S Survey on the management of patients treated with therapeutic hypothermia post cardiac arrest in London hospitals A Walecka, SC Robert, A Prasad The Royal Free Hospital, London, UK Critical Care 2012, 16(Suppl 1):P284 (doi: 10.1186/cc10891) Survey on the management of patients treated with therapeutic hypothermia post cardiac arrest in London hospitals A Walecka, SC Robert, A Prasad The Royal Free Hospital, London, UK Critical Care 2012, 16(Suppl 1):P284 (doi: 10.1186/cc10891) The target temperature varied from 32 to 35ºC with two (6.7%) ITUs targeting a temperature of 32ºC, 11 (36.7%) of 33ºC, six (20%) of 34ºC, one (10%) of 32 to 33ºC, seven (23.4%) of 32 to 34ºC and one (10%) of 34 to 35ºC. The time of cooling varied between 12 and 48 hours. The cooling period was measured from initiation of cooling by 22 (73.3%) ITUs and from achievement of target temperature by six (20%) ITUs. Two responders were not sure how it was measured. Twenty-fi ve (83.3%) units measured core temperature during the cooling. Passive rewarming was used by 20 (66.6%) responders. Twenty-four (80%) units maintain normothermia post therapeutic hypothermia. From additional aspects of the management of the induced hypothermia, 20 (66.6%) ITUs adjusted drug doses while starting TH, 15 (50%) monitored the depth of sedation, and 17 (56.6%) regularly checked train of four in paralyzed patients. Shivering was treated with sedation and paralysis by 25 (83.3%) of responders. Pregnancy status of all women younger than 50 years old was checked by 10 (33.3%) units. Fifteen (50%) units do not audit the practice regularly.i Results Of the 32 ITUs contacted, 30 (93.7%) had been using therapeutic hypothermia following cardiac arrest. Fifteen (50%) of them were teaching hospitals and the remaining 15 (50%) were district general hospitals. Twenty-two (73.3%) hospitals had a protocol in place. External cooling was the preferred method used by 28 (93.3%) hospitals. Acknowledgements The study was supported by grant MZO 00179906. References 1. Kim F, et al.: Circulation 2007, 115:3064-3070. 1. Kim F, et al.: Circulation 2007, 115:3064-3070. 2. Skulec R, et al.: Crit Care 2010, 14:R231. 2. Skulec R, et al.: Crit Care 2010, 14:R231. Cerebral oxygenation during induction of therapeutic hypothermia after cardiac arrest I Meex, J Dens, F Jans, C De Deyne I Meex, J Dens, F Jans, C De Deyne y Ziekenhuis Oost-Limburg, Genk, Belgium y Ziekenhuis Oost-Limburg, Genk, Belgium g g Critical Care 2012, 16(Suppl 1):P283 (doi: 10.1186/cc10890) Critical Care 2012, 16(Suppl 1):P283 (doi: 10.1186/cc10890) Introduction Induced mild hypothermia (32 to 34°C) improves survival and neurological outcome after CA. Near-infrared spectroscopy (NIRS) measures cerebral tissue oxygen saturation (SctO2). As of today, no data are available on SctO2 monitoring during therapeutic hypothermia (TH). Therefore, SctO2 was measured in this study during the fi rst 36 hours after CA. Methods After IRB approval, data were collected from 23 patients. Cold saline (30 ml/kg) was administered as soon as possible after hospital admission. TH (33°C) was induced by endovascular or surface cooling and maintained for 24 hours. All patients were sedated (propofol/ remifentanil) for the duration of TH. NIRS sensors were bilaterally applied to the frontotemporal area before start of TH. Patients were monitored during induction, maintenance and recovery of TH. Results Of 23 patients, 11 patients did not survive until hospital discharge due to post-ischemic brain damage. Twelve patients survived until hospital discharge, of whom eight without any neurological Methods After IRB approval, data were collected from 23 patients. Cold saline (30 ml/kg) was administered as soon as possible after hospital admission. TH (33°C) was induced by endovascular or surface cooling and maintained for 24 hours. All patients were sedated (propofol/ remifentanil) for the duration of TH. NIRS sensors were bilaterally applied to the frontotemporal area before start of TH. Patients were monitored during induction, maintenance and recovery of TH. Conclusion There are signifi cant variations in practice between London hospitals which probably refl ect the ongoing debate on the optimal management of patients treated with TH. Of note is that 50% of surveyed hospitals do not audit the current practice regularly which may have an impact on the quality and eff ectiveness of therapeutic cooling. g Reference y Results Of 23 patients, 11 patients did not survive until hospital discharge due to post-ischemic brain damage. Twelve patients survived until hospital discharge, of whom eight without any neurological Therapeutic hypothermia in an out-of-hospital arrest population: are we selecting appropriately? Conclusion Noninvasive monitoring revealed a decrease in cerebral oxygenation during induction of mild hypothermia in patients after cardiac arrest. We observed a diff erence in oxygenation between hospital survivors and nonsurvivors. Binks AC, et al.: Therapeutic hypothermia after cardiac arrest – implementation in UK intensive care units. Anaesthesia 2010, 65:260-265. Reference . Binks AC, et al.: Therapeutic hypothermia after cardiac arrest – implementation in UK intensive care units. Anaesthesia 2010, 65:260-265. S103 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P285 Simplifi ed EEG/aEEG to monitor the injured brain after cardiac arrest H Friberg1, M Rundgren1, E Westhall1, N Nielsen2, T Cronberg1 1Lund University, Lund, Sweden; 2Helsingborg Hospital, Helsingborg, Sweden Critical Care 2012, 16(Suppl 1):P285 (doi: 10.1186/cc10892) maintained for 48 hours and rewarmed to 36°C over another 48 hours. As induction of patients’ sedation, we injected 5 mg midazolam and 0.2 μg fentanyl intravenously just as we recognized patients’ movement or immediately before induction of TH. For maintenance of sedation, midazolam at dose 0.1 mg/kg/hour, dexmedetomidine at dose 0.4 μg/ kg/hour and fentanyl at doses 0.8 μg/kg/hour were administrated continuously. The midazolam and the dexmedetomidine infusion were adjusted to a target BIS value of 40 or less. BIS monitoring was ceased after completion of both rewarming and discontinuation of sedative drugs. maintained for 48 hours and rewarmed to 36°C over another 48 hours. As induction of patients’ sedation, we injected 5 mg midazolam and 0.2 μg fentanyl intravenously just as we recognized patients’ movement or immediately before induction of TH. For maintenance of sedation, midazolam at dose 0.1 mg/kg/hour, dexmedetomidine at dose 0.4 μg/ kg/hour and fentanyl at doses 0.8 μg/kg/hour were administrated continuously. The midazolam and the dexmedetomidine infusion were adjusted to a target BIS value of 40 or less. BIS monitoring was ceased after completion of both rewarming and discontinuation of sedative drugs. Introduction Once hemodynamics is stabilized, the main concern in the comatose cardiac arrest patient is the status of the brain and the potential recovery of brain functions. Approximately 30% of comatose cardiac arrest patients develop electrographic seizures, many of whom have associated clinical seizures that may be concealed by sedation and paralyzers. As part of the Lund coma project, we have continuously monitored and evaluated simplifi ed EEG/aEEG in consecutive hypothermia-treated cardiac arrest patients. g Results In all six patients, TH was completed without severe complication, especially shivering movement and serious hypostatic pneumonia. Three patients presenting unstable BIS values lower than 10 during TH showed poor neurological outcome, while the other three patients presenting stable BIS values about 40 showed favorable neurological outcome. Usefulness of a Bispectral index oriented sedative method without neuromuscular blocker for therapeutic hypothermia after cardiac arrest Usefulness of a Bispectral index oriented sedative method withou neuromuscular blocker for therapeutic hypothermia after cardiac arrest S Shiraishi, Y Ohta, T Tagami, Y Ono, T Masuno, H Yokota Nippon Medical School, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P286 (doi: 10.1186/cc10893) g p Results There were 33 PROSC patients: 27 (81%) survived and 14 (42%) achieved a favorable neurological outcome. The cause of the CPA was cardiac attack in 17, noncardiac attack in 10, and unknown in six patients. Average age in the favorable recovery group was signifi cantly younger than in the poor recovery group (62.5 vs. 70.3, P <0.05). The favorable group was all the proportion of patients with ventricular fi brillation (VF) at the scene. Of the 14 that achieved a favorable neurological outcome, the cause of the CPA was cardiac attack in 12 and unknown in two patients. On the other hand, electrocardiograms of poor neurological outcome showed VF, pulseless electrical activity, and asystole. The cause of the CPA was cardiac attack in fi ve, noncardiac attack in 10, and unknown in four. Average pH of artery blood gas (ABG) in the favorable recovery group was signifi cantly higher than in the poor recovery group (7.31 vs. 7.17, P <0.004). The receiver-operator characteristic curve for pH of ABG on arrival was analyzed. The area under the curve was 0.76. S Shiraishi, Y Ohta, T Tagami, Y Ono, T Masuno, H Yokota Nippon Medical School, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P286 (doi: 10.1186/cc10893) Introduction During therapeutic hypothermia (TH) after cardiac arrest (CA), neuromuscular blockers are often used to prevent or treat thermogenic shivering [1]. But the following risks due to neuromuscular paralysis are encountered: prolonged muscle weakness, hypostatic pneumonia and venous thromboembolism. So we evaluated the usefulness of Bispectral index (BIS) oriented sedation without neuromuscular blocker in six cases of post CA patients receiving TH. Methods Six consecutive patients admitted after CA and treated with TH by the same attending physicians’ group were included. BIS monitoring was applied immediately after the admission to ER. After initial resuscitation and radiological examination, including coronary angiography and angioplasty, patients were admitted to the ICU and cooled down to a target body temperature of 34°C using a surface cooling system with an external pad. Target body temperature was Introduction During therapeutic hypothermia (TH) after cardiac arrest (CA), neuromuscular blockers are often used to prevent or treat thermogenic shivering [1]. Reference Myoclonic movement or convulsion, regarded as signs of bad outcome, was observed in two poor neurological outcome patients. Cough refl ex was observed in two favorable neurological outcome patients throughout their TH. yp p Methods Needle electrodes corresponding to the F3 to P3 and F4 to P4 leads were applied at admission to the ICU. The Nervus NicoletOne® monitor (CareFusion Inc.) was used to display the continuous raw EEG curves as well as the amplitude integrated EEG (aEEG). The EEG data were available to the treating intensivist and were linked to the Department of Neurophysiology, where the accumulated data were interpreted once daily, 5 days a week. Conclusion BIS oriented sedation without neuromuscular blocker is feasible in maintaining TH for survivors from CA. By keeping muscular function, both noxious and benefi cial movements are preserved and these help us to predict neurological outcome and prevent patients from hypostatic disorders. R f Results Monitoring of aEEG was successfully applied in all patients. Four dominating patterns were defi ned; fl at, continuous, suppression- burst (SB) and electrographic status epilepticus (ESE) [1]. We identifi ed three groups of patients: one group with mild brain injury and a good outcome, characterized by a return of a continuous EEG pattern during the fi rst 24 hours. A second group with severe brain injury and a poor outcome had a fl at EEG or a SB pattern during the fi rst 24 hours, which evolved into alfa-coma or a treatment refractory ESE. In this group, early myoclonus was common. The third group with a presumed intermediate brain injury often developed a late ESE during rewarming, from a continuous and sometimes reactive background EEG. In this third group, which presented with low brain damage biomarkers and unremarkable MR brain imaging, there were survivors, some of whom received prolonged care in the ICU [2]. y Reference 1. Chamorro et al.: Anesth Analg 2010, 110:1328-1335. 1. Chamorro et al.: Anesth Analg 2010, 110:1328-1335. p References 1. Rundgren M, et al.: Crit Care Med 2010, 38:1838. 2. Cronberg T, et al.: Neurology 2011, 77:623. 1. Rundgren M, et al.: Crit Care Med 2010, 38:1838. Predictive factors of neurologic outcome in therapeutic hypothermia after prehospital return of spontaneous circulation Y Ohta, S Shiraishi, Y Ono, G Matsumoto, T Tagami, T Masuno, H Yokota Nippon Medical School, Tokyo, Japan Critical Care 2012 16(Suppl 1):P287 (doi: 10 1186/cc10894) Predictive factors of neurologic outcome in therapeutic hypothermia after prehospital return of spontaneous circulation Y Ohta, S Shiraishi, Y Ono, G Matsumoto, T Tagami, T Masuno, H Yokota Nippon Medical School, Tokyo, Japan C iti l C 2012 16(S l 1) P287 (d i 10 1186/ 10894) pp y p Critical Care 2012, 16(Suppl 1):P287 (doi: 10.1186/cc10894) Introduction Induction of hypothermia is generally accepted to improve neurologic recovery of out-of-hospital cardiopulmonary arrest (CPA). Early prognostication of post-CPA patients is challenging. The aim of the present study was to evaluate the predictive factors for neurologic outcome in out-of-hospital cardiac arrest patients who returned their spontaneous circulation in a prehospital setting (PROSC) and underwent therapeutic hypothermia (TH). p g Conclusion Simplifi ed EEG/aEEG is easily applied and well adapted to the ICU environment. In combination with the raw EEG, the aEEG serves as a trend monitor of the injured brain in the comatose patient after cardiac arrest. The simplifi ed EEG/aEEG helps detect ESE and is of importance for guiding anticonvulsive treatment. The evolution of the EEG pattern mirrors the natural recovery of cortical function after cardiac arrest and gives useful positive as well as negative prognostic information. Simplifi ed EEG/aEEG serves the needs of the intensivist and has the potential to become part of a standard monitoring regimen. References y Methods PROSC patients transported to our institution between January 2007 and May 2011 were retrospectively analyzed. TH was performed for all comatose PROSC patients admitted to the hospital for post-resuscitation care, regardless of the etiology of cardiac arrest or patient’s age, except for those whose hemodynamic and pulmonary status could not be maintained. Neurological outcome at 1 month was compared as a primary end-point using the Pittsburgh cerebral performance category (CPC) scale and patients were classifi ed into a favorable outcome group (CPC 1 and 2) or poor outcome group (CPC 3 to 5). Clinical parameters were compared between patients whose neurologic outcomes were favorable and poor. P290 P290 Noninvasive cerebral oxygenation monitoring during rapid ventricular pacing in transcutaneous aortic valve implant J Dens, I Meex, F Jans, H Gutherman, C De Deyne Ziekenhuis Oost-Limburg, Genk, Belgium Critical Care 2012, 16(Suppl 1):P290 (doi: 10.1186/cc10897) y Methods From 30 June 2004 to 30 June 2009, OHCA patients between 18 and 65 years of age treated with TIMH were identifi ed by the Danish National Patient Registry and intensive unit registrations. Data were collected from ambulance and hospital records. Employment status was registered prior to and 1 year after OHCA from the Danish Ministry of Employment and Welfare database, using fi ve work categories (WC): WC 1, working full-time and independent of any social welfare; WC 2, unemployed but able to work; WC 3, on sick leave and receiving social welfare; WC 4, substantially reduced ability to work: and WC 5, on early retirement.i Introduction Most recent attention in interventional cardiology is now directed towards treatment of valvular heart disease. In patients with high-risk cardiac surgery, transcutaneous aortic valve implantation (TAVI) could off er a therapeutic solution. Near-infrared spectroscopy (NIRS) has been introduced as a useful noninvasive cerebral monitoring technique assessing cerebral oxygenation. As of today, no reports have been published on the use of any NIRS technology during TAVI procedures. During valve prosthesis implantation, a cardiac standstill by rapid ventricular pacing (RVP) is induced to minimize cardiac motion. While RVP is advantageous for valve positioning, a combination of rapid heart rate and ventricular hypertrophy can induce a complete loss of cardiac output. In most cases, this hemodynamic defi cit is well tolerated, due to the brief duration of RVP. But as of today no data are available on cerebral oxygenation during these critical periods of RVP. Methods We report on 10 consecutive patients (>75 years, major comorbidities) suff ering from severe aortic stenosis. Bilateral ForeSight sensors were applied after induction of anesthesia. We were especially interested if any change in cerebral oxygenation (SctO2 monitoring) occurred during these RVP periods. Results One hundred and thirty-three patients were identifi ed. Forty eight patients were excluded from the fi nal analysis, of which 29 patients were not able to work at baseline (WC 3 to 5), 14 patients in WC 1 to 2 at baseline died in hospital, three patients died after hospital discharge and two patients had turned 65 years of age at follow-up and went on regular retirement. Usefulness of a Bispectral index oriented sedative method without neuromuscular blocker for therapeutic hypothermia after cardiac arrest But the following risks due to neuromuscular paralysis are encountered: prolonged muscle weakness, hypostatic pneumonia and venous thromboembolism. So we evaluated the usefulness of Bispectral index (BIS) oriented sedation without neuromuscular blocker in six cases of post CA patients receiving TH. p p g Methods Six consecutive patients admitted after CA and treated with TH by the same attending physicians’ group were included. BIS monitoring was applied immediately after the admission to ER. After initial resuscitation and radiological examination, including coronary angiography and angioplasty, patients were admitted to the ICU and cooled down to a target body temperature of 34°C using a surface cooling system with an external pad. Target body temperature was Conclusion A suitable pH at the time of hospital arrival was associated with a favorable neurologic outcome among post-cardiac arrest patients without presumed noncardiac etiology. S104 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P288 times the serum levels, and remained high after the peak of 23,500 pg/ ml at the time of admission. CSF and serum IL-10 levels were high, but not abnormally high as for IL-6 and IL-8, and decreased with time. The diff erence in CSF and serum levels, as seen for IL-6 and IL-8, was not seen for IL-10. P288 Employment status 1 year after out-of-hospital cardiac arrest in comatose patients treated with therapeutic hypothermia K Kragholm1, M Skovmoeller1, AL Christensen1, K Fonager2, HH Tilsted2, H Kirkegaard3, I De Haas1, BS Rasmussen1 1Cardiovascular Research Center, Aalborg, Denmark; 2Aarhus University Hospital, Aalborg, Denmark; 3Aarhus University Hospital, Skejby, Aarhus, Denmark Critical Care 2012, 16(Suppl 1):P288 (doi: 10.1186/cc10895) Conclusion We elucidated the following points concerning the acute infl ammatory response following severe traumatic brain injury. High levels of IL-6 and IL-8 are maintained in both CSF and serum. CSF levels of IL-6 and IL-8 are one or two orders of magnitude greater than serum levels. Upregulation of IL-10 is minimal in comparison with IL-6 and IL-8, suggesting that in neuroinfl ammation IL-10 functions poorly as an anti- infl ammatory cytokine. Introduction Therapeutic-induced mild hypothermia (TIMH) with a core temperature of 32 to 34°C for 12 to 24 hours for comatose survivors of out-of-hospital cardiac arrest (OHCA) with ventricular fi brillation or tachycardia has improved survival and neurologic outcome [1,2]. The aim of this study was to evaluate the incidence of patients returning to work 1 year after survival of OHCA treated with TIMH. References Results In all patients, the procedure was technically successfully performed. Mean SctO2 before RVP was 67% (59 to 71%) and immediately decreased during RVP to mean 54% (37 to 70%). In seven patients, RVP resulted in SctO2 decreases below 55% (mean 44%; range 37 to 52%). These decreases lasted for mean 20 minutes (14 seconds to 87 minutes). Systolic blood pressure before RVP was mean 135 mmHg (95 to 165 mmHg) and decreased to mean 74 mmHg (112 to 42 mmHg) during RVP. In six patients, RVP resulted in a decrease in systolic blood pressure below 90 mmHg, which was immediately countered by vasoactive drugs (adrenaline). In two patients, extensive hypotension persisted despite vasoactive support and CPR had to be initiated. In one patient, SctO2 values remained below 55% for 87 minutes and the patient was declared brain dead 48 hours later. 1. Bernard SA, Gray TW, Buist MD, Jones BM, Silvester W, Gutteridge G, Smith K: Treatment of comatose survivors of out of hospital cardiac arrest with induced hypothermia. N Engl J Med 2002, 346:557-563. 2. Hypothemia After Cardiac Arrest Study Group: Mild therapeutic hypothermia to improve the outcome after cardiac arrest. N Engl J Med 2002, 346:549-556. P291 P291 Changes in cerebrospinal fl uid and serum cytokine levels in severe traumatic brain injury patients Changes in cerebrospinal fl uid and serum cytokine levels in severe traumatic brain injury patients T Saito1, H Kushi2, J Sato1, A Yoshino1, K Tanjo1 1Nihon University, School of Medicine, Tokyo, Japan; 2Nihon University, College of Humanities and Sciences, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P289 (doi: 10.1186/cc10896) Conclusion Transcutaneous cardiac interventions, especially those with transient cardiac standstill, can induce longlasting intraprocedural inadequacy of cerebral perfusion, despite immediate restoration of normal blood pressure. Future strategies should therefore be focused on optimalizing cerebral oxygenation before RVP. T Saito1, H Kushi2, J Sato1, A Yoshino1, K Tanjo1 1Nihon University, School of Medicine, Tokyo, Japan; 2Nihon University, College of Humanities and Sciences, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P289 (doi: 10.1186/cc10896) Introduction Infl ammatory response following brain injury begins with brain tissue injury triggered neuroinfl ammation, which induces a systemic infl ammatory response syndrome. We investigated the characteristics of the acute infl ammatory response following severe traumatic brain injury through changes in cerebrospinal fl uid (CSF) and serum cytokine levels. P290 A total of 85 patients in WC 1 to 2 at baseline were included in the fi nal analysis, of which 55 (64.7%) of these initially comatose patients with OHCA treated with TIMH had returned to work 1 year after OHCA. Conclusion Approximately two-thirds of the survivors belonging to WC 1 to 2 at baseline have returned to work at 1 year follow-up after OHCA treated with TIMH. A larger study is needed to confi rm these results and to determine predictors of returning to work in comatose patients after OHCA treated with TIMH. References Table 1 (abstract P291). Model performance Elevated ICP GOS 1 to 2 static GOS 1 to 2 dynamic AUROC 0.87 0.72 0.90 HL P value 0.12 0.51 0.95 Brier scaled 39.4% 7.7% 46% Figure 1 (abstract P292). Receiver operating characteristic curve. Figure 1 (abstract P292). Receiver operating characteristic curve. Conclusion Dynamic data in continuous MAP and ICP monitoring allows prediction of elevated ICP. Adding information of the fi rst 24 hours of ICP and MAP to known risk factors allows accurate prediction of neurological outcome at 6 months. R f References 1. Piper I, et al.: Acta Neurochir 2003, 145:615-628. 2. Murray GD, et al.: J Neurotrauma 2007, 24:329-337. 3. CRASH Trial Collaborators: BMJ 2008, 336:425-429. 1. Piper I, et al.: Acta Neurochir 2003, 145:615-628. 2. Murray GD, et al.: J Neurotrauma 2007, 24:329-337. Novel models to predict elevated intracranial pressure during intensive care and long-term neurological outcome after TBI F Guiza1, B Depreitere1, I Piper2, G Van den Berghe1, G Meyfroidt1 1UZ Leuven, Belgium; 2Southern General Hospital, Glasgow, UK Critical Care 2012, 16(Suppl 1):P291 (doi: 10.1186/cc10898) Novel models to predict elevated intracranial pressure during intensive care and long-term neurological outcome after TBI F Guiza1, B Depreitere1, I Piper2, G Van den Berghe1, G Meyfroidt1 1UZ Leuven, Belgium; 2Southern General Hospital, Glasgow, UK Critical Care 2012, 16(Suppl 1):P291 (doi: 10.1186/cc10898) Methods The subjects were 24 patients with severe traumatic brain injury. We measured levels of the proinfl ammatory cytokines IL-6 and IL-8, and the anti-infl ammatory cytokine IL-10 in peripheral blood and CSF on four occasions, at the time of admission and after 24 hours, 72 hours and 1 week. Introduction Elevated intracranial pressure (ICP) episodes are associated with poor outcome and should be prevented. We developed models to predict these episodes 30 minutes in advance, and to predict long-term neurological outcome by using dynamic characteristics of continuous ICP and mean arterial pressure (MAP) monitoring. Introduction Elevated intracranial pressure (ICP) episodes are associated with poor outcome and should be prevented. We developed models to predict these episodes 30 minutes in advance, and to predict long-term neurological outcome by using dynamic characteristics of continuous ICP and mean arterial pressure (MAP) monitoring. Results CSF and serum IL-6 levels continued to rise until 72 hours after admission. CSF IL-6 levels were 50 to 400 times serum levels. Serum IL-8 levels remained at 20 to 30 pg/ml. CSF IL-8 levels were 100 to 800 S105 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 h // f / l /16/S1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P291). Elevated ICP episode. achieve favourable outcome. In the literature the value of noninvasive measurement of transcranial Doppler (TCD)-derived pulsatility index (PI) in predicting increased intracranial pressure remains questionable. The aim of this study was to examine the value of PI in predicting hydrocephalus in patients with aSAH. Methods The Brain-IT [1] dataset has records for 264 patients from 22 neuro-ICUs in 11 European countries. Logistic regression and Gaussian processes (machine learning method) were used. CRASH [2] and IMPACT [3] predictors were used together with dynamic data. Results Predictions of elevated ICP episodes (Figure 1) were externally validated with good calibration and discrimination (AUROC 0.87). Prediction of poor neurological outcome at 6 months (GOS 1 to 2) with static data had 0.72 AUROC; adding dynamic information increased performance to 0.9 (Table 1). Novel models to predict elevated intracranial pressure during intensive care and long-term neurological outcome after TBI F Guiza1, B Depreitere1, I Piper2, G Van den Berghe1, G Meyfroidt1 1UZ Leuven, Belgium; 2Southern General Hospital, Glasgow, UK Critical Care 2012, 16(Suppl 1):P291 (doi: 10.1186/cc10898) Methods In a retrospective cohort study from January 2010 to June 2011, 61 patients with aSAH were diagnosed with hydrocephalus on CT scan during treatment in our ICU. On 93 occasions of TCD recordings of the middle cerebral artery, PI was calculated on the same day. y, y Results See Table 1 and Figure 1. Ninety-three CT scans could be correlated with PI on the same day of the scan. Using a cut-off value Transcranial cerebral oximetry in newborn infants on mechanical ventilation as a method for prevention of hyperoxia and oxidative stress P295 Deoxyhaemoglobin as a biomarker of cerebral autoregulation D Highton1, A Ghosh1, I Tachtsidis2, C Kolyva2, J Panovska2, C Elwell2, M Smith1 1National Hospital for Neurology and Neurosurgery, London, UK; 2UCL, London, UK Critical Care 2012, 16(Suppl 1):P295 (doi: 10.1186/cc10902) Deoxyhaemoglobin as a biomarker of cerebral autoregulation D Highton1, A Ghosh1, I Tachtsidis2, C Kolyva2, J Panovska2, C Elwell2, M Smith1 1National Hospital for Neurology and Neurosurgery, London, UK; 2UCL, London, UK Critical Care 2012, 16(Suppl 1):P295 (doi: 10.1186/cc10902) Introduction Cerebral autoregulation (CA) maintains cerebral blood fl ow over a range of perfusion pressure. Continuous CA monitoring might defi ne pressure targets minimising secondary brain injury, but application is limited by available monitoring modalities. Near-infrared spectroscopy (NIRS) is a noninvasive optical technique characterising aspects of CA. The NIRS-derived tissue oxygenation index (TOI) is correlated with blood pressure (BP) to produce an index of vascular reactivity (TOx) [1]. The contribution from extracerebral tissues, optical complexity of injured brain and complex physiology represented by NIRS are likely to limit agreement with other techniques. NIRS- measured deoxyhaemoglobin (HHb) may have advantages as its physiological confounds are less complicated and are predominantly in the cerebral venous circulation. This study compares HHb with established indices of reactivity – the mean velocity index (Mx) and oxygen reactivity index (ORx). Results Determined by the age norm, TCO indicators for healthy infants amounted in the left hemisphere of the brain to 79.2 ± 4.06% (P <0.01), and in the right hemisphere to 84.89 ± 5.1% (P <0.01). We established in the group of infants where the mode selection ventilation and FiO2 were carried out on the basis of TCO indicators, an average FiO2 in the inspired mixture of 21% with an average pO2 in blood capillaries 61.95 ± 20.16%, in contrast to FiO2 55% with pO2 –78.01 ± 18.93% in patients of group 2. Patients of group 1 showed signifi cantly (in all cases P <0.01) decreased length of stay on mechanical ventilation (from 9.4 to 5.6 bed-days), compared with the control group. Investigation of the activity markers of oxidative stress showed three times reduction of the oxidation products of proteins (AOPP) and twofold reduction of peroxides in patients in the study group, compared with the control group, to 10 days of observation (P <0.05). Methods Thirteen brain-injured patients were studied. Transcranial cerebral oximetry in newborn infants on mechanical ventilation as a method for prevention of hyperoxia and oxidative stress V Estrin, A Simonova Scientifi c Research Institute of Obstetrics and Pediatrics, Rostov on Don, Russia Critical Care 2012, 16(Suppl 1):P293 (doi: 10.1186/cc10900) Critical Care 2012, 16(Suppl 1):P293 (doi: 10.1186/cc10900) Introduction The aim of this research was the eff ectiveness of respiratory therapy in infants with respiratory distress syndrome (RDS) who are ventilated by correcting the oxygen status and parameter optimization of ventilation by determining the oxygen saturation in the brain by transcranial cerebral oximetry (TCO). y Conclusion In this small cohort of patients, the onset of brain death was accompanied in all cases by a sharp and large decrease in SctO2 from 67% to 55% (mean, n = 5) and remained stable. SctO2 values only reached minimal values (25%, n = 3) at complete circulatory arrest. Our data suggest that SctO2 measurement may be helpful in the timing of the diagnosis of brain death, especially in those patients without ICP or continuous EEG monitoring. y y Methods A total of 24 infants born in the physiological department of a maternity hospital in RNIIAP was studied. All of the children were measured for the saturation of brain tissue oxygen (SctL, SctR) with a cerebral oximeter (ForeSight; USA) at 1, 3 and 5 days after birth. Later in the study, two groups of newborn infants on mechanical ventilation were included. Patients of group 1 (n = 38), modes of mechanical ventilation and FiO2 were determined under control TCO to bring rates of cerebral oxygenation to the age norm. Patients of group 2 (n = 37), mode selection and ventilator FiO2 were carried out under the control of pulse oximetry and partial oxygen tension (pO2) in acid–base balance, without regard for performance of TCO. In all patients were determined serum peroxides (Oxystat test; BIOMEDICA GRUPPE, Germany), as well as the oxidation products of proteins (AOPP) in the serum of a set of AOPP (Immunodiagnostik, USA) for 1, 5 and 10 days. We measured blood gas parameters with an automatic analyzer (ABL; Denmark). Transcranial Doppler pulsatility index is a poor predictor of hydrocephalus in patients with aneurysmal subarachnoid haemorrhage Negative and positive predictive values were 71.6% resp. 58.3%. The receiver operating characteristic curve showed an area under the curve of 0.67. The likelihood ratio for a negative (LR–) resp. positive (LR+) test was 0.83 resp. 2.94. Pretest probability of 32% increased to 57% post- test probability with PI >1.4 and decreased to 28% with PI ≤1.4. Conclusion PI with a cut-off value of 1.4 has a poor sensitivity and a high specifi city. PI has limited value in ruling in and out hydrocephalus in aSAH patients due to a low LR+ and LR–. yi Methods We retrospectively analyzed the cerebral oximetry data of seven patients with severe TBI or diff use cerebral edema who evolved to brain death while being treated in the ICU. Absolute SctO2 values were continuously measured with ForeSight technology (Casmed) with sensors applied bilaterally to the forehead. pp y Results Three patients (one TBI and two SAH) with continuous ICP and SctO2 monitoring suff ered, despite maximal medical treatment, a sudden (over 1 to 3 hours) increase in mean ICP from 32 mmHg to 91 mmHg (equalization of ICP and MAP). Over the same time period, a parallel decrease in mean SctO2 from 71% to 54% ICP was observed. One patient (cerebral edema after asphyxia) had continuous EEG and SctO2 monitoring: a sharp decrease in SctO2 from 67% to 56% over 30 minutes was accompanied by an increase in suppression ratio from 70% to 100%. The absence of cerebral blood fl ow was confi rmed by CT angiography. One patient (cerebral edema after prolonged CPR) had only SctO2 measurement for cerebral monitoring: during his stay in the ICU, there was a sudden decrease in SctO2 from 64% to 54% over a 90-minute period. Shortly after this, the pupils became dilated and fi xed. Brain death was confi rmed by full EEG. Three brain-dead patients with documented absence of cerebral blood fl ow were monitored for SctO2 during subsequent organ donation procedure: SctO2 remained at a mean value of 59% during the procedure, and fell sharply only at the onset of circulatory arrest to reach a stable value of 25%. P293 Transcranial cerebral oximetry in newborn infants on mechanical ventilation as a method for prevention of hyperoxia and oxidative stress V Estrin, A Simonova Scientifi c Research Institute of Obstetrics and Pediatrics, Rostov on Don, Russia Critical Care 2012, 16(Suppl 1):P293 (doi: 10.1186/cc10900) Transcranial Doppler pulsatility index is a poor predictor of hydrocephalus in patients with aneurysmal subarachnoid haemorrhage g MH Kiel, AW Oldenbeuving, M Sluzewski, JA Van Oers, D Ramnarain St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P292 (doi: 10.1186/cc10899) MH Kiel, AW Oldenbeuving, M Sluzewski, JA Van Oers, D Ramnarain St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2012, 16(Suppl 1):P292 (doi: 10.1186/cc10899) Introduction Hydrocephalus is a common complication of aneurysmal subarachnoid haemorrhage (aSAH). The increase in intracranial pressure is associated with increased mortality and morbidity. Early recognition and intervention in these patients is essential in order to Figure 1 (abstract P292). Receiver operating characteristic curve. S106 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P292). The 2×2 table for PI 1.4 Index test Hydrocephalus + Hydrocephalus – Total PI >1.4 7 5 12 PI ≤1.4 23 58 81 Total 30 63 93 of PI >1.4, sensitivity was low (23.3%) and specifi city was high (92.1%). Negative and positive predictive values were 71.6% resp. 58.3%. The receiver operating characteristic curve showed an area under the curve of 0.67. The likelihood ratio for a negative (LR–) resp. positive (LR+) test was 0.83 resp. 2.94. Pretest probability of 32% increased to 57% post- test probability with PI >1.4 and decreased to 28% with PI ≤1.4. Conclusion PI with a cut-off value of 1.4 has a poor sensitivity and a high specifi city. PI has limited value in ruling in and out hydrocephalus in aSAH patients due to a low LR+ and LR–. Table 1 (abstract P292). The 2×2 table for PI 1.4 Index test Hydrocephalus + Hydrocephalus – Total PI >1.4 7 5 12 PI ≤1.4 23 58 81 Total 30 63 93 P294 Cerebral oximetry and brain death in the ICU: data from seven cases N Billet1, I Meex2, M Vanderlaenen1, R Heylen1, W Boer1, C De Deyne1, FV Jans 1 1Ziekenhuis Oost-Limburg, Genk, Belgium; 2University of Hasselt, Diepenbeek, Belgium Critical Care 2012, 16(Suppl 1):P294 (doi: 10.1186/cc10901) g Critical Care 2012, 16(Suppl 1):P294 (doi: 10.1186/cc10901) Introduction Cerebral oximetry, using near-infrared spectroscopy to measure cerebral tissue oxygen saturation (SctO2), is being increasingly used in the ICU. We hypothesized that if a patient becomes brain dead in the ICU, this must be refl ected in SctO2 values. This might help in the timing of invasive procedures such as angiography, sometimes necessary in the confi rmation of brain death. of PI >1.4, sensitivity was low (23.3%) and specifi city was high (92.1%). Study of the acoustic stem evoked potentials in blood circulation disorder in the vertebral basilar basin I Vlasova, T Vizilo, V Tsiuriupai p Scientifi c Clinical Center of Miners’ Health Protection, Leninsk-Kuznetsky, Russia Methods ICU patients mechanically ventilated for at least 3 days were included. Exclusion criteria: polynomic or autonomic neuropathy, admission after stroke or cardiac arrest. HRV was investigated using power spectral analysis of continuous 5-minute ECG recordings [1]. The simulated SWT was used and wrinkling was assessed on a fi ve-point scale [2]. Under continuous ECG recording a cold pack was applied to measure the CFT [3]. Changes in SWT and CFT results over time were compared to the changes in the SOFA score. Studies procedures were also performed in 17 healthy controls. Critical Care 2012, 16(Suppl 1):P296 (doi: 10.1186/cc10903) Introduction Acoustic brainstem evoked potentials (ABEP) off er a possibility to objectivise disorder of the brain stem structure function.l Introduction Acoustic brainstem evoked potentials (ABEP) off er a possibility to objectivise disorder of the brain stem structure function. Methods There were fl icks of 9.5 Hz with intensity 70 dB higher than the hearing threshold. The latency time of the I to V peaks, the interpeak intervals (IPI), the peak amplitudes (PA) and the amplitude correlations were measured. The clinical neurophysiological assessment of 30 patients (16 men and 14 women, age from 40 to 70 years) with clinical presentation of ischemic stroke in the vertebral basilar basin (VBB) allowed us to determine the following forms of acute ischemic disorders of the brain circulation: transitory ischemic attacks (TIA) (n = 16), lacunar infarction (LI) (n = 10), and nonlacunar infarction (NLI) in VBB (n = 4). p y Results Twelve patients were included (mean age: 54 (SD: 15)). HRV analysis showed decreased heart rate variability in all patients (median total power: 32 ms2 (IQR: 11 to 320)). The SWT could be performed in 10 patients. SWT results were abnormal (score ≤2) in 60% of cases (6% in healthy controls; P <0.01). The CFT was done in nine patients. Critically ill patients showed a blunted response on the CFT (2.5% increase in RR length (95% CI: –0.2% to 5.2%) vs. 7.1% in healthy controls (95% CI: 3.7% to 10.5%; P = 0.03)). Figure 1 displays the CFT results over time. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Photonics). ORx and Mx were derived from continuous correlation between BP and neuromonitoring [1]. HHb was compared identically deriving HHBx. Comparisons used Pearson correlation, subsequent analysis characterised time lags between BP and monitored variables (0.05 to 0.003Hz) with wavelet lag coherence. of the brainstem were observed. There was a tendency to increase of the I to III and III to V intervals in 46 to 61% in TIA. The I to III and III to V IPI were signifi cantly increased in LI and NLI, in 35% and 47% cases respectively. The patients with NLI demonstrated an increase of the I to V IPI. There was such neurophysiological dynamics. The reconstruction of the amplitude and peak latency in TIA was observed in 100% of cases in the treatment process. This was not registered in LI and NLI. g Results There was correlation between HHBx (r = –0.62, P <0.01), ORx (r = 0.52, P <0.05) and Mx. TOx showed no signifi cant correlation (r = 0.18) as individual recordings demonstrated TOI fl uctuations paradoxical to other monitoring. The mean lag between BP and HHb (24 seconds) was shorter than PbrO2 (68 seconds). p g Conclusion All strokes in the VBB are characterized by functional changes on the part of the brain stem structures predominantly at the pontomedullary and pontomesencephalic levels. There is a dependence between stroke severity, brainstem structure damage and neurophysiological dynamics. ABEP allow one to objectivise the brain stem structure dysfunction in the VBB’s disturbed circulation. 2 Conclusion HHb may provide a surrogate to inform cerebrovascular reactivity assessment. Complexity in the oxyhaemoglobin component of TOI may be introduced by vasopressor-related skin artefact or arterial volume changes [2] explaining poor agreement of TOx. HHb is theoretically free of this eff ect but will vary with cerebral metabolism, venous dynamics and oxygenation and demonstrates lag behind BP changes. Future analyses might compensate using model-based analysis [3], potentially describing measures of vascular reactivity from multiple NIRS and neuromonitoring variables, incorporating widely diff erent aspects of cerebral physiology. P297 Examination of the autonomic nervous system in the ICU: a pilot study y L Wieske, E Kiszer, C Verhamme, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P297 (doi: 10.1186/cc10904) L Wieske, E Kiszer, C Verhamme, IN Van Schaik, MJ Schultz, J Horn Academic Medical Center, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P297 (doi: 10.1186/cc10904) f References References 1. Zweifel C, et al.: Stroke 2010, 41:1963-1968. 2. Ogoh S, et al.: Clin Physiol Funct Imaging 2011, 31:445-451. 3. Banaji M, et al.: PLoS Comput Biol 2008, 11:e1000212. Introduction The most widely used test for autonomic dysfunction in the ICU is the heart rate variability (HRV) test [1]. HRV is thought to be a very sensitive but less specifi c test [1]. Several other tests are available. For this pilot study we have investigated the ability of two tests, the skin wrinkle test (SWT), a test for postganglionic sympathetic function, and the cold face test (CFT), a refl ex slowing heart rate after cold application to the forehead, to detect autonomic dysfunction in critically ill patients alongside the HRV. Transcranial cerebral oximetry in newborn infants on mechanical ventilation as a method for prevention of hyperoxia and oxidative stress Ipsilateral 60-minute recordings included intracranial pressure, brain tissue oxygen (PbrO2), transcranial Doppler and NIRS (NIRO 100; Hamamatsu Conclusion Monitoring of oxygen saturation in the brain tissue by TCO in infants with RDS reduces the mortality rate and the term of mechanical ventilation and hyperoxia. S107 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review Among those, one was found to be a duplicate publication. Seven studies were thus included (n = 404). Four studies presented data on mortality (3 or 6 months) and four studies used the GOS (6 or 12 months) as an outcome measure. We found signifi cant associations between serum GFAP levels and mortality in pooled analysis of three studies (GMR 14.73 (95% CI 5.93 to 34.12); I2 = 79%), and between GFAP and GOS ≤3 in three studies (GMR 8.80 (95% CI 3.94 to 19.66); I2 = 77%). Two studies could not be used in pooled analyses: one presented means of GFAP levels from multiple samplings over time (GMR 1.98 (95% CI 1.06 to 3.70)) while the other presented the highest peak levels of GFAP during the acute phase of care (GMR 3.20 (95% CI 1.82 to 5.65)). i y Results Among 12,514 citations, we included 55 studies (4,648 patients). Patients suff ered from mild (11.9%, n = 555), moderate (7.9%, n = 367) and severe (30.4%, n = 1,415) TBI, others being of unknown severity. Prevalences of pituitary axis dysfunction are reported in Table 1. Few studies considering mainly moderate/severe TBI patients were at low risk of bias. Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review j y y F Lauzier1, O Lachance1, B Senay2, I Côté2, P Archambault2, F Lamontagne3, A Boutin1, L Moore1, F Bernard4, C Gagnon2, D Cook5, AF Turgeon1 1CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 2Université Laval, Québec, Canada; 3Université de Sherbrooke, Canada; 4Université de Montréal, Canada; 5McMaster University, Hamilton, Canada Critical Care 2012, 16(Suppl 1):P299 (doi: 10.1186/cc10906) Introduction Biomarkers have been proposed as potential prognostic indicators following a traumatic brain injury (TBI). Among those, glial fi brillary acidic protein (GFAP) has been one of the most studied. The objective of this study was to assess the prognostic value of GFAP levels in patients with moderate to severe TBI. Introduction Pituitary disorders are an often-neglected consequence of traumatic brain injury (TBI). We systematically reviewed their prevalence in studies with low risk of bias including moderate/severe TBI patients. Methods We systematically searched Medline, Embase, Cochrane Central, Scopus, BIOSIS, TRIP, conference abstracts, bibliography of selected studies and narrative reviews. Cohort studies including ≥4 patients with moderate or severe TBI and reporting GFAP levels (sampled within the fi rst 24 hours of care) from any biological tissue or fl uid, and mortality or Glasgow Outcome Scale (GOS), were eligible. Two independent reviewers screened all citations, selected eligible studies and extracted data using a standardized data extraction form. Pooled results from random eff ect models are presented using geometric mean ratios (GMRs). I2 tests were used to measure statistical heterogeneity. Methods We searched EMBASE, MEDLINE, Scopus, Cochrane Central Register, BIOSIS, Trip Database, references of included studies and narrative reviews. We included cohort studies, cross-sectional studies and RCTs that tested the integrity of ≥1 pituitary axis in adult victims of TBI. Two investigators independently reviewed selected citations, extracted data and assessed the risk of bias. Studies including <10% of mild TBI victims were considered as involving mainly moderate/severe TBI patients. Prevalence is reported as weighted mean (lowest and highest prevalence) in three time-frames: acute (<1 month post TBI), mid (3 to 12 months) and long-term setting (>12 months). Studies were considered at low risk of bias if the authors defi ned inclusion/exclusion criteria, avoided voluntary sampling, and tested >90% of included patients with proper detailed diagnostic criteria. Studies testing all pituitary axes were considered as evaluating hypopituitarism, which was defi ned as the dysfunction of at least one axis. Results We retrieved 4,709 citations and eight studies were deemed potentially eligible. Study of the acoustic stem evoked potentials in blood circulation disorder in the vertebral basilar basin P298 Predictive value of glial fi brillary acidic protein for prognosis in patients with moderate and severe traumatic brain injury: a systematic review and meta-analysis E Laroche1, AF Turgeon1, A Boutin1, E Mercier1, F Lauzier1, R Zarychanski2, L Moore1, J Granton3, P Archambault1, F Lamontagne4, F Rousseau1, F Légaré1, E Randell5, J Lapointe1, J Lacroix6, D Fergusson7 1Université Laval, Québec, Canada; 2University of Manitoba, Canada; 3University of Toronto, Ontario, Canada; 4Université de Sherbrooke, Québec, Canada; 5Memorial University, NewFoundland, Canada; 6Université de Montréal, Québec, Canada; 7Ottawa Hospital Research Institute, Ontario, Canada Critical Care 2012, 16(Suppl 1):P298 (doi: 10.1186/cc10905) Study of the acoustic stem evoked potentials in blood circulation disorder in the vertebral basilar basin Results According to the ABEP the common feature in all groups of patients was the decrease of the correlation of the V PA to I PA that was signifi cant in 56% cases in NLI, in 47% cases in LI and in 15% cases in TIA; the decrease of all PA (to 0.12 to 0.15 mkV) was signifi cant in 49% cases in NLI and in 39% cases in LI. A distinct tendency to the laterality of the peak latency increase in TIA and LI in 49% of cases, and a signifi cant laterality of the peak latency increase in 35% that refl ected the dissymmetric disorder of the neuronal acoustic activity y Conclusion CFT detected autonomic dysfunction in critically ill patients better than the SWT and was easier to perform. Diagnostic accuracy and prognostic value need to be investigated. References 1. Buchman TG, et al.: Curr Opin Crit Care 2002, 8:311-315. 2. Wilder-Smith EP, et al.: Clin Neurophysiol 2009, 120:953-958. 3. Reyners AK, et al.: Eur J Appl Physiol 2000, 82:487-492. 1. Buchman TG, et al.: Curr Opin Crit Care 2002, 8:311-315. 2. Wilder-Smith EP, et al.: Clin Neurophysiol 2009, 120:953-958. 3. Reyners AK, et al.: Eur J Appl Physiol 2000, 82:487-492. 1. Buchman TG, et al.: Curr Opin Crit Care 2002, 8:311-315. Figure 1 (abstract P297). Changes in cold face test (CFT) results over time. Figure 1 (abstract P297). Changes in cold face test (CFT) results over time. S108 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Serum GFAP levels following TBI were signifi cantly higher in patients showing an unfavourable prognosis (death or GOS ≤3). The small number of studies included precluded further exploration of statistical heterogeneity. More investigations of the association between serum GFAP levels and prognosis following TBI are needed before recommending for routine use for neuroprognostication. Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review Factors other than methodological quality and TBI severity are likely to explain the observed wide prevalence ranges. The clinical signifi cance of TBI- associated pituitary disorders also requires further rigorous evaluation. anticoagulants for 4 weeks along with the induction therapy. They were assessed for; their clinical presentation, disease severity (progressive or nonprogressive), hospital course, adverse eff ects of the used treatment and outcome. Reports of their neuroimaging studies were also collected. Results Studied patients were 42 (62.76%) boys and 26 (38.23%) girls. Their mean age was 8.5 ± 3.5 years. The commonest presenting symptoms were motor defi cit (70%), headache (64%) and fever (20%), while the commonest presenting neurological signs were hemiparesis (60%), seizure 55% (focal 35%, generalized 20%), and decreased level of consciousness (30%). Neuroradiological studies of the brain revealed: ischemic strokes in 50 children (73.5%), hemorrhagic strokes in 10 (14.7%) and ischemic–hemorrhagic lesions in eight (11.8%). Conventional angiography (CA) and/or magnetic resonance angiography (MRA) at the time of admission revealed that 51 (75%) patients had nonprogressive and 17 (25%) had evidence of progressive arteriopathy. Out of the studied patients, 56 (81.5%) survived and 12 (18.5%) died. Male sex, deep coma and intracerebral bleeding causing severe raised intracranial pressure were poor prognostic signs. Survivors were discharged on oral aspirin and 15 of them commenced also on azathioprine. On follow-up it was found that out of the 56 survivors, 11 were normal (19.65%), 14 (25%) had minor disabilities, another 11 (19.65%) had moderate disabilities and 20 (35.7%) had severe disabilities. P300 Mannose binding lectin defi ciency attenuates neurobehavioral defi cits following experimental traumatic brain injury L Longhi1, F Orsini2, N Fedele2, N Stocchetti1, MG De Simoni2 1University of Milano, Milan, Italy; 2Mario Negri Institute, Milan, Italy Critical Care 2012, 16(Suppl 1):P300 (doi: 10.1186/cc10907) Introduction Mannose binding lectin (MBL) is the activator of the lectin complement pathway. After cerebral ischemia it has been shown that MBL could be a mediator of secondary brain damage, in contrast after traumatic brain injury (TBI) there are data suggesting that it could be linked to neuroprotection. We tested the hypothesis that MBL is involved in the pathophysiology of TBI. We characterized (1) the temporal activation of MBL and (2) the eff ects of its inhibition in a model of experimental TBI. Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review Table 1 (abstract P299) Hypopituitarism GH ACTH TSH Gonadal ADH Acute phase All studies, 58.3% (32.3 to 76.1), 28.7% (8.8 to 77.2), 14.3% (0.7 to 45.7), 9.4% (0 to 40.6), 44.3% (7.7 to 91.7), 12.6% (0 to 27.2), n (patients) 6 (513) 9 (784) 11 (958) 12 (837) 10 (827) 13 (1821) Low risk of bias, 71.3% (52.9 to 76.1), 36.8% (8.8 to 77.2), 14.5% (0.7 to 23.6), 10.1% (1.6 to 32.6), 54.3% (23.5 to 80.0), 18.8% (0 to 27.2), n (patients) 3 (216) 5 (389) 4 (385) 6 (523) 5 (337) 5 (739) + moderate/severe, 70.0% (52.9 to 74.3), 36.1% (8.8 to 77.2), 14.5% (0.7 to 23.6), 5.2% (1.6 to 14.7), 61.4% (23.5 to 80.0), 23.8% (0 to 27.2), n (patients) 2 (170) 4 (321) 4 (385) 4 (406) 3 (220) 4 (303) Mid-term All studies, 32.1% (8.9 to 56.4), 14.8% (6.3 to 25.0), 9.7% (0 to 50.0), 4.3% (0 to 22.2), 18.8% (0 to 66.7), 3.8% (0 to 14.0), n (patients) 9 (608) 11 (643) 12 (669) 11 (629) 15 (792) 11 (691) Low risk of bias, – 12.1% (6.3 to 22.2), 16.7% (4.2 to 50.0), 6.1% (0 to 22.2), 25.2% (0 to 56.3), 11.2% (8.3 to 14.0), n (patients) 5 (231) 4 (215) 5 (231) 5 (218) 2 (98) + moderate/severe, – 12.1% (6.32 to 22.2), 16.7% (4.2 to 50.0), 6.1% (0 to 22.2), 25.2% (0 to 56.3), 8.3% (–), n (patients) 5 (231) 4 (215) 5 (231) 5 (218) 1 (48) Long-term All studies, 29.1% (0.9 to 73.3), 15.0% (0 to 51.8), 10.2% (0 to 64.4), 6.3% (0 to 31.8), 12.2% (0 to 50.0), 2.7% (0 to 18.2), n (patients) 19 (1418) 27 (1966) 26 (1782) 25 (1698) 25 (1798) 17 (1108) Low risk of bias, 31.1% (–), 16.6% (7.2 to 28.0), 6.8% (0 to 18.8), 8.2% (1.0 to 20.0), 12.9% (1.5 to 29.3), 5.0% (0 to 6.9), n (patients) 1 (45) 8 (499) 6 (369) 10 (734) 9 (707) 3 (200) + moderate/severe, 31.1% (–), 15.7% (7.2 to 21.7), 7.3% (1.4 to 18.8), 8.6% (1.0 to 20.0), 12.7% (1.5 to 29.3), 6.7% (6.3 to 6.9), n (patients) 1 (45) 5 (381) 4 (301) 7 (616) 6 (589) 2 (150) S109 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Pituitary disorders frequently arise after TBI, but prevalence remains uncertain due to low overall quality of available data. Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review Methods (1) Male C57/Bl6 mice were subjected to intraperitoneal anesthesia (pentobarbital, 65 mg/kg) followed by the controlled cortical impact brain injury model of experimental TBI (injury parameters: velocity of 5 m/second and 1 mm depth of deformation). MBL immunostaining was evaluated at various time points after TBI: 30 minutes, 1, 6, 12, 24, 48, 96 hours and 1 week using anti MBL-A and MBL-C antibodies (n = 3). (2) The eff ects of MBL inhibition were evaluated by comparing functional and histologic outcomes in C57/ Bl6 mice (WT) and in MBL knockout (–/–) mice. Functional outcome was tested using the Composite Neuroscore and Beam Walk test weekly up to 4 weeks postinjury (n = 11). Histologic outcome was evaluated by calculating the contusion volume at 4 weeks postinjury (n = 6). Sham- operated mice received identical anesthesia without brain injury. Conclusion The spectrum of cPACNS includes progressive and non- progressive forms. Characteristic features on presentation may predict later progression and outcome; identify a distinct high-risk cPACNS cohort; and guide the selection of patients for immunosuppressive therapy. Further studies are required to substantiate our fi ndings. Changes of ribosomal protein S3 immunoreactivity and its new expression in microglia in the mice hippocampus afte lipopolysaccharide treatment JH Cho, CW Park, HY Lee, MH Won Kangwon National University, Chuncheonsi, South Korea Critical Care 2012, 16(Suppl 1):P302 (doi: 10.1186/cc10909) p j y Results We observed a robust MBL-positive immunostaining in the injured cerebral cortex starting at 30 minutes postinjury and up to 1 week, suggestive of an activation of this pathway following TBI. MBL was observed both at endothelial and tissue levels. Consistently, injured WT and MBL (–/–) mice showed neurological motor defi cits up to 4 weeks postinjury when compared to their sham controls. Notably, MBL (–/–) mice showed attenuated behavioral defi cits when compared to their WT counterpart at 2 to 4 weeks postinjury (P <0.01 for both Neuroscore and Beam Walk test). In contrast we observed similar contusion volumes at 4 weeks postinjury (WT = 15.6 ± 3.2 cm3 and MBL KO = 13.9 ± 3.2 cm3, P = 0.3). Introduction Lipopolysaccharide (LPS) has been commonly used as a reagent for a model of systemic infl ammatory response. Ribosomal protein S3 (rpS3) is a multifunctional protein that is involved in transcription, metastasis, DNA repair and apoptosis. Prevalence of pituitary disorders associated with traumatic brain injury: a systematic review In the present study, we examined the changes of rpS3 immunoreactivity in the mouse hippocampus after systemic administration of 1 mg/kg LPS. Conclusion We observed that: (1) MBL deposition and/or synthesis is increased following TBI; and (2) MBL defi ciency is associated with functional neuroprotection, suggesting that MBL modulation might be a potential therapeutic target after TBI. Methods Six-week-old male ICR mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). LPS (Sigma, St Louis, MO, USA) was dissolved in saline, and administered intraperitoneally with 1.0 mg/ kg/10 ml dose. The control animals were injected with the same volume of saline. Mice (n = 7 at each time point) were sacrifi ced at designated times (3, 6, 12, 24, 48 and 96 hours after LPS treatment). The brain tissues were cryoprotected by infi ltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30-μm coronal sections, and they were then collected into six-well plates containing 0.1 M PBS. 30 Azathioprine and aspirin in treatment of childhood primary arterial stroke: therapeutic benefi ts and side eff ects A Alhaboob, G Ahmed P303 Neuronal damage using Fluoro-Jade B histofl uorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils JH Cho, CW Park, HY Lee, MH Won Kangwon National University, Chuncheonsi, South Korea Critical Care 2012, 16(Suppl 1):P303 (doi: 10.1186/cc10910) P303 Neuronal damage using Fluoro-Jade B histofl uorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils P303 Neuronal damage using Fluoro-Jade B histofl uorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils g JH Cho, CW Park, HY Lee, MH Won Kangwon National University, Chuncheonsi, South Korea Critical Care 2012, 16(Suppl 1):P303 (doi: 10.1186/cc10910) Introduction Ischemic damage occurs well in vulnerable regions of the brain, including the hippocampus and striatum. In the present study, we examined neuronal damage/death and glial changes in the striatum 4 days after 5, 10, 15 and 20 minutes of transient cerebral ischemia using the gerbil. Spontaneous motor activity was shown to be increased with the duration time of ischemia–reperfusion (I-R). y yp Conclusion The experimental model of bilateral carotid artery occlusion and systemic hypotension-induced cerebral ischemia in pigs is a useful tool to investigate the mechanism of cerebral ischemia and/ or neuroprotection (medications, hypothermia, and so forth). Methods To examine neuronal damage, we used Fluoro-Jade B (F-JB, a marker for neuronal degeneration) histofl uorescence staining. F-JB- positive cells were detected only in the 20-minute ischemia group, not in the other groups. In addition, we examined gliosis of astrocytes and microglia using antiglial fi brillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. P305 Molecular, histological and microcirculatory modeling of cerebral ischemia in pigs Results Both N2O and xenon administration reduced the number of ischaemic neurons in the cortex. In xenon-treated rats, fewer ischaemic neurons were also observed in the CA1 region of the hippocampus. The xenon group demonstrated a signifi cant reduction of c-fos expression compared to control and N2O groups. See Figure 1. Molecular, histological and microcirculatory modeling of cerebral ischemia in pigs O Suchadolskiene1, A Pranckunas1, B Kumpaitiene1, P Dobozinskas1, Z Dambrauskas1, V Veikutis2, K Stasaitis1, G Baliutyte3, D Vaitkaitis1, V Borutaite3 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Institute of Neurosciences, Lithuanian University of Health Sciences, Kaunas, Lithuania Critical Care 2012, 16(Suppl 1):P304 (doi: 10.1186/cc10911) O Suchadolskiene1, A Pranckunas1, B Kumpaitiene1, P Dobozinskas1, Z Dambrauskas1, V Veikutis2, K Stasaitis1, G Baliutyte3, D Vaitkaitis1, V Borutaite3 O Suchadolskiene1, A Pranckunas1, B Kumpaitiene1, P Dobozinskas1, Z Dambrauskas1, V Veikutis2, K Stasaitis1, G Baliutyte3, D Vaitkaitis1, V Borutaite3 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Institute of Neurosciences, Lithuanian University of Health Sciences, Kaunas, Lithuania Figure 1 (abstract P305). Regulation of c-fos expression after administration of N2O or xenon. Critical Care 2012, 16(Suppl 1):P304 (doi: 10.1186/cc10911) Introduction Ischemic brain injury due to stroke and/or cardiac arrest is a major health issue in modern society requiring urgent development of new eff ective therapies. The use of appropriate animal models is essential to study the mechanisms of ischemia-induced injury and neuroprotection. The goal of our study was to establish a reliable and reproducible model of brain ischemia in pigs (with the ischemia- induced microcirculatory, mitochondrial and structural alterations) for further research. Methods Eighteen pigs (18 to 22 kg) were anesthetized and randomly assigned to the one of the following groups: 1 – control, 2 – unilateral carotid occlusion, 3 – bilateral carotid occlusion, 4 – bilateral carotid occlusion + hypotension (MAP 40 to 50 mmHg). In order to investigate the eff ects and mechanisms of cerebral ischemia, we assessed the mitochondrial respiration (high-resolution respirometry), microcirculation (in vivo SDF videomicroscopy) and histological structure (light microscopy) of brain tissue in healthy control animals and after 3 hours of brain ischemia (three diff erent models). Results LEAK respiration (measured in the presence of pyruvate + malate but without ADP) was not aff ected by ischemia in any model. P305 Delayed post-ischaemic administration of xenon reduces brain damage in a rat model of global ischaemia V Metaxa1, R Lagoudaki2, S Meditskou2, O Thomareis2, A Sakadamis2 1St Bartholomew’s Hospital, London, UK; 2Aristotle University, Thessaloniki, Greece Critical Care 2012 16(Suppl 1):P305 (doi: 10 1186/cc10912) Results In the 5-minute ischemia group, GFAP-immunoreactive astro cytes were distinctively increased in number, and the immuno- reactivity was stronger than that in the sham group. In the 10-minute, 15-minute and 20-minute ischemia groups, GFAP immunoreactivity was more increased with the duration of I-R. On the other hand, the immunoreactivity and number of Iba-1-immunoreactive microglia were distinctively increased in the 5-minute and 10-minute ischemia groups. In the 15-minute ischemia group, microglia were largest in size, and the immunoreactivity was highest; however, in the 20-minute ischemia group, the immunoreactivity was low compared to the 15-minute ischemia group. The results of western blotting for GFAP and Iba-1 were similar to the immunohistochemical data.i Introduction Cerebral ischaemia is among the leading causes of death, disability and economic expense in the world. Xenon has been shown to be neuroprotective both in vivo and in vitro, predominantly when administered as a preconditioning agent. We have used a rat model of global ischaemia to investigate whether xenon-induced neuroprotection is observed following an ischaemic insult. Methods Adult male Wistar rats underwent bilateral common carotid artery occlusion and were ventilated for 1 hour with 21% O2/78% N2. The animals were randomized to receive 21% O2/78% N2, 50% O2/50% N2O or 50% O2/50% xenon (n = 10). After a further 45 minutes, they were killed and their brains were removed for histological, immunochemical and molecular analysis. The numbers of ischaemic neurons in the cortex and the hippocampus as well as the expression of c-fos were evaluated on adjacent brain sections. Conclusion These fi ndings indicate that neuronal death was detected only in the 20-minute ischemia group 4 days after I-R; in addition, the change pattern of astrocytes and microglia were apparently diff erent according to the duration time of I-R. 30 Azathioprine and aspirin in treatment of childhood primary arterial stroke: therapeutic benefi ts and side eff ects A Alhaboob, G Ahmed p g Results From 6 hours after LPS treatment, rpS3 immunoreactivity was decreased in pyramidale cells of the hippocampus proper and granule cells of the dentate gyrus. At this point in time, rpS3 immunoreactivity began to increase in nonpyramidal cells and nongranule cells in the hippocampus. From 1 day after LPS treatment, rpS3 immunoreactivity in pyramidal and granule cells was hardly detected, and nonpyramidal and nongranule cells showed strong rpS3 immunoreactivity. Based on double immunofl uorescence staining, microglia, not astrocytes, expressed strong rpS3 immunoreactivity at 1 and 2 days after LPS treatment. King Khalid University Hospital and College of Medicine, King Saud University, Riyadh, Saudi Arabia Critical Care 2012, 16(Suppl 1):P301 (doi: 10.1186/cc10908) Introduction The objectives were to describe a cohort of children presenting with medium/large vessel childhood primary angiitis of the central nervous system (PACNS); to report their short-term neurological outcome; and to evaluate effi cacy and safety of implemented management. g Methods The study included 68 patients, aged less than 16 years. They had their symptoms within 14 days of admission. They received induction therapy with pulses of intravenous steroids and/ or intravenous immunoglobulin followed by maintenance therapy with azathioprine and low-dose aspirin. They were also treated with Conclusion These results indicate that changes in rpS3 immunoreactivity in pyramidal and granule cells and rpS3 expression in activated microglia after LPS treatment may be associated with the neuroinfl ammatory responses in the brain. S110 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P303 respectively, resulting in the decrease of RCI (ADP/PM) by 14% and 73%. The OXPHOS capacity with succinate as substrate remained constant after unilateral carotid artery occlusion but decreased by 53% after bilateral carotid artery occlusion and hypotension compared to the control level (P <0.05, n = 3 to 6). Mitochondrial respiration rates after addition of atractyloside and cytochrome c were the same in all experimental groups, suggesting that intactness of mitochondrial outer membrane was not aff ected by cerebral ischemia. Microcirculatory and histological alterations also demonstrated increasing derangement and reversible structural changes after bilateral carotid occlusion and vascular occlusion combined with systemic hypotension. Seizures in the respiratory ICU: single-center study of patients with new-onset seizures D Talwar, V Nair, J Chudiwal Metro Center for Respiratory Diseases, Noida, India Critical Care 2012, 16(Suppl 1):P306 (doi: 10.1186/cc10913) Seizures in the respiratory ICU: single-center study of patients with new-onset seizures D Talwar, V Nair, J Chudiwal Metro Center for Respiratory Diseases, Noida, India Critical Care 2012, 16(Suppl 1):P306 (doi: 10.1186/cc10913) Methods In this prospective, randomized, double-blind and placebo- controlled trial critically ill patients with clinical evidence for incipient CIP, a diagnosis of SIRS/sepsis and failure of at least two organ systems were randomized to be treated either with IgM-enriched IVIG or with human albumin 1% as placebo over a period of 3 days. The primary objective was to demonstrate that administration of IVIG prevents and/or mitigates CIP in critically ill patients, measured by electrophysiological stimulation of the median, ulnar and tibial nerves on days 0, 4, 7 and 14. Electrophysiological measures were graded according to compound muscle action amplitude size (CIP score) of the respective nerve. Secondary objectives were mortality from any cause within a 28-day period and lengths of ICU stay. D Talwar, V Nair, J Chudiwal Critical Care 2012, 16(Suppl 1):P306 (doi: 10.1186/cc10913) Introduction New-onset seizures in the ICU are a diagnostic and management challenge as patients have multiple comorbidities and receive various antibiotics. In the respiratory ICU with diff erent patient profi les, etiopathogenesis of seizures is unreported.i i g Methods We retrospectively analyzed the profi le of 3,342 patients admitted to the RICU from 2006 to 2011. A computerized search revealed 79 patients (2.4%) with new-onset seizures. Complete clinical, laboratory, radiological and treatment profi les were recorded and statistically analyzed using the chi-square test, odds ratio and relative risk of individual variable. Results Thirty-eight critically ill patients were included and randomized to either receiving IgM-enriched IVIG (n = 19) or placebo (n = 19). Baseline characteristics including CIP score on day 0 were similar between the two groups. CIP could not be improved signifi cantly by IVIG treatment for three consecutive days, represented by similar CIP scores of all three measured nerves on days 4, 7 and 14 in the IVIG and the placebo group. Mean CIP score levels of all three nerves signifi cantly increased from baseline to day 4 in both groups. Results Of 79 patients, 44 patients (55.7%) were males and the mean age was 61.28 ± 19.57 years. Severe sepsis was diagnosed in 32 (40.5%) and multiorgan failure in 19 (24.1%). Head CT done in 65 (82.3%) patients was reported abnormal in 34 (52.3%; P = 0.072) patients. Intracranial pressure monitoring in acute liver failure: a retrospective cohort study Intracranial pressure monitoring in acute liver failure: a retrospective cohort study C Karvellas1, O Fix2, H Battenhouse3, V Durkalski3, C Sanders4, W Lee4 1University of Alberta, Edmonton, Canada; 2UCSF, San Francisco, CA, USA; 3Medical University of South Carolina, Charleston, SC, USA; 4University of Texas-Southwestern, Dallas, TX, USA Critical Care 2012, 16(Suppl 1):P308 (doi: 10.1186/cc10915) Table 1 (abstract P306). Attributable causes of seizures in RICU cases (n = 79) Table 1 (abstract P306). Attributable causes of seizures in RICU cases (n = 79) Anoxia 8 10.1% Metabolic 15 19.0% Drugs only 16 20.3% CNS infection 5 6.3% Trauma 2 2.5% Alcohol 5 6.3% Multiple 22 27.8% Miscellaneous 6 7.6% , , , Critical Care 2012, 16(Suppl 1):P308 (doi: 10.1186/cc10915 Introduction Intracranial hypertension (ICH) complicates roughly 25% of acute liver failure (ALF) patients with grade III/IV encephalopathy. Intracranial pressure (ICP) monitoring is controversial due to complications in 5 to 20% and absence of documented mortality benefi t. i Methods Using prospectively collected data from the US Acute Liver Study Group registry, we reviewed 630 ALF patients with severe encephalopathy (grade III/IV) and INR >1.5 enrolled between 1 March 2004 through 31 August 2011. ICP monitoring was used in 143 patients (23%); 487 control patients with grade III/IV hepatic coma (n = 487) were not monitored. Conclusion New-onset seizure in RICU cases is multifactorial in origin. Use of levofl oxacin in combination had the highest relative risk of developing seizure although when given alone the risk is rare (2.1%). Severe sepsis with multiorgan failure being seen in nearly one-half of RICU cases may decrease seizure threshold in these patients. Results The most common etiology of ALF was acetaminophen (51%, P = 0.13 between groups). Of ICP monitored (ICPM) patients, 85% (n = 121) received devices within 24 hours of admission to study. ICPM patients were signifi cantly younger (36 ± 6 years vs. 43 ± 15 years, P <0.001) than controls, more likely to be on renal replacement therapy (48% vs. 31%, P <0.001) but less likely to be on vasopressors (20% vs. 32%, P = 0.008). ICPM patients were given more ICH directed therapies (mannitol 43% vs. 13%, hypertonic saline 21% vs. 6%, hypothermia 29% vs. 11%, P <0.001 for each comparison). For ICPM patients, the median INR on the day of monitor insertion was 2.2 (1.6 to 2.9) and platelet count 116 (84 to 171); 74% were given FFP (vs. Molecular, histological and microcirculatory modeling of cerebral ischemia in pigs The OXPHOS capacity with pyruvate + malate as substrates decreased by 20% and 79% compared to the control level after bilateral carotid artery occlusion and bilateral carotid occlusion + hypotension, Methods Eighteen pigs (18 to 22 kg) were anesthetized and randomly assigned to the one of the following groups: 1 – control, 2 – unilateral carotid occlusion, 3 – bilateral carotid occlusion, 4 – bilateral carotid occlusion + hypotension (MAP 40 to 50 mmHg). In order to investigate the eff ects and mechanisms of cerebral ischemia, we assessed the mitochondrial respiration (high-resolution respirometry), microcirculation (in vivo SDF videomicroscopy) and histological structure (light microscopy) of brain tissue in healthy control animals and after 3 hours of brain ischemia (three diff erent models). Results LEAK respiration (measured in the presence of pyruvate + malate but without ADP) was not aff ected by ischemia in any model. The OXPHOS capacity with pyruvate + malate as substrates decreased by 20% and 79% compared to the control level after bilateral carotid artery occlusion and bilateral carotid occlusion + hypotension, Figure 1 (abstract P305). Regulation of c-fos expression after administration of N2O or xenon. S111 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 of the mobilization phase. Although the exact etiopathogenesis has not yet been fully elucidated, sepsis, systemic infl ammatory response syndrome, and multiple organ failure seem to play an important role. CIP is diagnosed by signs of denervation in electromyography. Although there is no causal treatment for CIP, retrospective data suggest that early IgM-enriched intravenous immunoglobulin (IVIG) application may prevent or mitigate CIP. Therefore we aimed to investigate the use of IVIG in the early treatment of CIP in critically ill patients in a prospective, randomized, double-blind and placebo- controlled setting. of the mobilization phase. Although the exact etiopathogenesis has not yet been fully elucidated, sepsis, systemic infl ammatory response syndrome, and multiple organ failure seem to play an important role. CIP is diagnosed by signs of denervation in electromyography. Although there is no causal treatment for CIP, retrospective data suggest that early IgM-enriched intravenous immunoglobulin (IVIG) application may prevent or mitigate CIP. Therefore we aimed to investigate the use of IVIG in the early treatment of CIP in critically ill patients in a prospective, randomized, double-blind and placebo- controlled setting. Molecular, histological and microcirculatory modeling of cerebral ischemia in pigs Conclusion In our model of global cerebral ischaemia, the administration of xenon reduced the number of ischaemic neurons compared to control, both in the cerebral cortex and in the hippocampus. 1. Wilhelm S, et al.: Eff ects of xenon on in vitro and in vivo models of neuronal injury. Anesthesiology 2002, 96:1485-1489. Seizures in the respiratory ICU: single-center study of patients with new-onset seizures D Talwar, V Nair, J Chudiwal Metro Center for Respiratory Diseases, Noida, India Critical Care 2012, 16(Suppl 1):P306 (doi: 10.1186/cc10913) Lumbar puncture was done in 40 (50.6%) with fi ve (12.5%) patients having meningitis. Thirteen of 37 (35.1%) patients showed focal activity on EEG (P = 0.27; OR = 1.73). Electrolyte abnormalities were: hypermagnesemia in 20 patients (25.3%), hypocalcemia in 17 patients (21.5%), and hypernatremia in 13 patients (16.5%), hyponatremia in three patients (3.8%) and hypomagnesia in four (5.17%) cases. The antibiotics received revealed 27 (34.2%; RR = 1.27) patients on levofl oxacin alone or in combination. Twenty-eight of 79 (35.4%) patients were on carbapenems with meropenem in 23/79 (29.1%; RR = 1.21) and imipenem in 5/79 (6.32%; RR = 0.41). See Table 1. y g p Conclusion Results suggest that early treatment with IVIG neither signifi cantly improves CIP nor infl uences the length of stay or mortality in critically ill patients. Consistent with the literature, CIP deteriorated during the course of disease in critically ill patients with a diagnosis of SIRS/sepsis and failure of two organ systems. P308 Intracranial pressure monitoring in acute liver failure: a retrospective cohort study 46% controls, P <0.001) and 19% (vs. 14% controls, P = 0.14) received platelets. ICP monitoring was also strongly associated with listing (78% vs. 27%, P <0.001) and receipt of liver transplant (42% vs. 18%, P <0.001). Twenty-one-day mortality was similar between ICPM patients (33%) and controls (37%, Retrospective observation of 6-month survival following decompressive craniectomy in a London major trauma and stroke centre We will now prospectively collect these data including quality-of-life measures. References 1. Cooper DJ, et al.: N Engl J Med 2011, 364:1493-1502. 2. Hofmeijer J, et al.: Lancet Neurol 2009, 8:326-333. P310 Feasibility of a multicenter prospective cohort study on the evaluation of prognosis in severe traumatic brain injury AF Turgeon1, F Lauzier1, M Thibodeau1, A Rigamonti2, M Meade3, F Bernard4, K Burns2, K Reddy3, D Scales2, L McIntyre5, R Green6, D Griesdale7, L Moore1, M Savard1, D Jichici3, J Paquet1, D Zygun8, D Fergusson5, for the Canadian Critical Care Trials Group5 1Université Laval, Québec, Canada; 2University of Toronto, Ontario, Canada; 3McMaster University, Ontario, Canada; 4Université de Montréal, Québec, Canada; 5University of Ottawa, Ontario, Canada; 6University Dalhousie, Nova Scotia, Canada; 7University of British Columbia, Canada; 8University of Calgary, Alberta, Canada Critical Care 2012, 16(Suppl 1):P310 (doi: 10.1186/cc10917) Introduction Conducting prospective research in severe traumatic brain injury (TBI) patients is challenging To prepare for a large-scale Table 1 (abstract P309) P311 Table 1 (abstract P309) Neurosurgical/ Total Hospital 6-month MCA Year total DC survival survival TBI stroke Other 2006 292/1,839 2 2 2 1 0 1 2007 298/1,652 2 2 2 0 2 0 2008 286/1,563 11 10 8 (1N/A) 7 4 0 2009 493/1,840 18 11 10 7 8 3 2010 505/1,835 21 16 15 (1N/A) 11 9 1 2011 274/918 17 7 5 (2N/A) 7 10 0 5.5-year 2,148/9,647 71 48 42 (4N/A) 33 33 5 data Predictive value of neuron-specifi c enolase following moderate and severe traumatic brain injury: a systematic review and meta-analysis E Mercier1, AF Turgeon1, A Boutin1, F Lauzier1, R Zarychanski2, P Archambault1, J Granton3, F Lamontagne4, L Moore1, F Rousseau1, F Légaré1, E Randell5, J Lacroix6, J Lapointe1, D Fergusson7 1Université Laval, Québec, Canada; 2University of Manitoba, Canada; 3Université of Toronto, Ontario, Canada; 4Université de Sherbrooke, Québec, Canada; 5Memorial University, NewFoundland, Canada; 6Université de Montréal, Québec, Canada; 7Ottawa Hospital Research Institute, Ontario, Canada Critical Care 2012, 16(Suppl 1):P311 (doi: 10.1186/cc10918) Introduction Biomarkers such as the neuron-specifi c enolase (NSE) have been proposed as potential prognostic markers following traumatic brain injury (TBI) [1,2]. However, the use of NSE is not currently recommended for prognostic evaluation. Our objective was to systematically review the prognostic value of NSE levels following moderate or severe TBI. Conclusion Survival following DC in this institution compares favourably with published data. Reduced survival in 2011 may be a case-mix eff ect related to increased tertiary referrals. We will now prospectively collect these data including quality-of-life measures. References Methods We systematically searched MEDLINE, Embase, Cochrane, Biosis, Scopus, Trip, references of eligible studies, reviews and conference proceedings. Eligible studies were cohort studies including ≥4 patients with moderate or severe TBI having measured the association between NSE levels (fi rst 24 hours) and mortality or the Glasgow Outcome Scale (GOS). Independently, two reviewers selected studies and extracted data using a standardized form. Pooled results using random-eff ect models were used using weighted mean diff erences (WMD); heterogeneity was assessed using I2 tests. Sensitivity analyses were planned to explain statistical heterogeneity (for example, extracerebral injuries). P307 P307 Early treatment with intravenous immunoglobulins in patients with critical illness polyneuropathy: a randomized controlled, double- blinded study R Brunner, W Rinner, R Kitzberger, T Sycha, J Warszawska, U Holzinger, C Madl Medical University of Vienna, Austria Critical Care 2012, 16(Suppl 1):P307 (doi: 10.1186/cc10914) Introduction Critical illness polyneuropathy (CIP) is a severe complication of critical illness. The clinical features of CIP are muscle weakness and atrophy causing delayed weaning and prolongation S112 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P = 0.33) when all or only nontransplanted patients (46% vs. 45%, 0.8) were considered. Of 66 ICPM patients with detailed information, 18 (29%) had evidence of ICH (ICP >25 mmHg) at the time of ICPM insertion (maximum ICP on day 1 ~18 (12 to 26) mmHg). Of 49 patients with a known ICPM device, 14 patients received epidural catheters, six subdural, 11 intraparenchymal, seven intraventricular and 11 lumbar monitors. In only one of 49 ICPM patients was intracranial hemorrhage reported, and this patient survived. multicenter study to evaluate long-term prognosis in severe TBI, we conducted a prospective pilot study evaluating the patterns of enrollment, the compliance to the schedule of prognostic tests and the completeness of follow-up for 6-month functional outcome measures. Methods We conducted a pilot study in nine level I trauma centers in Canada. Adult patients with severe TBI expected to require mechanical ventilation for ≥48 hours were enrolled on their fi rst day in the ICU. Prognostic tests were performed on arrival (CT scan), day 1 (serum biomarker), day 3 (serum biomarker, CT scan) and day 7 (serum biomarker, CT scan, MRI, SSEP, EEG) with time windows of 24 or 48 hours depending on the test. Prognostic measures were collected during the fi rst week in the ICU to examine the association with the extended Glasgow Outcome Scale score. We considered as appropriate a compliance to the schedule of prognostic tests ≥90% and a proportion of lost to follow-up <10%. We obtained REB approval from participating centers and written informed consent from SDMs. multicenter study to evaluate long-term prognosis in severe TBI, we conducted a prospective pilot study evaluating the patterns of enrollment, the compliance to the schedule of prognostic tests and the completeness of follow-up for 6-month functional outcome measures. P310 Feasibility of a multicenter prospective cohort study on the evaluation of prognosis in severe traumatic brain injury AF Turgeon1, F Lauzier1, M Thibodeau1, A Rigamonti2, M Meade3, F Bernard4, K Burns2, K Reddy3, D Scales2, L McIntyre5, R Green6, D Griesdale7, L Moore1, M Savard1, D Jichici3, J Paquet1, D Zygun8, D Fergusson5, for the Canadian Critical Care Trials Group5 1Université Laval, Québec, Canada; 2University of Toronto, Ontario, Canada; 3McMaster University, Ontario, Canada; 4Université de Montréal, Québec, Canada; 5University of Ottawa, Ontario, Canada; 6University Dalhousie, Nova Scotia, Canada; 7University of British Columbia, Canada; 8University of Calgary, Alberta, Canada C iti l C 2012 16(S l 1) P310 (d i 10 1186/ 10917) P307 Methods We conducted a pilot study in nine level I trauma centers in Canada. Adult patients with severe TBI expected to require mechanical ventilation for ≥48 hours were enrolled on their fi rst day in the ICU. Prognostic tests were performed on arrival (CT scan), day 1 (serum biomarker), day 3 (serum biomarker, CT scan) and day 7 (serum biomarker, CT scan, MRI, SSEP, EEG) with time windows of 24 or 48 hours depending on the test. Prognostic measures were collected during the fi rst week in the ICU to examine the association with the extended Glasgow Outcome Scale score. We considered as appropriate a compliance to the schedule of prognostic tests ≥90% and a proportion of lost to follow-up <10%. We obtained REB approval from participating centers and written informed consent from SDMs. p p Conclusion In ALF patients, ICP monitor placement is strongly associated with liver transplantation but not with overall or transplant free mortality. In the absence of ICP monitoring, ALF patients may be less aggressively treated for intracranial hypertension. The value of ICP monitoring in ALF remains to be determined but ICPM placement clearly aff ects the frequency of interventions for elevated ICP. Retrospective observation of 6-month survival following decompressive craniectomy in a London major trauma and stroke centre Results Among 116 consecutive eligible patients, 50 were enrolled over a total of 204 weeks of screening between May 2010 and May 2011. Two centers used a deferred consent approach. Patients were primarily male with a median age of 45 years and a GCS of 5 (25th to 75th: 3 to 7). The two main reasons for nonenrollment were the time window for inclusion being after regular working hours (35%, n = 23) and oversight (24%, n = 16). The compliance to the diff erent tests ranged from 93 (three missing tests) to 100%. All blood samples but one (day 7) were performed. The main reason for missing a test was the patient’s instability (hemodynamic or increased ICP) (n = 5). In six patients, the MRI had to be delayed due to the presence of material not compatible with the procedure. No patient was lost to follow-up at 6 months. J Dawson, P Hopkins, J Ling, D Walsh, C Tolias King’s Health Partners, London, UK Critical Care 2012, 16(Suppl 1):P309 (doi: 10.1186/cc10916) Introduction This study describes 5.5 years of retrospective data examining hospital and 6-month outcome of patients following decompressive craniectomy (DC). The eff ectiveness of DC remains uncertain with confl icting results in patients with TBI and stroke [1,2]. Methods Data were drawn (1 January 2006 to 30 June 2011) from three hospital databases following approval by the institutional board. Results There were 2,148 neurosurgical admissions with 71 undergoing DC. Forty-eight of 71 (67.6%) survived to hospital discharge and 21/33 in both TBI and stroke groups survived to 6 months. See Table 1. Conclusion These results demonstrate the feasibility of enrollment and complying to a structured protocol of prognostic tests in a prospective multicenter study in severe TBI patients. Table 1 (abstract P309) Neurosurgical/ Total Hospital 6-month MCA Year total DC survival survival TBI stroke Other 2006 292/1,839 2 2 2 1 0 1 2007 298/1,652 2 2 2 0 2 0 2008 286/1,563 11 10 8 (1N/A) 7 4 0 2009 493/1,840 18 11 10 7 8 3 2010 505/1,835 21 16 15 (1N/A) 11 9 1 2011 274/918 17 7 5 (2N/A) 7 10 0 5.5-year 2,148/9,647 71 48 42 (4N/A) 33 33 5 data Conclusion Survival following DC in this institution compares favourably with published data. Reduced survival in 2011 may be a case-mix eff ect related to increased tertiary referrals. P312 Blood–brain barrier permeability following traumatic brain injury M Jungner, P Bentzer Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P312 (doi: 10.1186/cc10919) Blood–brain barrier permeability following traumatic brain injury M Jungner, P Bentzer Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P312 (doi: 10.1186/cc10919) Introduction There is substantial evidence to suggest that oxidative stress is associated with cerebral vasospasm following subarachnoid hemorrhage (SAH). Urinary 8-OHdG is the most common biomarker of DNA damage by oxidative stress. The aim of this study was to determine whether 8-OHdG is a good indicator of vasospasm occurrence following SAH. y Critical Care 2012, 16(Suppl 1):P312 (doi: 10.1186/cc10919) Introduction Brain edema and intracranial hypertension is deleterious after traumatic brain injury (TBI), but the underlying pathophysiology is complex and poorly understood. One major subject of controversy is the time course and extent of blood–brain barrier dysfunction following trauma, and previous studies in humans have only provided semi-quantitative data. The objective of the present study was therefore to quantify changes in blood–brain barrier permeability in the early course of TBI. Methods The subjects were 23 patients who received surgical clipping or endovascular coiling within 24 hours after the onset of SAH. We classifi ed the patients according to the occurrence of angiographic vasospasm. We examined the urinary 8-OHdG levels with high- performance liquid chromatography for 10 days following SAH. The urinary 8-OHdG levels were adjusted according to serum creatinine levels. Methods Seventeen nonconsecutive brain trauma patients and two controls were included in this prospective observational study. Following i.v. injection of iohexol and CT perfusion scans, patients were scanned eight times from 4 to 25 minutes. The blood-to-brain transfer constant (Ki) for iohexol, refl ecting permeability and area available for diff usion, was calculated by Patlak plot analysis of the enhancement curves of intracerebral large venous vessels and pericontusional brain parenchyma. Results The urinary 8-OHdG levels were elevated on day 2 compared with those on day 1 only in the vasospasm (+) group. The urinary 8-OHdG levels in the vasospasm (+) group were signifi cantly higher than those in the nonvasospasm (–) group on days 1, 2, 8 and 9. Furthermore, we examined the correlations between the urinary 8-OHdG levels on admission to the ICU and the grades of the World Federation of Neurologic Surgeons and Fisher, but none were observed. Discussion An elevated urinary 8-OHdG level on day 2 was observed only in the vasospasm group. Therefore, we speculated that free radicals may have a role in inducing vasospasm in the early phase following SAH. Feasibility of a multicenter prospective cohort study on the evaluation of prognosis in severe traumatic brain injury Results We retrieved 4,711 citations and included 22 studies (n = 757). Seventeen studies used the GOS as an outcome measure while 10 studies reported mortality. Most studies evaluated outcomes at 6 months or beyond (range: 1 to 12 months). Ten studies could not be included in the pooled analyses: three reported mean levels of serial samplings, two presented peak levels, two reported medians, one did not report any measure of dispersion and data could not be extracted from two studies. We observed a signifi cant association between serum NSE levels and mortality (fi ve studies: WMD 25.90 (95% Critical Care 2012, 16(Suppl 1):P310 (doi: 10.1186/cc10917) Introduction Conducting prospective research in severe traumatic brain injury (TBI) patients is challenging. To prepare for a large-scale S113 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 CI 15.97 to 35.83), I2 = 60%) and GOS ≤3 (10 studies: WMD 17.69 (95% CI 12.14 to 23.24), I2 = 64%). Similar results were found with or without extracerebral injuries. The number of studies included in pooled analyses precluded performing relevant sensitivity analyses.i References References 1. Patlak et al.: Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. J Cereb Blood Flow Metab 1983, 3:1-3. 2. Maeda et al.: Ultra-early study of edema formation in cerebral contusion using diff usion MRI and ADC mapping. Acta Neurochir Suppl 2003, 86:329-331. Conclusion We observed a signifi cant association between serum NSE levels and unfavorable outcomes (mortality or GOS ≤3) not infl uenced by extracerebral injuries. Further studies need to evaluate the usefulness of serum NSE levels for prognosis assessment in TBI and its potential impact on clinical decision-making. R f 1. Papa L, et al.: Use of biomarkers for diagnosis and management of traumatic brain injury patients. Exp Opin Med Diagn 2008, 2:937-945. P313 P313 Can urinary 8-OHdG be a good indicator of vasospasm occurrence following subarachnoid hemorrhage? K Ikeda, T Ikeda, H Taniuchi, S Suda, Y Ikeda, H Jimbo Tokyo Medical University, Hachioji Medical Center, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P313 (doi: 10.1186/cc10920) 2. Zitnay GA, et al.: Traumatic brain injury research priorities: the Conemaugh International Brain Injury Symposium. J Neurotrauma 2008, 25:1135-1152. 2. Zitnay GA, et al.: Traumatic brain injury research priorities: the Conemaugh International Brain Injury Symposium. J Neurotrauma 2008, 25:1135-1152. P312 Blood–brain barrier permeability following traumatic brain injury M Jungner, P Bentzer Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P312 (doi: 10.1186/cc10919) The urinary 8-OHdG levels were higher in the vasospasm group than in the nonvasospasm group, but we did not fi nd any correlation with severity of SAH. We suspect that the higher urinary 8-OHdG levels on days 8 and 9 in the vasospasm group indicated ischemic brain injury after vasospasm. Results Fourteen patients were included within 1 day and three were included within 5 days of the injury. In nonischemic tissue surrounding contusions and hematomas, Ki was focally increased in 11 of all included trauma patients and in six of seven patients with raised intracranial pressure. In noninjured areas and in controls, Ki was about 0.06 ml/ minute/100 g and increased by 100 to 2,000% in pericontusional tissue. See Figure 1. g Conclusion TBI is associated with early focal increases in blood–brain barrier permeability. The results suggest that in the injured brain, capillary hydrostatic and oncotic pressures are likely to infl uence edema formation. Conclusion We believe that oxidative stress has a role in the development of cerebral vasospasm and that urinary 8-OHdG may be a good indicator of vasospasm occurrence following SAH. Figure 1 (abstract P312). CBF (left) and permeability (right) maps, and contrast-enhanced CT scan (middle). igure 1 (abstract P312). CBF (left) and permeability (right) maps, and contrast-enhanced CT scan (middle). S114 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P314 Cortical capillary recruitment by rosuvastatin in experimental brain trauma is associated with increased NO production P Bentzer, M Jungner Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P314 (doi: 10.1186/cc10921) P314 P314 Cortical capillary recruitment by rosuvastatin in experimental brain trauma is associated with increased NO production P Bentzer, M Jungner Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P314 (doi: 10.1186/cc10921) Results We excluded two patients with bleeding for more than 72 hours. There was no signifi cant change in the levels of CK total, renal or liver function. We included 21 patients, 11 in the SVT group and nine in the control group. The mortality was eight patients (38%), six patients in the control group and two of the SVT group. Vasospasm was confi rmed by cerebral arteriography examination in four patients in the control group and one patient in the SVT group. All patients that had a bad outcome (death) had Fisher IV scale. P312 Blood–brain barrier permeability following traumatic brain injury M Jungner, P Bentzer Lund University, Lund, Sweden Critical Care 2012, 16(Suppl 1):P312 (doi: 10.1186/cc10919) Introduction Microvascular dysfunction, characterized by edema formation secondary to increased blood–brain barrier (BBB) permeability and decreased blood fl ow, contributes to poor outcome following brain trauma. Recent studies have indicated that statins may counteract edema formation following brain trauma but little is known about other circulatory eff ects of statins in this setting. The objective of the present study was to investigate whether statin treatment improves brain microcirculation early after traumatic brain injury, and whether microvascular eff ects are associated with altered production of nitric oxide and prostacyclin.l Conclusion SVT at a dose of 80 mg was eff ective in reducing the mortality (18.1% against 66%) compared to the group that did not use SVT, and also decreased the incidence of cerebral vasospasm despite the APACHE II score being higher in the group that used SVT (14.3 vs. 10.7). There was less morbidity in the SVT group with an average Glasgow Outcome Scale of 3.25 vs. 2.1. Eff ects of sinvastatin in prevention of vasospasm in nontraumatic subarachnoid hemorrhage: preliminary data Introduction Some trials have shown that statins in the acute phase of aSAH reduce the incidence, morbidity and mortality of cerebral vasospasm. Independent of their cholesterol-lowering eff ect, statins have multiple biological properties, including downregulating infl ammation and upregulating endothelial NO synthase. The purpose of this study is to evaluate the potential of sinvastatin (SVT) as prevention against vasospasm. Conclusion In women with preeclampsia signifi cant changes in ophthalmic hemodynamics take place – mFV in arterial and venous ophthalmics increases while PI values go down. This might be evidence of orbital hyperperfusion in preeclamptic pregnant women. Low PI values may be used as the markers of severe preeclampsia. Reference 1. Gosling RG, King DH: Arterial assessment by Doppler shift ultrasound. Proc R Soc Med 1974, 67:447-449. 1. Gosling RG, King DH: Arterial assessment by Doppler shift ultrasound. Proc R Soc Med 1974, 67:447-449. g Methods We realized a prospective study, randomized, nonblind, with the use of 80 mg SVT (night) in the fi rst 72 hours of the beginning of bleeding, and a control group that did not use SVT, for 21 days, between January and December 2008. Informed consent was obtained for all patients. CT scans were performed as control and another CT scan in patients with altered neurological signals. In the presence of changes suggestive of vasospasm or correlation in clinical and CT scans, the patients were taken for cerebral arteriography examination followed by an angioplasty procedure if necessary. Liver and renal function and LDL cholesterol were evaluated every 3 days. Exclusion criteria: liver and renal disease, pregnancy, elevation of serum transaminases (three times the value of normality), creatinine ≥2.5, rhabdomyolysis or CK total ≥1,000 U/l. P315f P315 Eff ects of sinvastatin in prevention of vasospasm in nontraumatic subarachnoid hemorrhage: preliminary data S Macedo, V Aguiar, PF Rosa, IT Ladeia, YK Castro, LA Ferreira, DR De Melo, LG Rezende São Jose do Avai Hospital, Itaperuna, Brazil Critical Care 2012, 16(Suppl 1):P315 (doi: 10.1186/cc10922) Evaluation of arterial and venous ophthalmic hemodynamics in preeclamptic pregnant women p p g M Shifman, NV Khramchenko, SV Sokologorskiy EM Shifman, NV Khramchenko, SV So EM Shifman, NV Khramchenko, SV Sokologorskiy Federal Centre for Obstetrics, Gynecology & Neonatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P316 (doi: 10.1186/cc10923) y Results Trauma resulted in brain edema, BBB dysfunction, and reduced cortical blood fl ow, and no eff ect of treatment on these parameters could be detected. Trauma also induced a reduction in the number of perfused capillaries, which was improved by statin treatment. Statin treatment led to increased plasma NOx levels and reduced mean arterial blood pressure. The 6-keto-PGF-1α levels tended to increase after trauma, and were signifi cantly reduced by rosuvastatin. , y gy gy, Critical Care 2012, 16(Suppl 1):P316 (doi: 10.1186/cc10923) Introduction The aim of the study was to evaluate arterial and venous ophthalmic blood fl ow parameters in mild and severe preeclampsia pregnancies and in normotensive pregnancies. Introduction The aim of the study was to evaluate arterial and venous ophthalmic blood fl ow parameters in mild and severe preeclampsia pregnancies and in normotensive pregnancies. Methods A total of 117 women 25 to 30 years old with singleton pregnancies 30 to 40 weeks of gestation were recruited. Among them 40 pregnant women developed severe preeclampsia, 42 mild preeclampsia, and 35 were normotensive. Using color fl ow mapping (CFM) and pulse-wave Doppler imaging (PWD), maximum blood fl ow velocity (mFV) in the right/left arterial and venous ophthalmics along with Gosling’s Doppler pulsatility index (PI) [1] in both arterial ophthalmics were evaluated. Mean blood pressure in all patients was also registered. gi y y Conclusion Rosuvastatin treatment improves microcirculation after traumatic brain injury by increasing the number of perfused capillaries. This eff ect is associated with increased NO and reduced prostacyclin production. R f p References 1. Béziaud et al.: Crit Care Med 2011, 39:2300-2307. 2. Cherian, Robertson: J Neurotrauma 2003, 20:77-85. 3. Prinz, Endres: Anesth Analg 2009, 109:572-584. Results The highest mFV values (59.2 ± 4.61 and 23.6 ± 4.03 cm/second) were in the severe preeclampsia group while in the mild preeclampsia group mFV increased slightly or remained normal (35.6 ± 2.97 and 13.6 ± 0.81 cm/second). There was no mFV increase in the normotensive pregnancy group (31.5 ± 2.21 cm/second). No signifi cant correlation was found between gestation age and mentioned hemodynamic parameters in the normotensive pregnancy group. PI values in the arterial ophthalmic in normotensive pregnant women were 2.92 ± 0.59 and the highest in all groups. In group with mild preeclampsia this parameter was 1.47 ± 0.30 and the lowest one was in patients with severe preeclampsia – 1.17 ± 0.08. g References 1. Lynch JR, Wang H, et al.: Simvastatin reduces vasospasm after aneurysmal subarachnoid hemorrhage: results of a pilot randomized clinical trial. Stroke 2005, 36:2024-2026. 1. Lynch JR, Wang H, et al.: Simvastatin reduces vasospasm after aneurysmal subarachnoid hemorrhage: results of a pilot randomized clinical trial. Stroke 2005, 36:2024-2026. p y Methods After fl uid percussion injury, rats were randomized to intravenous treatment with 10 mg/kg rosuvastatin or vehicle. Brain edema (wet/dry weight), BBB integrity (51Cr-EDTA blood to brain transfer), cerebral blood fl ow (14C-iodoantipyrine autoradiography), and the number of perfused cortical capillaries (FITC-albumin fl uorescence microscopy) were measured at 4 and 24 hours. Production of NO and prostacyclin was estimated by measuring the stable degradation products nitrite and nitrate (NOx), and 6-keto-PGF-1α in plasma. Sham injured animals were treated with vehicle and analyzed at 4 hours. 2. McGirt MJ, Lynch JR: Simvastatin increases endothelial nitric oxide synthase and ameliorates cerebral vasospasm resulting from subarachnoid hemorrhage. Stroke 2002, 33:2950-2956. Clinical outcomes in neonates following maternal magnesium sulfate therapy in preeclampsia/eclampsia G Tikhova, E Shifman Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P319 (doi: 10.1186/cc10926) Combined injury in occipital– frontal lobes (29.3%) and occipital–temporal (27.6%) lobes were observed in almost one-third of patients. Synchronous injury in the temporal and frontal lobes was the least common (6.9%). Simultaneous damage of three and more lobes was observed quite rarely (14.6%). Most abnormalities were bilateral with frequency not less than 78.0%. Unsymmetrical injury observed in some patients was located in the right lobe in most cases. All analyzed cases include only 7.1% of single left injury and all of them were located in the occipital lobe. Vasogenic edema occurred in 83.5% of cases, while ischemic damage was observed in 10.4%. The incidence of hemorrhage was 6.1%. Methods Trials were searched for in the PubMed database among English-language articles published in 1990 to 2010. Analysis includes randomized controlled prospective clinical trials comparing MST with no treatment, placebo or other anticonvulsant. The following neonatal outcomes were chosen as the main endpoints of the study: neonatal death, neonatal hypotonia, Apgar score <7 at 1 and 5 minutes, intuba- tion at place of delivery, admission to the NICU, treatment in NICU >7 days. The total eff ect of MST was measured as the relative risk of adverse outcome in the MST group compared with control and its 95% CI. Meta-analysis of neonatal outcomes was performed under a random-eff ect model for seven endpoints and a fi xed-eff ect model for three endpoints. p Results Neonatal mortality in the MST group was compared with diff erent control groups. Each of these studies showed no signifi cant diff erence between two groups: MST/mixed (0.89, 95% CI 0.80 to 0.99), MTS/no treatment-placebo (0.99, 95% CI 0.93 to 1.05), MTS/diazepam (1.09, 95% CI 0.91 to 1.29), MTS/fenitoin (0.75, 95% CI 0.56 to 1.02). The neonatal hypotonia rate is signifi cantly higher in the MST group (3.57, 95% CI 2.89 to 4.42), although signifi cant heterogeneity of the control group may be a valuable confounding factor. There was no evidence for changing incidence of Apgar <7 at 1 and 5 minutes in the MTS group compared with control (0.79, 95% CI 0.70 to 0.89 and 0.80, 95% CI 0.64 to 0.99 correspondingly). The same results were observed for intubation at place of delivery (1.04, 95% CI 0.90 to 1.29) and admission to NICU (0.96, 95% CI 0.85 to 1.08). Clinical outcomes in neonates following maternal magnesium sulfate therapy in preeclampsia/eclampsia G Tikhova, E Shifman Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P319 (doi: 10.1186/cc10926) Clinical outcomes in neonates following maternal magnesium sulfate therapy in preeclampsia/eclampsia G Tikhova, E Shifman Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P319 (doi: 10.1186/cc10926) Critical Care 2012, 16(Suppl 1):P319 (doi: 10.1186/cc10926) ji Methods We collected cases of neurological complications of eclampsia reported in English-language medical journals from 1980 to 2008. The study methods include structural and frequency analysis of brain MRT/ CT image protocols. Introduction Magnesium sulfate therapy (MST) is the method of choice in prophylaxis and treatment of eclamptic seizures in many countries. A lot of high-quality clinical trials and meta-analyses proved its effi cacy and safety for mothers. But the eff ect of maternal MST on the fetus and neonate is still controversial. The goal of the study was to analyze available trials concerning this problem in order to prove statistically that maternal MST given as prophylaxis or treatment of eclamptic seizures has no adverse eff ects on the mature fetus and term neonate. Methods Trials were searched for in the PubMed database among English-language articles published in 1990 to 2010. Analysis includes randomized controlled prospective clinical trials comparing MST with no treatment, placebo or other anticonvulsant. The following neonatal outcomes were chosen as the main endpoints of the study: neonatal death, neonatal hypotonia, Apgar score <7 at 1 and 5 minutes, intuba- tion at place of delivery, admission to the NICU, treatment in NICU >7 days. The total eff ect of MST was measured as the relative risk of adverse outcome in the MST group compared with control and its 95% CI. Meta-analysis of neonatal outcomes was performed under a random-eff ect model for seven endpoints and a fi xed-eff ect model for three endpoints. g p Results The analyzed sample included 77 cases of neurological complications of eclampsia. We extracted the following positions from the plain texts of MRT/CT descriptions: brain injury areas (occipital, temporal, parietal and frontal lobes); injury depth (cortical and/or subcortical matter); brain structures undergoing injury (classifi cation was too complicated); injury nature (vasogenic/ischemic edema, hemorrhage). Abnormalities in occipital (84.6%) and parietal (70.7%) lobes were the most frequent, injuries in temporal lobes were quite rare (26.9%), but damage in frontal lobes was the most uncommon (24.4%). Combined injury in occipital and parietal lobes was recorded in more than two-thirds of cases (72.4%). P318 3 8 Eleven years of critical obstetric pathology: epidemiologic study L Calejman, V Nunes Velloso, E Canedo, M Deheza Hospital Rivadavia, Capital Federal, Argentina Critical Care 2012, 16(Suppl 1):P318 (doi: 10.1186/cc10925) Introduction The objective was to describe the characteristics of pregnant and puerperal women admitted to the ICU from February 2000 to February 2011. Conclusion Maternal MST given as prophylaxis or treatment of eclamptic seizures does not aff ect neonatal mortality and incidence of neonatal hypotonia, Apgar <7 at 1 and 5 minutes, intubation at place of delivery and admission to the NICU in a population of term newborns. Maternal MST signifi cantly reduces the risk of neonate treatment in NICU >7 days in this population. Methods Patients admitted between the mentioned periods were grouped by age, sex, nationality, APACHE II score, days in the ICU, cause of admission: hypertensive syndromes in pregnancy (HSP), preeclampsia (P), eclampsia (E), HELLP syndrome (H), gestational diabetes, sepsis and placentary disorders; need for mechanical ventilation (MV) and dialysis, maternal mortality, and if they had proper prenatal care. P320 Results A total of 3,568 patients were admitted, from which 471 patients (13.2%) were of obstetric cause; average age was 24 years, APACHE II score of 6. There were 39 cases of arterial hypertension and 26 of diabetes mellitus before pregnancy. Sixty-eight percent were fi rst pregnancy. The most frequent causes of admission were hypertension secondary to pregnancy (HSP) in 353 patients (75%): P 44% (n = 156), E 8% (n = 28), H 33% (n = 116), H/E combined 15% (n = 53). Other causes of admission: sepsis 16% (n = 75), placental disorders 7% (n = 33), and neurological deterioration (CVA/S SHEEHAN) 2% (n = 10). They required an average of two drugs to control blood pressure for the patients who needed it in 68% (n = 320). The average stay in the ICU was 6.5 days. From a total of 471 patients, 73 patients required mechanical ventilation (15%) and 118 (25%) patients presented high levels of urea and creatinine, 11 patients (2%) required dialysis. With respect to nationality 301 patients (64%) were Argentinean, the others reported were from Bolivia, Paraguay and Peru. Prenatal checks occurred in only 35% (n = 165) of the patients. The mortality rate was 6% (n = 28). Sleep monitoring by actigraphy in short-stay ICU patients AW Van der Kooi1, JH Tulen2, AW De Weerd3, MM Van Eijk1, MJ Van Uitert4, SE De Rooij4, BC Van Munster4, AJ Slooter1 1University Medical Center Utrecht, the Netherlands; 2Erasmus MC, University Medical Center Rotterdam, the Netherlands; 3Sein, Zwolle, the Netherlands; 4Academic Medical Center, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P320 (doi: 10.1186/cc10927) Introduction Sleep deprivation is common in ICU patients, but diffi cult to investigate [1]. The gold standard for sleep monitoring, polysomnography (PSG), is impractical for use in ICU patients [2]. Actigraphy proved to be a good alternative in non-ICU patients [3]. However, in prolonged mechanically ventilated patients, actigraphy was inaccurate, probably due to ICU-acquired weakness and resulting inactivity [2]. Short-stay ICU patients do not suff er from ICU-acquired weakness, and the accuracy of actigraphy in these patients has not yet been studied [4]. The aim of this study was to investigate actigraphy for sleep assessment in short-stay ICU patients. Introduction Sleep deprivation is common in ICU patients, but diffi cult to investigate [1]. The gold standard for sleep monitoring, polysomnography (PSG), is impractical for use in ICU patients [2]. P319 computer tomography (CT) examinations of the brain in patients with neurological complications of eclampsia; to defi ne the MRT/ CT examination data structure; and to perform frequency analysis of main MRT/CT characteristics and estimate their frequency distributions defi ned by studied pathology. The data included in the study were reported in medical journals and met defi nite criteria for inclusion. Methods We collected cases of neurological complications of eclampsia reported in English-language medical journals from 1980 to 2008. The study methods include structural and frequency analysis of brain MRT/ CT image protocols. computer tomography (CT) examinations of the brain in patients with neurological complications of eclampsia; to defi ne the MRT/ CT examination data structure; and to perform frequency analysis of main MRT/CT characteristics and estimate their frequency distributions defi ned by studied pathology. The data included in the study were reported in medical journals and met defi nite criteria for inclusion. computer tomography (CT) examinations of the brain in patients with neurological complications of eclampsia; to defi ne the MRT/ CT examination data structure; and to perform frequency analysis of main MRT/CT characteristics and estimate their frequency distributions defi ned by studied pathology. The data included in the study were reported in medical journals and met defi nite criteria for inclusion. Data classifi cation of magnetic resonance tomography and computer tomography images of brain in parturients with neurological complications of eclampsia kh h f Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P317 (doi: 10.1186/cc10924) Introduction The goal of the study was to classify protocol data recorded during magnetic resonance tomography (MRT) and S115 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Clinical outcomes in neonates following maternal magnesium sulfate therapy in preeclampsia/eclampsia G Tikhova, E Shifman Kulakov Scientifi c Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P319 (doi: 10.1186/cc10926) The incidence of treatment in the NICU >7 day was signifi cantly lower in MST group than in control (0.54, 95% CI 0.52 to 0.78). Conclusion The analysis reveals a general picture of the most distinctive features of brain damage following neurological complications of eclampsia. Quality and quantity of sleep in multipatient versus single-room ICUs M Van Eijk, A Slooter University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P321 (doi: 10.1186/cc10928) Quality and quantity of sleep in multipatient versus single-room ICUs M Van Eijk, A Slooter University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P321 (doi: 10.1186/cc10928) Methods Double-blind RCT between placebo and melatonin (3 mg bid, 8:00 and 12:00 p.m., from third ICU day until discharge). Inclusion: age >18, SAPS II >32, expected mechanical ventilation (MV) >4 days, practicability of the gastroenteric tract. Patients were treated according to local guidelines [2], titrating sedatives to a conscious target (Richmond Agitation Sedation Scale (RASS) = 0) as early as possible. Each day, the physician in charge stated the RASS target; nurses assessed the actual RASS. Introduction Sleep fragmentation and deprivation is common in ICU patients [1]. It is assumed that the ICU environment (overexposure to sound and light during night-time) leads to disturbed sleep [2]. In our hospital, a new ICU was built with quiet, single-patient rooms with much daylight. This created an opportunity to study the eff ects of nursing environment on sleep quality and quantity in ICU patients. Introduction Sleep fragmentation and deprivation is common in ICU patients [1]. It is assumed that the ICU environment (overexposure to sound and light during night-time) leads to disturbed sleep [2]. In our hospital, a new ICU was built with quiet, single-patient rooms with much daylight. This created an opportunity to study the eff ects of nursing environment on sleep quality and quantity in ICU patients. Methods We included 21 postcardiothoracic surgery patients: 11 subjects were admitted to the old, ward-like ICU, and 10 patients to the new, single-room ICU (see Figure 1). Hypnograms were derived from a polysomnography from 07:00 p.m. to 07:00 a.m. Results Eighty-two patients enrolled: age 72 (60 to 77), SAPS II 41 (34 to 54), MV length 11 (6 to 22) days. Fifteen pancreatitis, 33 acute lung diseases, 13 acute heart diseases, 21 other. The analgesic/sedative therapy during the fi rst 3 days was not diff erent between groups. Melatonin administration determined early weaning from sedatives and analgesics. The prevalence of conscious sedation (RASS = 0) was higher in the melatonin group (67.9 vs. 60.1%, P <0.01), while deeper levels of sedation (RASS = –3/–4) were lower in the melatonin group (RASS –3: 2.4 vs. 7.7%, P <0.01; RASS –4: 1.9 vs. 4.3%, P <0.01). Melatonin administration caused no oversedation (26.3 vs. 24.2%, P = 0.94), while decreased undersedation (18.6% vs. 26.2%, P = 0.05). References 1. Iapichino et al.: Crit Care Med 2006, 34:1039. 1. Iapichino et al.: Crit Care Med 2006, 34:1039. 2. Cigada et al.: J Crit Care 2008, 23:349. 1. Iapichino et al.: Crit Care Med 2006, 34:1039. 2. Cigada et al.: J Crit Care 2008, 23:349. Figure 1 (abstract P321). New, single-room ICU. 2. Cigada et al.: J Crit Care 2008, 23:349. Quality and quantity of sleep in multipatient versus single-room ICUs M Van Eijk, A Slooter University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P321 (doi: 10.1186/cc10928) RASS targets were joined more frequently in the melatonin group, even if not signifi cantly (55.1 vs. 49.6%, P = 0.12). of nursing environment on sleep quality and quantity in ICU patients. Methods We included 21 postcardiothoracic surgery patients: 11 subjects were admitted to the old, ward-like ICU, and 10 patients to the new, single-room ICU (see Figure 1). Hypnograms were derived from a polysomnography from 07:00 p.m. to 07:00 a.m.f y g y Results Both groups did not diff er with respect to age, duration of surgery or use of psychoactive medication. Polysomnography recordings showed no diff erences in total sleep time and awakenings (63 ± 26 in the old ICU and 56 ± 30 in the new ICU). The mean percentage of sleep stages in the old versus new situation did not essentially diff erent either: N1: 12.9% versus 8.0%, P = 0.21, ANOVA; N2: 80.3% versus 87.2%, P = 0.07, ANOVA; N3: 5.2% versus 2.5%, P = 0.18, ANOVA. Only REM sleep latency was longer in the old ICU: 314.7 versus 633.5 minutes, P = 0.02, ANOVA. Conclusion Oral melatonin increased the prevalence of conscious sedation in high-risk critically ill patients; it allowed a better achievement of RASS target, particularly decreasing undersedation episodes. Conclusion Except for REM onset latency, sleep improvement was not achieved by changing a ward-like into a single-patient-room ICU environment. When striving for more natural sleep, attitudes towards Oral melatonin in high-risk critically ill patients: quality of sedative eff ect f G Sabbatini, G Mistraletti, B Cerri, S Miori, I Galluccio, M Tozzi, C Villa, M Umbrello, F Fraschini, G Iapichino Università degli Studi di Milano, Milan, Italy Critical Care 2012, 16(Suppl 1):P322 (doi: 10.1186/cc10929) Conclusion Actigraphy is not reliable for one-night sleep–wake detection in short-stay postoperative ICU patients. References Conclusion Actigraphy is not reliable for one-night sleep–wake detection in short-stay postoperative ICU patients. 1. Figueroa-Ramos M, et al.: Intensive Care Med 2009, 35:781-795. 2. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 3. de Souza L, et al.: Sleep 2003, 26:81-85. 4. Schweickert WD, et al.: Chest 2007, 131:1541-1549. Introduction Analgesic/sedative therapy is necessary in ICU patients; however, it presents important side eff ects. Critically ill patients have altered circadian rhythm, delirium and agitation often requiring additional sedation. The dramatically reduced endogenous blood melatonin level (basal and night peaks) could play a role in this context. We evaluated the eff ects of oral melatonin administration on the adaptation to critical illness and invasive procedures in high-risk critically ill patients [1] consciously sedated [2]. 1. Figueroa-Ramos M, et al.: Intensive Care Med 2009, 35:781-795. 2. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 3. de Souza L, et al.: Sleep 2003, 26:81-85. 4. Schweickert WD, et al.: Chest 2007, 131:1541-1549. References References References 1. Cooper AB, et al.: Chest 2000, 117:809-818. 2. Gabor JY, et al.: Am J Respir Crit Care Med 2003, 167:708-715. g p y Results The only parameter that showed a signifi cant correlation between PSG and actigraphy was the number of awakenings (r = 0.76, P = 0.049, high threshold setting). Actigraphy underestimated wake time after sleep onset and overestimated total sleep time and sleep effi ciency. The median specifi city for actigraphy was below 19% and the median sensitivity above 94% for all threshold settings. Conclusion Actigraphy is not reliable for one-night sleep–wake detection in short-stay postoperative ICU patients. References Results The only parameter that showed a signifi cant correlation between PSG and actigraphy was the number of awakenings (r = 0.76, P = 0.049, high threshold setting). Actigraphy underestimated wake time after sleep onset and overestimated total sleep time and sleep effi ciency. The median specifi city for actigraphy was below 19% and the median sensitivity above 94% for all threshold settings. P320 Actigraphy proved to be a good alternative in non-ICU patients [3]. However, in prolonged mechanically ventilated patients, actigraphy was inaccurate, probably due to ICU-acquired weakness and resulting inactivity [2]. Short-stay ICU patients do not suff er from ICU-acquired weakness, and the accuracy of actigraphy in these patients has not yet been studied [4]. The aim of this study was to investigate actigraphy for sleep assessment in short-stay ICU patients. p y Conclusion Critical obstetrical pathology is common in the ICU, HSP as the main cause. A high number of fi rst pregnancy patients with little prenatal care was observed. This type of patient requires low levels of life support. y Conclusion Critical obstetrical pathology is common in the ICU, HSP as the main cause. A high number of fi rst pregnancy patients with little prenatal care was observed. This type of patient requires low levels of life support. Methods PSG and actigraphy measurements were conducted in seven postcardiothoracic surgery patients. Total sleep time, sleep S116 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 nursing and medication may play a more important role than ICU design. nursing and medication may play a more important role than ICU design. effi ciency, number of awakenings and wake time after sleep onset were determined with actigraphy and compared to PSG. The accuracy, sensitivity (percentage correctly scored as sleep) and specifi city (percentage correctly scored as awake) were calculated for actigraphy using high, medium, low and automatic threshold sensitivity settings of the actigraphy software. n in the ICU: nurses’ perceptions of practices and infl uenci Sedation in the ICU: nurses’ perceptions of practices and infl uencing factors B Sneyers1, PF Laterre2, MM Perreault3, A Spinewine1 1Université catholique de Louvain, Louvain Drug Research Institute, Brussels, Belgium; 2Université catholique de Louvain, Cliniques Universitaires St-Luc, Brussels, Belgium; 3Université de Montreal, Canada Critical Care 2012, 16(Suppl 1):P324 (doi: 10.1186/cc10931) Introduction Our goals are to describe adherence to sedation recommendations [1] in Belgian ICUs and to identify major factors infl uencing practices. l Methods A national survey including all nurses working in Belgian ICUs was conducted with seven nurses sampled per hospital. A validated self-administered paper survey was designed based on a literature review and data from a previous qualitative study. Topics addressed were current practices and reasons for (non)compliance to sedation recommendations such as use of sedation scales and daily sedation interruption (DSI). Four postal reminders were sent. Conclusion In mechanically ventilated patients of a medical ICU including also patients with neurologic diseases, a sedation goal of RASS 0 to –2, as recommended by a current guideline, could only be achieved in a minority of patients despite intensive instructions and a motivated team. In most cases the nurses were able to provide reasonable medical explanations for a deeper sedation or an otherwise impaired consciousness. Results The response rate was 70% (n = 587/840 nurses from 99/120 hospitals). Sedation scales are available to 89% of nurses and frequency of use is variable (≤1×/day: 13%, 3 to 4×/day: 31%, ≥6×/day: 56%). When sedation scales are available, perceived indications are monitoring of sedation and analgesia (96% and 31% of nurses respectively) and dosing adjustment for sedatives and analgesics (14% and 28% of nurses respectively). DSI is infrequently used (never used: 38% of respondents, used for <25% of patients: 47% of respondents, used for 25 to 75% of patients: 12% of respondents, used for >75% of patients: 3% of respondents). Numerous barriers for wide implementation are identifi ed, mainly lack of outcome expectancy, as DSI is perceived to impair patient outcomes. It is perceived that DSI increases the risk of complications such as unplanned extubation and pulling of lines and tubes (79% of nurses agree), impairs patients’ comfort (59% of nurses agree), and creates traumatic memories in the intubated patients (36% of nurses agree). Moreover, 63% of nurses agree that they would prefer no DSI if they were an intubated patient. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Sedation scales are widely used in Belgium, while use of DSI is low. Barriers impairing adherence to recommendations were identifi ed. Perception that sedation scales are not used for sedative dosing adjustments is present, as well as inadequate use for analgesia. Fear of worsening patient outcomes using DSI is present, contrasting with current literature. A similar survey addressing physicians’ perceptions is ongoing. Reference Conclusion Sedation scales are widely used in Belgium, while use of DSI is low. Barriers impairing adherence to recommendations were identifi ed. Perception that sedation scales are not used for sedative dosing adjustments is present, as well as inadequate use for analgesia. Fear of worsening patient outcomes using DSI is present, contrasting with current literature. A similar survey addressing physicians’ perceptions is ongoing. Reference a multicentre prospective longitudinal cohort study in 11 centers in Malaysia. Critically ill patients ventilated and sedated ≥24 hours were followed from ICU admission to hospital discharge. The administration of all sedatives was measured daily. Four-hourly RASS assessments were conducted and delirium assessed daily (CAM-ICU during light sedation RASS –2 to +1). Multivariable Cox regression proportional hazard was used to quantify relationships between early deep sedation and time to extubation and delirium occurring after 48 hours and hospital mortality adjusting for diagnosis, age, gender, APACHE II score, operative, elective, early use of vasopressors and dialysis. 1. Jacobi et al.: Crit Care Med 2002, 30:119-141. 1. Jacobi et al.: Crit Care Med 2002, 30:119-141. p y p y Results We studied 259 patients with mean (SD) age 53.1 (15.9) years and APACHE II score 21.3 (8.2), ventilated for median (IQR) 5 (3 to 8.8) days. Hospital mortality was 82 (31.7%). Midazolam and morphine were the commonest agents used, given to 241 (93.1%) and 201 (77.6%) patients respectively. Over 2,657 study days, 13,836 assessments were conducted. Deep sedation was recorded in 187 (72%) patients within 4 hours of commencing ventilation and in 159 (61%) patients at 48 hours. Daily interruption was used on 20% of study days. Delirium occurred in 114 (43%) of assessed patients with a mean (SD) duration of 1.3 (2.2) days. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Early deep sedation independently predicted time to hospital death (HR 1.11, 95% CI 1.05 to 1.18, P <0.001) and time to extubation (HR 0.93, 95% CI 0.89 to 0.96, P = 0.001) but not time to delirium occurring after 48 hours (HR 0.98, 95% CI 0.93 to 1.03, P = 0.46). Midazolam cumulative dose in the fi rst 48 hours was signifi cantly associated with the number of RASS assessments ≤–3 (P <0.001).i P324 Results The independent observer documented only in 13% (47/364) of all measurements a RASS of 0 to –2. We analyzed 295 questionnaires, in which 368 reasons for a deviation from a RASS of 0 to –2 were stated (multiple answers were possible). In 113 questionnaires (38%) the nurses mentioned that a short-term increase in sedation depth was required for nursing procedures or medical interventions. In 89 questionnaires (30%) a RASS of 0 to –2 was considered reasonable but could not be achieved at the time of measurement with the current medication (n = 32) or the consciousness was impaired by CNS diseases (n = 52). In 100 questionnaires (34%) a RASS of 0 to –2 was not considered reasonable. The following reasons were stated: disease with coma (n = 25), controlled ventilation (n = 32), distressed patient (n = 12), increased intracranial pressure (n = 7), status epilepticus (n = 7), hypothermia (n = 4), dying patient (n = 4), delirium/(auto) aggression (n = 4). Other reasons were mentioned in 66 questionnaires (22%), most commonly a physician order for a deeper sedation (n = 19) or a missing sedation goal (n = 14). Reference 1. Devlin JW: The pharmacology of over sedation in mechanically ventilated adults. Curr Opin Crit Care 2008, 14:403-407. P325 3 5 Implementation of a national guideline for analgesia and sedation: how often can a RASS of 0 to –2 be achieved? R Riessen, P Tränkle, R Pech, G Blumenstock, M Haap University Hospital Tübingen, Germany Critical Care 2012, 16(Suppl 1):P325 (doi: 10.1186/cc10932) Introduction Based on a new national guideline we implemented in our medical ICU an interdisciplinary algorithm for the management of analgosedation, in which nurses had to adjust the dose of the analgesics and sedatives based on sedation goals given by the physicians. Within this project we investigated in what portion of mechanically ventilated patients a sedation level of Richmond Agitation and Sedation Scale (RASS) of 0 to –2, which is generally recommended by the guideline, can be achieved. We also asked the nurses for an explanation when this goal was not reached. Conclusion Early ICU sedation depth is a modifi able risk factor for delayed extubation and increased risk of death and should be considered in future sedation trials. g Methods After an educational program the level of sedation was measured 364 times in 37 mechanically ventilated patients at diff erent time points by an independent observer. In all cases in which the RASS was outside the desired level of 0 to –2, the nurse in charge was asked to fi ll out a structured as well as open questionnaire, in which the reasons for this deviation could be stated. Reference P323 Sedation depth and mortality in mechanically ventilated critically ill adults Y Shehabi1, S Kadiman2, L Chan3, W Ismail4, M Saman5, A Alias6 1University New South Wales, Randwick, Australia; 2National Heart Institute, Kuala Lumpur, Malaysia; 3University Malaya, Kuala Lumpur, Malaysia; 4Raja Perempuan Zainab II Hospital, Kota Bharu, Malaysia; 5Sarawak General Hospital, Kuching, Malaysia; 6Malacca General Hospital, Malacca, Malaysia Critical Care 2012, 16(Suppl 1):P323 (doi: 10.1186/cc10930) Introduction Deep sedation is common in ventilated patients, particularly in the fi rst 48 hours in the ICU, which may adversely aff ect outcomes such as mortality. This period is usually unobserved in clinical trials due to late randomisation. We investigated the relationship between early sedation depth, sum of Richmond Agitation Sedation Scale (RASS) –3 to –5 and clinical outcomes, including mortality. Figure 1 (abstract P321). New, single-room ICU. Methods A waiver of consent was granted. In collaboration with the Australian New Zealand Intensive Care Research Centre, we conducted S117 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Cerebral ischemia–reperfusion model in rabbits: relationship between dexmedetomidine and biochemical parameters in lowering intraparenchymal pressure g y Results In the baseline phase (36 patients/422 measurements) using the RAMSAY score, sedation depth documented by the nurses and the observer matched in only 39% of the measurements. The nurses documented in 246 (58%) measurements a lighter sedation and in 12 measurements (3%) a deeper sedation than the observer. In the post- implementation phase (37 patients/346 measurements) using the RASS, we found a signifi cantly higher matching rate of 76% between nurses and observer compared to RAMSAY (P <0.001). Nurses documented in 47 measurements a lighter (14%) and in 37 measurements (11%) a deeper sedation than the observer. The nurses evaluated the RASS in terms of the ability to describe the actual depths of sedation with a mean of 1.7 on the six-point Likert scale signifi cantly better than the RAMSAY score with 3.2 (P <0.001). Similar results were found regarding the discrimination between diff erent levels of sedation (RASS 1.7, RAMSAY 3.1, P <0.001). g y A Tavlan1, ME Ustun1, A Yosunkaya1, A Ak1, A Kiyici1, HK Bardakcı2, F Gok1 1Selcuk University, Meram Medical Faculty, Konya, Turkey; 2Farabi Hospital, Konya, Turkey Critical Care 2012 16(Suppl 1) P328 (doi 10 1186/cc10935) y y Critical Care 2012, 16(Suppl 1):P328 (doi: 10.1186/cc10935) Introduction The eff ect of dexmedetomidine in two diff erent doses on the levels of endothelin-1 (ET-1) and prostoglandin I2 (PGI2) in blood and cerebrospinal fl uid (CSF) of rabbits via the transient global cerebral ischemia model was studied to determine its intraparenchymal pressure (IPP) reduction mechanism. Methods Twenty-four New Zealand type rabbits were employed and randomly distributed into four groups. Group I (sham group, n = 6): craniotomy was performed only. Group II (control group, n = 6): bilateral carotid arteries were clamped for 60 minutes after craniotomy, then reperfusion was performed for 60 minutes. In Group III (n = 6) and Group IV (n = 6), 80 μkg–1 and 320 μkg–1 dexmedetomidine was administered within the fi rst 10 minutes of the reperfusion procedure respectively. Blood and CSF samples were collected 120 minutes after craniotomy. Mean arterial pressures (MAP), heart rates (HR), IPP and temperature values were recorded. Conclusion In routine use the RAMSAY score showed a poor performance regarding the measurement of sedation depth. After implementation of the RASS, measurement of sedation depth appeared signifi cantly improved. Results There was no signifi cant diff erence in MAP values between groups (P ≥0.05). n in the ICU: nurses’ perceptions of practices and infl uenci Other barriers are related to knowledge, as 26% of nurses do not know the practice, and to behaviour, as 53% of respondents feel DSI is diffi cult to implement because of organizational constraints. P326 Comparison of the RAMSAY score and the Richmond Agitation Sedation Score for the measurement of sedation depth R Riessen, R Pech, P Tränkle, G Blumenstock, M Haap University Hospital Tübingen, Germany Critical Care 2012, 16(Suppl 1):P326 (doi: 10.1186/cc10933) Introduction We implemented an interdisciplinary algorithm for the management of analgosedation in mechanically ventilated patients based on a new national guideline. As part of this project we investigated whether the newly introduced Richmond Agitation Sedation Score (RASS) allowed a better monitoring of sedation depth than the formerly used RAMSAY score. Introduction We implemented an interdisciplinary algorithm for the management of analgosedation in mechanically ventilated patients based on a new national guideline. As part of this project we investigated whether the newly introduced Richmond Agitation Sedation Score (RASS) allowed a better monitoring of sedation depth than the formerly used RAMSAY score. S118 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods During the baseline phase of the study we investigated the RAMSAY score, which had been routinely used for several years in our unit. Following an educational program the RAMSAY was replaced by the RASS. During both study phases the actual sedation score was determined within a short period of time by the nurse in charge and an independent observer. In addition, the nurses were asked to evaluate on a six-point Likert scale whether the score appeared to be suitable to describe the actual state of sedation or to discriminate between diff erent levels of sedation (1 = very good). The measurements took place at three defi ned time points (7, 9 and 12 o’clock) during the morning shift on weekdays. P328 P328 3 8 Cerebral ischemia–reperfusion model in rabbits: relationship between dexmedetomidine and biochemical parameters in lowering intraparenchymal pressure A Tavlan1, ME Ustun1, A Yosunkaya1, A Ak1, A Kiyici1, HK Bardakcı2, F Gok1 1Selcuk University, Meram Medical Faculty, Konya, Turkey; 2Farabi Hospital, Konya, Turkey Critical Care 2012, 16(Suppl 1):P328 (doi: 10.1186/cc10935) Dexmedetomidine is associated with better outcomes in patients undergoing cardiac surgery PG Brandão1, S Lobo1, M Nassau Machado1, J Duarte1, F Lobo1, Y Sakr2 1Hospital de Base de São José do Rio Preto, Brazil; 2Friedrich Schiller University Hospital, Jena, Germany Critical Care 2012, 16(Suppl 1):P327 (doi: 10.1186/cc10934) Introduction Cardiac anesthesia has changed over the years from high- dose opioids to fast-track surgery. The use of high doses of opioids was justifi ed based on the hemodynamic stability [1] at a cost of prolonged mechanical ventilation support. Our study aims to analyze the use of dexmedetomidine as an anesthesia adjuvant during the induction and maintenance of anesthesia for patients undergoing coronary artery bypass graft (CABG) and valvular heart surgeries. Conclusion Dexmedetomidine could decrease intraparenchymal pressure in the transient global cerebral ischemia model when administered at low doses [1,2]. It probably contributed to this reduction by preventing an increase of endothelin levels in blood and CSF as well as decreasing PGI2 levels in CSF. g 2 Referencesf References yp g g Methods This study is a retrospective analysis from a prospective database collected from January 2003 to April 2011. The patients were divided into two groups, based on the use of dexmedetomidine (DEX group) intraoperatively or conventional opioid-based technique (Control group). Isofl urane was used for anesthesia maintenance in both groups. 1. Zornow MH, Scheller MS, Sheehan PB: Intracranial pressure eff ects of dexmedetomidine in rabbits. Anesth Analg 1992, 75:232-237. 2. Jolkkonen J, Puurunen K, Koistinaho J, et al.: Neuroprotection by the alpha 2-adrenoceptor agonist, dexmedetomidine in rat focal cerebral ischemia. Eur J Pharmacol 1999, 7:31-36. g p Results We included 1,302 consecutive patients undergoing cardiac surgery during the study period (63% male; median age = 57 years), 796 patients in the DEX group and 506 patients in the control group. CABG was the most commonly performed surgery (63%) followed by valve surgeries (37%). The overall 30-day hospital mortality rate was 5.8%. Length of stay was signifi cantly lower for patients in the Dex group (3.7 ± 4.4 days) than for patients in the control group (4.5 ± 6.3 days) (P = 0.02). Thirty-day mortality rates were 3.4% in the Dex group and 9.7% in the control group (P <0.001). In the multivariable Cox regression analysis with in-hospital death as the dependent variable, dexmedetomidine (OR = 0.39, 95% CI: 0.23 to 0.64, P ≤0.001), a high L-EuroSCORE (OR= 1.05, 95% CI: 1.01 to 1.10, P = 0.004) and older age (OR = 1.03, 95% CI: 1.01 to 1.05, P = 0.003) were independently related to in-hospital death. Need for reoperation (2.0% vs. 2.8%, P = 0.001), neurologic lesion type 1 (2.0% vs. 4.7%, P = 0.005) and prolonged hospitalization (3.1% vs. 7.3%, P = 0.001) were signifi cantly less frequent in the DEX group than in the control group. P327 Dexmedetomidine is associated with better outcomes in patients undergoing cardiac surgery PG Brandão1, S Lobo1, M Nassau Machado1, J Duarte1, F Lobo1, Y Sakr2 1Hospital de Base de São José do Rio Preto, Brazil; 2Friedrich Schiller University Hospital, Jena, Germany Critical Care 2012, 16(Suppl 1):P327 (doi: 10.1186/cc10934) Cerebral ischemia–reperfusion model in rabbits: relationship between dexmedetomidine and biochemical parameters in lowering intraparenchymal pressure A decrease of HR in Group IV was signifi cantly lower after reperfusion (P <0.05). IPP values after the reperfusion in Groups II and IV were signifi cantly higher than Group I (P <0.05), but no signifi cant increase in Group III (P ≥0.05). ET-1 levels of both blood and CSF were increased in the group with performed ischemia and reperfusion and no treatment (Group II) and the group administered high-dose dexmedetomidine (Group IV) (P <0.05), while the group administered low-dose dexmedetomidine (Group III) was similar to the sham group (P ≥0.05). However, PGI2 levels of CSF were signifi cantly decreased in the group administered low-dose dexmedetomidine (P <0.05). References 1. Lowenstein E, et al.: Cardiovascular response to large doses of intravenous morphine in man. N Engl J Med 1969, 281:1389-1393. 2. Jalonen J, et al.: Dexmedetomidine as an anesthetic adjuvant in coronary artery bypass grafting. Anesthesiology 1997, 86:331-345. 1. Lowenstein E, et al.: Cardiovascular response to large doses of intravenous morphine in man. N Engl J Med 1969, 281:1389-1393. 2. Jalonen J, et al.: Dexmedetomidine as an anesthetic adjuvant in coronary artery bypass grafting. Anesthesiology 1997, 86:331-345. 1. Lowenstein E, et al.: Cardiovascular response to large doses of intravenous morphine in man. N Engl J Med 1969, 281:1389-1393. 2. Jalonen J, et al.: Dexmedetomidine as an anesthetic adjuvant in coronary artery bypass grafting. Anesthesiology 1997, 86:331-345. 2. Jalonen J, et al.: Dexmedetomidine as an anesthetic adjuvant in coronary artery bypass grafting. Anesthesiology 1997, 86:331-345. P329 Evaluation of sedation using pupilometry in ICUs: a pilot study O Rouche, A Wolak-Thierry, Q Destoop, L Milloncourt, T Floch, P Raclot, J Cousson Centre Hospitalier Universitaire, Reims, France Critical Care 2012, 16(Suppl 1):P329 (doi: 10.1186/cc10936) Evaluation of sedation using pupilometry in ICUs: a pilot study O Rouche, A Wolak-Thierry, Q Destoop, L Milloncourt, T Floch, P Raclot, J Cousson Centre Hospitalier Universitaire, Reims, France Critical Care 2012, 16(Suppl 1):P329 (doi: 10.1186/cc10936) Centre Hospitalier Universitaire, Reims, France Critical Care 2012, 16(Suppl 1):P329 (doi: 10.1186/cc10936) Introduction The depth of hypnosis is correlated with the decrease in photomotor refl ex (PMR) [1]. It would be benefi cial to develop an automated, noninvasive, simple and reproducible technique allowing one to effi ciently evaluate the depth of sedation in ICUs. The objective of this observational study is to evaluate the eff ectiveness of pupilometric video in comparison to the Bispectral index (BIS). Introduction The depth of hypnosis is correlated with the decrease in photomotor refl ex (PMR) [1]. It would be benefi cial to develop an automated, noninvasive, simple and reproducible technique allowing one to effi ciently evaluate the depth of sedation in ICUs. The objective of this observational study is to evaluate the eff ectiveness of pupilometric video in comparison to the Bispectral index (BIS). Methods Sedation level was based on the Richmond Score (RASS between –4 and –5). Exclusion criteria were neurological pathologies interfering with the PMR. Following a 320 lux fl ash of light, the PMR was measured by the Neurolight (IDmed). Three measurements a day Conclusion Use of dexmedetomidine as anesthesia adjuvant was asso- ciated with better outcomes in patients undergoing cardiac surgery. S119 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 correlated with EEG frequency, with maximal slowing in the delta (≤4 Hz) range. were taken during 48 hours along with the collection of the BIS value (Bis Vista Anandic Medical Systems). The data collected included the variation of pupillary diameter (PD), latency time (LT) and maximal speed of pupillary constriction (Vmax). These parameters were analyzed after having classifi ed BIS values into three groups. Conclusion Midazolam concentrations while on continuous infusion were associated with EEG tracings suggestive of deep sedation. Although clearance was relatively preserved, it varied over a wider range than found in healthy populations. Eff ect of critical illness on the pharmacokinetics and dose–response relationship of midazolam p g p y Results We included 15 patients; APACHE II score was (median) 17.5 points and SOFA score 9 points. The Bispectral index was lower in the midazolan group (43 vs. 48.5 points, P = 0.005), although RASS was the same for both groups. Large-vessel perfusion was similar for both groups. The small perfusion vessel proportion was signifi cantly reduced with propofol (92 vs. 96.3%, P = 0.003). The microvascular fl ow index was also lower during propofol infusion (MFI – 2.4 vs. 2.7, P = 0.002). We observed a higher heterogeneity index when patients were sedated with propofol (0.4 vs. 0.19, P = 0.01). Introduction Critically ill patients require sedation to tolerate the interventions necessary to facilitate their care. There is growing evidence, however, that use of sedatives, such as the benzodiazepine midazolam, is associated with delirium and other complications that can lead to prolonged ICU stay and increased mortality. The pharmacokinetics of midazolam in healthy populations has been well characterized, and pharmacodynamic studies demonstrate a predictable dose–response relationship. However, in critical illness, where midazolam is often administered as a continuous infusion, the pharmacokinetic properties are often altered. We sought to investigate whether analysis of midazolam plasma concentrations in combination with electroencephalography (EEG) will better defi ne the eff ect of critical illness on the pharmacokinetics and clinical response to midazolam, while providing a method to assess the adequacy of sedation thereby minimizing the risks associated with prolonged or over-sedation. Conclusion Propofol reduces small-vessel perfusion and increases the heterogeneity of circulation in the sublingual mucosa, when compared with the use of midazolan in septic shock patients. P329 The apparent lower threshold for onset of coma may be a refl ection of illness severity, concomitant medication use, and variable clearance during the course of illness. These preliminary results suggest that the combination of continuous bedside EEG and therapeutic drug monitoring may be useful for titrating midazolam infusions and to guide tapering to avoid prolonged coma in patients with variable clearance of midazolam. i Results A total of 186 analyses of PMR and BIS were conducted on 31 patients. The averages and standard deviations for each class of BIS were as shown in Table 1. We conducted an analysis of variance in order to compare these three groups of BIS. For the values Vmax and the PD, the ANOVA was signifi cant. Therefore, we proceeded to compare the groups two by two using Bonferroni tests. They revealed signifi cant diff erence between the BIS <40 and 40 ≤ BIS ≤ 60 group (P <0.0001 for both variables) and between BIS <40 and BIS >60 (Vmax P <0.0001 and PD P <0.05). There was no correlation between any of the BIS groups and the LT variable. P331 Eff ect of propofol and midazolan on microcirculation of septic shock patients G Penna1, F Fialho2, A Japiassu3, D Salgado4, P Kurtz1, G Nobre1, M Kalichsztein1, N Villela5, E Bouskela5 1Casa de Saúde São José, Rio de Janeiro, Brazil; 2IFF-FIOCRUZ, Rio de Janeiro, Brazil; 3IPEC-FIOCRUZ, Rio de Janeiro, Brazil; 4UFRJ, Rio de Janeiro, Brazil; 5UERJ, Rio de Janeiro, Brazil Critical Care 2012, 16(Suppl 1):P331 (doi: 10.1186/cc10938) 33 Eff ect of propofol and midazolan on microcirculation of septic shock patients G P 1 F Fi lh 2 A J i 3 D S l d 4 P K t 1 G N b 1 Table 1 (abstract P329). Values of Vmax, PD and TL BIS <40 (n = 68) 40 ≤ BIS ≤ 60 (n = 62) BIS >60 (n = 37) Vmax (mm/second) 0.98 ± 0.44 1.45 ± 0.73 1.66 ± 0.95 TL (ms) 253.8 ± 68.6 241.6 ± 41.8 240.6 ± 52.2 PD% 12.95 ± 5.58 18.3 ± 6.12 17.7 ± 6.72 Conclusion The Vmax and the PD seem to be relevant criteria when compared to the BIS. This noninvasive technique of monitoring the depth of sedation could be benefi cial especially with patients under myorelaxant drugs. A larger study is necessary in order to confi rm these results and enable one to set cut-off values for the Vmax and PD. Reference 1 Leslie K et al : Anesthesiology 1996 84:52 63 Table 1 (abstract P329). Values of Vmax, PD and TL BIS <40 (n = 68) 40 ≤ BIS ≤ 60 (n = 62) BIS >60 (n = 37) Vmax (mm/second) 0.98 ± 0.44 1.45 ± 0.73 1.66 ± 0.95 TL (ms) 253.8 ± 68.6 241.6 ± 41.8 240.6 ± 52.2 PD% 12.95 ± 5.58 18.3 ± 6.12 17.7 ± 6.72 Introduction Septic shock patients are submitted to many therapeutic strategies, including sedation. It is unknown if diff erent sedative drugs infl uence microcirculation. Methods We performed a prospective observational study, using sidestream dark-fi eld imaging (SDF), to evaluate sublingual mucosa of septic shock patients admitted to our ICU. SDF was applied in two settings: continuous sedation with propofol and with midazolan. We repeated each examination after an interval of 30 minutes. Eight fi elds (videos) were analyzed during propofol and midazolan infusion. Two videos were obtained from each side of the tongue. The Bispectral index was monitored along with the Richmond Agitation Sedation Scale: the dose of both sedatives was titered to maintain light sedation. All demographic and severity of illness data were collected. Vasopressor agents were maintained to a mean arterial pressure of 70 mmHg and the cardiac index was kept stable through the protocol study. 1. Leslie K, et al.: Anesthesiology 1996, 84:52-63. P330f P330 Eff ect of critical illness on the pharmacokinetics and dose–response relationship of midazolam D Ovakim, KJ Bosma, GB Young, M Sen, LE Norton, F Priestap, RG Tirona, R Kim, GK Dresser University of Western Ontario, London, Canada Critical Care 2012, 16(Suppl 1):P330 (doi: 10.1186/cc10937) P335 Methods Ninety patients admitted to the RRCEM urgently with chest traumatic injuries have been examined. They were divided into two groups against the applied method of anesthesia. First (control) group (47 patients, 38.5 ± 2.4 years): IPA was done before the induction of anesthesia into the second intercostal space from the damaged side with bupivakain at a dose of 75 to 100 mg. Analgesic component maintained by the abovementioned IPA and phentanyl bolus dosing. The second group (43 patients, 36.8 ± 5.4 years): one-sided TPVB maintained before the induction at ThIV, ThVII levels 0.5% – 5 ml (25 mg) bupivakain dosing (at the average total 75 to 100 mg) with posterior paravertebral area catheterization. Analgesic component maintained by paravertebral analgesia and phentanyl bolus dosing.f Long-term adverse neuropsychological functioning in children who survived meningococcal septic shock: is there a relationship with sedation and analgesia during paediatric ICU admission? HL Van Zellem1, E Utens1, SN De Wildt1, WC Hop2, NJ Vet1, KF Joosten1, C Buysse1 1Erasmus MC – Sophia Children’s Hospital, Rotterdam, the Netherlands; 2Erasmus MC, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P335 (doi: 10.1186/cc10942) Long-term adverse neuropsychological functioning in children who survived meningococcal septic shock: is there a relationship with sedation and analgesia during paediatric ICU admission? HL Van Zellem1, E Utens1, SN De Wildt1, WC Hop2, NJ Vet1, KF Joosten1, C Buysse1 y 1Erasmus MC – Sophia Children’s Hospital, Rotterdam, the Netherlands; 2Erasmus MC, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P335 (doi: 10.1186/cc10942) Introduction Our objective was to evaluate the association between the use of sedative and analgesic agents during paediatric intensive care unit (PICU) treatment and long-term neuropsychological outcome in children who survived meningococcal septic shock (MSS). Results The diff erences in hemodynamics indexes appeared at the traumatic moment of operation. In the group using IPA, medium hypertension with ABP rise in 25.5%, higher rate of HR in 26.1% and GPVR in 22% were observed and were followed by the decrease of SV on 24.6% and EF on 13% compared with the second group. Conducting anesthesia in the fi rst group, hyperdynamic reactions of the systemic hemodynamics at the separate traumatic levels of operation were followed by unbalance of hemodynamic rhythms indicating insuffi cient prevention from surgical aggression. In the second group, as the result of development of segmental sympathetic block the indexes of ABP, HR and GPVR were not higher than normal. P334 and nonteaching hospitals (85%) in comparison to academic hospitals (15%). Most ICUs (94%) use a standardized pain score in the group of patients who are capable of verbal communication: the Visual Analogue Scale (57%), Numerical Rating Scale (48%) and Faces Pain Scale (5%) being the most frequently used scores. In the group of patients who are unable to communicate, ICUs less frequently use pain scores (19%), with the Critical-Care Pain Observation Tool (6%) and Behaviour Pain Scale (5%) being used most frequently. Measurement of pain was considered most important for patients with burn wounds (67%), trauma patients (64%), postoperative patients (57%) and those who receive end-of-life care (64%). Barriers to use pain measurements included the patient’s inability to communicate (82%), interference with pain assessment due to sedation (79%), hemodynamic instability (64%), insuffi cient dosages of analgesics (60%) and the unavailability of a standard pain scoring system (51%). In addition, guidelines for management of sedation and analgesics from the Netherlands Association for Intensive Care (NVIC) had been read by only 20% of the respondents. Factors that were mentioned to be useful in contributing to an improvement in pain assessment and eff ective pain control included adequate analgesic dosage (87%), utilization of protocols and directives (86%), enthusiastic and motivated personnel (81%) and the utilization of standardized pain measurement tools. Effi ciency estimation of intrapleural and thoracic paravertebral block in combination with general anesthesia at thoracoscopic interventions g g Conclusion Preoperatively diclofenac reduces postcraniotomy head- ache compared to placebo, and reduces postoperative analgesic requirements. Introduction Chest injuries and traumas have become one of the most common reasons for admitting patients to emergency surgical hospitals in recent years. P333fi Effi ciency estimation of intrapleural and thoracic paravertebral block in combination with general anesthesia at thoracoscopic interventions J Sabirov, A Khadjibaev, V Sharipova Republican Reseach Centre of Emegency Medicine, Tashkent, Uzbekistan Critical Care 2012, 16(Suppl 1):P333 (doi: 10.1186/cc10940) Preoperative diclofenc reduces postcraniotomy headache: a randomized, placebo-controlled trial Preoperative diclofenc reduces postcraniotomy headache: a randomized, placebo-controlled trial p C Molnár, É Simon, J Gál, P Siró, Á Kazup, B Fülesdi University of Debrecen, Hungary Critical Care 2012, 16(Suppl 1):P334 (doi: 10.1186/cc10941) p C Molnár, É Simon, J Gál, P Siró, Á Kazup, B Fülesdi University of Debrecen, Hungary Critical Care 2012, 16(Suppl 1):P334 (doi: 10.1186/cc10941) Introduction We tested the hypothesis that 100 mg oral preoperative diclofenac reduces postcraniotomy headache. Methods A total of 145 patients having elective craniotonomies were randomly assigned to diclofenac or placebo. Severity of pain was assessed by an independent observer using a visual analogue scale on the day of surgery, on the fi rst postoperative day, and on the fi fth postoperative day. The total amount of analgesics administered during the fi rst fi ve postoperative days was converted to intramuscular morphine equivalents. Results were compared using unpaired, two- tailed t tests; P <0.05 was considered statistically signifi cant. Results In total, 104 patients had supratentorial and 41 had infratentorial interventions. Sixty-two patients were assigned to placebo and 83 were assigned to diclofenac. The results of VAS scores are shown in Table 1. Table 1 (abstract P334). Results of VAS scores Placebo DICLO P value Day of surgery 4.9 ± 3.5 2.2 ± 3.5 <0.001 First postoperative day 5.5 ± 3.4 3.7 ± 3.5 <0.01 Fifth postoperative day 4.3 ± 3.8 2.6 ± 2.9 <0.01 Table 1 (abstract P334). Results of VAS scores Table 1 (abstract P334). Results of VAS scores p Conclusion Most Dutch ICUs measure pain frequently (94%) in patients who are able to communicate. However, in the group of patients who cannot communicate only 19% of the Dutch ICUs use a standardized pain score. This fi nding applied to both academic and nonacademic ICUs, which suggests that eff orts should be put into implementing pain measures in Dutch ICUs. The relative effi cacy of diclofenac was similar in patients having supratentorial and infratentorial surgery. Diclofenac also appeared to be comparably eff ective in both men and women. Systemic analgesic requirements were reduced during the initial fi ve postoperative days in patients assigned to diclofenac (intramuscular morphine equivalents: placebo = 5.3 ± 4.3 mg vs. diclofenac = 3.6 ± 3.3 mg). P332 Current use of pain scores in Dutch ICUs: a postal survey in the Netherlands M Van der Woude1, L Bormans1, J Hofhuis2, P Spronk2 1Atrium Medical Center, Heerlen, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2012, 16(Suppl 1):P332 (doi: 10.1186/cc10939) Methods For this observational study, patients admitted to the ICU with a diagnosis of sepsis and receiving a continuous infusion of midazolam were screened for inclusion. Upon enrollment, a continuous subhairline EEG was applied and blood samples were collected daily for plasma midazolam quantifi cation. Clinical data and laboratory parameters were followed. Plasma midazolam levels were quantifi ed using liquid chromatography with tandem mass spectroscopy. Critical Care 2012, 16(Suppl 1):P332 (doi: 10.1186/cc10939 Introduction Pain is a common problem for patients admitted to the ICU, causing patient discomfort, agitation and accidental self- extubation. For this reason the recognition of pain and its severity is extremely important. Several pain scores and protocols are in use. We aimed to elucidate current practice of pain measurements and treatment in Dutch ICUs. Results Data were available for nine patients. Midazolam clearance demonstrated wide intersubject variability (range 31 ml/minute to 1,157 ml/minute) although average clearance among all patients (418 ml/minute) was comparable to that of healthy controls. Mean midazolam concentrations for patients with coma were signifi cantly higher than for patients without coma (218 ± 185 ng/ml vs. 106 ± 107 ng/ml). The plasma midazolam concentration inversely Methods In March 2011, a questionnaire was sent to all Dutch adult ICUs irrespective of the number of ICU beds with active follow-up by telephone calls to optimize the participation rate. Results A total of 84 ICUs (84/107) returned the survey, representing a response rate of 87%. Most ICUs are community teaching hospitals S120 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P336 Delirium could be an indicator of sepsis in patients under 65 years old with urinary tract infections U Yamada, K Yokota, D Ohta, K Furukawa St Luke’s International Hospital, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P336 (doi: 10.1186/cc10943) Introduction Delirium, known as sepsis-associated encephalopathy, is a frequent complication of sepsis and may be an independent predictor of mortality of septic patients [1]. A recent study reported delirium could be a predictor or an early marker of sepsis in CABG patients [2]. Urinary tract infection (UTI) often complicates sepsis and delirium; however, relations between delirium and sepsis in UTI patients have not been well investigated. We assessed the relationship between delirium and sepsis in patients with UTI. Figure 1 (abstract P337). ROC curve: CAM-ICU (solid line and circles) and ICDSC (dashed line and squares). Methods This study was conducted at St Luke’s International Hospital in Tokyo, Japan between January 2009 and October 2011. UTI and sepsis were diagnosed based on positive bacterial cultures and clinical symptoms. Delirium was screened with the Delirium Screening Tool (the 11-item questionnaire, sensitivity 98% and specifi city 76%) by trained physicians and nurses. Medical records of patients were reviewed to collect information including age, sex and complications. The association between possible risk factors and delirium was analyzed by chi-squared tests and t tests. Statistical analysis was performed using SPSS software version 15.0J. had to be diagnosed based on appropriate criteria by a delirium expert. The outcomes assessed were: sensitivity, specifi city, likelihood ratios and summary receiver-operating characteristic (ROC) curves.i had to be diagnosed based on appropriate criteria by a delirium expert. The outcomes assessed were: sensitivity, specifi city, likelihood ratios and summary receiver-operating characteristic (ROC) curves. Results Fifteen studies covering 1,404 participants and fi ve screening tools were included in the systematic review. The pooled sensitivities and specifi cities for CAM-ICU for detection of delirium in critically ill patients were 76.0% and 95.7% and for ICDSC were 74.4% and 75.2%, respectively. All but one study was performed in a research setting, and that one study suggested that, with routine use of CAM-ICU, one-half of the patients with delirium were not detected. See Figure 1. Conclusion The CAM-ICU was the most specifi c bedside tool for assessment of delirium in critically ill patients. However, there was signifi cant heterogeneity of the results. p References 1. Ebersoldt M, Sharshar T, Annane D: Sepsis-associated delirium. Intensive Care Med 2007, 33:941-950. 1. Ebersoldt M, Sharshar T, Annane D: Sepsis-associated delirium. Intensive Care Med 2007, 33:941-950. 2. Martin BJ, Buth KJ, Arora RC, Baskett RJ: Delirium as a predictor of sepsis in post-coronary artery bypass grafting patients: a retrospective cohort study. Crit Care 2010, 14:R171. 2. Martin BJ, Buth KJ, Arora RC, Baskett RJ: Delirium as a predictor of sepsis in post-coronary artery bypass grafting patients: a retrospective cohort study. Crit Care 2010, 14:R171. Introduction Up to 80% of patients experience delirium during their ICU stay [1,2]. The most sensitive screenings tool for delirium in a research setting is the Confusion Assessment Method for the ICU (CAM- ICU), but the low sensitivity of the CAM-ICU in daily practice (47%) hampers early detection of delirium and thereby delays treatment [3,4]. Therefore, there is a need for an objective tool for continuous delirium monitoring. Diagnosis of delirium can also be conducted using electroencephalography (EEG) [5]. EEG with a limited number of electrodes and automatic processing may be a more sensitive approach for delirium monitoring. The aim of this systematic review is to explore opportunities for automatic detection of delirium by summarizing EEG characteristics of delirium. P335 and adverse outcome on multiple domains of neuropsychological functioning (full-scale IQ (P = 0.02; Z = –2.28), verbal IQ (P = 0.02; Z = –2.32), verbal reasoning (P = 0.02; Z = –2.34), social comprehension (P = 0.01; Z = –2.56), visual–motor integration (P = 0.03; Z = –2.17)). After univariate analysis, correcting for socioeconomic status, age at follow- up and severity of illness, these correlations remained signifi cant.i and adverse outcome on multiple domains of neuropsychological functioning (full-scale IQ (P = 0.02; Z = –2.28), verbal IQ (P = 0.02; Z = –2.32), verbal reasoning (P = 0.02; Z = –2.34), social comprehension (P = 0.01; Z = –2.56), visual–motor integration (P = 0.03; Z = –2.17)). After univariate analysis, correcting for socioeconomic status, age at follow- up and severity of illness, these correlations remained signifi cant.i Conclusion The use of opioids during PICU admission was signifi cantly associated with long-term adverse neuropsychological outcome in MSS survivors. P335 g Methods This study is part of a medical and psychological follow- up study of all consecutive MSS survivors requiring PICU treatment between 1988 and 2001 at the Erasmus MC – Sophia Children’s Hospital, a tertiary-care university hospital. This follow-up study revealed that MSS survivors showed long-term (at least 4 years after PICU admission) impairments on several domains of neuropsychological functioning. Severity of illness was no signifi cant predictor of adverse neuropsychological outcome. The use (type, number and dose) of sedatives and analgesics was retrospectively evaluated. Conclusion Both methods of regional anesthesia cut short pain syndrome suffi ciently and safely in patients with chest injuries before an operative intervention. Introduction of the TPVB component into the anesthesia scheme of thoracoscopic operative interventions allows one to provide additional antinociceptive protection in the intraoperative period with minimal stress of central and peripheral parameters and promotes the reduction of narcotic analgesic use due to signifi cant analgesic effi ciency and neurovegetative protection. Results The study population consisted of 77 patients (52% male (n = 40), median age 25 months at time of PICU admission). In 45 patients (58%) one or more analgesic and/or sedative drugs were administered during PICU admission. Benzodiazepines were the most commonly used drugs (n = 39; 51%), followed by opioids (n = 23; 30%). In total 15 diff erent kinds of analgesic or sedative drugs were given. There was a statistically signifi cant correlation between the use of opioids (both as continuous (cumulative dose) and dichotomous variable) S121 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 and adverse outcome on multiple domains of neuropsychological functioning (full-scale IQ (P = 0.02; Z = –2.28), verbal IQ (P = 0.02; Z = –2.32), verbal reasoning (P = 0.02; Z = –2.34), social comprehension (P = 0.01; Z = –2.56), visual–motor integration (P = 0.03; Z = –2.17)). After univariate analysis, correcting for socioeconomic status, age at follow- up and severity of illness, these correlations remained signifi cant. Conclusion The use of opioids during PICU admission was signifi cantly associated with long-term adverse neuropsychological outcome in MSS survivors. Figure 1 (abstract P337). ROC curve: CAM-ICU (solid line and circles) and ICDSC (dashed line and squares). P338 Electroencephalography-based monitoring of delirium in the ICU: what are the opportunities? AW Van der Kooi, FS Leijten, RJ Van der Wekken, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P338 (doi: 10.1186/cc10945) P338 Electroencephalography-based monitoring of delirium in the ICU: what are the opportunities? AW Van der Kooi, FS Leijten, RJ Van der Wekken, AJ Slooter University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P338 (doi: 10.1186/cc10945) P336 These fi ndings were largely obtained in research settings, and the low sensitivity of the CAM-ICU in routine, daily practice may limit its use as a screening test. y p g Results Fifteen studies covering 1,404 participants and fi ve screening tools were included in the systematic review. The pooled sensitivities and specifi cities for CAM-ICU for detection of delirium in critically ill patients were 76.0% and 95.7% and for ICDSC were 74.4% and 75.2%, respectively. All but one study was performed in a research setting, and that one study suggested that, with routine use of CAM-ICU, one-half of the patients with delirium were not detected. See Figure 1.i Results Of all 1,727 UTI patients, 905 were men and the mean age was 73.65 ± 14.1. In total, 425 patients (24.6%) became delirious, and 247 patients (14.3%) had sepsis. There was no signifi cant association between sepsis and delirium (P = 0.051). However, in the younger population (age <65) delirium occurred signifi cantly more frequently in septic patients than in nonseptic patients (22.9% vs. 10%, P <0.001). Conclusion Among UTI patients, sepsis may increase the complication of delirium. Especially in patients under 65 years old with UTI, delirium symptoms can be a marker for complication of sepsis. In contrast, delirium of patients aged 65 or over could be associated with not only sepsis but also other factors such as dementia, aging and UTI itself. p g Conclusion The CAM-ICU was the most specifi c bedside tool for assessment of delirium in critically ill patients. However, there was signifi cant heterogeneity of the results. These fi ndings were largely obtained in research settings, and the low sensitivity of the CAM-ICU in routine, daily practice may limit its use as a screening test. P338 Delirium screening in critically ill patients: a systematic review and meta-analysis A Serpa Neto1, AP Nassar Júnior2, SO Cardoso1, JA Manetta1, VG Pereira1, DC Esposito1, MC Damasceno1, AJ Slooter3 1ABC Medican School, Santo André, Brazil; 2São Camilo Hospital, São Paulo, Brazil; 3University Medical Center Utrecht, the Netherlands Critical Care 2012, 16(Suppl 1):P337 (doi: 10.1186/cc10944) Introduction Despite its frequency and impact, delirium in critically ill patients is poorly recognized. Our aim was to systematically review the accuracy of delirium screening instruments in critically ill patients. Introduction Despite its frequency and impact, delirium in critically ill patients is poorly recognized. Our aim was to systematically review the accuracy of delirium screening instruments in critically ill patients. Methods Systematic review and meta-analysis of publications between 1966 and 2011. The Medline and Embase databases were searched for studies on delirium in critically ill patients in ICUs, surgical wards or emergency rooms. The delirium screening tool had to be feasible in a clinical setting for use by a nonexpert. As the gold standard, delirium Methods A systematic literature search was conducted in Embase and Medline. Articles concerning quantitative EEG and delirium were included. Per article, the diff erences between delirious and nondelirious subjects in EEG characteristics were noted. Methods Systematic review and meta-analysis of publications between 1966 and 2011. The Medline and Embase databases were searched for studies on delirium in critically ill patients in ICUs, surgical wards or emergency rooms. The delirium screening tool had to be feasible in a clinical setting for use by a nonexpert. As the gold standard, delirium Results Fourteen studies were included, which were predominantly conducted in older patients. The relative power of the theta frequency band was most often and without exception signifi cantly diff erent (7/14 studies) in delirious subjects. Other frequently measured parameters S122 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods A prospective observational study in a university hospital including patients over 18 years old, in the fi rst 48 hours of ICU admission, with an expected ICU stay of at least 72 hours and signed informed consent. Pregnancy, cognitive impairment prior to admission, hepatic encephalopathy, Glasgow Coma Scale ≤9, active psychiatric illness, need for sedation or neuromuscular blockade, aphasia, foreign language, deafness and brain death were exclusion criteria. CAM-ICU was applied and doctors and nurses asked about the presence of delirium. Demographic data, SOFA score, mechanical ventilation and drugs used were determined. g p Reference y Results A total of 225 patients were included. The incidence of delirium was 24%. Patients who develop delirium during the ICU stay were older (OR 1.04, 1.02 to 1.07) and more likely to have a previous diagnosis of hypertension (OR 2.36, 1.24 to 4.52). The SAPS 3 (OR 1.09, 1.06 to 1.13) score, SOFA (OR 1.23, 1.09 to 1.39) score, and mechanical ventilation requirement (OR 3.6; 1.35 to 9.60) were higher among patients with delirium. These patients had longer ICU and hospital length of stay, and a higher crude mortality rate (24.07 vs. 7.02%). In a multivariate analysis, age (OR 1.03, 1.00 to 1.05), use of mechanical ventilation (OR 3.91, 1.22 to 12.96) and SAPS 3 score (OR 1.08, 1.04 to 1.12) were independently associated with delirium. SAPS 3 performed well in predicting delirium with an AUR ROC of 0.785 (0.714 to 0.856, best cut-off value ≥54 points) and a GoF of 0.175. 1. Ely EW, et al.: Crit Care Med 2001, 29:1370-1379. P341 Performance of SAPS 3 in predicting delirium among critically ill patients p T Cosentino, J Biatto, I Souza, M Dutra, L Ilnicki, P Martins, G Schettino, F Machado Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P339 (doi: 10.1186/cc10946) Introduction Delirium is a common complication in critically ill patients, occurring in up to 80% of patients on mechanical ventilation [1]. Recent studies showed that sicker patients at ICU admission, assessed by severity scores, are more susceptible to developing delirium [2]. To further evaluate this hypothesis, we undertook this study to assess the performance of SAPS 3 in predicting delirium, among adult patients admitted to a general ICU. Table 1 (abstract P340). κ values Health provider Delirium Hypoactive Attending physicians 0.530 0.019 Medical residents 0.615 0.018 Nurses 0.588 0.025 g Methods This was a prospective observational cohort study performed between June 2010 and June 2011, in a 26-bed ICU at Hospital Sírio- Libanês, São Paulo, Brazil. All consecutive adult patients admitted to the ICU were included. Patients with a previous diagnosis of advanced dementia and those with acute neurological disease (Glasgow <13) were excluded. The evaluation of delirium was performed using the CAM-ICU during routine bedside rounds in the morning. Discrimination and calibration of SAPS 3 in predicting delirium were assessed by the area under the receiver operating curve (AUR ROC) and the goodness of fi t (GoF) test, respectively. Secondary outcomes were hospital mortality and lengths of stay among patients with delirium. Conclusion Delirium had a higher incidence in intensive care patients and was related to longer hospital stay and higher mortality. Specifi c tests should be used for diagnosis, since the clinical suspicion has low sensitivity, especially in cases of hypoactive delirium and among attending physicians. References 1. Ely EW, et al.: JAMA 2001, 286:2703-2710. 1. Ely EW, et al.: JAMA 2001, 286:2703-2710. 1. Ely EW, et al.: JAMA 2001, 286:2703-2710. 2. Van Rompaey B, et al.: Crit Care 2009, 13:R77. 1. Ely EW, et al.: JAMA 2001, 286:2703 2710. 2. Van Rompaey B, et al.: Crit Care 2009, 13:R77. 2. Van Rompaey B, et al.: Crit Care 2009, 13:R77. y Methods A prospective cohort study over 8 weeks in a 25-bed ICU setting. Daily CAM-ICU assessments were done by three trained doctors. It was noted whether the ICU team had assessed the individual patient for delirium. If the patient was delirious, the team was informed and their management was noted. Eligible patients had to have a RASS score above –4 and be able to comply with the assessment. The Fisher’s exact test was used to calculate statistical signifi cance of detection and treatment. Delirium screening in critically ill patients: a systematic review and meta-analysis Patients were followed for 14 days or until discharge from the ICU. The agreement between CAM-ICU and clinical diagnosis was assessed using Cohen’s kappa statistic (κ). Risk factors were assessed by a multivariate regression model. were the relative power of the delta and alpha frequency band and the peak frequency. None of these studies addressed the optimal electrode deviation or the question of how to distinguish sleep from delirium. Conclusion Given the feasibility for continuous EEG monitoring in ICU, EEG delirium monitoring in ICU patients seems to be promising. References 1. Ely EW, et al.: JAMA 2004, 291:1753-1762. 2. Girard TD, et al.: Lancet 2008, 371:126-134. 3. van Eijk MM, et al.: Am J Respir Crit Care Med 2011, 184:340-344. 4. van Eijk MMJ, et al.: Crit Care Med 2009, 37:1881-1885. 5. Romano J, et al.: Arch Neurol Psychiatry 1944, 51:356-377. Investigation into detection and treatment rates of hyperactive and hypoactive delirium in the ICU setting S Kudsk-Iversen, J Wong, H Kingston, L Poole S Kudsk-Iversen, J Wong, H Kingston, L Poole g g The Royal Liverpool University Hospital, Liverpool, UK y p y p , p , Critical Care 2012, 16(Suppl 1):P341 (doi: 10.1186/cc10948) Introduction We aimed to investigate the link between the type of delirium (that is, hyperactive or hypoactive), its detection by the day staff and the subsequent treatment. The morbidity related to delirium is well known to critical care medical staff ; however, some fi ndings suggest insuffi cient and inconsistent recognition and management of delirium [1]. Hypoactive delirium, despite being more common in the ICU setting, often goes undetected and undertreated due to its withdrawn and drowsy presentation [2]. Conclusion We found that SAPS 3 was a good parameter for predicting delirium during the ICU stay. Future studies are needed to confi rm our results in a larger and diff erent patient sampling. References P339 y g Results In the 119 patients included, the incidence of delirium was 24.4% (29 patients) and time to development of delirium was 68.3 ± 63.6 hours. The agreement between clinical diagnoses and CAM- ICU was better for medical residents (Table 1). Patients with delirium had a longer ICU (10.83 ± 15.08 and 4.98 ± 9.57, P = 0.015) and hospital (36.93 ± 31.33 and 19.10 ± 19.48, P = 0.0004) length of stay, higher ICU mortality (13.79% and 2.22%, OR = 7.04 (1.22 to 40.7)) and hospital mortality (27.6% and 6.66%, OR = 5.33 (1.67 to 17.04)) than patients without delirium. Risk factors were: mechanical ventilation (P = 0.018, OR = 3.09 (1.21 to 7.86)) and APACHE II score greater than 8.5 (P = 0.011, OR = 5.35 (1.48 to 19.43)). Incidence of delirium and inadequacy of the clinical diagnosis in patients in intensive carei Hyperactive and hypoactive cases and their treatment rate P343 Using tramadol to monitor hepatic drug metabolism in the critically ill K Lane1, JJ Dixon1, D McKeown2, A Johnston2, I MacPhee1, BJ Philips1 1Acute Kidney Injury Research Group, St George’s, University of London, UK; 2Analytical Services International Ltd, St George’s, University of London, UK Critical Care 2012, 16(Suppl 1):P343 (doi: 10.1186/cc10950) the delirium was recognised, 76% of the hyperactive and 20% of the hypoactive cases were started on targeted treatment (P = 0.0038). See Tables 1 and 2. Conclusion Although the study had a higher rate of hyperactive delirium compared to otherwise available research, the fi ndings confi rmed that a signifi cant proportion of hypoactive delirium goes undetected and remains largely undertreated. Introduction Previously, we have demonstrated signifi cant inhibition of hepatic drug metabolism by the enzymes cytochrome P450 (CYP) 3A4 and 3A5 in acute kidney injury (AKI) using midazolam as a probe drug [1,2]. We are now developing the use of tramadol as a probe drug to test the hypothesis that CYP2D6 function is also inhibited by AKI in critical illness. In this study we sought to determine whether a single timepoint tramadol concentration could be identifi ed as a reliable surrogate for measurement of a full area under the concentration time curve after intravenous administration in adults. Introduction Previously, we have demonstrated signifi cant inhibition of hepatic drug metabolism by the enzymes cytochrome P450 (CYP) 3A4 and 3A5 in acute kidney injury (AKI) using midazolam as a probe drug [1,2]. We are now developing the use of tramadol as a probe drug to test the hypothesis that CYP2D6 function is also inhibited by AKI in critical illness. In this study we sought to determine whether a single timepoint tramadol concentration could be identifi ed as a reliable surrogate for measurement of a full area under the concentration time curve after intravenous administration in adults. Methods We conducted a study of 10 critically ill patients in our hospital’s general critical care unit. Tramadol 10 mg was given intravenously, and serum was taken at 0.5, 1, 2, 3, 4 and 8 hours for determination of concentrations of tramadol ([tramadol]) and its two main metabolites. Inclusion criteria: age >18 years, predicted ICU stay >48 hours. Exclusion criteria: recent receipt of tramadol or major CYP2D6 inhibitors, hepatic failure, pregnancy/breastfeeding. P342 Memories and post-traumatic stress-related symptoms in older, post-cardiac surgery patients: substudy of an RCT N Hammond, F Bass, Y Shehabi Prince of Wales Hospital, Randwick, Australia Critical Care 2012, 16(Suppl 1):P342 (doi: 10.1186/cc10949) N Hammond, F Bass, Y Shehabi g y g Results There was a strong correlation between the area under the curve (AUC) of the [tramadol]–time graph and t = 4 hours [tramadol], P <0.0001, r = 0.983. See Figure 1. The [tramadol] at other timepoints correlated less strongly with the AUC. The mean [tramadol] at 4 hours was 29.7 ng/ml (24.3 to 35.1) and the mean AUC was 257 ng/hour/ml (211 to 303). Analysis of tramadol metabolites confi rmed that CYP2D6 was predominantly responsible for tramadol metabolism. Introduction The majority of ICU survivors display little evidence of severe psychological sequelae. However, there is evidence of post- traumatic stress disorder (PTSD)-related symptoms such as anxiety, depression, panic attacks, distressing memories and fl ashbacks within the fi rst 3 months post ICU discharge [1,2]. This substudy of the DEXCOM trial 3 (randomised controlled trial of neurobehavioural eff ects of dexmedetomidine or morphine for sedation and analgesia in patients 60 years or older, undergoing coronary artery bypass grafting and/or valve replacement) aims to explore any negative memories of the ICU and development of PTSD-related symptoms between treatment groups of patients at high risk of developing delirium. Conclusion A single blood sample, taken 4 hours post-intravenous tramadol injection, reliably predicts integral tramadol exposure in critically ill adults and may be useful for assessing CYP2D6 function.l A larger study of the infl uences of AKI and CYP genotype on hepatic drug metabolism in the critically ill is underway. Incidence of delirium and inadequacy of the clinical diagnosis in patients in intensive carei Results A total of 139 patients were included, providing a total of 507 patient-days. On 32 occasions (6%) the patient assessed was found to be delirious. Twelve patients in ITU (19%) and nine in HDU (9%) were delirious at least once. Of the 32 cases of delirium, 53% were hyperactive. Seventy-six percent of the hyperactive and 27% of the hypoactive cases had been detected by the day team (P = 0.0118). Once Introduction This study aims to assess the incidence, risk factors and impact of delirium on outcome and to analyze the concordance between the Confusion Assessment Method for the Intensive Care (CAM-ICU) and clinical diagnosis. S123 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 a convenience sample so was not powered to detect a signifi cant diff erence. No diff erences in factual or delusional memories or PTSD- related symptoms between the treatment groups were found. These data could be the basis of a sample size calculation for a larger study. References 1. Schelling G, et al.: Crit Care Med 1998, 26:651-659. 2. Stoll C, et al.: J Thorac Cardiovasc Surg 2000, 120:505-512. 3. Shehabi Y, et al.: Anesthesiology 2009, 111:1074-1083. Table 1 (abstract P341). Hyperactive and hypoactive cases and their detection rate Number of Number of hyperactive (%) hypoactive (%) Detected 13 (76) 4 (27) Not detected 4 (24) 11 (73) T bl 2 ( b P3 ) H i d h i d h i Table 1 (abstract P341). Hyperactive and hypoactive cases and their detection rate a convenience sample so was not powered to detect a signifi cant diff erence. No diff erences in factual or delusional memories or PTSD- related symptoms between the treatment groups were found. These data could be the basis of a sample size calculation for a larger study. References Schelling G, et al.: Crit Care Med 1998, 26:651-659. Stoll C, et al.: J Thorac Cardiovasc Surg 2000, 120:505-512. Shehabi Y, et al.: Anesthesiology 2009, 111:1074-1083. Table 2 (abstract P341). 1. Kirwan CJ, et al.: Intensive Care Med 2009, 35:1271-1275. 2. Kirwan CJ, et al.: Intensive Care Med 2012, 38:76-84. P344 Data mining techniques for predicting acute kidney injury after elective cardiac surgery J Van Eyck, J Ramon, F Guiza, G Meyfroidt, M Bruynooghe, G Van den Berghe K.U. Leuven, Heverlee, Belgium Critical Care 2012, 16(Suppl 1):P344 (doi: 10.1186/cc10951) P344 change in creatinine). Nine patients (33% of all patients, 64% of AKI patients) received RRT. ICU mortality was three out of 14 (21%) patients with AKI and one out of 13 (8%) patients without AKI. This diff erence was not statistically signifi cant. Thirteen out of 20 (65%) ventilated patients developed AKI, compared with one out of seven (14%) nonventilated patients. This diff erence was statistically signifi cant (P = 0.0329). Excluding fatalities, the duration of IPPV was longer in patients with AKI (median 11 days, range 0 to 54 days) than in patients without AKI (median 1 day, range 0 to 20 days). This diff erence was statistically signifi cant (P <0.05). Excluding fatalities, the length of stay was longer in patients with AKI (median 19 days, range 10 to 68 days) than in patients without AKI (median 5 days, range 2 to 29 days). This diff erence was statistically signifi cant (P <0.02). P344 Data mining techniques for predicting acute kidney injury after elective cardiac surgery J Van Eyck, J Ramon, F Guiza, G Meyfroidt, M Bruynooghe, G Van den Berghe K.U. Leuven, Heverlee, Belgium Critical Care 2012, 16(Suppl 1):P344 (doi: 10.1186/cc10951) Introduction Development of acute kidney injury (AKI) during the postoperative period is associated with increases in both morbidity and mortality. The aim of this study is to develop a statistical model capable of predicting the occurrence of AKI in patients after elective cardiac surgery. f y gi Conclusion We noted a higher incidence of AKI in critical illness associated with A/H1N1 (52%) compared to that of a larger study [1]. AKI was associated with the incidence as well as duration of mechanical ventilation and length of stay in the ICU. The use of RRT in the current study (60%) was much higher than in the modeling study (16%). We found a trend towards greater mortality with AKI, although (unlike Petillä and colleagues [1]) this failed to reach signifi cance. Methods A total of 810 adult (>18 years) elective cardiac surgery patients, admitted to the surgical ICU of the University Hospital of Leuven between 18 January 2007 and 8 January 2009, were retrospectively selected for this study. Patients with an ICU stay of less than 24 hours, as well as patients suff ering from chronic kidney disease, were excluded. Relevant patient records were extracted from an electronic database system and analyzed using data mining techniques [1]. Reference 1. Pettilä V, et al.: Acute kidney injury in patients with infl uenza A (H1N1) 2009. Intensive Care Med 2011, 37:763-767 . Results In this study, two separate models were developed for predicting the occurrence of AKI (defi ned as RIFLE stage three or need for renal replacement therapy) within a week after the patient’s admission. An initial model was built using only readily available admission data (including demographic information, previous treatments and pre- admission values for physiological variables). This resulted in an AUC of 0.6056 (95% CI, 0.4874 to 0.7239) on the validation cohort. The initial model was then extended by adding information on administered medication, measurements of physiological parameters and laboratory results available during the fi rst four hours of the patient’s ICU stay. This new model resulted in an AUC of 0.8339 (95% CI, 0.7364 to 0.9315) on the validation cohort. A RIFLE score-based trigger for renal replacement therapy and survival after cardiac surgery A Schneider, G Eastwood, S Seevanayagam, G Matalanis, R Bellomo Austin Health, Heidelberg, Australia Critical Care 2012, 16(Suppl 1):P346 (doi: 10.1186/cc10953) Acute kidney injury in critically ill patients with A/H1N1 pneumonitis in 2010/11 Acute kidney injury in critically ill patients with A/H1N1 pneumonitis in 2010/11 M Atkinson, A Krige, S Chukkambotla East Lancashire Hospitals NHS Trust, Blackburn, UK Critical Care 2012, 16(Suppl 1):P345 (doi: 10.1186/cc10952) Acute kidney injury in critically ill patients with A/H1N1 pneumonitis in 2010/11 Acute kidney injury in critically ill patients with A/H1N1 pneumonitis in 2010/11 M Atkinson, A Krige, S Chukkambotla East Lancashire Hospitals NHS Trust, Blackburn, UK Critical Care 2012, 16(Suppl 1):P345 (doi: 10.1186/cc10952) p M Atkinson, A Krige, S Chukkambotla g East Lancashire Hospitals NHS Trust, Blackburn, UK y Conclusion After cardiac surgery, RRT is typically applied to patients with the most severe clinical presentation irrespective of creatinine levels. A RIFLE score-based trigger for RRT is unlikely to improve patient survival. p Critical Care 2012, 16(Suppl 1):P345 (doi: 10.1186/cc10952) Introduction A/H1N1 infection is a major seasonal cause of illness requiring critical care admission. A high proportion of these patients develop acute kidney injury (AKI) [1]. P344 Data mining techniques for predicting acute kidney injury after elective cardiac surgery J Van Eyck, J Ramon, F Guiza, G Meyfroidt, M Bruynooghe, G Van den Berghe K.U. Leuven, Heverlee, Belgium Critical Care 2012, 16(Suppl 1):P344 (doi: 10.1186/cc10951) The main advantage of these techniques is that they are capable of automatically selecting the variables that are relevant to a particular problem. Using such a data mining algorithm, predictive models were built on a development cohort of 385 patients and validated on a separate cohort of 425 patients. A RIFLE score-based trigger for renal replacement therapy and survival after cardiac surgery A Schneider, G Eastwood, S Seevanayagam, G Matalanis, R Bellomo Austin Health, Heidelberg, Australia Critical Care 2012, 16(Suppl 1):P346 (doi: 10.1186/cc10953) Introduction It is controversial whether all critically ill patients with RIFLE-F class acute kidney injury (AKI) should receive renal replacement therapy (RRT). We reviewed the outcome of open-heart surgery patients with severe AKI who did not receive RRT.i Methods We identifi ed all patients who developed AKI after cardiac surgery during a 4-year period, and obtained baseline characteristics, intraoperative details and in-hospital outcomes. We analyzed physiological and biochemical features at the time of RRT initiation or at peak creatinine if no RRT was provided. Conclusion In this study, we have shown that data mining techniques are a viable option for developing predictive models in a clinical setting. Furthermore, we have shown that by adding information gathered during the patient’s stay, a model’s performance can drastically improve compared to a model using only admission data. Thus, it might be possible to further improve existing scoring systems such as the Thakar score [2] and the simplifi ed renal index [3]. References p p Results We reviewed 1,504 patients. Of these, 137 (9.1%) developed postoperative AKI with 71 meeting RIFLE-F criteria and 23 (32.4% of RIFLE-F cases) not receiving RRT. Compared with RRT-treated RIFLE-F patients, no-RRT patients had lower APACHE III scores, less intra- aortic balloon pump requirements, shorter intensive care stay and a trend toward lower mortality. At peak creatinine, their urinary output, arterial pH and PaO2/FIO2 ratio were all signifi cantly higher. Their serum creatinine was also higher (304 vs. 262 μmol/l, P = 0.02). Only three died in-hospital. Detailed review of cause and mode of death was consistent with non-RRT-preventable deaths. In contrast, 27 patients with RIFLE-R or RIFLE-I class received RRT. Compared with RRT-treated RIFLE-F patients, they had a trend towards a more severe presentation and a higher mortality (51.8% vs. 29.2%, P = 0.02). See Figure 1. 1. Meyfroidt G, et al.: Best Pract Res Clin Anaesthesiol 2009, 23:127-143. 2. Thakar C, et al.: J Am Soc Nephrol 2005, 16:162-168. 3. Duminda N, et al.: JAMA 2007, 297:1801-1809. P347f Methods We studied all A/H1N1-positive admissions to a district general hospital (DGH) ICU during the months of December 2010 and January 2011. The study aimed to describe the incidence of AKI using the creatinine score from the RIFLE criteria and its associations with mortality, incidence and duration of intermittent positive pressure ventilation (IPPV), length of stay in the ICU and provision of renal replacement therapy (RRT). Eff ect of off -pump versus on-pump coronary artery bypass grafting in patients with chronic kidney disease ME Schroeder1, L Chawla1, Y Zhao2, F Lough1, F Najam1, M Seneff 1, JM Brennan3 1George Washington University Hospital, Washington, DC, USA; 2Duke Clinical Research Institute, Raleigh, NC, USA; 3Duke University Medical Center, Raleigh, NC, USA Critical Care 2012, 16(Suppl 1):P347 (doi: 10.1186/cc10954) References 1. Kirwan CJ, et al.: Intensive Care Med 2009, 35:1271-1275. 2. Kirwan CJ, et al.: Intensive Care Med 2012, 38:76-84. g p p g p g Methods At 8 weeks post ICU discharge, patients completed three assessment tools, by mail or telephone. Tools used were Depression Anxiety Stress Scale, ICU memory assessment tool and impact of events scale. Figure 1 (abstract P343). Correlation of [tramadol] at t = 4 hours and AUC [tramadol]–time graph. iv, intravenous. Results A total of 153 patients completed the substudy; 72 patients in the [M]orphine group and 81 in the [D]exmedetomidine group. The mean age (years) in the M group was 72 (SD 5) and in the D group 69 (SD 6), with 71% (n = 51) males in the M group and 84% (n = 68) in the D group. The mean ICU hours for M and D were 58 (SD 40) and 48 (SD 32) respectively. No signifi cant diff erences of memories or PTSD- related symptoms between the two treatment groups, for each of the three assessment tools, were found. From the ICU memory tool, 21% (n = 15/70) of M group patients and 15% (n = 12/81) of the D patients remember being in the ICU. Just over one-half of the patients in both groups did not remember all of their ICU stay with clarity (M group: 54%, n = 39/72; D group: 51%, n = 40/78). Furthermore, 23% (n = 15/64) of M patients and 14% (n = 10/73) of D patients had intrusive memories whilst in the ICU. Figure 1 (abstract P343). Correlation of [tramadol] at t = 4 hours and AUC [tramadol]–time graph. iv, intravenous. Conclusion Patients undergoing cardiac surgery with ICU stay do not have clear memories of this episode. A small number had intrusive memories, which are more common in M patients. The study used S124 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Eff ect of off -pump versus on-pump coronary artery bypass grafting in patients with chronic kidney diseasef Results Twenty-seven patients were admitted to the ICU who tested positive for A/H1N1. Fourteen (52%) met the RIFLE criteria for AKI. Of these, three (11%) met the RIFLE criterion for Risk (>150% change in creatinine), three (11%) met the criterion for Injury (>200% change in creatinine), and eight (30%) met the criterion for Failure (>300% Critical Care 2012, 16(Suppl 1):P347 (doi: 10.1186/cc10954) Introduction Patients with chronic kidney disease (CKD) have been largely excluded from clinical trials of off -pump coronary artery bypass S125 Critical Care 2012, Volume 16 Suppl 1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P346). Flow chart. ASCTS, Australian Society of Cardio Thoracic Sugery. Figure 1 (abstract P346). Flow chart. ASCTS, Australian Society of Cardio Thoracic Sugery. dopamine to protect the kidneys against hypoperfusion injury following cardiac surgery remains controversial. Cystatin C has been described as a sensitive biomarker of early renal tubular injury. We aimed to evaluate the eff ect of renal-dose dopamine on renal tubular functions in patients undergoing coronary artery bypass grafting (CABG) surgery. Methods Thirty-six patients undergoing CABG surgery were prospec- tively randomized to receive either 2 μg/kg/minute dopamine infusion (Group D, n = 19) or saline as placebo (Group P, n = 17) starting from induction of anesthesia for 48 hours. Serial blood and urine samples after induction of anesthesia and 2, 12, 24, 48 hours post CPB were collected to measure serum cystatin C, creatinine levels and urinary β2-microglobulin. Intraoperative and daily measurements of hemodynamic parameters and urine output were recorded. grafting (OPCAB). We sought to determine if the pump status aff ected outcomes in patients with CKD. Methods Using a nonrandomized cohort of 742,909 nonemergent, isolated CABG cases (including 158,561 OPCAB cases) in the Society of Thoracic Surgery Database from 2004 through 2009, we evaluated the association between pump status (off -pump vs. on-pump) and in-hospital death or incidence of renal replacement therapy (RRT) across strata of preoperative renal function. We used both propensity methods and an instrumental variable (IV) approach to account for imbalances in baseline patient risk.f Results Compared with on-pump cases, off -pump cases were of similar age (65.6 vs. 64.9 years) with a similar distribution of preoperative estimated glomerular fi ltration rate (eGFR). Eff ects of renal-dose dopamine on renal tubular functions following coronary artery bypass grafting surgery Eff ects of renal-dose dopamine on renal tubular functions following coronary artery bypass grafting surgery P Zeyneloglu, H Ozdemir, O Komurcu, N Bayraktar, A Sezgin, A Pirat, G Arslan Baskent University, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P348 (doi: 10.1186/cc10955) Eff ects of renal-dose dopamine on renal tubular functions following coronary artery bypass grafting surgery P Zeyneloglu, H Ozdemir, O Komurcu, N Bayraktar, A Sezgin, A Pirat, G Arslan Baskent University, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P348 (doi: 10.1186/cc10955) y y yp g g g y P Zeyneloglu, H Ozdemir, O Komurcu, N Bayraktar, A Sezgin, A Pirat, G Arslan Baskent University, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P348 (doi: 10.1186/cc10955) Conclusion The results suggest that renal-dose dopamine does not exacerbate the severity of renal tubular injury when compared with the untreated controls during the early postoperative period of patients undergoing CABG surgery. Reference y y Critical Care 2012, 16(Suppl 1):P348 (doi: 10.1186/cc10955) Introduction Cardiopulmonary bypass (CPB) is regarded as an important contributor to acute kidney injury and use of renal-dose Eff ect of off -pump versus on-pump coronary artery bypass grafting in patients with chronic kidney diseasef In a propensity weighted analysis, OPCAB was associated with a reduction in composite in- hospital death or RRT, with a progressively increased benefi t among those with lower preoperative renal function (eGFR ≥90 ml/minute: risk diff erence = 0.05 per 100 patients (on-pump minus off -pump), 95% confi dence interval = –0.06 to 0.16; 60 to 89 ml/minute: 0.14, 0.05 to 0.23; 30 to 59 ml/minute: 0.66, 0.45 to 0.87; and 15 to 29 ml/minute: 3.66, 2.14 to 5.18). A similar trend was observed for both component endpoints. However, while the IV analysis confi rmed the protective eff ect of OPCAB on composite in-hospital death or RRT among patients with a reduced eGFR, this result was driven by an eff ect on RRT and not mortality. y p p Results The groups were similar in terms of physical characteristics, perioperative hemodynamic measurements, urine outputs and surgical times. Serum cystatin C levels demonstrated similar increases during 12, 24 and 48 hours post CPB in the dopamine and placebo groups (P >0.05 for all). See Table 1. No diff erences were detected with respect to serum creatinine and urine β2-microglobulin levels between the groups (P >0.05 for both). GFR was preserved equally in both groups on postoperative day 2 (104.1 ± 23.1 vs. 101.4 ± 35.8 ml/minute, P >0.05). Table 1 (abstract P348). Serum cystatin C levels (ng/ml) of the patients Group D (n = 19) Group P (n = 17) P value Induction 803 ± 173 789 ± 285 0.987 2 hours CPB 857 ± 236 861 ± 347 0.664 12 hours CPB 807 ± 239 1,132 ± 396 0.052 24 hours CPB 906 ± 211 1,158 ± 432 0.149 48 hours CPB 1,296 ± 341 1,129 ± 350 0.296 Table 1 (abstract P348). Serum cystatin C levels (ng/ml) of the patients yf y Conclusion Patients with CKD experience less death or incidence of RRT when treated with off -pump versus on-pump CABG; however, this composite eff ect is driven by a reduction in incidence of RRT (not death) among low eGFR patients. Prospective trials comparing these procedures in patients with impaired preoperative renal function are warranted. P351 P351 Plasma and urine neutrophil gelatinase-associated lipocalin as markers of acute kidney injury in critically ill adults R Matsa1, E Ashley2, J Osypiw2, V Sharma1, A Walden2, L Keating2 1Oxford University Hospitals NHS Trust, Oxford, UK; 2Royal Berkshire NHS Foundation Trust, Reading, UK Critical Care 2012, 16(Suppl 1):P351 (doi: 10.1186/cc10958) Methods This was a multicenter, prospective, longitudinal study. The study population included 216 nurses who work in the ICU, inpatient care unit, and emergency unit at six public and private hospitals. Data collection was performed from October 2010 to February 2011 using a 10-question questionnaire related to prevention, diagnosis, and treatment of AKI. Introduction Acute kidney injury (AKI) has signifi cant impact both on the morbidity and mortality in patients on the ICU. The current defi nition and classifi cation of AKI [1] uses changes in both the serum creatinine and urine output. This occurs late in the evolution of AKI and so the diagnosis can be delayed. Early detection of AKI could allow earlier recognition and treatment of the condition. Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein produced in response to infl ammation, infection and kidney injury [2] and is found in blood and urine samples obtained from patients soon after the onset of AKI [3]. Earlier studies have shown that NGAL can be detected as early as 2 hours following AKI [2]. The predictive value of NGAL in the ICU may help the earlier recognition of AKI. The aim of the study is to determine whether plasma and/or urine NGAL levels can predict the earlier incidence of AKI (as defi ned by RIFLE criteria) in critically ill patients. Results Data showed that 81.7% of the nurses gave correct answers regarding the association of urine volume rate in the identifi cation of AKI; 57.2% did not know how to identify the clinical manifestations of AKI; 67.1% made a mistake by answering that the subtle increase of creatinine has no great impact on a mortality rate; 66.8% answered the question incorrectly on measures to prevent AKI; 60.4% were correct when they answered that the use of loop diuretics in the prevention of AKI is not recommended; and 92.5% said they had no knowledge of the Acute Kidney Injury Network classifi cation. Conclusion The results showed that most nurses do not have enough knowledge for the early identifi cation of AKI. References References 1. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. 2. Mishra J, et al.: Lancet 2005, 365:1231-1238. 3. Mishra J, et al.: Pediatr Nephrol 2006, 21:856-863. References 1. Bellomo R, et al.: Crit Care 2004, 8:R204-R212. 2. Mishra J, et al.: Lancet 2005, 365:1231-1238. h l d h l 3. Mishra J, et al.: Pediatr Nephrol 2006, 21:856-863. Results Postoperative fl uid overload was present in 15% of patients with a mean positive fl uid balance of 12 ± 9 kg. Patients who survived the hospital stay had a lower positive fl uid balance of 2.8 l (25th to 75th percentiles: 1.5 to 5.5) compared to patients who died (23.0 l (25th to 75th percentiles: 14.5 to 24.0)); P = 0.010. A positive fl uid balance predicted in-hospital mortality with AUC 0.94 (95% CI 0.83 to 0.99), sensitivity 100% and specifi city 80% at a cut-off >6 l. Urine NGAL predicted fl uid overload (AUC-ROC 0.80 (95% CI 0.64 to 0.93)) and mortality (AUC-ROC 0.88 (95% CI 0.78 to 0.97)). Serum creatinine did not predict fl uid overload (AUC-ROC 0.51 (95% CI 0.24 to 0.78)) or mortality (AUC-ROC 0.61 (95% CI 0.16 to 0.99)).i P352 P352 Plasma and urine neutrophil gelatinase-associated lipocalin in septic and nonseptic ICU patients CS Vrettou1, S Kokkoris2, K Apostolou2, M Parisi2, E Haritatos2, S Dimopoulos2, S Nanas1 1National and Kapodistrian University of Athens, Greece; 2Evaggelismos Hospital, Athens, Greece Critical Care 2012, 16(Suppl 1):P352 (doi: 10.1186/cc10959) g g Reference Sumeray M, et al.: J Nephrol 2001, 14:397-402. Sumeray M, et al.: J Nephrol 2001, 14:397-402. S126 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P349 Nurses’ knowledge regarding the early identifi cation of acute kidney injury S Agege Lobo, RM Matheus Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, Brazil Critical Care 2012, 16(Suppl 1):P349 (doi: 10.1186/cc10956) P351 This highlights the importance of training programs for nurses who work at hospital units, with the purpose of developing professional competences and aptitudes regarding both prevention and detection of AKI. Methods This single-centre prospective observational study is currently recruiting 200 consecutive adult patients with no AKI on presentation to the ICU. Serial samples of plasma and urine are collected on all patients included in the study at 0 hours and then every 24 hours in the ICU stay up to 72 hours and assayed for NGAL using a turbidimetric assay on the standardised automated analyser.i P349 Nurses’ knowledge regarding the early identifi cation of acute kidney injury Critical Care 2012, 16(Suppl 1):P349 (doi: 10.1186/cc10956) Critical Care 2012, 16(Suppl 1):P349 (doi: 10.1186/cc10956) Introduction The objective was to evaluate nurses’ knowledge on the early identifi cation of acute kidney injury (AKI) in an ICU, inpatient care unit, and emergency unit. P351 P350 Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload, a potentially modifi able risk factor for mortality after cardiac surgery M Haase1, P Mertens1, M Plaß2, R Bellomo3, A Haase-Fielitz1 1Otto-von-Guericke University Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3Austin Health, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P350 (doi: 10.1186/cc10957) Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload, a potentially modifi able risk factor for mortality after cardiac surgery Neutrophil gelatinase-associated lipocalin predicts postoperative fl uid overload, a potentially modifi able risk factor for mortality after cardiac surgery y y Results Results on the fi rst 27 patients are currently available. The predictive performance of pNGAL at admission for AKI (24 hours prior to creatinine-based (RIFLE) diagnosis) was good (AUC-ROC 0.8 (95% CI 0.88 to 1.03)). The predictive performance of uNGAL at admission for the occurrence of AKI (24 hours prior to creatinine-based (RIFLE) diagnosis) (AUC-ROC 0.77 (95% CI 0.47 to 1.07)) was fair. See Table 1. M Haase1, P Mertens1, M Plaß2, R Bellomo3, A Haase-Fielitz1 1Otto-von-Guericke University Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3Austin Health, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P350 (doi: 10.1186/cc10957) Introduction In most previous studies, neutrophil gelatinase- associated lipocalin (NGAL), measured immediately following cardiac surgery, has been demonstrated to predict postoperative increases in serum creatinine and decline in urine output. In patients undergoing cardiac surgery, postoperative fl uid overload is a typical complication. In this study, we investigated the early postoperative value of NGAL to predict subsequent fl uid overload, a potentially modifi able risk factor in these patients. Table 1 (abstract P351). Sensitivity and specifi city of pNGAL (time ‘0) to diagnose AKI occurrence at 24 hours Cut-off (ng/ml) Sensitivity (95% CI) Specifi city (95% CI) <270.0 82.6 (61.2 to 95) 100 (37.6 to 100) <316.5 86.9 (66.4 to 97.2) 100 (37.6 to 100) <381.0 91.3 (71.9 to 98.9) 100 (37.6 to 100) Table 1 (abstract P351). Sensitivity and specifi city of pNGAL (time ‘0) to diagnose AKI occurrence at 24 hours p Methods We studied 100 adult cardiac surgery patients assigned to the control arm of a randomized controlled trial. Urine and serum were sampled immediately after admission to the ICU. Urine NGAL was measured on the ARCHITECT laboratory platform (Abbott Diagnostics) and serum creatinine using an enzymatic assay. Postoperative fl uid overload was defi ned as positive fl uid balance with >10% excess of preoperative body weight within 48 hours. An area under the curve of the receiver-operating characteristics (AUC-ROC) of 0.5 indicates the predictive ability equaling the toss of a coin and >0.7 of a useful biomarker.l Conclusion Early results on pNGAL suggest that it could be an independent predictor of AKI in an unselected population of critically ill adults. Further results are awaited. P349 Nurses’ knowledge regarding the early identifi cation of acute kidney injury S Agege Lobo, RM Matheus Faculdade de Medicina de São José do Rio Preto – FAMERP, São José do Rio Preto, Brazil Critical Care 2012, 16(Suppl 1):P349 (doi: 10.1186/cc10956) P349 mortality. Early postoperatively measured urine NGAL is a good predictor of fl uid overload and mortality whereas measurement of serum creatinine at the same time equals the toss of a coin. Early NGAL- guided adjustments of fl uid management might reduce organ edema and potentially improve patient outcomes after cardiac surgery. Our fi ndings should be validated in larger patient cohorts. Additive value to clinical judgement of blood neutrophil gelatinase- associated lipocalin in diagnosis of acute kidney injury and prediction of mortality in patients hospitalized from the emergency department p L Magrini1, B De Berardinis1, R Marino1, G Gagliano1, E Ferri1, P Moscatelli2, P Ballarino2, B Gliozzo3, G Carpinteri3, S Di Somma1 1S. Andrea Hospital ‘Sapienza’ University, Rome, Italy; 2S. Martino Hospital, Genoa, Italy; 3S. Elia Hospital, Catania, Italy Critical Care 2012, 16(Suppl 1):P354 (doi: 10.1186/cc10961) p L Magrini1, B De Berardinis1, R Marino1, G Gagliano1, E Ferri1, P Moscatelli2, P Ballarino2, B Gliozzo3, G Carpinteri3, S Di Somma1 1S. Andrea Hospital ‘Sapienza’ University, Rome, Italy; 2S. Martino Hospital, Genoa, Italy; 3S. Elia Hospital, Catania, Italy Critical Care 2012, 16(Suppl 1):P354 (doi: 10.1186/cc10961) i y Methods Ninety-six patients consecutively admitted to the ICU were included in the study. Exclusion criteria were chronic renal failure, AKI prior to ICU admission, brain death, pregnancy, age <18 years and predicted ICU stay less than 48 hours. Patients’ demographic characteristics, APACHE II and SOFA score, existing comorbidities, primary reason for admission to intensive care, pNGAL, uNGAL, white cell count and C-reactive protein levels were recorded on admission, while the RIFLE score and sepsis status were recorded until day 7 post admission. The Mann–Whitney U test was used to compare pNGAL and uNGAL levels in septic and nonseptic patients. Introduction Acute kidney injury (AKI) is a common and diffi cult to diagnose complication among hospitalized patients with increasing incidence. Introduction Acute kidney injury (AKI) is a common and diffi cult to diagnose complication among hospitalized patients with increasing incidence. Methods A total of 665 (357 M:308 F; mean age 74 ± 14 years) emergency department (ED) patients designated for hospitalization were included in a multicenter prospective study to evaluate the utility of blood neutrophil gelatinase-associated lipocalin (NGAL) assessments as an aid in the early risk evaluation for AKI. NGAL and serum creatinine (sCr) were determined at ED presentation (T0), 6, 12, 24 and 72 hours after hospitalization. The clinical certainty of AKI was determined by ED physician (Ph) while blinded to NGAL results. p p p Results Out of 96 patients included, 56 were male, 12 had AKI and 30 had sepsis on admission. The mean age was 55.5 ± 19.6 years, the mean APACHE II score was 14.8 ± 5.6 and the mean admission SOFA score was 6.6 ± 2.9. There were 43 medical admissions, 17 elective surgical, and 36 emergency surgical including trauma. Both pNGAL and uNGAL were higher in patients with AKI on admission (P <0.001). Additive value to clinical judgement of blood neutrophil gelatinase- associated lipocalin in diagnosis of acute kidney injury and prediction of mortality in patients hospitalized from the emergency department Their levels were also found to be higher in septic compared with nonseptic patients (septic pNGAL = 153.13 ± 144.86 vs. nonseptic pNGAL = 102.45 ± 95.65, P = 0.076; septic uNGAL = 306.66 ± 532.88 vs. nonseptic uNGAL = 123.41 ± 354.07, P = 0.002). When patients with AKI as well as patients who developed AKI within the fi rst 7 days post admission were excluded from the analysis, higher uNGAL and pNGAL values in the group of septic patients were not signifi cant at the level of 5%. The estimated sample size for signifi cance 5% and power 80% is 74 for uNGAL (2,200 for pNGAL). Moreover pNGAL and uNGAL had a similar area under the ROC curve (0.773 and 0.779 respectively) for predicting AKI in septic patients. y Results Preliminary diagnosis of AKI by the ED Ph occurred in 218/665 patients (33%). Final adjudicated AKI clinical diagnosis was confi rmed in 49/665 patients (7%). The AUC for NGAL alone in the fi nal diagnosis of AKI was 0.80 (± 0.07). When NGAL was added to the ED Ph’s clinical judgement in a logistic model, the AUC was increased to 0.89 (± 0.06). The AUCs for the additional endpoints are shown in Table 1. When the same model combining NGAL with the ED Ph’s clinical judgement was compared to a clinical model combining T0 sCr results with the ED Ph’s clinical judgement, the net reclassifi cation index was 32.4%, meaning that the correction classifi cation of AKI improved 32.4 percentage points. Conclusion Both biomarkers are increased in the case of sepsis in our population. Septic AKI aff ecting uNGAL more than pNGAL could explain the smaller P value for uNGAL in the group of patients with sepsis. Table 1 (abstract P354) Event No event AUC (95% CI) Diagnosis of AKI 49 616 0.80 (0.07) RIFLE 25 640 0.72 (0.12) sCr bump 10 655 0.85 (0.10) Oliguria 14 651 0.81 (0.14) Mortality 27 638 0.76 (0.11) P354 (pNGAL and uNGAL) levels are aff ected by the presence of sepsis in a general ICU population. These novel biomarkers are currently being evaluated for acute kidney injury (AKI) prediction. However, they are also increased in sepsis, which can be a confounding factor regarding their specifi city for AKI [1,2]. References 1. Int Care Med 2010, 36:1333-1340. 2. Am J Respir Crit Care Med 2011, 183:907-914. 2. Am J Respir Crit Care Med 2011, 183:907-914. Urinary neutrophil gelatinase-associated lipocalin as an early marker of acute kidney injury complicating circulatory shock H Sherif, M Foda, M Shehata, A Ibrahim Urinary neutrophil gelatinase-associated lipocalin as an early marker of acute kidney injury complicating circulatory shock H Sherif, M Foda, M Shehata, A Ibrahim Urinary neutrophil gelatinase-associated lipocalin as an early marker of acute kidney injury complicating circulatory shock H Sherif, M Foda, M Shehata, A Ibrahim Faculty of Medicine, Cairo University, Cairo, Egypt Critical Care 2012, 16(Suppl 1):P353 (doi: 10.1186/cc10960) Conclusion Our study demonstrated that blood NGAL measurements in patients hospitalized from the ED for critical conditions may improve the clinical diagnosis of AKI development. The routine use of NGAL in the ED may provide utility in deciding the appropriate treatment and management strategies for patients at risk for AKI development. Faculty of Medicine, Cairo University, Cairo, Egypt y y gyp Critical Care 2012, 16(Suppl 1):P353 (doi: 10.1186/cc10960) Introduction We evaluated the novel urinary neutrophil gelatinase- associated lipocalin (NGAL) as an early biomarker that rapidly releases in acute kidney injury (AKI) complicating circulatory shock. y j y p g y Methods We measured the urinary NGAL level from collected urine in 45 patients with circulatory shock, during the fi rst 6 hours and after 24 hours. Eleven patients responded to fl uid infusion ± vasopressors and were considered as a separate control group. P352 Plasma and urine neutrophil gelatinase-associated lipocalin in septic and nonseptic ICU patients p p p CS Vrettou1, S Kokkoris2, K Apostolou2, M Parisi2, E Haritatos2, S Dimopoulos2, S Nanas1 1National and Kapodistrian University of Athens, Greece; 2Evaggelismos Hospital, Athens, Greece Critical Care 2012, 16(Suppl 1):P352 (doi: 10.1186/cc10959) Introduction In this prospective cohort study we investigate how admission plasma and urine neutrophil gelatinase-associated lipocalin Conclusion Fluid overload frequently occurs during the fi rst 48 hours after cardiac surgery and is strongly correlated with postoperative S127 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P355 P355 Is cystatin C reliable in the anesthetized pig? An experimental study with special reference to septic shock M Eriksson1, E Söderberg1, M Lipcsey1, J Sjölin2, M Castegren3, M Sjöquist4, A Larsson5 1Surgical Sciences, Anesthesia & Intensive Care, Uppsala, Sweden; 2Medical Sciences, Infectious Disease, Uppsala, Sweden; 3Centre for Clinical Research, Eskilstuna, Sweden; 4SLU, Uppsala, Sweden; 5Medical Sciences, Clinical Chemistry, Uppsala, Sweden Critical Care 2012, 16(Suppl 1):P355 (doi: 10.1186/cc10962) Is cystatin C reliable in the anesthetized pig? An experimental study with special reference to septic shock p g p Results The estimated urinary NGAL at day 1 and day 2 post circulatory shock could predict AKI presented at days 2 and 3 and days 3 and 4 (P <0.05, P <0.001 and P <0.001, P <0.001) respectively. Apart from all conventional kidney parameters and biomarkers, signifi cant inverse correlations could be detected only between urinary NGAL at days 1 and 2 with the corresponding urine output in the patient group (r = –0.51 and –0.64, P <0.05 and P <0.001, respectively). The best cut- off value of urinary NGAL at day 1 was 26 ng/ml, for which sensitivity was 62% and 69% and specifi city was 75% and 80% for prediction of AKI presented at days 2 and 3, respectively. While the best cut-off at day 2 was 29 ng/ml, for which sensitivity was 70% and 74% and specifi city was 90% and 80% for prediction of AKI presented at days 3 and 4, respectively. Urinary NGAL at day 2 could signifi cantly predict mortality complicating AKI rather than at day 1 (P <0.05). Introduction Our aim was to investigate renal function during 24 hours of endotoxemic shock with special focus on the reliability of analysis options in kidney damage. Methods Twenty anesthetized pigs received randomly a continuous 24-hour endotoxin infusion at 0.063 μg/kg/hour (n = 8) or 0.25 μg/kg/ hour (n = 9) or NaCl (controls n = 3). Boluses (10 ml/kg) of succinylated gelatin were given when the arterial blood pressure was 50 mmHg or below. Samples for analysis of cystatin C as well as clearances of inulin, PAH and creatinine were noted and urine was collected. Conclusion Urinary NGAL seems to be a potential early and sensitive biomarker for AKI and a persistently increased level at day 2 can predict mortality following circulatory shock. S128 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results Cystatin C was, already at baseline, not normally distributed. This was in contrast to the other renal variables. Five pigs had baseline values of cystatin C in plasma >0.6 mg/l (one control; four endotoxemic pigs), whereas 15 pigs had plasma levels <0.3. Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate J Dixon1, K Lane1, N Dalton2, I MacPhee1, B Philips1 1St George’s Hospital, London, UK; 2King’s College, London, UK Critical Care 2012, 16(Suppl 1):P358 (doi: 10.1186/cc10965) Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate Introduction The aim of our study was to evaluate the utility of two cystatin-C-based equations, as a surrogate of the renal function (glomerular fi ltration rate (GFR)) in a group of critically ill patients. i y Methods This was a monocentric, prospective and observational study including 146 samples respecting 22 ICU patients. Daily evaluation of seric creatinine, seric cystatin C (CC) and 24-hour creatinine clearance (24CrCL) was performed during the ICU stay. Comparisons were done between two CC-based equations (Hoek (H) and Larsson (L) formulas) and: 24CrCL; Cockroft–Gault (CG); modifi ed Cockroft–Gault (mCG); and six-variable Modifi cation of Renal Disease (MDRD6) formulas. Patients with chronic renal failure were excluded. Correlation, precision, bias and discrimination power were assessed using Spearman coeffi cient, Bland–Altman plots and receiver operating characteristic curves. Introduction We have designed a method of continuous measurement of the glomerular fi ltration rate (GFR) with the intention of applying the method in patients with acute kidney injury (AKI). The aim of the study was to prove the method in healthy volunteers (HV) and patients with chronic kidney disease (CKD). Methods HV and patients with CKD were randomly allocated to measurement of GFR using iohexol, either by the established method of single injection and measurement of its rate of elimination (gold standard), or by the continuous infusion of a very low dose of iohexol (0.5 ml/hour for 12 hours). The GFR was measured again, using the other method, after a washout period of 4 to 28 days. Plasma iohexol concentration was measured at 10 time points and plotted on a two- phase exponential decay graph. The GFR was calculated by dividing the infusion concentration by the plateau concentration. The t test compared results with 4-hour creatinine clearance (4-CrCl), and the CKD-EPI equation. Results The average age of the patients was 63.4 years and male gender was predominant (68%). The APACHE II score was 16.8 ± 5.7. Is cystatin C reliable in the anesthetized pig? An experimental study with special reference to septic shock When individual values were noted over time, it became obvious that, with the exception of the four endotoxemic animals, which shifted considerably over time, there appears to be two subgroups of pigs regarding their cystatin C values. There were only minor diff erences in cystatin C over time for each individual pig compared with the baseline value, except for the four pigs that shifted considerably over time. There were no major diff erences in urinary output between untreated controls and any of the two endotoxemic groups of pigs during the 24-hour experimental period. There was no obvious relation between the administration of bolus doses of gelatin and subsequent diuretic response. Cystatin C did not correlate to creatinine clearance (r2 = 0.06), PAH clearance (r2 = 0.05), inulin×urine (r2 = 0.04) or diuresis (r2 = 0.004). No similar subgroupings were noted for any of the other renal variables, although it should be noted that correlations between all variables were weak. Are serum cystatin-C-based estimates better than those derived from serum creatinine in critically ill patients? JP Baptista, SC Teixeira, J Pimentel Coimbra Universitary Hospital, Coimbra, Portugal Critical Care 2012, 16(Suppl 1):P356 (doi: 10.1186/cc10963) JP Baptista, SC Teixeira, J Pimentel p Coimbra Universitary Hospital, Coimbra, Portugal P358 Assessment of glomerular fi ltration rate in trauma patients in early resuscitation phase Lipcsey, et al.: Crit Care Med 2009, 37:2782-2790. 2. Benes J, et al.: Crit Care 2011, 15:R256. Conclusion Incidence of glomerular hyperfi ltration is relatively common in critically ill multitrauma patients in the fi rst 24 hours. This should be taken into account while deciding drug dosing in this group of patients. 2. Benes J, et al.: Crit Care 2011, 15:R256. 2. Benes J, et al.: Crit Care 2011, 15:R256. Assessment of glomerular fi ltration rate in trauma patients in early resuscitation phase M Bhattacharyya1, R Kumar2, S Todi1 1AMRI Hospitals, Kolkata, India; 2Jadavpur University, Kolkata, India Critical Care 2012, 16(Suppl 1):P357 (doi: 10.1186/cc10964) M Bhattacharyya1, R Kumar2, S Todi1 1AMRI Hospitals, Kolkata, India; 2Jadavpur University, Kolkata, India Critical Care 2012, 16(Suppl 1):P357 (doi: 10.1186/cc10964) Introduction An estimate of the glomerular fi ltration rate (GFR) is important to individualize drug dosages. Trauma leads to systemic infl ammatory response syndrome, which has an eff ect on GFR. The main objective of this study was to assess the GFR in trauma patients during the fi rst 24 hours of admission. gi Methods A prospective observational study of 50 trauma patients aged between 18 and 90 years admitted to the ICU. Exclusion: patients with chronic kidney disease and structural kidney damage. The study population was assessed for GFR by the measurement of creatinine clearance from 24-hour urine creatinine and from serum creatinine. Demographic parameters were documented. g p p Results Total patients admitted to the ITU during July 2010 to April 2011 with trauma were 67, of which 50 patients were included in the study. The mean age of the study group was 39 years, male 86%, mean APACHE IV score 32 and mean Injury Severity Score 10. Out of 50 trauma patients, 13 (26%) patients developed glomerular hyperfi ltration (GHF) within 24 hours of admission. Mean creatinine clearance in the GHF group was 177.92 ± 29 and minimum/maximum values were 151.4 and 254.3 ml/minute/1.73 m2 respectively. Compared to the GHF group, mean creatinine clearance levels were considerably lower in non- GHF patients (86.03 ± 29) with a range of values from 41 to 138.5 ml/ minute/1.73 m2.i Conclusion In this experiment, we noted that there appears to be two populations of pigs regarding their cystatin C values. This result is in contrast to a previous study from our group [1]. Our fi ndings may be explained by the alterations that occur in renal vascular resistance [2], although these fi ndings may also indicate a genetic variation infl uencing either the levels of cystatin C or the antigen determinants of cystatin C. Until our data have been confi rmed or disproved, we strongly suggest that porcine Cystatin C values should be interpreted with great care as a marker for glomerular fi ltration rate in pigs. R f 1. Lipcsey, et al.: Crit Care Med 2009, 37:2782-2790. 1. Lipcsey, et al.: Crit Care Med 2009, 37:2782-2790 1. P358 y p g Critical Care 2012, 16(Suppl 1):P356 (doi: 10.1186/cc10963) Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate S129 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods The Critical Care Minimum Dataset records of patients admitted to our mixed general ICU were investigated. Those patients who received renal organ support were investigated further. The change in serum creatinine levels in the 48 hours prior to institution of RRT was used to determine the AKIN score. Patients in whom there was not a signifi cant rise in creatinine, but who received RRT, were staged zero. Unfortunately, urine output data were not available to improve accuracy. to reach steady state. However, given the simplicity of the method we hypothesise that changes in iohexol concentration may provide valuable real-time information about the GFR in AKI. Changes are likely to occur before serum creatinine rises. In conclusion, the continuous iohexol infusion method of measuring GFR appears to be accurate and precise. In stable subjects, a steady plasma concentration is achieved before it is observed with creatinine changes. y Results There were a total of 276 patients whose records were adequate for this audit. Several records were incomplete and not used. Demography and APACHE II scores were similar across all groups. Length of stay and days of RRT were similar across the groups. ICU survival was as follows: AKIN stage: (0) 42.2%, (1) 50.6%, (2) 51.7%, (3) 70.4%. Pearson chi-square P <0.001. P359 Investigation into the eff ects of commencing haemodialysis in the critically ill R Docking1, L Moss1, M Sim1, D Sleeman2, J Kinsella1 1University of Glasgow, UK; 2University of Aberdeen, UK Critical Care 2012, 16(Suppl 1):P359 (doi: 10.1186/cc10966) q Conclusion We were not able to demonstrate improved survival when RRT was initiated at an earlier AKIN stage. A small nonsignifi cant trend was observed with increasing stage and the diff erences between groups were signifi cant. Very early initiation of RRT was associated with increased mortality. Stage (3) included patients with chronic kidney disease, which probably skewed the results in this group. We cannot recommend the use of the AKIN score as a pointer to when to initiate RRT, based on these data. Introduction We aimed to describe haemodynamic changes when haemodialysis is instituted in the critically ill. Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate The medians of H and L estimates were 50.5 (28 to 77.6) and 47.7 (24.5 to 79.2) respectively, as compared to 69.8 (29.8 to 115.7), 60.7 (42.6 to 101.4), 58.9 (42.6 to 65.1) and 59.2 (40.6 to 106.8) ml/minute/1.73 m2, respectively to 24CrCL (reference method), CG, mCG and MDRD6. Correlation (r) between H, F, CG, mCG, MDRD6 and 24CrCL was 0.83/0.83/0.73/0.70/0.74, respectively. H and L formulas showed the smallest bias and limits of agreement, when compared with formulas based on serum creatinine, respectively –17.5/±52 ml/minute/1.73 m2 and –21/±52.8 ml/minute/1.73 m2. The sensibility for the identifi cation of acute renal dysfunction (24CrCL under 60 ml/minute/1.73 m2) was high for H and L formulas (area under the curve of 0.94 for both). In the subgroup of 29 samples with 24CrCL above 130 ml/minute/1.73 m2 (patients with hyperfi ltration) these two formulas had low sensibility (between 8 and 22%) for identifi cation of this condition. q Results Six HV and seven CKD patients volunteered, with fi ve in each group completing the study. There was no diff erence between the two groups (P = 0.79). In HV, the mean GFR measured by single injection was 105 ± 7.3 and 109.4 ± 9.9 ml/minute/1.73 m2 by infusion (Pearson r = 0.95, P = 0.0002). In CKD patients, the mean GFR measured by single injection was 40 ± 5.4 and 44.8 ± 6.2 ml/minute/1.73 m2 by infusion (Pearson r = 0.99, P <0.0001). The infusion method depends on reaching a steady plasma concentration, which took 165 ± 84 minutes in HV and 483 ± 127 minutes in CKD patients to be within 10% of the steady state. The GFR is overestimated by 4-CrCl (by 13.9 ± 12.9 ml/minute/1.73 m2, P <0.0001) and by CKD-EPI (by 8.4 ± 9.6 ml/minute/1.73 m2, P <0.0001). Conclusion In future work, we aim to validate this method in critically ill patients with AKI. We predict the steady state achieved will be increased. Anticipated problems include increased time or failure Conclusion In this population of critically ill patients, cystatin-C-derived Hoek and Larsson equations underestimated 24CrCL; however, they have a better performance than the classic estimates (CG and MDRD6). Nevertheless, they are inaccurate when applied to ICU patients with hyperfi ltration (GFR >130 ml/m/1.73 m2). Validation of a continuous low-dose iohexol infusion to measure the glomerular fi ltration rate Three hypotheses are tested: (1) the initial session is associated with cardiovascular instability; (2) the initial session is associated with more cardiovascular instability compared to subsequent sessions; and (3) looking at unstable sessions alone, there will be a greater proportion of potentially harmful changes in the initial sessions compared to subsequent ones. References References 1. Uchino S, et al.: Intensive Care Med 2007, 33:1563-1570. 2. Bagshaw SM, et al.: J Crit Care 2009, 24:129-140. 3. Mehta RL, et al.: Crit Care 2007, 11:R31. 1. Uchino S, et al.: Intensive Care Med 2007, 33:1563-1570. 2. Bagshaw SM, et al.: J Crit Care 2009, 24:129-140. 3. Mehta RL, et al.: Crit Care 2007, 11:R31. Methods Data were collected for 209 patients, identifying 1,605 dialysis sessions. Analysis was performed on hourly records, classifying sessions as stable/unstable by a cut-off >±20% change in baseline physiology (HR/MAP). Data from 3 hours prior to and 4 hours after dialysis were included, and average and minimum values derived. Three time comparisons were made (pre-HD:during, during HD:post, pre- HD:post). Initial sessions were analysed separately from subsequent sessions to derive two groups. If a session was identifi ed as being unstable, then the nature of instability was examined by recording whether changes crossed defi ned physiological ranges. The changes seen in unstable sessions could be described as to their eff ects: being harmful/potentially harmful, or benefi cial/potentially benefi cial. Timing for initiation of continuous renal replacement therapy in patients with septic shock and acute kidney injury Conclusion Our results reject the claims that using haemodialysis is an inherently unstable choice of therapy. Although proportionally more of the initial sessions are classed as unstable, the majority of MAP and HR changes are benefi cial in nature. Table 1 (abstract P361). Baseline characteristics and parameters on initiation of CRRT Early Late P value Age 70.7 ± 15.1 69.3 ± 13.1 0.614 APACHE IV 119 ± 31 131 ± 37 0.110 Starting GFR 36.2 ± 20.9 18.1 ± 8.2 <0.001 Start SOFA 11.6 ± 3.3 13.3 ± 2.7 0.006 Table 2 (abstract P361). Outcome parameters Early Late P value NR SOFA 0 to 48 –0.52 ± 3.91 –0.71 ± 3.57 0.827 Hospital death 17 (54.8%) 48 (53.9%) 0.931 28-day survival 16 (51.6%) 46 (51.7%) 0.994 Table 1 (abstract P361). Baseline characteristics and parameters on initiation of CRRT P361 P361 Timing for initiation of continuous renal replacement therapy in patients with septic shock and acute kidney injury HP Shum, KC Chan, MC Kwan, WT Yeung, WS Cheung, WW Yan Pamela Youde Nethersole Eastern Hospital, Hong Kong Critical Care 2012, 16(Suppl 1):P361 (doi: 10.1186/cc10968) Timing for initiation of continuous renal replacement therapy in patients with septic shock and acute kidney injury Introduction The optimal timing for initiation of renal replacement therapy (RRT) in septic acute kidney injury (AKI) remains controversial. The aim of this study is to investigate the impact of early versus late initiation of continuous RRT (CRRT), as defi ned using the simplifi ed RIFLE classifi cation, on organ dysfunction among patients with septic shock and AKI. Results Discarding incomplete data, 181 initial and 1,382 subsequent sessions were analysed. A session was deemed to be stable if there was no signifi cant change (>±20%) in the time-averaged or minimum MAP/ HR across time comparisons. By this defi nition 85/181 initial sessions were unstable (47%, 95% CI SEM 39.8 to 54.2). Therefore Hypothesis 1 is accepted. This compares to 44% of subsequent sessions (95% CI 41.1 to 46.3). Comparing these proportions and their respective CI gives a 95% CI for the standard error of the diff erence of –4% to 10%. Therefore Hypothesis 2 is rejected. In initial sessions there were 92/1,020 harmful changes. This gives a proportion of 9.0% (95% CI SEM 7.4 to 10.9). In the subsequent sessions there were 712/7,248 harmful changes. This gives a proportion of 9.8% (95% CI SEM 9.1 to 10.5). Comparing the two unpaired proportions gives a diff erence of –0.08% with a 95% CI of the SE of the diff erence of –2.5 to +1.2. Hypothesis 3 is rejected. Fisher’s exact test gives a result of P = 0.68, reinforcing the lack of signifi cant variance. Methods Patients were divided into early (sRIFLE Risk) or late (sRIFLE Injury or Failure) initiation of RRT. Patients with chronic kidney disease stage 5 or on dialysis were excluded. Results One hundred and twenty patients admitted within a 3.5-year period fulfi lled inclusion criteria. Thirty-one (26%) underwent early, 89 (74%) had late CRRT. No signifi cant diff erence was noted between the two groups with respect to change in total SOFA score/non-renal SOFA score in the fi rst 24/48 hours after initiation of CRRT, vasopressor use, dialysis requirement and mortality (at 28 days, 3 months and 6 months). The change of nonrenal SOFA score 48 hours after CRRT correlated with the SOFA score at the start of CRRT (P = 0.034) and the APACHE IV risk of death (P = 0.000), but not the glomerular fi ltration rate (GFR) at the start of CRRT (P = 0.348). See Tables 1 and 2. Amino acid concentrations in serum, urine and dialysate/ultrafi ltrate solutions of continuous venovenous hemodiafi ltration patients JM Lee, YJ Lee, J Hong Amino acid concentrations in serum, urine and dialysate/ultrafi ltrate solutions of continuous venovenous hemodiafi ltration patients JM Lee, YJ Lee, J Hong Ajou University School of Medicine, Suwon, Kyeonggi-do, South Korea Critical Care 2012, 16(Suppl 1):P364 (doi: 10.1186/cc10971) Amino acid concentrations in serum, urine and dialysate/ultrafi ltrate solutions of continuous venovenous hemodiafi ltration patients JM Lee, YJ Lee, J Hong Ajou University School of Medicine, Suwon, Kyeonggi-do, South Korea Critical Care 2012, 16(Suppl 1):P364 (doi: 10.1186/cc10971) Table 1 (abstract P362) Variable First group Second group Third group Age 42 ± 15 39 ± 13 47 ± 16 BMI 31 ± 5 29 ± 4 29 ± 5 APACHE II score 17 (5) 17 (9) 15 (7) SOFA score 5 (4) 5 (3) 5 (3) Ranson score 8 (6) 8 (7) 10 (9) Early mortality (%) 27 10* 42 Infection (%) 47 35 29 Overall mortality (%) 49 25* 51 Data presented as median (IQR). P <0.05, second group versus third group. Introduction A prospective study was performed for evaluating the amino acid losses during continuous venovenous hemodiafi ltration (CVVHDF). Introduction A prospective study was performed for evaluating the amino acid losses during continuous venovenous hemodiafi ltration (CVVHDF). Methods Serum, 24-hour urine and dialysate/ultrafi ltrate solutions of CVVHDF were obtained on days 1, 3, and 5 from 11 critically ill patients (fi ve males, six females, mean age 63.0 ± 18.1 (24 to 90)) in the surgical ICU. We analyzed 40 kinds of amino acid concentrations in serum (34 samples), urine (15 samples) and dialysate/ultrafi ltrate solutions (30 samples) by high-performance liquid chromatography analysis. The mean dialysate amount was 918.2 ml (600 to 1,500 ml), mean replacement fl uid amount 1,136.4 ml (1,000 to 2,000 ml) and mean blood fl ow rate 175 ml (100 to 200 ml), respectively. Nutritional support for CVVHDF patients was guided as protein intake at 1.2 to 1.5 g/kg/ day, caloric intake at 30 kcal/kg/day.i Results Among the analyzed 40 amino acids, the fi ve highest mean concentration levels of 24-hour dialysate/ultrafi ltrate solutions were glutamine (65,178.3 μmol/l (hereafter, all units for amino acids are μmol/l)), alanine (48,633.3), glycine (33,959.5), proline (27,701.5), lysine (26,519.4); of serum were glutamine (694.4), alanine (438.1), glycine (349.7), lysine (275.7), proline (262.4); and of 24-hour urine were glycine (1,523.0), histidine (957.5), alanine (920.7), glutamine (904.6), lysine (699.1), respectively. Early application of CVVH In the complex treatment of patients with early severe acute pancreatitis y p I Aleksandrova, M Ilynsky, S Rei, G Berdnikov, L Marchenkova, V Kiselev Hospital Research Institute for Emergency Medicine N.V. Sklifosovsky, Moscow, Russia Critical Care 2012 16(Suppl 1):P362 (doi: 10 1186/cc10969) Introduction A large population-based study of 1,024 deaths from acute pancreatitis (AP) has revealed that the median time lapse between the onset of AP and death was 6 days [1]. A number of authors considered the patients with persistent or progressive early multiple organ failure (MOF) as patients with early severe acute pancreatitis (ESAP) [2].fi g g Conclusion Timing of RRT, stratifi ed into early and late by RIFLE and BUN, showed no signifi cant diff erence in 28-day mortality in patients with severe sepsis and septic shock. Methods The aim of current study was to evaluate the effi ciency of early CVVH in a complex treatment of ESAP. The retrospective analysis involved 106 patients. The patients were divided into three groups: the fi rst group (n = 45) received CVVH dose <30 ml/kg/hour, the second group (n = 20) received the dose >30 ml/kg/hour, and in the third group (n = 41) CVVH was not used during the early phase of disease (Table 1). In the fi rst and second groups the median time interval between admission and start of CVVH was 2 (2; 3) days. P364 y References y Conclusion The highest concentration level of 24-hour dialysate/ ultrafi ltrate solution was glutamine. The amount of amino acid loss after CVVHDF was correlated with the serum amino acid amount and increased according to CVVHDF progression. 1. Mole DJ, et al.: HPB 2009, 11:166-170. 2. Isenmann R, et al.: Pancreas 2001, 22:274-278. Amino acid concentrations in serum, urine and dialysate/ultrafi ltrate solutions of continuous venovenous hemodiafi ltration patients JM Lee, YJ Lee, J Hong Amino acid concentrations of 24-hour dialysate/ ultrafi ltrate solutions showed signifi cant correlation with amino acid concentrations of serum (P = 0.000). The mean amount of total amino acid loss on day 5 of CVVHDF was 2.8 times that of day 1 and 1.7 times that of day 3. The increase of amino acid loss according to CVVHDF progression was most prominent in glutamic acid (8.9 times from day 1 to day 5). Results As compared to reference group 3, signifi cant (P = 0.022) reduction of early mortality (14 days) was observed in the second group, and decreasing tendency (P = 0.093) of mortality rate was detected in the fi rst group. The median time interval between admission and death was 14 days (in the fi rst and second groups) and 5 days in the third group. g p Conclusion The early application of the CVVH increases time interval for care delivery and allows reducing early mortality. The best results were obtained in the group of patients who were treated with the higher dose of CVVH (earlier restoration of homeostasis and decreased severity of early MOF). P360 The timing of RRT was categorized into early (Risk, and Injury) and late (Failure) by RIFLE criteria and also categorized into early (BUN <75 mg/dl) and late (BUN ≥75 mg/dl). Comparing the relationship between RIFLE criteria (Risk and Injury vs. Failure) and 28-day mortality showed no signifi cant diff erence (70.8% vs. 73.3%, P = 0.81). The timing of RRT by serum BUN also showed no signifi cant diff erence in 28-day mortality before start of RRT by BUN ≥75 mg/dl versus BUN <75 mg/dl (77.3% vs. 69.6%, P = 0.50).i Methods All patients diagnosed with severe sepsis and septic shock and treated at the medical ICU in a university-affi liated, tertiary-referral center, from January 2005 to December 2006 were reviewed. Timing of RRT was stratifi ed into early and late by RIFLE (Risk, Injury, Failure, Loss, and End-stage) criteria and blood urea nitrogen (BUN) at the time RRT was started. The primary outcome was 28-day death from any cause. P362 Results Of the 326 patients diagnosed with severe sepsis and septic shock and admitted to the medical ICU during the study period, 78 patients received RRT. The mean age of the patients was 61.5 ± 14.7 years and 54 patients were male (69.2%). The timing of RRT was categorized into early (Risk, and Injury) and late (Failure) by RIFLE criteria and also categorized into early (BUN <75 mg/dl) and late (BUN ≥75 mg/dl). Comparing the relationship between RIFLE criteria (Risk and Injury vs. Failure) and 28-day mortality showed no signifi cant diff erence (70.8% vs. 73.3%, P = 0.81). The timing of RRT by serum BUN also showed no signifi cant diff erence in 28-day mortality before start of RRT by BUN ≥75 mg/dl versus BUN <75 mg/dl (77.3% vs. 69.6%, P = 0.50). P360 P360 Is the AKIN score useful as an indicator of the optimum time for intervention with renal replacement therapy in critically ill patients? S Mousdale, J Bannard-Smith Royal Blackburn Hospital, Blackburn, UK Critical Care 2012, 16(Suppl 1):P360 (doi: 10.1186/cc10967) Introduction Acute kidney injury represents a signifi cant workload and economic burden for critical care units. In the critical care setting AKI is usually associated with a variety of aetiologies such as septic shock, major surgery and heart failure [1]. Controversy exists as to the optimal time for the institution of renal replacement therapy (RRT) [2]. Scoring systems such as AKIN have used the rise in serum creatinine combined with reduced urine output over a period of 48 hours as indicative of the degree of injury [3]. We used this scoring system to see if ITU mortality correlated with increasing AKIN score. S130 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 is to evaluate the relationship between timing of RRT and 28-day mortality in patients with severe sepsis and septic shock. Conclusion For septic shock with AKI, no signifi cant diff erence in organ function and outcome was noted when the timing of initiation of CRRT was classifi ed using sRIFLE criteria. Subsequent improvement of organ function correlated with initial SOFA and APACHE scores instead of the GFR (which determine sRIFLE class) on starting of CRRT. The use of more global assessment tools, such as the SOFA score, for stratifi cation purposes on appropriate timing of CRRT warrants further investigation. is to evaluate the relationship between timing of RRT and 28 day mortality in patients with severe sepsis and septic shock. Methods All patients diagnosed with severe sepsis and septic shock and treated at the medical ICU in a university-affi liated, tertiary-referral center, from January 2005 to December 2006 were reviewed. Timing of RRT was stratifi ed into early and late by RIFLE (Risk, Injury, Failure, Loss, and End-stage) criteria and blood urea nitrogen (BUN) at the time RRT was started. The primary outcome was 28-day death from any cause. Results Of the 326 patients diagnosed with severe sepsis and septic shock and admitted to the medical ICU during the study period, 78 patients received RRT. The mean age of the patients was 61.5 ± 14.7 years and 54 patients were male (69.2%). P365 Conclusion Although needing confi rmation in an adequate number of patients, protocol B was able to provide a buff er balance more positive than protocol A and allowed one to adequately control the A–B status without additional NaHCO3 infusion and in the absence of alkalosis, despite the use of a standard HCO3 – concentration HF solution. Furthermore, the combination of a phosphate-containing replacement fl uid appeared eff ective to prevent hypophosphatemia. Finally, the use of a mathematical model allowed predicting the eff ects of diff erent replacement solutions and/or RCA-CVVH settings on the mass balance of the main solutes. (P = NS), BE –2.8 ± 2.1 versus –1.6 ± 1.2 (P <0.01), serum phosphate 0.85 ± 0.2 versus 1.3 ± 0.5 mmol/l (P = 0.027), serum K+ 4 ± 0.2 versus 4.2 ± 0.3 mmol/l (P = NS) with KCl infusion 4 ± 0.2 versus 1.4 ± 1.5 mmol/ hour (P <0.0001). Protocol A required NaHCO3 and Na-phosphate infusion (8.9 ± 2.8 mmol/hour and 5g/day, respectively) while protocol B allowed one to stop both supplementations. Systemic and circuit Ca2+ were easily maintained in the target range with both protocols.i py Methods Fourteen mongrel dogs were anesthetized, instrumented, and received CVVH with the test (n = 6) or negative control article (n = 8) for 6 hours. The test article was Accusol 35 with induced precipitate formation prior to CVVH. The test article contained visible particles and subvisible particles 36× higher than the maximum concentration specifi ed in the European Pharmacopoeia (EP). The negative control article was Accusol 35 containing no visible particles and subvisible particles within EP specifi cation. One-half of the dogs in the negative control article group received a central venous injection of Sephadex G-50 beads (10 mg/kg) following CVVH as a positive control. Select cardiovascular (CV) parameters were monitored continuously or were calculated at predetermined times. Arterial samples were obtained at predetermined times for analysis of blood gases and electrolytes. Samples of the test and negative control articles were obtained hourly during CVVH for determination of pH and subvisible particles. Dogs were euthanized and lung tissue samples were examined histologically. Results All CV parameters remained stable and no diff erences were observed between the test and negative control articles. Sephadex beads caused an increase (P <0.01) in mean pulmonary arterial pressure due solely to a similar increase (P <0.01) in pulmonary vascular resistance. P366 P366 Regional citrate anticoagulation in CVVH: a new protocol combining citrate solution with a phosphate-containing replacement fl uid S Morabito1, V Pistolesi1, L Tritapepe1, E Vitaliano2, E Strampelli1, F Polistena1, L Zeppilli1, A Pierucci1 1Policlinico Umberto I, Rome, Italy; 2Pertini H, Rome, Italy Critical Care 2012, 16(Suppl 1):P366 (doi: 10.1186/cc10973) Results In 30 patients at high bleeding risk (age 70.5 ± 9.3, SOFA score 13.7 ± 2.5) the AC modality was switched to RCA-CVVH from no AC or Hep. CVVH initial settings: dialysis dose 33.6 ± 3.4 ml/kg/hour; Qb 135 ± 14 ml/minute; Q Citr 1,703 ± 250 ml/hour; Q postdilution 761 ± 181 ml/hour; Citr load 11.6 ± 2.1 mmol/hour; CaCl2 3.7 ± 1.5 ml/ hour. Target circuit Ca2+ and s-Ca2+ were maintained (0.37 ± 0.09 and 1.18 ± 0.13 mmol/l) with few modifi cations of Citr and CaCl2 infusion rates. We used 146 RCA-CVVH circuits with fi lter life 50.5 ± 35.8 hours (median 41; total 7,372). RCA-stopping causes: 34% CVC malfunction, 24% alarm handling/technical issues, 20% scheduled, 14% medical procedures, 8% others. Before starting RCA, we used 69 Hep circuits (2,015 hours) and 74 no-AC circuits (1,827 hours) with a fi lter life of 29.2 ± 20.7 hours (median 22) and 24.7 ± 20.6 hours (median 20), shorter than RCA (P <0.0001). Circuits running at 24, 48 and 72 hours (%): RCA 73, 42 and 28; Hep 43, 23 and 10; and no-AC 38, 12 and 5 (log-rank test P <0.0001). During RCA-CVVH no patients had bleeding complications and the transfusion rate was lower if compared to other AC modalities (0.29 vs. 0.69 blood units/day, P = 0.001). PLT count (P = 0.018) and AT-III activity (P = 0.009) increased throughout days of RCA, reducing supplementation needs. RCA has been stopped for Citr accumulation in one patient (calcemia/s-Ca2+ >2.5). Introduction Regional citrate anticoagulation (RCA) is a highly eff ective anticoagulation (AC) method in CRRT and diff erent combinations of citrate (Citr) and CRRT solutions can aff ect the acid–base (A–B) balance. Regardless of the AC protocol, hypophosphatemia occurs frequently in CRRT (80%). The aim was to evaluate safety and eff ects on A–B balance of a new RCA-CVVH protocol using 18 mmol/l Citr solution combined with a phosphate-containing hemofi ltration (HF) solution. P367 Regional citrate anticoagulation with a low-concentration solution in predilution–postdilution CVVH V Pistolesi, S Morabito, L Tritapepe, L Cibelli, M Ambrosino, F Polistena, L Zeppilli, E Strampelli, MI Sacco, A Pierucci Policlinico Umberto I, Rome, Italy Critical Care 2012, 16(Suppl 1):P367 (doi: 10.1186/cc10974) Introduction Systemic anticoagulation (AC) can increase the bleeding risk in CRRT. However, regional citrate anticoagulation (RCA) is a valid alternative to heparin (Hep) in patients at high risk of bleeding. The aim was to evaluate effi cacy and safety of RCA-CVVH using a low- concentration citrate (Citr) solution. g Conclusion CVVH performed on anesthetized dogs for 6 hours using Accusol 35 containing visible and subvisible particles 36× higher than the maximum concentration specifi ed in the EP resulted in no adverse eff ects on CV parameters, blood gases and electrolytes, and lung histology as compared with Accusol 35 containing no visible particles and subvisible particles that were within EP specifi cation. Methods In cardiac surgery patients with AKI we adopted RCA-CVVH as an alternative to Hep or no-AC CRRT. Criteria for switching to RCA: early circuit clotting (24 hours) or Hep-related complications. RCA- CVVH was performed with a predilution Citr solution (12 mmol/l) and a postdilution hemofi ltration solution (HCO3 – 32 mEq/l). In relation to blood fl ow rate (Qb), the Citr solution rate was set to meet a circuit Citr concentration of 3 mmol/l and modifi ed to obtain circuit Ca2+ <0.4 mmol/l. CaCl2 (10%) was infused to maintain systemic Ca2+ (s-Ca2+) of 1.1 to 1.25 mmol/l. To facilitate CVVH settings, we developed a mathematical model to estimate the metabolic Citr load, buff er balance and Ca2+ loss. P366 P365 P365 Evaluation of the potential adverse eff ects associated with calcium carbonate precipitate during continuous venovenous hemofi ltration J McKee, B Brooks, J Daller, J Gass, D Pantaleone, P Zieske Baxter, Round Lake, IL, USA Critical Care 2012, 16(Suppl 1):P365 (doi: 10.1186/cc10972) P365 Evaluation of the potential adverse eff ects associated with calcium carbonate precipitate during continuous venovenous hemofi ltration J McKee, B Brooks, J Daller, J Gass, D Pantaleone, P Zieske Baxter, Round Lake, IL, USA Critical Care 2012, 16(Suppl 1):P365 (doi: 10.1186/cc10972) Evaluation of the potential adverse eff ects associated with calcium carbonate precipitate during continuous venovenous hemofi ltration J McKee, B Brooks, J Daller, J Gass, D Pantaleone, P Zieske Baxter, Round Lake, IL, USA Critical Care 2012, 16(Suppl 1):P365 (doi: 10.1186/cc10972) Seoul Asan Hospital, Seoul, South Korea Seoul Asan Hospital, Seoul, South Korea p Critical Care 2012, 16(Suppl 1):P363 (doi: 10.1186/cc10970) Critical Care 2012, 16(Suppl 1):P363 (doi: 10.1186/cc10970) , , , Critical Care 2012, 16(Suppl 1):P365 (doi: 10.1186/cc10972) Introduction Timing of renal replacement therapy (RRT) in critically ill severe sepsis and septic shock patients with acute kidney injury is highly subjective and may infl uence outcome. The aim of this study Introduction This study evaluated the potential adverse eff ects associated with exposure to calcium carbonate precipitate during S131 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 continuous venovenous hemofi ltration (CVVH). The clinical use of Accusol 35 Solution (Accusol 35) has been associated with occasional formation of calcium carbonate precipitate in the tubing set during therapy. (P = NS), BE –2.8 ± 2.1 versus –1.6 ± 1.2 (P <0.01), serum phosphate 0.85 ± 0.2 versus 1.3 ± 0.5 mmol/l (P = 0.027), serum K+ 4 ± 0.2 versus 4.2 ± 0.3 mmol/l (P = NS) with KCl infusion 4 ± 0.2 versus 1.4 ± 1.5 mmol/ hour (P <0.0001). Protocol A required NaHCO3 and Na-phosphate infusion (8.9 ± 2.8 mmol/hour and 5g/day, respectively) while protocol B allowed one to stop both supplementations. Systemic and circuit Ca2+ were easily maintained in the target range with both protocols. P365 No diff erences in blood gases or electrolytes were observed between the test and negative control articles. Sephadex beads caused a decrease (P >0.05) in arterial blood PO2 and an increase (P >0.05) in arterial blood PCO2. No diff erences in lung histology were observed between the test and negative control articles. The lungs from all dogs given Sephadex beads contained multiple intravascular particles in large-caliber blood vessels. y g g p Conclusion Although needing confi rmation in an adequate number of patients, protocol B was able to provide a buff er balance more positive than protocol A and allowed one to adequately control the A–B status without additional NaHCO3 infusion and in the absence of alkalosis, despite the use of a standard HCO3 – concentration HF solution. Furthermore, the combination of a phosphate-containing replacement fl uid appeared eff ective to prevent hypophosphatemia. Finally, the use of a mathematical model allowed predicting the eff ects of diff erent replacement solutions and/or RCA-CVVH settings on the mass balance of the main solutes. Exposure to intermittent hemodialysis and renal recovery after acute kidney injury: a systematic review A Schneider1, M Bagshaw2, NJ Glassford1, R Bellomo1 1Austin Health, Heidelberg, Australia; 2University of Alberta, Edmonton, Canada Critical Care 2012, 16(Suppl 1):P368 (doi: 10.1186/cc10975) Introduction Sustained low-effi ciency dialysis (SLED) as primary renal replacement therapy in acute renal failure is still not widely used compared to continuous venovenous hemodiafi ltration (CVVHDF), despite possible economical advantages. Based on one key paper [1] we use SLED as primary renal replacement therapy. However, since medical and economical data with SLED are scarce, we evaluated costs and outcome in a 5-year retrospective study on our ICU. Introduction Renal replacement therapy (RRT) in critically ill patients can be applied in a continuous (CRRT) or intermittent (IRRT) fashion. To date, there is no systematic comparison on the impact of these two modalities on renal recovery after an episode of acute kidney injury (AKI). We sought to compare the rates of renal recovery with RRT independence between CRRT and IRRT as an initial modality for RRT in AKI. y p y Methods During 2006 to 2010 we performed a search on our KIS selecting all patients with the diagnoses N17 and N18 who were treated with SLED or CVVHDF on our ICU. We excluded all patients with a stay <2 days or with an extrarenal indication for dialysis or with pre- existing chronic dialysis. The following variables were extracted from the chart: number of SLED, stay in ICU and hospital, mortality in ICU and hospital, SAPS II, TISS 28, blood urea and creatinine, C-reactive protein, mechanical ventilation, and diagnoses. We evaluated the long-term outcome by sending all discharged patients a questionnaire. y Methods We searched MEDLINE and EMBASE. We retrieved all studies published between 2000 and 2010 that report original data on renal recovery to RRT dependence after AKI in adults. Authors of studies with incomplete data were contacted. Search date: January 2011. Two authors independently assessed the trial quality and extracted data. Pooled analyses were performed and a chi-square test performed. Sensitivity analyses were performed after stratifi cation by premorbid chronic kidney disease, number of centers, type of study and illness severity index. In a subsequent analysis we pooled the studies according to the percentage of patients exposed to IHD into low-exposure (<50% of patients exposed) or high-exposure (>50% patients exposed).i y Results During the period from 2006 to 2010, 3,247 SLED treatments in 421 patients (mean SAPS II was 52 patients) were performed. P368 P369 Sustained low-effi ciency dialysis for renal replacement therapy in the ICU: a cost–benefi t analysis of the years 2006 to 2010 T Neuenfeldt, HB Hopf Asklepios Klinik, Langen, Germany Critical Care 2012, 16(Suppl 1):P369 (doi: 10.1186/cc10976) p Reference p Reference 1. Vinsonneau C, et al.: Lancet 2006, 368:379-385. 1. Vinsonneau C, et al.: Lancet 2006, 368:379-385. Exposure to intermittent hemodialysis and renal recovery after acute kidney injury: a systematic review ICU mortality was 36% and hospital mortality was 46%. A persistent need for dialysis (end-stage kidney disease) was registered in 9%. Total costs for SLED were €518.431 and total reimbursements amount to €734.996 (Figure 1). Assuming for cost comparisons also 3,247 CVVHDF-days, we estimated costs of €722.734 with reimbursements of €690.876 for CVVHDF. Results We identifi ed 50 studies (14,796 patients). Overall, as compared with those that received IRRT as an initial modality (IRRT group), those that received CRRT (CRRT group) had higher average illness severity scores (mean APACHE III equivalent 88 vs. 72, P <0.01) and higher in- hospital mortality (57.7% vs. 37.9%, P <0.0001). When reported at 28 days after initiation of RRT (outcome reported in 25 studies), 19.4% of survivors were RRT dependent in the CRRT group versus 26.9% in the IRRT group (P = 0.004). At hospital discharge (reported in 26 studies), RRT dependence was present in 10.9% of the CRRT group versus 20.8% in the IRRT group (P <0.0001). At day 90 (reported in 22 studies), RRT dependence was 7.8% in the CRRT group versus 36.1% in the IRRT group (P <0.0001). The sensitivity analyses confi rmed these fi ndings in all subgroups. The rates of RRT dependency in the low-exposure group and the high-exposure group at days 28, 90 and hospital discharge were 19.6%, 8.8% and 12.4% versus 43.2%, 26.8% and 14.0% respectively (all P <0.0001, except for hospital discharge P = NS). Conclusion Thus, since short-term and long-term outcome of our patients was comparable to published outcome data with CVVHDF, SLED is at least comparable to CVVHDF even in a busy ICU environment. Moreover, in view of costs, SLED is the preferable dialysis form for renal replacement therapy also in the ICU. P366 p p gi Methods In our center, RCA-CVVH is routinely performed with a 12 mmol/l predilution Citr solution (Prismocitrate 10/2) and a postdilution HF solution (HCO3 – 32, Ca2+ 1.75, Mg2+ 0.5, K+ 2 mmol/l) (protocol A). In the case of persistent acidosis, not related to Citr accumulation, NaHCO3 infusion is started. In order to optimize the buff er balance, a new protocol has been designed throughout a mathematical model developed to estimate Citr and HCO3 – mass transfer. Recently introduced solutions have been adopted: 18 mmol/l predilution Citr solution (Prismocitrate 18), postdilution HF solution (Phoxilium, HCO3 – 30, phosphate 1.2, Ca2+ 1.25, Mg2+ 0.6, K+ 4 mmol/l) (protocol B). In relation to Qb, the Citr solution rate was set to meet the target circuit Citr concentration (3 mmol/l). To maintain systemic Ca2+ (1.1 to 1.25 mmol/l), CaCl2 10% was started according to estimated Ca2+ loss. Conclusion In this experience, RCA allowed one to safely prolong the fi lter life, decreasing the transfusion rate and supplementation needs for AT-III and PLT. The use of a mathematical model allowed one to simplify the CVVH settings. Therefore, RCA should be worthy of more consideration as the fi rst-choice CRRT AC modality in patients at high risk of bleeding. Results In a cardiac surgery patient with AKI, A–B status and electrolytes have been evaluated comparing protocol A (fi ve circuits, 301 hours) versus protocol B (two circuits, 97 hours): pH 7.39 ± 0.03 versus 7.44 ± 0.03 (P <0.0001), blood HCO3 – 22.3 ± 1.8 versus 22.6 ± 1.4 mmol/l S132 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P369). Cost–benefi t ratio for SLED compared to CVVHDF. Figure 1 (abstract P369). Cost–benefi t ratio for SLED compared to CVVHDF. P372 P372 Evaluation of microcirculation before and during continuous renal replacement therapy and the impact of dose prescription C Pipili1, CS Vrettou2, S Poulaki3, A Papastylianou3, M Parisi3, ES Tripodaki3, S Ioannidou3, S Kokkoris3, E Douka3, S Nanas2 1Aretaieion University Hospital, Athens, Greece; 2National and Kapodistrian University of Athens, Greece; 3Evaggelismos Hospital, Athens, Greece Critical Care 2012, 16(Suppl 1):P372 (doi: 10.1186/cc10979) Table 1 (abstract P370). Dialysis methods and clinical parameter CRRT PD Low Qb HD Mini-SLED (n = 25) (n = 3) (n = 21) (n = 12) NIHSS (score) 30.8 ± 17.2 34.6 ± 16.4 31.7 ± 20.8 32 ± 19.8 Rebleed (n, %) 6, 24% 1, 33% 4, 19.0% 0, 0% Mortality (n, %) 5, 20% 1, 33% 5, 23.8% 2, 16% Kt/V (daily) 0.68 ± 0.32 0.25 ± 0.16 0.86 ± 0.23 0.72 ± 0.23 Cost ($/1 treat) 498 ± 30.2 92.4 ± 22.6 82.5 ± 12.5 86.7 ± 14.3 Conclusion Our Mini-SLED methods are eff ective and safe for dialyzing acute brain stroke patients. Table 1 (abstract P370). Dialysis methods and clinical parameter Introduction Microcirculation (MC) might provide evidence for the solute exchange taking place during the dialysis process. Near-infrared spectroscopy (NIRS) with combination of vascular occlusion technique (VOT) allows evaluation of peripheral tissue oxygen utilization and restoration mainly depending on integrity and functionality of vascular endothelium. Our purpose was to evaluate the acute eff ect of continuous renal replacement therapy (CRRT) on the MC as assessed by NIRS and VOT and to explore the impact of delivered CRRT dose on MC alterations. Methods A total of 43 critically patients who underwent CRRT were eligible to participate in the study. The mean age of our population was 66 ± 17 years and 40% were females. The APACHE II score was 20 ± 6, the mean serum creatinine before the CRRT initiation was 2.6 ± 1.6 mg/dl and the mean CRRT delivered dose was 23 ± 6 ml/kg/hour. The median value of dose was used to form groups of high (>22.5 ml/kg/ hour) and low (≤22.5ml/kg/hour) delivered dose. NIRS parameters were evaluated before CRRT initiation (H0), at 6 hours (H6) and at 24 hours (H24) during the CRRT process. Tissue oxygen saturation (StO2, %), defi ned as the percentage of hemoglobin saturation in the microvasculature compartments, was measured with a probe placed on the thenar muscle. Investigation into haemodynamic stability during intermittent haemodialysis in the critically ill Introduction Studies that have reported cardiovascular (CVS) instability with haemodialysis (HD) are outdated and small. By analysing sessions in detail it will be possible to identify the frequency and nature of CVS instability. Hypothesis 1: haemodialysis is associated with CVS instability in the majority of sessions. Hypothesis 2: the majority of CVS changes in unstable sessions will be harmful/potentially harmful. 2 Results Two-way repeated-measures ANOVA were performed for StO2, OCR, RS and hyperemia recovery area at H0, H6 and H24. StO2 correlated with RIFLE on admission and at the time of CRRT initiation (r = 0.283, P = 0.03 and r = 0.45, P <0.0001 respectively). There was a signifi cant decrease in OCR with time (hours on CRRT process) (within- subjects ANOVA F = 4.83, P = 0.014) and especially between H0 and H24 (–10.5 ± 9.4 vs. –12 ± 8.3, P = 0.008). Furthermore, a signifi cant increase in RS was found in patients who received a high CRRT dose (between- subjects ANOVA F = 4.5, P <0.05), especially at H6 post CRRT initiation (76 ± 117 vs. 86 ± 128, P = 0.05). g y Methods Data were collected for 209 patients, identifying 1,605 dialysis sessions. Analysis was performed on hourly records, classifying sessions as stable/unstable by a cut-off >±20% change in baseline physiology (HR/MAP). Data from 3 hours prior to and 4 hours after dialysis were included, and average and minimum values derived. Three time comparisons were made: pre-HD:during, during HD:post, pre-HD:post-HD. If a session was identifi ed as being unstable, then the nature of instability was examined by recording whether changes crossed defi ned physiological ranges. The changes seen in unstable sessions could be described as to their eff ects: being harmful/ potentially harmful, or benefi cial/potentially benefi cial. Conclusion Critically ill patients, receiving a dialysis dose higher than 22.5 ml/kg/hour, showed improved MC. Further studies are needed to investigate the role of NIRS technology as a tool to assess the need for CRRT initiation in acute renal failure. ii Results Discarding incomplete data, 1,563 sessions were analysed. A session was deemed to be stable if there was no change >±20% in time-averaged or minimum MAP/HR across three time comparisons. In 1,563 sessions there was stability in 874 sessions (55.8%, 95% CI for SEM 53.2 to 58.4). Hypothesis 1 is rejected. The new dialysis method Mini-SLED is useful for dialyzing acute brain stroke patients F Taki, Y Komatsu St Luke’s International Hospital, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P370 (doi: 10.1186/cc10977) Conclusion The preponderance of the available evidence suggests that CRRT is associated with a higher rate of renal recovery in AKI survivors compared with IRRT. p F Taki, Y Komatsu Introduction Hemodialysis (HD) patients are known to be a high-risk population for brain stroke. On acute phase of stroke, standard HD treatment may increase cerebral damage by changing serum and S133 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 of 7,464/8,268 (90.3%, 95% CI SEM 89.6 to 90.9). Therefore Hypothesis 2 is rejected. tissue osmolarity. For low clearance dialysis, CRRT, PD or low Qb HD were used but there are some complications. To dialyze these patients more safely and simply, we modifi ed a new dialysis method, Mini-SLED (sustained low-effi ciency dialysis). Conclusion The results above are encouraging, especially given the stringent defi nitions of instability used. By making multiple time-period comparisons the validity of the claims of haemodynamic stability are enforced, compared to previous papers. The number of sessions and measurement points combine to add weight to our fi ndings, supported by robust confi dence interval data. fi Methods We conducted a retrospective observational study from June 2006 to October 2011. Maintenance HD patients who onset acute brain stroke, including hemorrhage and ischemic infarction, were observed. We divided patients into four groups by dialysis modality and compared the clinical parameters. Determination of Mini-SLED was Qb 200 ml/minute, QD 100 to 200 ml/minute, duration for 2 to 3 hours. Results Sixty-one patients were observed in this study. Mean age 72.5 years, 39 patients were male, 45 patients had diabetes. Major clinical parameters and outcomes are presented in Table 1. Patients treated with Mini-SLED have lower risk of rebleeding compared to low Qb HD or CRRT, and were more cost-eff ective than PD. Delivered Kt/V of Mini- SLED was 0.72 ± 0.23. Modality diff erence did not aff ect mortality. P372 A 3-minute brachial VOT was applied to evaluate the oxygen consumption rate (OCR, %/minute), the recovery slope (RS, %/minute), and the hyperemia recovery area as the area (units/minute) under the StO2% curve above baseline values. Conclusion Our Mini-SLED methods are eff ective and safe for dialyzing acute brain stroke patients. Investigation into haemodynamic stability during intermittent haemodialysis in the critically ill Each session had 12 potential comparisons of MAP, HR and time, therefore in the 689 unstable sessions there were 8,268 potential changes ±20% (689×12). There were 804/8,268 harmful/potentially harmful changes, 922/8,268 benefi cial/potentially benefi cial changes and 6,542/8,268 opportunities for change where none occurred. Therefore, looking at harmful/potentially harmful changes there were 804/8,268 (9.7%, 95% CI for SEM 9.1 to 10.4). Looking at potentially benefi cial changes this occurred in 922/8,268 (11.2%, 95% CI for SEM 10.5 to 11.9), and if these were combined with the nonsignifi cant changes this gave a proportion P373i P373 Ultrafi ltration during continuous hemofi ltration in stabilized ICU patients is not associated with microcirculatory perfusion derangements B Scheenstra, G Veenstra, M Koopmans, WP Kingma, H Buter, HM Hemmelder, EC Boerma Medical Centre Leeuwrden, the Netherlands Critical Care 2012, 16(Suppl 1):P373 (doi: 10.1186/cc10980) Degree of impaired kidney function at hospital discharge has a major impact on long-term survival of critically ill patients recovered from renal failure S Stads, G Fortrie, J Van Bommel, R Zietse, M Betjes Erasmus MC, Rotterdam, the Netherlands Critical Care 2012, 16(Suppl 1):P375 (doi: 10.1186/cc10982) Table 1 (abstract P373). (Micro)circulatory variables during ultrafi ltration T1 T2 P value RR mean 71 (65 to 94) 66 (63 to 95) 0.87 Heart rate 97 (78 to 126) 94 (75 to 123) 0.03 MFI 2.9 (2.7 to 3) 3 (3 to 3] 0.34 TVD 20 (18 to 22) 21 (17 to 23) 0.5 Conclusion A negative net fl uid balance of 50 ml/hour during ultra- fi ltration in CVVH is not associated with microcirculatory perfusion alterations. References 1. Bemelmans et al.: Nephrol Dial Transplant 2009, 24:3487-3492. 2. Pottecher et al.: Intensive Care Med 2010, 36:1867-1874. P374 Plasmapheresis without apparatus in complex care of victims with crush syndrome during the fi rst hours after extrication in a fi eld hospital of EMERCOM of Russia in emergency areas A Popov1, I Yakirevich1, A Skorobulatov1, V Shabanov2 1EMERCOM of Russia, Zhukovsky, Moscow Region, Russia; 2All-Russian Centre of Disaster Medicine, Moscow, Russia Table 1 (abstract P373). (Micro)circulatory variables during ultrafi ltration T1 T2 P value RR mean 71 (65 to 94) 66 (63 to 95) 0.87 Heart rate 97 (78 to 126) 94 (75 to 123) 0.03 MFI 2.9 (2.7 to 3) 3 (3 to 3] 0.34 TVD 20 (18 to 22) 21 (17 to 23) 0.5 Conclusion A negative net fl uid balance of 50 ml/hour during ultra- fi ltration in CVVH is not associated with microcirculatory perfusion alterations. References 1. Bemelmans et al.: Nephrol Dial Transplant 2009, 24:3487-3492. 2. Pottecher et al.: Intensive Care Med 2010, 36:1867-1874. Table 1 (abstract P373). (Micro)circulatory variables during ultrafi ltration Table 1 (abstract P373). (Micro)circulatory variables during ultrafi ltration T1 T2 P value RR mean 71 (65 to 94) 66 (63 to 95) 0.87 Heart rate 97 (78 to 126) 94 (75 to 123) 0.03 MFI 2.9 (2.7 to 3) 3 (3 to 3] 0.34 TVD 20 (18 to 22) 21 (17 to 23) 0.5 Introduction Renal replacement therapy (RRT) in critically ill patients with acute kidney injury (AKI) is associated with high mortality. However, little is known about the prognosis of renal function after ICU discharge and the eff ect of persisting impaired kidney function on long- term survival. Ultrafi ltration during continuous hemofi ltration in stabilized ICU patients is not associated with microcirculatory perfusion derangements g B Scheenstra, G Veenstra, M Koopmans, WP Kingma, H Buter, HM Hemmelder, EC Boerma Medical Centre Leeuwrden, the Netherlands Critical Care 2012, 16(Suppl 1):P373 (doi: 10.1186/cc10980) Introduction Ultrafi ltration during intermittent haemodialysis has been associated with reduction in microcirculatory perfusion, as observed with sidestream dark-fi eld (SDF) imaging [1]. This technique has also been useful in the evaluation of volume status in critically Introduction Ultrafi ltration during intermittent haemodialysis has been associated with reduction in microcirculatory perfusion, as observed with sidestream dark-fi eld (SDF) imaging [1]. This technique has also been useful in the evaluation of volume status in critically S134 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 ill patients [2]. To date no data are available on the infl uence of ultrafi ltration during continuous venovenous hemofi ltration (CVVH) on microcirculatory perfusion. ill patients [2]. To date no data are available on the infl uence of ultrafi ltration during continuous venovenous hemofi ltration (CVVH) on microcirculatory perfusion. with 25 to 30% hematocrit; correction of the blood coagulation system; detoxication with the application of active methods of homeostasis correction; prevention and elimination of purulent and septic complications; primary surgical debridement and excision of necrotic mass areas carried out with general anesthesia, no excision conducted; and transport immobilization before evacuation. y p Methods In this single-centre, prospective, observational study patients with acute renal failure on CVVH were included after hemodynamic stabilization and written informed consent A fi xed dose of net ultrafi ltration was calculated for each patient, aiming at a negative total fl uid balance of 50 ml/hour. Microcirculatory perfusion was observed with sublingual SDF imaging after 1 hour of zero balance CVVH (T1) and additionally after 1 hour of negative fl uid balance ultrafi ltration (T2). The primary outcome was a change in microvascular fl ow index (MFI) between T1 and T2. Data are presented as median (IQR). Diff erences are calculated with a nonparametric test for paired data. Results Among all victims, hemodynamics stabilization was noted in 28 ± 6 hours, and dieresis increased up to 1,200 ± 100 ml/day in 18 ± 8 hours. Acute renal failure cases were not noted. All victims in stable condition were evacuated to specialized hospitals by helicopter. No mortality rate during medical aid rendering was noted. References References 1. Bemelmans et al.: Nephrol Dial Transplant 2009, 24:3487-3492. 2 Pottecher et al : Intensive Care Med 2010 36:1867 1874 1. Bemelmans et al.: Nephrol Dial Transplant 2009, 24:3487-3492. 1. Bemelmans et al.: Nephrol Dial Transplant 2009, 24:3487 3492. 2. Pottecher et al.: Intensive Care Med 2010, 36:1867-1874. Results In-hospital mortality was 54.9%. After hospital discharge, the overall mortality was 75.3% after a median follow-up of 8.5 years (range 1 to 17 years). Univariate analysis showed that age, surgical or nonsurgical reason for ICU admission and kidney function at discharge were associated with overall survival. Multivariate Cox regression analysis of the association of kidney function at hospital discharge with patient survival was performed, adjusting for age, sex and surgical or nonsurgical admission type. The eGFR at hospital discharge remained independently associated with long-term survival (P <0.001). Only 87 (15.8%) patients were discharged with an eGFR >90 ml/minute (using the MDRD formula). In this group 5-year and 10-year survival were respectively 77.6% and 66.7%. The mortality risk increased for every increase in stage of chronic kidney disease (hazard ratio 1.25, P <0.001). Patients discharged with an eGFR <30 ml/minute (CKD 4 to 5, 37.3% of patients at hospital discharge) had a 5-year and 10-year survival of only 42.5% and 28.5%. P374 Plasmapheresis without apparatus in complex care of victims with crush syndrome during the fi rst hours after extrication in a fi eld hospital of EMERCOM of Russia in emergency areas y A Popov1, I Yakirevich1, A Skorobulatov1, V Shabanov2 A Popov1, I Yakirevich1, A Skorobulatov1, V Shabanov2 1EMERCOM of Russia, Zhukovsky, Moscow Region, Russia; 2All-Russian Centre of Disaster Medicine, Moscow, Russia Critical Care 2012, 16(Suppl 1):P374 (doi: 10.1186/cc10981) Introduction This is the generalization of the experience of membranous plasmapheresis without apparatus (MPPA) application in the complex care of victims with crush syndrome (CS) in the fi eld hospital (FH) of EMERCOM of Russia during elimination of medical consequences of earthquakes (Pakistan, 2005; China, 2008; Haiti, 2010). Methods Thirty-eight victims with CS (19 males, 19 females, age 34.5 ± 4) were in the resuscitation department of the FH. Compound fractures of tubular bones and crushed tissues necrosis were observed. Joint movement was severely restricted and artery pulsation was uncertain. Condition severity: according to the Glasgow Coma Scale 12 ± 1, according to APACHE II score 29 ± 4. The tendency to hypotension and tachycardia, increase of body temperature and dyspnea intensifi cation were observed, diurnal diuresis decreased. Plasmapheresis treatment was carried out by the MPPA method. A total of 2 ± 1 procedures were conducted to each patient with the removal of 70 ± 10% of the plasma circulation volume per session. Removed plasma volume was calculated for each victim individually on the basis of average volume before plasma exchange. The procedure frequency was once per day. Substitution means: crystalloids, hydroxyethylized starch, proteins. The MPPA procedure time was from 60 to 120 minutes. MPPA was carried out in all victims during complex care for CS: elimination of painful impact and stressful situation; restoration of acid–alkaline conditions and water–electrolytic balance of blood, maintenance of hemodilution Conclusion ICU patients with AKI who received CRRT have a high mortality risk. This is more outspoken for patients who experience incomplete recovery of renal function at hospital discharge. Impaired kidney function at discharge has a major negative impact on their long- term survival. These results stress the importance of preserving kidney function in ICU patients and the need for long-term nephrological follow-up. Future research will have to identify possible determinants in the period following hospital discharge that can be used to prolong survival in these patients. Degree of impaired kidney function at hospital discharge has a major impact on long-term survival of critically ill patients recovered from renal failure The objective of this study was to evaluate the overall long-term mortality in a cohort of ICU patients with AKI necessitating RRT. We hypothesized that both patient characteristics and the degree of renal insuffi ciency at hospital discharge will infl uence long-term mortality. y Methods A retrospective cohort study was performed including all patients older than 18 years admitted to the ICU of a tertiary-care center between 1994 and 2010, who underwent continuous RRT during their ICU stay (n = 1,220). Ultrafi ltration during continuous hemofi ltration in stabilized ICU patients is not associated with microcirculatory perfusion derangements y g g Conclusion MPPA application allows one to reduce the rate of compli- cations and mortality. MPPA application is the method of extra- corporeal homeostasis correction option for victims with CS in a FH in emergencies. Results Eleven patients were eligible for the study; one denied informed consent. One patient could not be evaluated due to the unavailability of the research team and in two patients we were unable to obtain images of proper quality. The median APACHE II score was 26 (21 to 29); at baseline LOS ICU was 5 (3 to 6) days and fl uid balance +7.9 (5.1 to 14.2) l. Hemodynamic and microcirculatory variables are depicted in Table 1. P375 P376 Long-term survival for ICU patients with acute kidney injury D Scott1, F Cismondi2, J Lee1, T Mandelbaum3, LA Celi1, RG Mark1, D Talmor2 1MIT, Cambridge, MA, USA; 2Beth Israel Deaconess Medical Center, Boston, MA, USA; 3Sheba Medical Center, Tel Hashomer, Israel Critical Care 2012, 16(Suppl 1):P376 (doi: 10.1186/cc10983) Introduction A recently published study [1] validated the criteria used in the Acute Kidney Injury Network (AKIN) defi nitions [2] of the S135 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 immunoglobulins (κ-FLC) (23 kDa) and albumin (68 kDa) clearances were measured at 15 minutes, 1 hour, 4 hours, 12 hours, 24 hours and 48 hours. β2-M and κ-FLC were chosen as a middle molecular weight marker. A linear mixed-eff ects model compared clearances between groups. three stages of acute kidney injury (AKI) using in-hospital mortality. In the present study, we validate the clinical applicability of the AKIN classifi cations through long-term survival analysis of AKI patients. three stages of acute kidney injury (AKI) using in-hospital mortality. In the present study, we validate the clinical applicability of the AKIN classifi cations through long-term survival analysis of AKI patients. i y Methods From over 17,000 adult ICU patients in the MIMIC II database [3,4] (V2.5), we excluded patients having end-stage renal disease and those with insuffi cient data and determined AKI stages for each patient. Multivariate Cox regression was used to determine hazard ratios (HRs) for 2-year survival, controlling for: age, sex, nonrenal Sequential Organ Failure Assessment (SOFA) score and selected co-morbidities. g p Results Twenty-four patients were included, 12 in the SHF-HD group (32 sessions) and 12 in CVVH (30 sessions). κ-FLC and albumin clearances were higher in the SHF-HD group over time. No diff erence was observed for creatinine (P = 0.18) and β2-M (P = 0.98) clearances. Plasma albumin levels and the amount of albumin infused did not diff er between groups. See Figure 1. Results Among the fi nal cohort of 14,525 patients, 43% had no AKI and 39%, 14% and 4% developed AKI 1, 2 and 3 respectively. The results of the regression analysis show that AKI 1 (HR 1.12, P <0.05), AKI 2 (HR 1.10, P = 0.05) and AKI 3 (HR 1.64, P <0.001) were signifi cantly associated with increased 2-year mortality. Super high-fl ux continuous hemodialysis: an effi cient compromise for blood purifi cation in sepsis p p Methods Thirty-fi ve patients diagnosed with sepsis (ARDS (n = 10), pyelonephritis (n = 5), cholangitis (n = 5), tsutsugami in Scrub typhus disease (n = 1), mamushi snake bite (n = 1), haemophagocytic syn- drome (n = 1), antineutrophil cytoplasmic antibody lung disease (n = 1), beriberi heart disease (n = 1) and unknown causes (n = 8)) were enrolled in this study between August 2010 and November 2011.The common cause for ARDS in older patients was aspiration pneumonia. Our study group comprised 15 men and 20 women, aged 35 to 85 years (median age 68 years). i T Rimmelé, M Page, C Ber, F Christin, J Baillon, J Crozon, C Chapuis-Cellier, R Ecochard, B Allaouchiche Edouard Herriot Hospital, Hospices Civils de Lyon, France Critical Care 2012, 16(Suppl 1):P377 (doi: 10.1186/cc10984) i T Rimmelé, M Page, C Ber, F Christin, J Baillon, J Crozon, C Chapuis-Cellier, R Ecochard, B Allaouchiche Edouard Herriot Hospital, Hospices Civils de Lyon, France Critical Care 2012, 16(Suppl 1):P377 (doi: 10.1186/cc10984) Introduction High cut-off membranes are proposed for blood purifi - cation therapy in septic shock. However, albumin loss related to these membranes is a major drawback limiting their clinical acceptance. Super High-Flux membranes with an optimized cut-off may combine enhanced middle molecule clearances (infl ammatory mediators) with limited albumin loss. The aim of our study was to compare small, middle molecule clearances and albumin loss between continuous hemodialysis using a Super High-Flux membrane (SHF-HD) and conventional continuous hemofi ltration (CVVH). ( g y ) Results Before initiating treatment with the PMMA-CHDF, the average APACHE score of these patients was 17.5 ± 3.6, whereas the average Sepsis-related Organ Failure Assessment score was 6.5 ± 1.3. The duration of PMMA-CHDF treatment was 5.2 ± 2.3 days. Following initiation of PMMA-CHDF treatment, early improvement of haemodynamics was observed, along with an increase in the urine output. The average survival rates of patients were 75.6%. The lowest survival rate among diseases (35%) belonged to the unknown group. The highest survival rate for patients with ARDS was 95%. Moreover, the urine output signifi cantly increased in the survival group. i Methods After approval by the ethics committee, patients were enrolled in a single-blind RCT. Effi cacy of continuous haemodiafi ltration using a polymethylmethacrylate membrane haemofi lter in the treatment of sepsis and acute respiratory distress syndrome y p Conclusion AKI stages 1, 2 and 3 are signifi cant indicators of 2-year mortality. The diff erence between AKI 1 and 2 is smaller than that between AKI 2 and 3 and it may be prudent to re-examine the criteria used to defi ne AKI to provide better separation among the three classes. Shintakeo Hospital, Takeo, Japan Critical Care 2012, 16(Suppl 1):P378 (doi: 10.1186/cc10985) Shintakeo Hospital, Takeo, Japan Critical Care 2012, 16(Suppl 1):P378 (doi: 10.1186/cc10985) Super high-fl ux continuous hemodialysis: an effi cient compromise for blood purifi cation in sepsis Patients with septic shock and acute kidney injury received either SHF-HD (EMiC2® fi lter; Fresenius Medical Care) (cut- off = 40 kDa, dialysate fl ow rate of 40 ml/kg/hour) or conventional CVVH (cut-off = 30 kDa, UF fl ow rate of 40 ml/kg/hour). Each patient received a maximum of three sessions of 48 hours each. Creatinine (113 Da), β2-microglobulin (β2-M) (11.8 kDa), kappa free light chain of i Conclusion The present study suggests that cytokine-oriented critical care using PMMA-CHDF might be eff ective in the treatment of sepsis and ARDS, particularly in the treatment of ARDS associated with aspiration pneumonia in older patients. Figure 1 (abstract P377). Figure 1 (abstract P377). References Introduction CHDF using a polymethylmethacrylate membrane is currently widely applied for nonrenal indications in Japan; this tech- nique is used in the treatment not only of patients with sepsis but also of those with cytokine-induced critical illness such as acute respiratory distress syndrome (ARDS) and pancreatitis. This study aimed to investigate the clinical effi cacy of continuous haemodiafi ltration using a polymethylmethacrylate membrane haemofi lter (PMMA-CHDF) in the treatment of patients with sepsis and ARDS. 3. Saeed M, et al.: Crit Care Med 2011, 39:952-960. 4. MIMIC II databases [http://physionet.org/mimic2] P376 In addition, age (HR 1.04, P <0.001), gender (M) (HR 0.93, P <0.05), nonrenal SOFA score (HR 1.05, P <0.001) and all co-morbidities were signifi cant predictors. Adjusted and unadjusted Kaplan–Meier curves for patients with AKI 3 are remarkably diff erent from each other, suggesting that in these most severely ill patients AKI is only one aspect of their illness.i f g p g Conclusion The removal of middle molecular weight molecules is higher with SHF-HD. Albumin loss was limited in both groups, even with SHF-HD. Therefore, SHF membranes seem to represent an alternative to high cut-off membranes for blood purifi cation therapies. P378fi P379 However, we have shown that greater than 2 hours duration of PMX treatment signifi cantly improved hemodynamics and signifi cantly decreased administration of norepinephrine than 2 hours duration of PMX treatment. Our hypothesis was that PMX treatment had the ability of endotoxin removal during 24 hours. Therefore, the purpose of this study was to evaluate the endotoxin adsorption ability of 24 hours duration of PMX treatment. Introduction Endotoxin plays an important role in the pathogenesis of septic shock. Endotoxin adsorption therapy by Polymyxin B-immobilized fi ber column (PMX) hemoperfusion has been used for the treatment of septic shock patients in Japan. According to the company’s recommendation, the standard duration of PMX treatment for patients with septic shock is 2 hours. However, we have shown that greater than 2 hours duration of PMX treatment signifi cantly improved hemodynamics and signifi cantly decreased administration of norepinephrine than 2 hours duration of PMX treatment. Our hypothesis was that PMX treatment had the ability of endotoxin removal during 24 hours. Therefore, the purpose of this study was to evaluate the endotoxin adsorption ability of 24 hours duration of PMX treatment. Figure 2 (abstract P379). Immunoelectron microscopy using anti- HMGB1 polyclonal antibodies. contributes to HMGB1, with a heparin-binding protein, adsorption on AN69ST. Methods The test solution contained 100 g HMGB1 and 35 g albumin in 1,000 ml substitution fl uid. We executed three diff erent experimental hemofi ltrations with solution fl ow of 100 ml/minute and: ultrafi ltrate fl ow 1,000 ml/hour using AN69ST primed with a heparinized saline, F(+) and H(+); ultrafi ltrate fl ow 1,000 ml/hour using AN69ST with no heparinized saline, F(–) and H(+); and ultrafi ltrate fl ow of 0 ml/hour using AN69ST with no heparinized saline, F(–) and H(–). In addition, AN69ST membrane was immunostained using an antibody that confi rmed dying on human kidney tissue. Methods In this study, we measured plasma endotoxin concentrations of blood drawn from the radial artery and the outlet circuit of the PMX column after 24 hours duration of PMX treatment in septic shock patients. The assay for endotoxin was performed with separated plasma from heparinized whole blood samples centrifuged at 3,000 rpm for 40 seconds. The high-sensitivity assay was performed by kinetic turbidimetric Limulus assay using a MT-358 Toxinometer (Wako Pure Chemical Industries, Ltd, Japan). This Limulus assay test is specifi c to endotoxin and has no cross-reaction to β-glucan. High mobility group box 1 levels in septic disseminated intravascular coagulation patients undergoing Polymixin-B immobilized fi ber-direct hemoperfusion High mobility group box 1 levels in septic disseminated intravascular coagulation patients undergoing Polymixin-B immobilized fi ber-direct hemoperfusion Y Ishibe, Y Suzuki, H Sato, G Takahashi, M Kojika, N Matsumoto, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2012, 16(Suppl 1):P380 (doi: 10.1186/cc10987) i p Y Ishibe, Y Suzuki, H Sato, G Takahashi, M Kojika, N Matsumoto, Y Inoue, S Endo Iwate Medical University, Morioka, Japan Critical Care 2012, 16(Suppl 1):P380 (doi: 10.1186/cc10987) Conclusion These fi ndings suggest that 24 hours duration of PMX treatment is eff ective to remove endotoxin. Further studies are needed to confi rm this ability. y, , p Critical Care 2012, 16(Suppl 1):P380 (doi: 10.1186/cc10987) P379 3 9 Possible adsorption mechanism of high mobility group box 1 protein on a polyacrylonitrile (AN69ST) membrane fi lter O Nishida1, M Yumoto1, K Moriyama1, Y Shimomura1, T Miyasho2, S Yamada3 1Fujita Health University School of Medicine, Toyoake, Japan; 2Rakuno Gakuen University, Ebetsu, Japan; 3Shino-Test Corporation, Sagamihara, Japan Critical Care 2012, 16(Suppl 1):P379 (doi: 10.1186/cc10986) Introduction At ISICEM 2011, we reported that AN69ST showed the highest capacity to adsorb high mobility group box 1 protein (HMGB1) when compared with polymethylmethacrylate, polysulfone and high cut-off membrane [1]. Here we focus on whether fi ltration or surface heparin on AN69ST by a priming circuit with a heparinized saline Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 S136 Figure 1 (abstract P379). Time course of HMGB1 levels in the test solution. Figure 2 (abstract P379). Immunoelectron microscopy using anti- HMGB1 polyclonal antibodies. Figure 1 (abstract P379). Time course of HMGB1 levels in the test solution. coagulation (DIC) undergoing Polymixin-B immobilized fi ber-direct hemoperfusion (PMX-DHP). Methods The subjects were 16 patients with serum endotoxin levels of 1.1 pg/ml or over. The average APACHE II score was 32.2, the average SOFA score 12.4, and the average DIC score 5.5. Results Following PMX-DHP, the serum endotoxin level decreased to below the limit of detection in all patients. The serum HMGB1 level decreased signifi cantly to 31.2, 16.6 and 7.9 ng/ml on days 0, 1, and 2, respectively. The average of the DIC score improved from 5.6 to 3.9 to 2.9. Overall, the 30-day, 60-day, 90-day and 180-day mortality rates were 0, 6.3%, 12.5% and 12.5%, respectively. y Conclusion Following initiation of PMX-DHP, the serum HMGB1 level decreased and the DIC score also decreased accordingly. Figure 1 (abstract P379). Time course of HMGB1 levels in the test solution. Figure 2 (abstract P379). Immunoelectron microscopy using anti- HMGB1 polyclonal antibodies. P381 Polymyxin B-immobilized fi ber column hemoperfusion has the ability of endotoxin removal during 24 hours C Mitaka, Y Ueda, Y Miyawaki, M Yamauchi, T Toyofuku, G Haraguchi, T Kudo Tokyo Medical and Dental University, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P381 (doi: 10.1186/cc10988) Introduction Endotoxin plays an important role in the pathogenesis of septic shock. Endotoxin adsorption therapy by Polymyxin B-immobilized fi ber column (PMX) hemoperfusion has been used for the treatment of septic shock patients in Japan. According to the company’s recommendation, the standard duration of PMX treatment for patients with septic shock is 2 hours. P379 The endotoxin removal rate was defi ned by the equation: ((radial artery endotoxin concentration – outlet circuit of PMX column endotoxin concentration) / radial artery endotoxin concentration)×100%. The endotoxin removal rate represents endotoxin adsorption ability. Five patients with septic shock were studied. i Results The concentration decreases of HMGB1 at 0, 60 and 360 minutes indicated no signifi cant diff erences among the three diff erent hemofi ltration experiments (Figure 1). At 60 minutes, reduction rates of HMGB1 were: F(+) and H(+), 97.3%; F(+) and H(–), 94.8%; and F(–) and H(–), 96.4% respectively. HMGB1 was not detected in bulk layers by immunostaining (Figure 2). g g Conclusion Surface heparin or fi ltration might not contribute to HMGB1 adsorption on the AN69ST membrane. Remarkable adsorption on AN69ST is likely to be infl uenced by material characteristics, hydrogel structure with moisture content, or negative electric charge and may occur not in bulk layers but on large surfaces of membranes. Reference Results The APACHE II scores of these patients were 26.2 ± 5.9 (mean ± SD, range 18 to 34) at admission to the ICU. Three patients survived and two patients died. Before the start of PMX treatment, heart rates were 119 ± 19 bpm, mean arterial pressures were 60 ± 19 mmHg, and plasma endotoxin concentrations of radial arterial blood were 91.4 ± 7.4 pg/ml (mean ± SD). After 24 hours duration of PMX treatment, plasma endotoxin concentrations decreased from 55.0 ± 58.9 pg/ml (radial arterial blood) to 19.4 ± 29.5 pg/ml (outlet circuit of PMX column). The endotoxin removal rate was 62.8 ± 22.1%, suggesting that endotoxin adsorption ability is still retained during 24 hours PMX treatment.i 1. Yumoto M, et al.: Ther Apher Dial 2011, 15:385-393. 1. Yumoto M, et al.: Ther Apher Dial 2011, 15:385-393. coagulation (DIC) undergoing Polymixin-B immobilized fi ber-direct hemoperfusion (PMX-DHP). coagulation (DIC) undergoing Polymixin-B immobilized fi ber-direct hemoperfusion (PMX-DHP). Methods The subjects were 16 patients with serum endotoxin levels of 1.1 pg/ml or over. The average APACHE II score was 32.2, the average SOFA score 12.4, and the average DIC score 5.5. Results Following PMX-DHP, the serum endotoxin level decreased to below the limit of detection in all patients. The serum HMGB1 level decreased signifi cantly to 31.2, 16.6 and 7.9 ng/ml on days 0, 1, and 2, respectively. The average of the DIC score improved from 5.6 to 3.9 to 2.9. Overall, the 30-day, 60-day, 90-day and 180-day mortality rates were 0, 6.3%, 12.5% and 12.5%, respectively. C l i F ll i i iti ti f PMX DHP th HMGB1 l l 1. Yumoto M, et al.: Ther Apher Dial 2011, 15:385-393. coagulation (DIC) undergoing Polymixin-B immobilized fi ber-direct hemoperfusion (PMX-DHP). Methods The subjects were 16 patients with serum endotoxin levels of 1.1 pg/ml or over. The average APACHE II score was 32.2, the average SOFA score 12.4, and the average DIC score 5.5. Results Following PMX-DHP, the serum endotoxin level decreased to below the limit of detection in all patients. The serum HMGB1 level decreased signifi cantly to 31.2, 16.6 and 7.9 ng/ml on days 0, 1, and 2, respectively. The average of the DIC score improved from 5.6 to 3.9 to 2.9. Overall, the 30-day, 60-day, 90-day and 180-day mortality rates were 0, 6.3%, 12.5% and 12.5%, respectively. Conclusion Following initiation of PMX DHP the serum HMGB1 level Methods The subjects were 16 patients with serum endotoxin levels of 1.1 pg/ml or over. The average APACHE II score was 32.2, the average SOFA score 12.4, and the average DIC score 5.5. P383 Methods Ten critically ill patients were studied who were on SHEDD- fA, at QB = 150 ml/minute, QF = 1,500 ml/hour (post dilution) and QD = 300 to 500 ml/minute as a nonrenal indication. In order to maximize cytokine adsorption effi ciency, we used a large-size (2.1 m2) PMMA dialyzer. Blood samples were taken to measure the CL of plasma cytokines (HMGB-1, IL-6, IL-8, IL-10, G-CSF, MCP-1 and MIP-1) at 1 hour and 3 hours after initiation (in one cytokine by 62 to 107 samples). Results The median values of CL with interquartile ranges of each cytokine (molecular weight: kDa) were: HMGB1 (30 kDa), 53.1 ml/ minute (2.1 to 12.5); IL-6 (21 kDa), 39.9 ml/minute (12.4 to 70.6); IL-8 (8 kDa), 64.1 ml/minute (–0.5 to 82.0); IL-10 ml/minute (35 to 40 kDa), 45.6 ml/minute (0.5 to 88.3); G-CSF (19 kDa), 33.2 ml/minute (9.3 to 60.8); MCP-1 (8.7 kDa), 68.5 ml/minute (–14.4 to 125.4); and MIP-1 (7.8 kDa), 66.5 ml/minute (18.6 to 100.0). In particular, CL of HMGB1 was positively correlated with pre-SHEDD-fA blood levels, indicating the mechanism of HMGB1 removal was through adsorption. As a result of enhancing the intensity of the dosage, CL (53 ml/minute) of HMGB1 was higher than that (25 ml/minute) of an in vitro experiment that we reported at the 31st ISICEM 2011. See Figure 1. Results One hundred and sixty-six patients (98 men, 68 women; age range 24 to 92 years (mean 64.7 ± 13.3)) were studied. The mortality rate was 34.9% at 28 days after PMX-DHP. There were 129 (77.7%) emergency surgical patients and 37 (22.3%) medical patients. The APACHE II score on the day of PMX-DHP was not signifi cantly diff erent between surgical and medical patients (20.3 ± 7.0 vs. 19.2 ± 8.1, P = 0.417). Mean arterial pressure (MAP) signifi cantly improved in emergency surgical patients before and after PMX-DHP therapy (73.7 ± 24.8 vs. 79.7 ± 26.0 mmHg, P = 0.017), while MAP was not statistically diff erent in medical patients (69.7 ± 24.2 vs. 76.7 ± 27.1 mmHg, P = 0.178). The inotropic score had no statistical diff erence between before and after PMX-DHP in both surgical and medical patients (13.2 ± 19.8 vs. 12.6 ± 19.2, P = 0.61; 16.8 ± 27.3 vs. 13.8 ± 23.6, P = 0.65, respectively). P383 P382 P382 Polymyxin B-direct hemoperfusion therapy could contribute to hemodynamics and outcomes in emergency surgical patients M Yokota, T Goto, T Harada, M Takeda, R Moroi, M Namiki, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P382 (doi: 10.1186/cc10989) P383 Clinical impact of enhanced cytokine clearance with sustained high- effi ciency daily diafi ltration using a mediator-adsorbing membrane (SHEDD-fA) in patients with severe sepsis O Nishida1, T Nakamura1, N Kuriyama1, K Moriyama1, T Miyasho2, S Yamada3 1Fujita Health University School of Medicine, Toyoake, Japan; 2Rakuno Gakuen University, Ebetsu, Japan; 3Shino-Test Corporation, Sagamihara, Japan Critical Care 2012, 16(Suppl 1):P383 (doi: 10.1186/cc10990) Clinical impact of enhanced cytokine clearance with sustained high- effi ciency daily diafi ltration using a mediator-adsorbing membrane (SHEDD-fA) in patients with severe sepsis O Nishida1, T Nakamura1, N Kuriyama1, K Moriyama1, T Miyasho2, S Yamada3 1Fujita Health University School of Medicine, Toyoake, Japan; 2Rakuno Gakuen University, Ebetsu, Japan; 3Shino-Test Corporation, Sagamihara, Japan Critical Care 2012, 16(Suppl 1):P383 (doi: 10.1186/cc10990) 1Fujita Health University School of Medicine, Toyoake, Japan; 2Rakuno Gakuen University, Ebetsu, Japan; 3Shino-Test Corporation, Sagamihara, Japan Critical Care 2012, 16(Suppl 1):P383 (doi: 10.1186/cc10990) Introduction Polymyxin B-direct hemoperfusion (PMX-DHP) (Toraymyxin®; Toray Medical Co., Tokyo, Japan) has been approved to treat patients with endotoxemia and/or severe sepsis due to Gram- negative infection since 1994 in Japan. However, its effi cacy and indication are still controversial. Recently, randomized controlled studies were performed in other countries. Our hypothesis is that PMX-DHP may be useful for emergency-operated patients to eliminate endotoxins from the systemic circulation after removal of the source of infection. Introduction SHEDD-fA is an eff ective modality that makes the best use of three principles in the treatment of severe sepsis: diff usion, convection and adsorption. We reported the effi cacy of SHEDD-fA for the treatment of severe sepsis at the 31st ISICEM 2011 [1]. Here we present the blood clearance (CL) of seven important cytokines with SHEDD-fA. Methods From July 1994 to May 2011, all adult patients treated with PMX-DHP in our ICU were included in this retrospective observational study. Patients’ clinical and microbiological data were collected from medical archives. The emergency postoperation patients and the medical patients were compared for severity, mortality, and hemodynamic status. Values are expressed as mean ± SD. Data were analyzed by Mann–Whitney U test, chi-square test and Fisher’s exact probability test. P <0.05 was considered statistically signifi cant. 1. Nishida O, et al.: Contrib Nephrol 2011, 173:172-181. i References Introduction The serum levels of high mobility group box 1 (HMGB1) were examined in patients with septic disseminated intravascular Introduction The serum levels of high mobility group box 1 (HMGB1) were examined in patients with septic disseminated intravascular . Mitaka C, et al.: Shock 2009, 32:478-483. Kambayashi J, et al.: J Biochem Biophys Methods 1991, 22:93-100. Kambayashi J, et al.: J Biochem Biophys Methods 1991, 22:93-100. 2. Kambayashi J, et al.: J Biochem Biophys Methods 199 S137 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P383 This study was performed over 10 years (from January 2000 to July 2010) and included 231 consecutive patients hospitalized for major trauma, requiring intubation at the roadside and in whom prehospital petCO2 has been recorded. Patients younger than 16 years and those with severe hypothermia were excluded from the study. There were 156 males and 75 females, age range 16 to 84, mean 43.6 ± 17.8 years. In hospital we calculated the APACHE II scoring system for each patient. For every scoring system, the sensitivity, specifi city, correct outcome prediction and area under the ROC curve were determined.f Introduction In El Salvador there are a limited number of ICU beds. The ICU bed per inhabitant ratio is only 0.7 per 100,000 in a country with a population of 6,071,774 [1]. The aim of this study was to show the impact that the ICU bed defi cit has on the mortality of the patients admitted to the internal medicine fl oor. l Methods We conducted a descriptive, cross-sectional study. A nonprobabilistic sample was estimated using EPIDAT 4.0 (mortality rate 16%, 95% CI, P <0.05). We enrolled 513 patients admitted to the Internal Medicine ward, from June to November 2011. All patients were evaluated using the ICU admission priority criteria of the Society of Critical Care Medicine (SCCM). We divide the patients into high priority (SCCM priority levels 1 and 2) and low priority (SCCM priority levels 3 and 4) for ICU admission. The probability of death using APACHE II score and mortality rate was calculated for each group, in order to obtain the Standardized Mortality Ratio (SMR). A t test and a Mantel–Haenszel test were used for statistical analysis between groups. Results For prediction of mortality, the best cut-off points were 19 for MEES and 22 for MEESc. The area under the ROC curve was 0.63 for MEES, 0.81 for MEESc (P = 0.02 vs. MEES) and 0.84 for APACHE II (P <0.01 vs. MEES).if y Results A total of 513 patients were included in the study; 101 patients in the high priority group and 412 patients in the low priority group. There was a signifi cantly higher mortality (P = 0.048) in the high priority level group especially with an APACHE score less than 9.0 (Figure 1). Conclusion There were signifi cant diff erences between MEES and MEESc. P383 The mortality rates at 28 days, 90 days, 0.5 year and 1 year after PMX-DHP were signifi cantly diff erent between surgical and medical patients (28.7 vs. 56.8, 43.8 vs. 83.3, 52.2 vs. 85.7, 54.5 vs. 91.2%, P <0.0001, respectively). Conclusion Taking into account the fact that the creatinine CL of native kidney function is 100 ml/minute, our fi ndings suggest that SHEDD-fA is a feasible adjusted modality for the treatment of patients with severe sepsis, with or without acute kidney injury. Considering our other laboratory fi ndings, deep fi ltration may enhance blood clearance. Reference Conclusion MAP increased in surgical patients but did not change in medical patients after PMX-DHP, and the inotropic score was not signifi cantly diff erent in both sets of patients. The mortality was signifi cantly lower in surgical patients than in medical patients. Figure 1 (abstract P383). Correlation between clearance and blood level of cytokines. Figure 1 (abstract P383). Correlation between clearance and blood level of cytokines. S138 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P384 systems in trauma patients. We present an improved Mainz Emergency Evaluation Scoring (MEES) in combination with capnometry (MEESc). MEESc is a new scoring system. We compared the prognostic role of outcome of these two prehospital descriptive scoring systems with the prognostic scoring system APACHE II. systems in trauma patients. We present an improved Mainz Emergency Evaluation Scoring (MEES) in combination with capnometry (MEESc). MEESc is a new scoring system. We compared the prognostic role of outcome of these two prehospital descriptive scoring systems with the prognostic scoring system APACHE II. P384 Mortality and priority level for ICU admission in the setting of limited critical care beds in El Salvador V Segura, NR Reyes, ME Tejada, EM Zolano Hospital San Rafael, Santa Tecla, El Salvador Critical Care 2012, 16(Suppl 1):P384 (doi: 10.1186/cc10991) p g g y Methods In a prehospital setting, the values of MEES and capnometry (initial and fi nal) were collected from each patient. We added fi nal values of petCO2 to the MEES scoring system and ranked from 0 to 2 so that the fi nal maximum sum of the scoring system would be 30 without any change in the minimal score being 10. P385 Introduction The aim of this study was to investigate and compare Ranson criteria (RC) and RC + serum CRP levels as a feasible, practical and precise method in acute pancreatitis (AP) cases admitted to the ICU in respect of length of stay (LOS) predicting severity of disease. Predicting outcome in the ICU: comparison of Ranson criteria and Ranson + CRP levels in acute pancreatitis V Inal L Yamanel B Comert Predicting outcome in the ICU: comparison of Ranson criteria and Ranson + CRP levels in acute pancreatitis V Inal, L Yamanel, B Comert GATA, Ankara, Turkey Critical Care 2012, 16(Suppl 1):P386 (doi: 10.1186/cc10993) Reference 1. Boletin de indicadores del Sistema Nacional de_Salud 2009 [http://www. salud.gob.sv/archivos/pdf/Boletin_de_indicadores_del_Sistema_Nacional_ de_Salud_2009.pdf] P383 MEESc improved the results of MEES in predicting outcome for severe trauma patients. The prehospital use of the improved MEESc could be an effi cient communication protocol between the prehospital and hospital settings (MEESc is comparable with APACHE II). y Conclusion The study shows that there is an increased mortality rate in patients with high priority level for admission to the ICU with an APACHE II score less than 9 points. This represents 90 patients/year whose survival and prognosis could be improved by increasing the number of ICU beds available. Mainz Emergency Evaluation Scoring in combination with capnometry predicts outcome in trauma patients The most frequent comorbidity was hypertension (41.8%), followed by dyslipemia (24.6%), cardiac disease (17.2%), DM and pulmonary pathology (13.1%). Solid or hematologic malignancy (10.6%), chronic renal failure (9%) and hepatic pathology (5.7%) were other comorbidities. Biliary etiology was the most frequent (48.5%), followed by alcoholic AP (20.5%) and unknown etiology (17.2%); 3.3% were post- biliary manipulation (surgery or ERCP) AP. The mean APACHE II score at admission was 16.42 ± 7.64. In total, 56.6% patients needed mechanical ventilation, 50.8% vasopressors and 40.2% renal support during their ICU stay. The ICU length of stay (LOS) was 16.55 ± 21.6, hospital LOS 45.39 ± 45.42 days. A total of 28.7% patients died in the ICU, and 38.5% during their hospital stay. We did not fi nd any relation between comorbidities or AP etiology and outcome. Mortality predictors in AP patients were: PaFi relation (–0.007, P = 0.006), mean and systolic arterial pressure (–0.39, P = 0.019 and –0.038, P = 0.001 respectively), pH (–5.641, P = 0.001), HCO3 (–0.081, P = 0.050), creatinine (0.347, P <0.001), urea (0.008, P = 0.002), 24-hour diuresis (–0.001, P = 0.002) and Glasgow Coma Scale (–0.312, P = 0.050). results. In addition, necrotizing cases were assumed to increase CRP levels more than predicted and were also excluded. After the exclusion of cases, 89 patients’ data were collected and compared for LOS in the ICU between 2005 and 2009. Results Statistical analysis of patients’ data for signifi cance and receiver operating curve (ROC) analysis to predict LOS, therefore pointing to disease severity, was executed. All of the statistical comparisons were found signifi cant for predicting LOS; RC (P <0.05), RC + CRP together (P <0.01) and CRP alone (P <0.04). Severity of the disease and therefore LOS were increased for RC score >3 and CRP levels >50 mg/l. ROC analysis resulted in RC (AUC 0.895), RC + CRP (AUC 0.901) and CRP (AUC 0.823) for LOS. Conclusion AP cases usually require ICU care and treatment. There are some consented scoring systems such as RC, APACHE II and Glasgow in predicting disease severity and guiding the physician’s approach. Although the most sensitive and specifi c method seemed to be APACHE II scoring, it is time consuming and complex. On the other hand, RC and Glasgow scorings need to be evaluated in 48 hours. System biology prediction model based on clinical data: highly accurate outcome prediction in patients with acute-on-chronic liver failure System biology prediction model based on clinical data: highly accurate outcome prediction in patients with acute-on-chronic liver failure MJ McPhail, DL Shawcross, RD Abeles, T Chang, GL Lee, MA Abdulla, C Willars, E Sizer, G Auzinger, W Bernal, JA Wendon King’s College Hospital, London, UK Critical Care 2012, 16(Suppl 1):P389 (doi: 10.1186/cc10996) Introduction The aim of the study was to establish if the number of organs failing at admission to the ICU and the response to support had a bearing on outcome in patients with severe acute pancreatitis (SAP). Methods Only SAP patients requiring organ support were included in the analysis. Gallstones (55%) and alcohol were the commonest aetiologies. The proportion of patients with one, two or three system failures at baseline, 24, 48, and 72 hours were calculated and related to outcome. Introduction Present outcome prediction tools for patients with acute- on-chronic liver failure during critical illness are only of moderate accuracy. Regression methods on latent variables (usually applied to top-down system biology applications with spectroscopic data) may off er signifi cant advantages over logistic regression techniques as multiple cross-correlations are acceptable in this form of modelling. Results A total of 123 patients (85 male and 38 female) with a mean age of 58 years met the study criteria. The numbers of patients presenting with one, two and three organ failures were 29, 70 and 24 respectively, of which the mortality was six (21%), 29 (41%) and 14 (48%). Subsequent fi gures were 24, 57 and 39 with mortalities of four (17%), 19 (33%), and 24 (62%) at 24 hours; 21, 53 and 43 with mortalities of two (10%), 18 (34%), and 26 (60%) at 48 hours; and 17, 49 and 45 with mortalities of zero (0%), 16 (33%), and 28 (62%) at 72 hours. Methods Between 1 January 2000 and 31 December 2010 all patients admitted to the Liver Intensive Therapy Unit (LITU) at King’s College Hospital had daily prospective collection of demographic, biochemistry and bedside physiology. Logistic regression modelling (LRM) and partial least-squares discriminant analysis (PLSDA), Model for End- stage Liver Disease (MELD) and APACHE II scores were compared using receiver operating characteristic (ROC) curve analysis. Conclusion These data allow prognostication of patients with SAP requiring organ support. At 72 hours, the prognosis of patients with single organ failure is excellent and that of patients with three-organ failure remains poor. Mainz Emergency Evaluation Scoring in combination with capnometry predicts outcome in trauma patients Mainz Emergency Evaluation Scoring in combination with capnometry predicts outcome in trauma patients EH Hajdinjak1, ŠG Grmec2, MK Križmarić3, ET Torkar2, DB Buić-Rerečić2, MZ Zelinka2, MŠ Škufca2 1Center for Emergency Medicine, University of Ljubljana, University of Maribor, Maribor, Slovenia; 2Community Health Centre Ljubljana, University of Ljubljana, University of Maribor, Ljubljana, Slovenia; 3Faculty of Medicine, University of Maribor, Slovenia Critical Care 2012, 16(Suppl 1):P385 (doi: 10.1186/cc10992) p y p p EH Hajdinjak1, ŠG Grmec2, MK Križmarić3, ET Torkar2, DB Buić-Rerečić2, MZ Zelinka2, MŠ Škufca2 g y g y Methods This study was based on determination of RC scores in AP cases in a retrospective manner. On the other hand, this study included only the patients’ zero-time RC scores, not the 48-hour scores, for the sake of more practical precision. Serum CRP levels were found to have prognostic importance in AP, signifi cantly more than 150 mg/l in necrotizing AP, at 50 mg/l in this study. Therefore, patients’ were evaluated for RC and RC + CRP scores for comparison. However, RC had been etiologically modifi ed for presence of gall bladder stones (GBS); only the cases without GBS were included in order to prevent bias of 1Center for Emergency Medicine, University of Ljubljana, University of Maribor, Maribor, Slovenia; 2Community Health Centre Ljubljana, University of Ljubljana, University of Maribor, Ljubljana, Slovenia; 3Faculty of Medicine, University of Maribor, Slovenia y , Critical Care 2012, 16(Suppl 1):P385 (doi: 10.1186/cc10992) y , Critical Care 2012, 16(Suppl 1):P385 (doi: 10.1186/cc10992) Introduction This prospective study assessed the effi cacy of the predicting power for mortality of two diff erent prehospital scoring Figure 1 (abstract P384). SMR according to APACHE II and SCCM criteria. Figure 1 (abstract P384). SMR according to APACHE II and SCCM criteria. S139 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 and readmissions were excluded. Demographic characteristics, co- morbidities and parameters included in severity scores (APACHE II, SAPS II, SOFA) were studied. A Cox proportional hazard regression model was used to assess the eff ect of each variable on patient survival. Results A total of 122 patients diagnosed with AP were admitted to our ICU between January 2000 and December 2009 (68.9% men, mean age: 60.5 ± 14 years); 43.4% were smokers and 41.8% alcohol consumers. System biology prediction model based on clinical data: highly accurate outcome prediction in patients with acute-on-chronic liver failure y Results A total of 986 patients (median age 52 (range 16 to 90) years; 603 (62%) male) with cirrhosis and emergency LITU admission were identifi ed. The median APACHE II score was 21 (5 to 50) and the median MELD score 23 (3 to 50). Overall LITU survival was 63% and survival to hospital discharge 51%. Predictive accuracy at day 3 was improved in all models over admission values. The AUROC for LITU survival for MELD and APACHE scores on day 3 was 0.78 (95% CI 0.75 to 0.82, sensitivity 72%, specifi city 75%) and 0.83 (0.78 to 0.83, sensitivity 83%, specifi city 63%) respectively. A LRM utilising nine variables had an AUROC of 0.85 (95% CI 0.82 to 0.87, sensitivity 72%, specifi city 83%). Two-component PLSDA identifi ed 30 variables with independent prognostic signifi cance. Performance in outcome prediction was improved over logistic regression at day 3 – sensitivity 86%, specifi city 81%, AUROC 0.91 (0.89 to 0.93, P <0.001 for all comparisons) in a model incorporating 30 variables. Cross-validation and permutation analysis confi rmed the internal validity of this method. Mainz Emergency Evaluation Scoring in combination with capnometry predicts outcome in trauma patients In the end, in the hardworking hours on the ICU, we need a more practical method of provision. In this study, we have found no priority of RC, RC + CRP and CRP alone in predicting AP outcome, excluding GBS disease and necrotizing cases. We conclude that, practically, ICU physicians could substantially depend on CRP levels alone in the evaluation and approach in these specifi c cases of AP. Conclusion Comorbidities and AP etiology are not predictors of ICU mortality. Of the variables included in severity scores, only those related to organ dysfunction (hemodynamic – SAP, MAP, pH, HCO3 –; respiratory –PaFi relation; and renal – Cr, urea and 24-hour diuresis) are ICU mortality predictors in AP patients. Number of failed organs and response to therapy determine outcome in patients with acute pancreatitis requiring level 1 organ supportf Number of failed organs and response to therapy determine outcome in patients with acute pancreatitis requiring level 1 organ support G Morris-Stiff , A Baker, A Breen, A Smith Leeds Teaching Hospitals, Leeds, UK Critical Care 2012, 16(Suppl 1):P387 (doi: 10.1186/cc10994) P389 Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure Results A total of 1,150 patients were admitted, 129 cases (11.2%) were identifi ed as having excess alcohol consumption. Of these cases 34% were women, whilst 48% of the controls were female. The median age of the cases was 54 years versus 68 years for the controls (P <0.001). The cases had a lower APACHE II score, 14.3 vs. 15.8 (P = 0.002). Twenty- four (18.6%) of the cases with excess alcohol consumption died on the ICU compared to 141 controls (13.8%) (P >0.1). The hospital mortality was similar between the two groups, 28 (21.7%) against 215 (21.1%) controls (P >0.5). The cases spent longer on the ICU, median 3.95 days versus 2.9 in the controls (P <0.001). On admission the cases required a median of 2.0 organ supportive therapies compared to 1.8 in the control group (P <0.001). The cases were ventilated for a mean of 4.1 days compared to 2.4 days in the controls (P <0.001). There was no diff erence in the rate of sepsis between either group, 10% in the cases and 9.8% in the controls. Twenty-six patients were admitted with known alcoholic cirrhosis (0.23%), 10 with oesophageal varices and three with acute pancreatitis related to alcohol. liver failure E McCarron, I Welters Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P391 (doi: 10.1186/cc10998) E McCarron, I Welters Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P391 (doi: 10.1186/cc10998) Introduction Patients with liver failure in the critical care unit frequently provide physicians with problems about management and prognosis. Alcoholic liver disease (ALD) in particular is showing an increase in admission and mortality in the UK [1]. Current biochemical tests make it diffi cult to diff erentiate between types and severity of liver damage and fail to give a true idea about prognosis and outcome, often only showing low-grade derangements before hyperacute decompensation of liver function. The aim of this study was to look at various liver function tests (LFTs) routinely recorded in patients admitted to critical care with liver failure, to see whether they diff ered between ALD and nonalcoholic aetiologies (NALD); that is, drug overdose and nonalcoholic steatohepatitis, and so forth. References 1. Thomson SJ, et al.: Alcohol Alcohol 2008, 43:416-422. 1. Thomson SJ, et al.: Alcohol Alcohol 2008, 43:416-422. 2. Antoine DJ, et al.: Keratin-18 and HMGB1 as predictive biomarkers for mode of cell death and clinical prognosis during acetaminophen hepatotoxicity in man. J Hepatol 2012. [Epub ahead of print] Methods One-hundred and ninety-seven critically ill patients were studied at the Medical University Vienna: 72 patients with HH, 22 with ALF, 58 with AoCLF and 45 critically patients without evidence for liver disease. Arterial ammonia concentrations were assessed on a daily basis in all patients and compared among the four study groups as well as between 28-day survivors and nonsurvivors. p y p p 3. Zhou RR, et al.: BMC Gastroenterol 2011, 11:21. P388 Mortality predictors in acute pancreatitis admitted to the ICU P Vidal-Cortés1, P Lameiro-Flores1, A Aller-Fernández2, M Mourelo-Fariña2, R Gómez-López1, P Fernández-Ugidos1, M Alves-Pérez1, E Rodríguez-García1 1CHU Ourense, Spain; 2CHU A Coruña, Spain Critical Care 2012, 16(Suppl 1):P388 (doi: 10.1186/cc10995) p p Critical Care 2012, 16(Suppl 1):P388 (doi: 10.1186/cc10995) Introduction Patients diagnosed with acute pancreatitis (AP) are usually admitted to our units. Despite using a lot of scores, none has proved an acceptable yield to identify patients with higher mortality risk. Our purpose is to identify mortality predictors of patients admitted to our ICU diagnosed with AP. Methods We performed a retrospective study in which we analyzed patients diagnosed with AP admitted to a 24-bed ICU between January 2000 and December 2009. Postcardiopulmonary bypass pancreatitis i Conclusion This application of latent variable regression modelling techniques to intensive care datasets demonstrates high accuracy of S140 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 deranged LFTs (P <0.001) and clotting studies (P <0.001). ALD patients also had longer ITU stays (P <0.001) and higher mortality rates (45.45% ALD vs. 13.2% NALD). Receiver-operated curve analysis revealed that current biochemical markers (ALT, PT, GGT, albumin) are not sensitive and specifi c enough in detecting ALD. The prothrombin time yielded the best area under the curve with 80.4% in ALD versus 71.7% in NALD. None of the markers was discriminatory for determining the type of liver damage. prediction. Liver-specifi c outcome schema based on logistic regression may not fully describe the complex cross-correlating interactions that PLS techniques are designed to incorporate. Further validation in other centres and disease groups is warranted. prediction. Liver-specifi c outcome schema based on logistic regression may not fully describe the complex cross-correlating interactions that PLS techniques are designed to incorporate. Further validation in other centres and disease groups is warranted. P390 P392 y Results The 28-day mortality rates in HH, ALF, AoCLF and in the control group were 54% (n = 39), 27% (n = 6), 53% (n = 31) and 27% (n = 12), respectively. Peak arterial ammonia levels in patients with HH were signifi cantly higher in 28-day nonsurvivors compared to survivors (P <0.01). Cox regression identifi ed peak arterial ammonia concentrations as an independent predictor for 28-day mortality (P <0.01). Peak arterial ammonia levels in 28-day transplant-free ALF survivors were signifi cantly lower compared to ALF patients who died or underwent liver transplantation (P <0.05). There was no association between outcome and arterial ammonia in AoCLF patients and in the control group. Incidence, morbidity and mortality of admissions related to alcohol consumption on critical care: a single-centre experience A Retter, F Tait, M Stockwell St Helier Hospital, London, UK Critical Care 2012, 16(Suppl 1):P392 (doi: 10.1186/cc10999) Introduction Excessive alcohol consumption is a major challenge to public health. In 2000 it accounted for 4% of the global disease burden. However, the relationship between alcohol and health is complex and the burden it places on admissions to critical care is uncertain. p g p Conclusion Elevated arterial ammonia levels are frequently observed in critically ill patients with liver injury but not in patients of comparable severity of illness without hepatic impairment. They indicate poor prognosis in HH and ALF, but not AoCLF. Methods We conducted a retrospective analysis of prospectively collected data on the infl uence of excess alcohol consumption on the outcome of patients admitted from July 2009 to July 2011. The admitting physician determined the relationship between alcohol use and admission. No patients were excluded. All continuous data are expressed as medians and were compared using the Wilcoxon Mann– Whitney U test. Categorical data were compared using the chi-squared test. P391 Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure E McCarron, I Welters Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P391 (doi: 10.1186/cc10998) Prognostic relevance of arterial ammonia levels in diff erent acute and acute-on-chronic liver diseases Prognostic relevance of arterial ammonia levels in diff erent acute and acute-on-chronic liver diseases V Fuhrmann, A Drolz, B Jaeger, M Wewalka, R Saxa, T Horvatits, T Perkmann, C Zauner, P Ferenci Medical University Vienna, Austria Critical Care 2012, 16(Suppl 1):P390 (doi: 10.1186/cc10997) g Conclusion Our results suggest that currently used markers of liver disease are neither sensitive nor specifi c enough in patients with failure secondary to ALD. Research is needed to develop novel biomarkers to better prognosticate outcome. Aetiology of acute-on-chronic liver failure plays a major role in determining outcome, and subgroups of liver patients should be analysed individually. Studies [2,3] have shown that various markers are released depending on the type of damage and diff er in acute liver damage of diff erent origin. Better understanding of their role could prove useful in these patients. References V Fuhrmann, A Drolz, B Jaeger, M Wewalka, R Saxa, T Horvatits, T Perkmann, C Zauner, P Ferenci Medical University Vienna, Austria Critical Care 2012, 16(Suppl 1):P390 (doi: 10.1186/cc10997) Introduction Increased levels of arterial ammonia in patients with acute liver failure (ALF) are associated with increased mortality. There is a lack of data for prognostic impact of arterial ammonia in patients with acute-on-chronic liver failure (AoCLF) and hypoxic hepatitis (HH). We evaluated arterial ammonia levels and their prognostic relevance in patients with HH, ALF, AoCLF and without evidence for any liver disease. Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure We also aimed to assess their prognostication value and relation to severity of disease scores. Methods A total of 119 patients admitted to the ITU with liver failure (66 ALD and 53 NALD) between 2008 and 20011 were included. Each patient had admitting electrolytes, haematology, LFTs and clotting studies along with APACHE II score, length of stay and ventilation and vital organ support requirement. Conclusion To our knowledge this is the largest single-centre assessment of the burden of excess alcohol consumption on patients admitted to critical care. Eleven per cent of all admissions to the ICU were complicated by excess alcohol consumption. The ITU mortality of these patients was increased when compared to the controls, despite the patients having an equivalent APACHE score on admission and tending to be younger. The cases spent less time in hospital than the controls. This was due to a bimodal distribution of their survival curve. Our study is limited by its retrospective design and the risk of selection bias. g y Methods A total of 119 patients admitted to the ITU with liver failure (66 ALD and 53 NALD) between 2008 and 20011 were included. Each patient had admitting electrolytes, haematology, LFTs and clotting studies along with APACHE II score, length of stay and ventilation and vital organ support requirement. Results ALD patients were found to have lower sodium (mean 135.56; P = 0.004) and be hypocalcaemic (P = 0.015), as well as having more S141 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P393 P393 Changing outcomes in patients with chronic liver disease in intensive care: a decade of experience MJ McPhail, DL Shawcross, RD Abeles, G Huei-Lee, M Abdulla, T Chang, C Willars, E Sizer, G Auzinger, W Bernal, J Wendon King’s College Hospital, London, UK Critical Care 2012, 16(Suppl 1):P393 (doi: 10.1186/cc11000) haemofi ltration), lactate and APACHE II score on ICU admission and at the time of transplantation were also analysed. The primary outcome measure was patient survival at 3 months. Statistical analysis was by Mann–Whitney test, logistic regression and area under the receiver- operator curve analysis. p y Results Eighty-one patients were transplanted from the ICU with cirrhosis complications. Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure Statistical signifi cance was demonstrated for admission lactate (P = 0.032), transplant lactate (P <0.000), transplant APACHE II score (P = 0.001), admission inotropic support (P = 0.019), transplant inotropic support (P <0.000) and transplant renal support (P <0.000) when comparing 3-month survival with death on univariate analysis. On multivariate logistic regression analysis, high lactate (OR 1.28, 95% CI 1.08 to 1.51, P = 0.003) and use of renal replacement therapy (OR 3.52, 95% CI 1.42 to 8.74, P = 0.006) at the time of trans- plantation were independently associated with poor outcome. A combination of these two measures had an AUROC of 0.883 (0.791 to 0.945, P <0.001, sensitivity 86%, specifi city 86%) for prediction of death within 3 months. Introduction Patients with chronic liver disease requiring intensive care are thought to carry a poor prognosis in comparison with noncirrhotic patients with similar severity of illness. During the last decade improvements in multiple areas of management in patients in the ICU have occurred but improvement in outcomes in patients with cirrhosis has not been shown. Methods Between 1 January 2000 and 31 December 2010 all patients admitted to the Liver Intensive Therapy Unit (LITU) at King’s College Hospital had daily prospective collection of demographic, biochemistry and bedside physiology. These data were used to quantify the severity of illness (APACHE II and Model for End-stage Liver Disease (MELD)) and outcomes in these patients. Conclusion Patients with chronic liver disease transplanted from the ICU have a worse outcome if they require renal support or demonstrate hyperlactataemia on the day of transplantation. f p Results A total of 958 patients (median age 52 (range 16 to 90) years; 603 (62%) male) with cirrhosis and emergency LITU admission were identifi ed. Aetiology of cirrhosis was alcohol in 43%, viral in 10%, autoimmune disease in 10% and nonalcoholic fatty liver disease/ metabolic in the remainder. The pattern of aetiology of cirrhosis did not change over time and a viral aetiology was associated with improved outcome (OR 0.53, 95% CI 0.34 to 0.81, P = 0.003); alcohol was not associated with poorer outcome (P = 0.09). The primary reasons for admission were bleeding (33%), sepsis (27%), hepatic encephalopathy (17%), metabolic (7%) and other (16%). The median APACHE II score was 21 (5 to 50) and the median MELD score 23 (3 to 50). Overall LITU survival was 63% and survival to hospital discharge 51%. P394 Multivariate regression analysis of outcomes following orthotopic liver transplantation in decompensated cirrhotics transplanted from the ICU T Hughes, M McPhail, M Al-Freah, D Abeles, W Bernal, G Auzinger, J Wendon, C Willars Institute of Liver Studies, King’s College Hospital, London, UK Critical Care 2012, 16(Suppl 1):P394 (doi: 10.1186/cc11001) Results A total of 198 cirrhotics (mean age 53 years, 66% male) were reviewed. The most common etiologies were hepatitis C (31%) and alcohol (15%). LT occurred a median time of 29 (5 to 101) days from listing and 5 (3 to 10) days from ICU admission. In total, 88% of patients required vasopressors, 56% received RRT prior and 87% were ventilated prior to LT. The median MELD score was 34 (26 to 39) on ICU admission and 34 (27 to 40) on the day of LT respectively. SOFA scores were 12 (10 to 15) and 13 (10 to 17) on ICU admission and on the day of LT respectively. Comparing patients who were alive (n = 166, 84%) versus dead (n = 32, 16%) at 90 days, there were no statistically signifi cant diff erences in MELD score on admission or day of LT (P >0.6 for both). There were also no statistically signifi cant diff erences between SOFA score on admission or day of LT (P >0.17 for both). Patients alive at 90 days were signifi cantly younger (52 vs. 56 years, P = 0.007). Patients over 60 had signifi cantly higher 90-day mortality (27% vs. 13%, P = 0.04) and a trend towards increased 1-year mortality (37% vs. 23%, P = 0.09). There were no signifi cant diff erences in donor characteristics (donor age >60, cold ischemia time >8 hours, split graft, donor cerebrovascular event) comparing patients alive at 90 days to nonsurvivors. Introduction Patients listed for orthotopic liver transplantation (OLT) frequently develop complications resulting in transfer to the ICUs of tertiary centres. The ICU mortality for cirrhotics has been variously reported from 38% to in excess of 90% [1]. The APACHE II score, MELD score and bacteraemia are independent predictors of mortality [2]. The aim of this study was to identify the risk factors relating to early mortality after OLT in cirrhotics transplanted from the ICU. Methods A retrospective analysis of 1,284 patients transplanted between the dates of 1 January 2000 and 31 December 2008 in a major UK liver transplant centre was performed. References 1. Austin MJ, et al.: Curr Opin Crit Care 2008, 14:202-207. 2. Karvellas CJ, et al.: Crit Care Med 2010, 38:121-126. 1. Austin MJ, et al.: Curr Opin Crit Care 2008, 14:202-207. 1. Austin MJ, et al.: Curr Opin Crit Care 2008, 14:202-207. 2. Karvellas CJ, et al.: Crit Care Med 2010, 38:121-126. 2. Karvellas CJ, et al.: Crit Care Med 2010, 38:121-126. Liver failure secondary to alcoholic liver disease carries a worse prognosis than other aetiologies of liver failure: retrospective analysis of routine biochemical markers in critically ill patients with liver failure LITU survival increased from 47% to 73% over the study period (2000 to 2010) with hospital outcome improving from 40% to 63%. The median admission APACHE II score fell from 23.4 to 21.9 over the study period (P <0.001) with the MELD score falling from 23.4 to 18.3 (P <0.001). Length of LITU stay did not change signifi cantly over the study period (P = 0.092). The reduction in illness severity was predominantly due to a smaller percentage of patients with renal failure and those with three or more organs in failure (32% up to 2005 and 24% post 2005, P = 0.004). The reduction in MELD score related to decreased renal dysfunction; creatinine over the study period (1.9 mg/dl to 1.6 mg/dl, P <0.001) with no change in bilirubin, and by contrast a small rise in international normalised ratio (INR 1.8 to 2.2, P = 0.07). P395 Liver transplantation in the critically ill: a Canadian collaboration C Karvellas1, T Lescot2, H Vahidy1, P Goldberg3, P Chaudhury3, P Metrakos3, N Kneteman1, G Meeberg1, M Sharpe4, J Ronco5, E Renner6, E Cook7, S Bagshaw1 1University of Alberta, Edmonton, Canada; 2Hôpital Saint-Antoine, Paris, France; 3McGill University, Montreal, Canada; 4University of Western Ontario, London, Canada; 5University of British Columbia, Vancouver, Canada; 6University of Toronto, Canada; 7Harvard School of Public Health, Boston, MA, USA Critical Care 2012, 16(Suppl 1):P395 (doi: 10.1186/cc11002) Introduction Critically ill cirrhotic patients awaiting liver transplan- ta tion (LT) often receive prioritization for organ allocation. Outcomes in these patients are multifactorial, and identifi cation of patients most likely to benefi t is essential. Despite the need for evidence-based allocation criteria based on patient factors and physiology scores, few data currently exist on outcomes. Scoring systems such as MELD and SOFA (Sequential Organ Failure Assessment) are in use, but have not been evaluated in predicting outcome with LT. Conclusion Survival of patients with cirrhosis admitted to the specialist LITU is improving over time. The factors relating to this may be resultant upon earlier admission to critical care and a lower incidence of renal dysfunction. Alcohol aetiology is not relevant to outcome. Methods In a fi ve-center Canadian study (Edmonton, Montreal, Toronto, London and Vancouver), all cirrhotics admitted to the ICU requiring organ support (mechanical ventilation, vasopressors or renal replacement therapy (RRT)) prior to undergoing LT between January 2000 and December 2009 were examined. MELD and SOFA scores were evaluated at ICU admission and the day of LT along with other donor factors. Acute respiratory distress syndrome: analysis of incidence and mortality in a university hospital critical care unit Introduction The aim was to determine the incidence of acute respiratory distress syndrome (ARDS) in patients admitted to a university hospital ICU, analyse the ICU and the in-hospital mortality, and evaluate the associated factors. Methods A prospective study in an ICU from October 2008 to January 2011. The ICU comprises 20 beds in a medical–surgical area, 10 in a critical burns area. All patients who underwent mechanical ventilation (MV) during 48 hours or more and who fulfi lled ARDS criteria as defi ned by the 1994 American–European Consensus Conference on ARDS were included. All patients were ventilated following the protective MV strategy recommended. Conclusion In this large multicenter cohort, severe sepsis and septic shock were independently associated with an increased risk of death. Our data underscore the regional variability in the epidemiology and outcome of sepsis syndromes and may be useful for resource allocation. gy Results During this period 1,900 patients were admitted, 697 needed MV for at least 48 hours and 108 fulfi lled the ARDS criteria (5.6% of those admitted, 17% of the group on MV); 63% were male. The patients’ age was 52 ± 12. The APACHE II score on admission was 23 ± 7, in survivors (S) 20 ± 7 and 24 ± 6 in nonsurvivors (NS) (P = 0.002). ARDS was primary in 70% and secondary in 30%. The most common aetiology was pneumonia (53%) followed by sepsis of intra-abdominal origin (15%). Duration of MV was 32.7 ± 30.2 days in S, 20.79 ± 20.73 in NS (P = 0.019). Survivors’ mean length of stay was 35 ± 24 days, 23 ± 20 for NS (P = 0.007). ICU mortality was 49% and in-hospital mortality was 55%. Primary ARDS had an ICU mortality of 47%, an in- hospital mortality of 52%. Secondary ARDS had a 55% ICU mortality, an in-hospital mortality of 64%. Duration of primary ARDS was longer, 15.3 ± 12.2 versus 8.7 ± 79. Globally the main cause of death was multiple organ dysfunction, predominantly respiratory failure (55%). In primary ARDS the main cause of death was chiefl y pulmonary (69%), while in secondary ARDS it was mainly multiple organ dysfunction associated with septic shock (71%). Factors associated with increased mortality were APACHE II score >23 and the presence of multiple organ dysfunction. P398 Outcome of faecal peritonitis in the ICU J S G Si L M I W lt J Sayer, G Simpson, L Mccrossan, I Welters Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P398 (doi: 10.1186/cc11005) Introduction Faecal peritonitis often leads to intensive care admission. Anecdotally, patients with co-existing malignancy had an improved outcome. A retrospective analysis of all patients admitted to intensive care over 7 years was conducted to investigate this observation and identify factors that are associated with outcome from faecal peritonitis in intensive care. Methods A retrospective analysis of all cases of faecal peritonitis admitted to the Royal Liverpool University Hospital ICU over 7 years. Clinical records, laboratory results, histology reports and radiological data were accessed. Statistical analysis was performed using chi- squared and Student’s t tests. Results A total of 133 patients were admitted to intensive care in 7 years. Thirty-six patients had underlying malignancy. Predicted mortality, indicated by APACHE II score, was similar in both groups (malignancy: 17.1, nonmalignancy: 16.2). Inpatient mortality was lower in patients with malignancy than those without (malignancy: 21.6%, nonmalignancy: 38.1%, P <0.1) and shorter ITU stay (malignancy: 6.8 days, nonmalignancy: 12.7 days, P ≤0.0005). Cancer patients required a shorter period of TPN or NG feeding (malignancy: 4.29 days, nonmalignancy: 7.7 days, P <0.05), and a shorter duration of inotropic support (malignancy: 2.54 days, nonmalignancy: 4.44 days, P <0.05). Peak infl ammatory markers are lower in patients with malignancy, notably neutrophil count (malignancy: 21.15, nonmalignancy: 24.9, P <0.05). y Conclusion Certain controversy remains regarding a decrease in ARDS- related mortality. Despite the fact that its incidence is not very high, it is still a clinical entity with a high mortality, and with a prognosis infl uenced not only by the degree of pulmonary involvement but by the association with multiple organ dysfunction. References 1. Roca O, et al.: Estudio de cohortes sobre incidencia de SDRA en pacientes ingresados en UCIy factores pronósticos de mortalidad. Med Intensiva 2006, 30:6-12 . 1. Roca O, et al.: Estudio de cohortes sobre incidencia de SDRA en pacientes ingresados en UCIy factores pronósticos de mortalidad. Med Intensiva 2006, 30:6-12 . 2. Zambon M, Vincent JL: Mortality for patients with ALI/ARDS have decreased over time. Chest 2008, 133:151-161 . 2. Zambon M, Vincent JL: Mortality for patients with ALI/ARDS have decreased over time Chest 2008 133:151-161 3. Frutos-Vivar et al.: Epidemiology of ALI and ARDS. Curr Opin Crit Care 2004, 10:1-6. The mean APACHE II score was signifi cantly lower in cases who survived, compared to those who did not (nondeaths: 15.3, deaths: 19.3, P <0.005). Mean albumin at admission was similar for patients who survived compared to those who did not (deaths: 18.2, nondeaths: 18.6); however, minimum albumin during admission is signifi cantly lower in patients who died than those who survived (deaths: 10.33, nondeaths: 13.24, P <0.005). Duration of feeding support (TPN or NG feeding) and time to commencement of feeding showed no diff erence between patients who survived and those who did not. P397 E id Epidemiology and outcome of sepsis syndromes in Italian ICUs: a regional multicenter observational cohort L Laudari1, Y Sakr2, C Elia1, L Mascia1, B Barberis3, S Cardellino4, S Livigni5, G Fiore6, C Filippini1, VM Ranieri1 1San Giovanni Battista-Molinette Hospital, University of Torino, Turin, Italy; 2Friedrich Schiller University Hospital, Jena, Germany; 3Ospedale degli Infermi, Revoli, Italy; 4Ospedale Cardinal Massaia, Asti, Italy; 5Ospedale Giovanni Bosco, Turin, Italy; 6Ospedale Santa Croce, Moncalieri, Italy Critical Care 2012, 16(Suppl 1):P397 (doi: 10.1186/cc11004) P394 Patient characteristics were recorded at transplant assessment and on the day of transplant: age, MELD score, UKELD score, serum sodium, creatinine, bilirubin, albumin, INR. Organ support (including ventilation, inotropic support and S142 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Older critically ill cirrhotics (over 60 years) undergoing liver transplantation have signifi cantly worse post-LT outcomes. MELD and SOFA scores do not appear to predict outcome post LT in this cohort. Methods In this prospective, multicenter, observational study, all 3,902 patients (mean age ± SD: 64.3 ± 15.7 years, 63.5% male) admitted to one of 24 medical or surgical ICUs between 3 April and 29 September 2006 were included. Results Four hundred and forty-six of the patients had sepsis, including 160 patients with severe sepsis (4.1%) and 145 patients (3.7%) with septic shock. ICU mortality was 20% (n = 780) and median ICU length of stay was 3 (1 to 9) days. ICU mortality was higher (41.3 vs. 17.2%, P <0.001) and the median ICU LOS longer (15 (7 to 26) vs. 2 (1 to 7), P <0.001) in patients with sepsis than in those without sepsis. The mortality rate increased with the severity of sepsis (sepsis without organ failure, severe sepsis, and septic shock: 19.9, 44.4, and 58.6%, respectively). ICU-acquired sepsis was associated with higher ICU mortality rates than sepsis occurring within 48 hours of ICU admission (49.8 vs. 33.0%, P <0.001). In multivariate logistic regression analysis, the occurrence of severe sepsis (OR, 1.70 (1.06 to 2.72); P = 0.026) and septic shock (OR, 2.25 (1.49 to 3.49); P <0.001) were independently associated with an increased risk of ICU death. Epidemiology and outcome of sepsis syndromes in Italian ICUs: a regional multicenter observational cohort P399 HIV patients in the ICU: our experience V Nunes Velloso, L Calejman, E Canedo, M Deheza Hospital Rivadavia, Capital Federal, Argentina Critical Care 2012, 16(Suppl 1):P399 (doi: 10.1186/cc11006) P399 HIV patients in the ICU: our experience V Nunes Velloso, L Calejman, E Canedo, M Deheza Hospital Rivadavia, Capital Federal, Argentina Critical Care 2012, 16(Suppl 1):P399 (doi: 10.1186/cc11006) Critical Care 2012, 16(Suppl 1):P399 (doi: 10.1186/cc11006) Introduction The objective was to describe characteristics of HIV- positive patients admitted to the ICU. Methods HIV-positive patients admitted between February 2000 and February 2011, and demographic data, APACHE II score, cause of admission, days of internment, need for mechanical ventilation (MV), previous antiretroviral therapy of high effi cacy before admission (HAART), viral load and CD4 count. Conclusion About three-quarters of survivors of severe sepsis/septic shock with congestive heart failure died after 1 year of hospital discharge. Many of them (70%) died within 3 months of hospital discharge. The majority had poor performance status and only 14% were able to carry on normal activity at 1 year after hospital discharge. These data highlight the need for diff erent strategies to care for sepsis survivors with congestive heart failure. ( ), Results A total of 3,568 patients were admitted; 715 patients (20.03%) were HIV-positive, 413 patients (57.76%) were masculine and 302 patients (42.23%) feminine, and average age was 33 for men and 35 for women. The APACHE II average score was 13 versus 15.28 for the general population. The most frequent cause of admission was respiratory failure in 329 patients (46%), 57% due to Pneumocystis jivoreci and bacterial pneumonias in 35%, the most frequent bacteria isolated were Streptococcus, Staphylococcus aureus and Haemophilus infl uenzae. There were two cases of respiratory Kaposi sarcoma and 26 cases of Mycobacterium tuberculosis. Other causes were decrease in mental state in 157 patients (22%), with the most frequent causes reported being toxoplasmosis, cryptococcus neoformans and brain lymphoma, immediately post surgery in 79 patients (11%), COPD reagudization and asthma (9%), digestive bleeding in 36 patients (5%) and renal insuffi ciency in 50 patients (7%). From the 715 HIV-positive patients admitted, 479 required MV (67%). Regarding nationality, 276 (38.6%) patients were Argentinean, and the other nationalities were Bolivian, Paraguayan, Peruvian and Korean. The average length of stay was 10.5 days and the mortality was 43%. Predictive value of N-terminal pro-brain natriuretic peptide among critically ill patients critically ill patients M Cubrilo-Turek, N Maric, I Mikacic, N Tolj Karaula, N Budinski, M Mackovic Clinical Hospital Sveti Duh, Zagreb, Croatia Critical Care 2012, 16(Suppl 1):P401 (doi: 10.1186/cc11008) M Cubrilo-Turek, N Maric, I Mikacic, N Tolj Karaula, N Budinski, M Mackovic Clinical Hospital Sveti Duh, Zagreb, Croatia Critical Care 2012, 16(Suppl 1):P401 (doi: 10.1186/cc11008) Introduction N-terminal pro-brain natriuretic peptide (NT-proBNP) represents a useful cardiac marker in evaluating heart failure. However, its role in the assessment of critically ill patients is not clear. The aim of this study was to evaluate survival of infected and noninfected patients according to the measurements of NT-proBNP. g Methods Serum NT-proBNP measurements were done in 89 (46 males/43 females, 68.20 ± 13.80 years) consecutive critically ill patients within 6 hours after admission to the ICU. NT-proBNP was determined with a sandwich immunoassay on an Elecsys 2010 (Roche Diagnostics, Mannheim, Germany). Logarithmic transformation of data was required because of the skewed distribution of NT-proBNP. Conclusion HIV-positive patients have a high frequency of admission to the ICU, and they have a lower risk score in comparison with non-HIV patients. The two main causes of admission where respiratory disease and infectious CNS disease. Signifi cant results were the prevalence of patients from limited countries, high mortality and prolonged stay in the ICU, and poor adherence to antiretroviral therapy. p Results The median NT-proBNP (pg/ml) was 2,485.1 pg/ml (range 31.5 to 12,041 pg/ml) (log NT-proBNP mean 3.34 ± 0.71 pg/ml). Mean log NT- proBNP levels were higher at admission to the hospital in nonsurvivors (3.73 ± 0.67 pg/ml) compared with survivors (3.12 ± 0.65 pg/ml), which was statistically signifi cant (P <0.0001). Higher concentrations were found in proven infection (X ± SD) (3.43 ± 0.68) than in bacteriological negative patients (3.30 ± 0.72), but it was statistically insignifi cant (P <0.42). From 57 survivors seven were mechanically ventilated (12.28%) while 14 (43.75%) from 32 nonsurvivors were ventilated, which was statistically signifi cant (P <0.001). More nonsurvivors were taking vasoactive medications (n = 12 or 37.5%) than survivors (n = 3 or 5.26%), which was statistically signifi cant (P <0.001). NT-proBNP showed no correlation for any analyzed parameters (age, erythrocytes, leucocytes, body temperature, systolic and diastolic blood pressure, C-reactive protein, fi brinogen, lactates or procalcitonin). Epidemiology and outcome of sepsis syndromes in Italian ICUs: a regional multicenter observational cohort The viral load average was inferior to 104 RNA/ml in just 44 known patients and the CD4 count was determined in 75 patients, from which the average was 400/mm3. The proportion of patients receiving HAART was just 26%. P400 Impact of congestive heart failure on severe sepsis and septic shock survivors: outcomes and performance status after 1-year hospital discharge M Alkhalaf1, N Abd-Aziz2, Y Arabi3, B Tangiisuran1 1School of Pharmaceutical Sciences, Penang, Malaysia; 2University Technology MARA, Puncak Alam, Malaysia; 3National Guard Hospithal, Riyadh, Saudi Arabia Critical Care 2012, 16(Suppl 1):P400 (doi: 10.1186/cc11007) Epidemiology and outcome of sepsis syndromes in Italian ICUs: a regional multicenter observational cohort L Laudari1, Y Sakr2, C Elia1, L Mascia1, B Barberis3, S Cardellino4, S Livigni5, G Fiore6, C Filippini1, VM Ranieri1 Conclusion Underlying malignancy is associated with an increased survival, shorter ITU stay, less requirement for inotropic support and decreased infl ammatory markers potentially due to a less aggressive infl ammatory response as a consequence of the presence of malig- nancy. In this series, delay to introduction of nutrition and length of nutritional support are not associated with outcome; however, low albumin is associated with a poor outcome, although it is not clear if this is secondary to nutrition or infl ammation. Introduction We assessed the epidemiology of sepsis syndromes in patients admitted to ICUs of the Piedmont region in northern Italy and investigated the impact of sepsis on ICU mortality in these patients. S143 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 congestive heart failure cases died, 70% of them dead within 3 months. Patients with CHF as compared to patients without CHF had a higher percentage of comorbidity disease (P <0.01) and poor performance status (P <0.05). The majority of these patients (85.7%) who were older (P <0.001), and required a higher dose of dobutamine (P <0.0001), had higher urine output (P <0.001) and prolonged INR (P <0.01) were unable to care for self at 1 year of hospital discharge. Survivors with CHF who died (OR 4.7, CI 1.52 to 14.33) had higher dose of dopamine (P <0.045) and poor performance status pre sepsis (P <0.028). congestive heart failure cases died, 70% of them dead within 3 months. Patients with CHF as compared to patients without CHF had a higher percentage of comorbidity disease (P <0.01) and poor performance status (P <0.05). The majority of these patients (85.7%) who were older (P <0.001), and required a higher dose of dobutamine (P <0.0001), had higher urine output (P <0.001) and prolonged INR (P <0.01) were unable to care for self at 1 year of hospital discharge. Survivors with CHF who died (OR 4.7, CI 1.52 to 14.33) had higher dose of dopamine (P <0.045) and poor performance status pre sepsis (P <0.028). Impact of congestive heart failure on severe sepsis and septic shock survivors: outcomes and performance status after 1-year hospital discharge M Alkhalaf1, N Abd-Aziz2, Y Arabi3, B Tangiisuran1 1School of Pharmaceutical Sciences, Penang, Malaysia; 2University Technology MARA, Puncak Alam, Malaysia; 3National Guard Hospithal, Riyadh, Saudi Arabia Critical Care 2012, 16(Suppl 1):P400 (doi: 10.1186/cc11007) Introduction The objective of this study was to evaluate the impact of CHF on severe sepsis and septic shock survivor outcomes after 1 year of hospital discharge. Conclusion Our results showed that cardiac NT-proBNP levels can be elevated in critically ill patients and may also serve as markers of severity and prognosis for survival. Mean baseline levels of log NT-proBNP were diff erent in critically ill patients with proved bacteriological infection than in patients without proven infection. p g Methods A retrospective cohort and cross-sectional study was conducted at a tertiary-care hospital in Saudi Arabia. All patients (≥18 years) with severe sepsis/septic shock admitted for more than 1 day to the medical–surgical and trauma ICU between April 2007 and March 2010 and alive at hospital discharge were included in the study. Patients who died during admission, could not be contacted and with multiple ICU admission within the same hospitalization were excluded. Data were collected using the electronic ICU database, hospital information system and systematic review of medical records to determine hospital outcomes and performance status pre sepsis. Assessment of the vital status and performance at 1-year hospital discharge were performed via structured telephone interviews using the Karnofsky Performance Status Scale. Predictive value of N-terminal pro-brain natriuretic peptide among critically ill patients The use of ROC curve analysis reveals for serum NT-proBNP high sensitivity (75%), low specifi city (57.9%) and low accuracy (64%) for discriminating survivors from nonsurvivors. Correlation between APACHE II score and quality of life among patients discharged from the ICU p g L Zubek1, L Szabó1, L Horváth1, A Mesterházi2, J Gál1, G Élő1 1Semmelweis University, Budapest, Hungary; 2Markusovszky Hospital, Szombathely, Hungary p g L Zubek1, L Szabó1, L Horváth1, A Mesterházi2, J Gál1, G Élő1 1Semmelweis University, Budapest, Hungary; 2Markusovszky Hospital, Szombathely, Hungary y g y Critical Care 2012, 16(Suppl 1):P404 (doi: 10.1186/cc11011) Introduction The goal of intensive therapy is not only saving the patient’s life, but also to restore their quality of life. Based on expected quality of life improvement, a fair allocation of limited available resources can be provided. The assessment scores for the physical state of ICU patients, which correlate with survival, are widely known. However, it would be useful to know if these score systems also correlate with the long-term quality of life. The aim of our study was to investigate the correlation between the APACHE II score and the long- term quality of life after ICU treatment. q y Methods We have collected data retrospectively from patients treated in our department during the fi rst quarter of 2008. The APACHE II score was calculated for all patients, after which we examined the correlation between this value and the survival of the patients. One year after ICU therapy, the Hungarian version of the EQ-5D questionnaire (measurement consist of fi ve dimensions: mobility, self-care, usual activities, pain/discomfort, anxiety/depression and a visual analog scale about health state) developed by EuroQol Group was sent out by post. The correlation between the APACHE score and quality of life was calculated, the Spearmann rank-order correlation was used. g Conclusion Our fi ndings suggest that low preoperative hepcidin concentration indicates mortality but not renal endpoints in patients undergoing cardiac surgery. Thereby, hepcidin may contribute to early risk stratifi cation. Findings should be validated in independent patient cohorts with a larger number of events. Results During this period, 190 patients were treated in our department. The average of the APACHE II score was 13.23 (±6.99). In total, 25.3% of patients died during treatment; 22.1% died during the fi rst post- treatment year; 27.9% surely survived and 24.7% of patients were unattainable. In our cohort, every patient below 11 points survived and none above 24. The average APACHE score of patients completing the questionnaire was 9.30 (±3.85). They assessed their health as 66% at VAS, although correlation between this value and the APACHE score could not be shown. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 a mean of 3.6 days, and 41% needed ARS for a mean of 3.8 days. See Table 1. biomarker that early postoperatively predicts protection from acute kidney injury (AKI). biomarker that early postoperatively predicts protection from acute kidney injury (AKI). Conclusion CC and hospital mortality was 17% and 33% respectively. This study concurs with another which demonstrated that age is not a good predictor of outcome post surgery [1]. These patients did not have a signifi cant impact on RRT or ARS resources or CC LOS. Reference Methods We studied 100 adult patients at increased risk of AKI (RIFLE) after cardiac surgery. Plasma and urine were sampled before induction of anesthesia and hepcidin 25-isoforms were quantifi ed by competitive enzyme-linked immunoassay. Our objective was to assess the predictive indices of preoperatively measured urine and plasma hepcidin for the development of postoperative AKI and other patient- related outcomes, including the need for renal replacement therapy (RRT) and in-hospital mortality. 1. Ford P, et al.: Determinants of outcome in critically ill octogenarians after surgery. Br J Anaesth 2007, 99:824-829. P404 Results Preoperatively, patients not developing AKI presented with nonsignifi cantly higher urine and plasma hepcidin concentrations compared to patients that developed AKI which did not translate into a good predictive value for postoperative AKI (AUC-ROC <0.70 for both biomarkers). Also, the preoperative urine and plasma hepcidin concentrations as well as serum creatinine concentration did not distinguish patients requiring postoperative RRT from those who did not require RRT (urine: AUC-ROC 0.62 (95% CI 0.38 to 0.86), plasma: AUC- ROC 0.63 (95% CI 0.34 to 0.91), serum creatinine: AUC-ROC 0.61 (95% CI 0.22 to 0.99)). However, a low preoperative hepcidin concentration in urine (median 5 ng/ml, 25th to 75th percentiles 4 to 15 ng/ml) and in plasma (median 50 ng/ml, 25th to 75th percentiles 30 to 55 ng/ml) was a good predictor for postoperative mortality with an AUC-ROC for urine hepcidin of 0.89 (95% CI 0.73 to 0.99) (cut-off : 130 ng/ml, sensitivity 73% and specifi city 100%) and an AUC-ROC for plasma hepcidin of 0.90 (95% CI 0.80 to 0.99) (cut-off : 55 ng/ml, sensitivity 83% and specifi city 100%). Preoperative serum creatinine did not predict mortality (AUC- ROC 0.50 (95% CI 0.10 to 0.94). Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Patients who survived the hospital stay had a median preoperative hepcidin concentration in urine of 330 ng/ ml (25th to 75th percentiles 140 to 760 ng/ml), and plasma of 115 ng/ ml (25th to 75th percentiles 80 to 200 ng/ml).i 0 Correlation between APACHE II score and quality of life among patients discharged from the ICU L Zubek1, L Szabó1, L Horváth1, A Mesterházi2, J Gál1, G Élő1 1Semmelweis University, Budapest, Hungary; 2Markusovszky Hospital, Szombathely, Hungary Critical Care 2012, 16(Suppl 1):P404 (doi: 10.1186/cc11011) Correlation between APACHE II score and quality of life among patients discharged from the ICU However, we found statistically signifi cant correlation between the APACHE score and the current mobility of the patients (P = 0.021). Based on our data, 34% of the patients had problems with mobility, 36% with usual activity, 62% of patients complained about pain or discomfort, 50% felt anxiety or depression and 18% had problems with self-care. P402 P402 Low preoperative hepcidin concentration is a risk factor for mortality but not for acute kidney injury after cardiac surgery A Haase-Fieiltz1, P Mertens1, M Plaß2, M Westerman3, R Bellomo4, M Haase1 1Otto-von-Guericke University, Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3IntrinsicLifeSciences, La Jolla, CA, USA; 4Austin Health, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P402 (doi: 10.1186/cc11009) Low preoperative hepcidin concentration is a risk factor for mortality but not for acute kidney injury after cardiac surgery A Haase-Fieiltz1, P Mertens1, M Plaß2, M Westerman3, R Bellomo4, M Haase1 1Otto-von-Guericke University, Magdeburg, Germany; 2German Heart Center, Berlin, Germany; 3IntrinsicLifeSciences, La Jolla, CA, USA; 4Austin Health, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P402 (doi: 10.1186/cc11009) Introduction Hepcidin – expressed in renal proximal tubular cells – is a key regulator of iron homeostasis and was recently described as a renal Results A total of 195 hospital survivors from 364 patients were included in the fi nal analysis. More than 70% of severe sepsis/septic shock with S144 Predicting hospital mortality: comparing accuracy of SAPS II and clinical staff prognosis Predicting hospital mortality: comparing accuracy of SAPS II and clinical staff prognosis I Patrício1, M Marques1, A Costa-Pereira2, O Ribeiro2, I Aragão1, T Cardoso1 1Hospital Geral de Santo António, University of Porto, Portugal; 2Faculty of Medicine, University of Porto, Portugal Critical Care 2012, 16(Suppl 1):P407 (doi: 10.1186/cc11014) f p g I Patrício1, M Marques1, A Costa-Pereira2, O Ribeiro2, I Aragão1, T Cardoso1 1Hospital Geral de Santo António, University of Porto, Portugal; 2Faculty of Medicine, University of Porto, Portugal Critical Care 2012, 16(Suppl 1):P407 (doi: 10.1186/cc11014) Results The mortality rate of surgical patients was 12.29%. We detected no statistical diff erence between the two groups according to age (P = 0.27), heart rate (P = 0.13), temperature (P = 0.57), Na (P = 0.44), K (P = 0.18), WBC (P = 0.56), Ht (P = 0.7), PaO2 (P = 0.28), PaCO2 (P = 0.7), albumin (P = 0.21), glucose (P = 0.68) and GCS (P = 0.26). We detected statistically signifi cant higher group B values according to BUN (P = 0.015), creatinine (P = 0.005), bilirubin (P = 0.0032), APACHE II score (P = 0.0018), LOS (P <0.0001) and VD (P <0.0001). We detected statistically signifi cant higher group A values according to mean arterial pressure (P = 0.0052) and PH (P = 0.0027). Introduction The purpose of this study is to compare the accuracy of Simplifi ed Acute Physiology Score (SAPS) II with the subjective opinion of clinical staff in predicting hospital mortality, in critically ill adult patients. Introduction The purpose of this study is to compare the accuracy of Simplifi ed Acute Physiology Score (SAPS) II with the subjective opinion of clinical staff in predicting hospital mortality, in critically ill adult patients. Methods A prospective study in a mixed ICU, at a university hospital, using SAPS II to assess the risk of death. Patient outcome was also predicted subjectively by the clinical staff (consultants, residents and nurses), including the possibility of return to prior physical activity. The subjective predictions were compared with SAPS II predictions using logistic regression analysis and receiver operating characteristic curve (ROC) measurement, as well as sensitivity and specifi city analysis for each group of participants. Conclusion According to our data, surgical patients who died (group B) had higher severity score on admission. Relationship between illness severity scores in the ICU Relationship between illness severity scores in the ICU A Schneider1, M Lipcsey1, M Bailey2, D Pilcher3, R Bellomo1 1Austin Health, Heidelberg, Australia; 2Monash University, Melbourne, Australia; 3ANZICS, Melbourne, Australia Critical Care 2012, 16(Suppl 1):P406 (doi: 10.1186/cc11013) Outcomes and resource use for over 80 year olds admitted to a UK critical care unit after an emergency laparotomy over a 3-year period V Banks, C Scott Northern General Hospital, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P403 (doi: 10.1186/cc11010) Outcomes and resource use for over 80 year olds admitted to a UK critical care unit after an emergency laparotomy over a 3-year period V Banks, C Scott Northern General Hospital, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P403 (doi: 10.1186/cc11010) V Banks, C Scott V Banks, C Scott pfi Critical Care 2012, 16(Suppl 1):P403 (doi: 10.1186/cc11010) Introduction There are few data on older people emergency surgical critical care (CC) admissions and the potential implications for future resource demands and service planning. Methods Retrospective data were collected from a cohort of patients >80 years old admitted after emergency surgery between 2009 and 2011. CC and hospital information databases were used. Data included mortality, length of stay (LOS) and duration of renal replacement therapy (RRT) and advanced respiratory support (ARS). Conclusion ICU admission is associated with a high mortality, a poor physical quality of life and low quality-adjusted life-years for 1 year after discharge. We found that the APACHE II score did not show signifi cant correlation with patient’s long-term quality of life, but we detected signifi cant correlation between the APACHE II score and the current mobility of the patients. py p y pp Results A total of 118 patients were admitted; 52% female: mean age 85 years, male mean age: 84 years. In total, 69% were general surgical, 22% vascular, and 9% hepatobiliary. Outcomes and resource use for over 80 year olds admitted to a UK critical care unit after an emergency laparotomy over a 3-year period V Banks, C Scott Northern General Hospital, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P403 (doi: 10.1186/cc11010) Eleven per cent required RRT for Table 1 (abstract P403) Patient group CC mortality, n (%) Hospital mortality, n (%) LOS CC (days) LOS hospital (days) All patients, n = 118 20 (17%) 39 (33%) 1 to 34, 4.5 mean 1 to 247, 29 mean Age 80 to 84, n = 70 15 (21%) 29 (41%) 1 to 34, 4.9 mean 1 to 247, 28 mean Age >85, n = 48 5 (10%) 10 (21%) 1 to 32, 4.1 mean 1 to 171, 31 mean ARS, n = 49 15 (30%) 22 (45%) 1 to 34, 7.5 mean 1 to 79, 20 mean No ARS, n = 69 5 (7%) 17 (25%) 1 to 24, 3.8 mean 1 to 247, 35 mean RRT, n = 13 7 (54%) 9 (69%) 1 to 34, 8 mean 1 to 247, 35 mean No RRT, n = 105 13 (12%) 30 (29%) 1 to 32, 4 mean 1 to 247, 30 mean S145 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 coeffi cient (SE) was 1.47 (0.001) for the whole cohort, 1.49 (0.001) after exclusion of cardiac surgery patients and 1.55 (0.006) after exclusion of patients with an absolute diff erence in ROD >1% between the two scores. Finally, the correlation between the APII and SAPS II scores was moderate (r2 = 0.63). The overall model was APII = 0.36×SAPS II + 4.4. The APII/SAPS II coeffi cient (SE) was 0.36 (0.0003) for the whole cohort, 0.37 (0.0004) after exclusion of cardiac surgery patients and 0.39 (0.002) after exclusion of patients with an absolute diff erence in ROD >1%. coeffi cient (SE) was 1.47 (0.001) for the whole cohort, 1.49 (0.001) after exclusion of cardiac surgery patients and 1.55 (0.006) after exclusion of patients with an absolute diff erence in ROD >1% between the two scores. Finally, the correlation between the APII and SAPS II scores was moderate (r2 = 0.63). The overall model was APII = 0.36×SAPS II + 4.4. The APII/SAPS II coeffi cient (SE) was 0.36 (0.0003) for the whole cohort, 0.37 (0.0004) after exclusion of cardiac surgery patients and 0.39 (0.002) after exclusion of patients with an absolute diff erence in ROD >1%. Conclusion Simple and robust translational formulas can be developed to allow clinicians to compare illness severity in intensive care studies of similar patients when such illness severity is expressed with diff erent scoring systems. Predicting hospital mortality: comparing accuracy of SAPS II and clinical staff prognosis Nevertheless, the main diff erence between surgical patients who died and who survived the ICU was hemodynamic instability, which was severe enough to cause hypoperfusion, metabolic acidosis, early acute kidney injury and early multiple organ dysfunction. As a result, the length of stay and the ventilation days were higher in group B patients, assuming that early and eff ective surgical management is important in order to avoid early multiple organ dysfunction on ICU admission. g p p p Results Over the study period 72 patients were included, with a mean age of 56.5 ± 16.8 years; 55% were male. The mean SAPS II was 47.3 ± 15.4. Eighteen patients died in hospital (25%). Discriminations analysis showed the following areas under ROC: SAPS II 0.84 (95% CI: 0.741 to 0.945); consultants 0.77 (95% CI: 0.632 to 0.908); residents 0.67 (95% CI: 0.513 to 0.828); nurses 0.62 (95% CI: 0.453 to 0.777). See Figure 1. Conclusion In our study, contrary to previous descriptions of similar studies, SAPS II was more accurate in predicting hospital mortality than clinical staff opinion. Diff erences were also found between diff erent groups of clinical staff , partially related to previous ICU clinical experience. Outcomes and resource use for over 80 year olds admitted to a UK critical care unit after an emergency laparotomy over a 3-year period V Banks, C Scott Northern General Hospital, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P403 (doi: 10.1186/cc11010) coeffi cient (SE) was 1.47 (0.001) for the whole cohort, 1.49 (0.001) after exclusion of cardiac surgery patients and 1.55 (0.006) after exclusion of patients with an absolute diff erence in ROD >1% between the two scores. Finally, the correlation between the APII and SAPS II scores was moderate (r2 = 0.63). The overall model was APII = 0.36×SAPS II + 4.4. The APII/SAPS II coeffi cient (SE) was 0.36 (0.0003) for the whole cohort, 0.37 (0.0004) after exclusion of cardiac surgery patients and 0.39 (0.002) after exclusion of patients with an absolute diff erence in ROD >1%. Parameters that aff ect outcome in surgical ICU patients A Vakalos, M Petkopoulou, D Jannussis Xanthi General Hospital, Xanthi, Greece Critical Care 2012, 16(Suppl 1):P405 (doi: 10.1186/cc11012) Introduction Surgical ICU patients have a lower severity illness score on ICU admission day. The aim of our study was to compare the length of stay (LOS), ventilation days (VD) and parameters that aff ect the APACHE II–III scoring system between surgical patients who died in the ICU and surgical patients who survived and discharged from the ICU. pf Conclusion Simple and robust translational formulas can be developed to allow clinicians to compare illness severity in intensive care studies of similar patients when such illness severity is expressed with diff erent scoring systems. g p g Methods During November 2005 and May 2011, 310 patients were admitted to our medical and surgical ICU. From these, 122 were surgical patients (39.35%) and were included retrospectively in our study. Mean age was 64 years, mean APACHE II score 14.5, actual mortality rate 12.29%. The patients were separated into two groups. Group A involved 107 surgical patients who survived the ICU and group B 15 surgical patients who died in the ICU. We looked for statistical signifi cant diff erence (two-tailed P value) between the mean APACHE values at admission of group A and group B, using the unpaired Mann– Whitney test (nonparametric) or the unpaired t test Welch corrected (parametric), according to the normality test. p References S146 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P408 Predictors of mortality in patients from a hematological ICU in Brazil OB Silva, L Correa, P Loureiro, E Araujo, D Teles, LA Vasconcelos, T Salvattori, P Schwambach, GT Henriques-Filho HEMOPE, Recife, Brazil Critical Care 2012, 16(Suppl 1):P408 (doi: 10.1186/cc11015) Results The last 50 patients were admitted between January 2004 and August 2011. Overall the number of admissions increased throughout this period, with only one admission in 2004, peaking at 10 in 2009. In 2011, patients with a hematological malignancy represented 0.5% of all the ICU admissions. The commonest malignancies were acute myeloid leukemia (43%) and lymphoma (31%). The primary reason for admission was sepsis (61%), with pneumonia the commonest source (27%) and 42% admitted with neutropenic sepsis. Compared to the 2010/11 cohort the patients admitted with a hematological malignancy had signifi cantly higher mean APACHE II scores (24 (SD 8) vs. 15 (SD 6) P <0.0001), a longer mean ICU stay (10 days (SD 17) vs. 6 days (SD 10) P <0.0001) and greater ICU (50% vs. 27% P <0.0001) and hospital mortality (61% vs. 29% P <0.0001). However, the overall trend was a considerable fall in mortality from 91% (2004 to 2007) to 36% (2008 to 2011). The mean SOFA score on admission for the hematological patients was 9 (SD 3). Twenty patients required two levels of organ support with only three patients receiving renal replacement therapy. No independent risk factors for outcome were identifi ed. Introduction The study was designed to analyze the factors responsible for increased mortality in an ICU specialized in hematological patients. There are few ICUs specialized in hematological diseases, with reports of high mortality rates (45 to 85%) [1], mostly related to severity of patients with blood cancer [2], mechanical ventilation (MV) and multiple organ failure [2-4]. The most prevalent disease diff ers among studies [1-4] and acute leukemia seems to have the worst prognosis [2]. Methods A retrospective cohort was conducted at HEMOPE’s ICU. Data were collected from the medical records of patients admitted from January 2006 to December 2009. y Results Of the 576 admissions, 396 (68.75%) could be analyzed. The average age was 48.3 ± 19.4 years (11 to 88 years), 54% were female and there was no association between mortality and age or gender. p References 1. Scholz N, et al.: Eur J Anaesthesiol 2004, 21:606-611. 2. Sinuff T, et al.: Crit Care Med 2006, 34:878-885. 1. Scholz N, et al.: Eur J Anaesthesiol 2004, 21:606-611.f 1. Scholz N, et al.: Eur J Anaesthesiol 2004, 21:606-611. 2. Sinuff T, et al.: Crit Care Med 2006, 34:878-885. 2. Sinuff T, et al.: Crit Care Med 2006, 34:878-885. Figure 1 (abstract P407). ROC curve for SAPS II, consultants, nurses and residents, for hospital mortality. Introduction Many diff erent illness severity scores are used to report the estimated risk of death (ROD) of patients in clinical research. Such variability makes mortality comparison between studies diffi cult. Accordingly, it would be desirable to establish a methodology to translate the value obtained from one scoring system into an estimated equivalent value for another scoring system. Methods We used the adult patient database of the Australian and New Zealand (ANZ) Intensive Care Society to obtain simultaneous APACHE II (APII), APACHE III (APIII) and SAPS II scores. We used linear regression analyses to create models enabling translation of one score into another. These analyses were performed for the whole cohort, after exclusion of cardiac surgery patients and after matching for similar risk of death. Results We obtained complete data for three illness severity scores (SAPS II, APII, and APIII) in 636,431 admissions. There was a good correlation between the APIII and APII scores (r2 = 0.76). The overall model was APIII = 3.09×APII + 5.8. The APIII/APII coeffi cient (SE) was 3.09 (0.002) for the whole cohort, 3.1 (0.002) after exclusion of cardiac surgery patients and 2.98 (0.01) after exclusion of patients with an absolute diff erence in ROD >1% between the two scores. There was a similar correlation between the APIII and the SAPS II scores (r2 = 0.76). The overall model was APIII = 1.47×SAPS II + 8.6. The APIII/SAPS II Figure 1 (abstract P407). ROC curve for SAPS II, consultants, nurses and residents, for hospital mortality. p References Acute leukemia occurred in 43% (65.3% acute myeloid leukemia). Sepsis was the major cause of admission (55.3%). The overall mortality rate was 57.5% and the specifi c one was 42.7%. The mean APACHE II score for this population was 13.4 ± 1.0 (7 to 43) and was statistically higher in the group that died (14.6 ± 0.7 vs. 11.8 ± 0.8; P = 0.013). Mean SOFA at day 1 (D1) and day 3 (D3) was 2.8 ± 0.2 and 2.1 ± 0.2 respectively, also signifi cantly higher in those that died (D1 3.9 ± 0.3 and D3 2.9 ± 0.3; P <0.0001). Almost 60% used vasoactive drugs (VAD) on admission and had a higher mortality rate (P <0.0001). MV was used in 86% and 69% died (P <0.001). Of those with renal substitutive therapy (RST), 81.9% died (OR = 3.12; 99% CI = 1.5 to 6.91). Mortality was also associated with the completion of chemotherapy before ICU admission (P = 0.003) and severe neutropenia (P <0.0001). In multivariate analysis, MV (RR = 13.1; 99% CI = 5.14 to 33.45) and a one-unit increase in SOFA D1 (RR = 1.26; 99% CI = 1.15 to 1.37) were associated with an increase in mortality. i Conclusion The outcomes of patients with hematological malignancies admitted to the ICU are improving with rates approaching that of our general ICU population. Patients with hematological malignancy requiring ICU admission continue to increase and admission should be based on their physiological derangement and overall prognosis. Further prospective studies are required to investigate potential predictors of outcomes in these patients. R f 1. Hampshire P, et al.: Admission factors associated with hospital mortality in patients with haematological malignancy admitted to UK adult, general critical care units: a secondary analysis of the ICNARC Case Mix Programme Database. Crit Care 2009, 13:R137. References References 1. Shelton BK: Crit Care Clin 2010, 26:1-20. 2. Kress JP, et al.: Am J Respir Crit Care Med 1999, 160:1957-1961. 3. Taccone FS, et al.: Crit Care 2009, 13:R15. 4. Thiéry G, et al.: J Clin Oncol 2005, 23:4406-4413. Methods All patients with nonresectable lung cancer admitted to our ICU between 1 January 2008 and 31 December 2010 were included in a retrospective study. Postoperative patients were not included. 4. Thiéry G, et al.: J Clin Oncol 2005, 23:4406-4413. Results Twenty-two patients were included. Seventeen had nonsmall- cell lung cancer (NSCLC). One had small cell lung cancer. Fifteen patients (65%) had metastatic disease. Twelve patients were in palliative therapy. The reason for ICU admission was acute respiratory failure in 12 patients (55%), hemorrhage in fi ve patients (23%). Nine patients (41%) had an infection. Fourteen patients (64%) needed invasive mechanical ventilation. One-month survival was 45% (10/22). Six-month survival was 13% (3/22). One-year survival was 0%. One- month survivors showed a nonsignifi cant trend to lower performance status and severity of disease. All 6-month survivors had metastatic disease. Six-month survivors had nonsignifi cantly lower performance status (1.7 ± 0.6 vs. 2.7 ± 1.2; P = NS). IGS II, SOFA score and duration of mechanical ventilation were signifi cantly shorter in survivors (see Table 1). Six-month survival of patients with lung cancer admitted to a medical ICU: a retrospective study O Keller, GL Laplatte, H Lessire y Conclusion For this population, in univariate analysis mortality was related to SOFA, RST, MV, use of VAD on admission, chemotherapy before ICU admission, and severe neutropenia. Although there was a relation between APACHE II score and mortality, this score underestimates it. In multivariate analysis, needing MV and a high SOFA D1 were independent predictors of death. CH Pasteur, Colmar, France Critical Care 2012, 16(Suppl 1):P410 (doi: 10.1186/cc11017) Introduction ICU admission of patients with lung cancer remains debated because of the poor short-term prognosis. We evaluated the duration of survival of patients admitted to our ICU and looked for factors associated with better survival. P409 Retrospective study of the outcomes of patients admitted to the ICU with a hematological malignancy Retrospective study of the outcomes of patients admitted to the ICU with a hematological malignancy H Lewis, J Patel, N Lonsdale Birmingham Heartlands Hospital, Birmingham, UK Critical Care 2012, 16(Suppl 1):P409 (doi: 10.1186/cc11016) Birmingham Heartlands Hospital, Birmingham, UK g g Critical Care 2012, 16(Suppl 1):P409 (doi: 10.1186/cc11016) Introduction The UK prevalence of haematological malignancy is increasing. Seven percent of these patients become critically ill, necessi- tating ITU care [1]. The past decade has seen signifi cant advances in the treatment and outcomes of patients with hemato logical malignancies. This has challenged the preconception that these patients are poor candidates for ICU admission. This study evaluated the trends in admission and outcomes of patients admitted to a general ICU with a diagnosis of hematological malignancy. g [ ] p gi treatment and outcomes of patients with hemato logical malignancies. This has challenged the preconception that these patients are poor candidates for ICU admission. This study evaluated the trends in admission and outcomes of patients admitted to a general ICU with a diagnosis of hematological malignancy. Conclusion Prognosis of patients with nonresectable lung cancer admitted to the ICU was poor. Metastatic disease did not infl uence Table 1 (abstract P410) Number of IGS SOFA ventilation days Nonsurvivors 53.2 ± 6.5 5.6 ± 3.8 6.1 ± 6.4 6-month survivors 36.3 ± 9.8 1.3 ± 2.3 0.7 ± 1.2 P <0.05 P <0.05 P <0.01 Table 1 (abstract P410) Methods A retrospective study of the last 50 consecutive admissions of patients with a hematological malignancy admitted to the ICU. Patients were identifi ed from the ICNARC database. Demographic data, APACHE II, SOFA scores on admission, baseline neutrophil count and organ support data were collected. The primary outcome was ICU and hospital mortality. Data were compared against the cohort of patients admitted between April 2010 and April 2011. S147 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 total admissions). Sixty-eight percent were male, with a mean age of 60.21 ± 14.31 years and with an APACHE score of 22.21 ± 9.13. Solid cancer was more frequent, 76.6% (urogenital 20%, lung 15.4% and low intestinal 15.4% were the most common). In the hematologic cancers (23.5%), the most frequent were non-Hodgkin lymphoma and acute leukemia (both 7%). Active cancer (new diagnosis, recurrence or progression) was presented in 75.3%. References 1. Toff art AC, et al.: Use of intensive care in patients with nonresectable lung cancer. Chest 2011, 139:101-108. 2. Roques S, et al.: Six month prognosis of patients with lung cancer admitted to the intensive care unit. Intensive Care Med 2009, 35:2044-2050. 2. Roques S, et al.: Six month prognosis of patients with lung cancer admitted to the intensive care unit. Intensive Care Med 2009, 35:2044-2050. Health-related quality of life and survival of cancer patients admitted to ICUs: results of the QALY study AB Cavalcanti1, UV Silva2, KN Normílio-Silva1, AN Silva1, R Zancani1, MJ Giorgi1, AD Dias1, AT Simone1, PL Safra1, AC Figueiredo1, G Tunes-da-Silva3, AC Lima3, LA Hajjar1, JO Auler1, J Eluf-Neto4, FR Galas1 1São Paulo State Cancer Institute, São Paulo, Brazil; 2Barretos Cancer Hospital, Barretos, Brazil; 3Instituto de Matemática e Estatística – Universidade de São Paulo, Brazil; 4Faculdade de Medicina da Universidade de São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P411 (doi: 10.1186/cc11018) Introduction Very limited data are available regarding postdischarge health-related quality of life (HRQL) of cancer patients needing intensive care. Our objective is to describe HRQL and survival in an unselected population of cancer patients who were admitted to ICUs. y Conclusion The mortality was associated with organ failure and greater need for resources. Hematologic cancer develops more organ failure without aff ecting resource consumption or their outcome in our series. Septic patients have higher ICU and hospital mortality, and neurological patients lower. Methods In this prospective cohort study conducted at two cancer hospitals in Brazil, we enrolled a random sample of adult patients with cancer admitted to the ICUs. We collected data at ICU admission, including HRQL before the acute process that led to ICU admission, and followed patients up on 15, 90 and 180 days after ICU admission to assess HRQL and vital status. We determined HRQL with the EQ-5D questionnaire, and the results were presented as summary measures with values between –1 and 1, with 0 meaning HRQL similar to death and 1 perfect HRQL. Summary measures were calculated using time- trade-off value sets obtained from the UK population. Survival was calculated with the Kaplan–Meier estimator. P409 Retrospective study of the outcomes of patients admitted to the ICU with a hematological malignancy The main reason for admission was respiratory failure (52.9%), shock (18.8%) or neurological impair- ment (16.5%). The most common diagnoses were pulmonary sepsis (23.5%), other sepsis (21.2%) and heart failure (8.2%). The ICU stay was 7.20 ± 12.32 days; with a mortality of 41.2% (hospital mortality 50.6%). The mortality was higher in the active disease (91% vs. 64%), P <0.01. Patients who died developed more respiratory (88.6% vs. 48%), hemodynamic (91.4% vs. 44%), renal (68.6% vs. 16%) or hematologic failure (45.7% vs. 16%), P <0.03. Septic patients were those with higher ICU mortality (55.3% vs. 29.8%) and hospital mortality (63.2% vs. 40.4%), P <0.05. By contrast, the patients with the longest survival were the neurological (90% vs. 54.7%) and cardiology patients (88.9% vs. 55.3%), P <0.05. Patients who died needed more MV (88.6% vs. 52%), vasopressors (91.4% vs. 46%) or dialysis (34.3% vs. 4%), P <0.01. The hematologic cancer had more cardiovascular (85% vs. 56.9%) or hematologic failure (65% vs. 16.9%) and neutropenia (45% vs. 9.2%) with P <0.03, but this is not refl ected in more consumption of resources or mortality. survival in our survey. Patients admitted for a critical illness requiring more than a few days of mechanical ventilation were very unlikely to survive over 6 months. Managing critically ill oncological patients in hospital: a survey across all ICUs in the UK p Results We enrolled 805 patients. Mean age was 61.4 ± 14.3 and 42.5% were female. Elective surgeries represented 52.2% of admissions, urgent surgeries represented 5.0% and 42.8% were admitted due to clinical reasons. Survival at 180 days was 51.2% (95% CI 47.4 to 54.9). The HRQL summary measure (median (interquartile range)) before ICU admission was 0.64 (0.12 to 0.81), on the 15-day follow-up 0.73 (0.19 to 0.92), on the 90-day follow-up 0.73 (0.20 to 0.85) and on the 180-day follow-up 0.70 (0.35 to 0.89). Introduction The survival rates for oncology patients admitted to the ICU have improved signifi cantly. The prognostic infl uence of the pre- admission oncological and treatment history is being questioned, the most signifi cant impact being related to acute physiological status. In Figure 1 (abstract P413). (a) Hematological cancer. (b) Solid tumor. Checked bars, not proven in the literature. p Conclusion HRQL is, on average, moderately impaired before ICU admission and through the 180-day follow-up in cancer patients needing intensive care. Only about one-half of the patients were alive after 180 days. However, there is large variability on both HRQL and length of survival; thus, methods to estimate quality-adjusted life-years on an individual basis are necessary. Characteristics, resource consumption and outcome of cancer patients admitted to ICUs New severity score of acute respiratory failure S Allal, A Khedher, I Ben Saida, A Azouzi, A Farjallah, I Chouchen, S Bouchoucha, M Boussarsar CHU Farhat Hached Hospital, Sousse, Tunisia Critical Care 2012, 16(Suppl 1):P415 (doi: 10.1186/cc11022) Methods We surveyed intensive care lead clinicians in December 2011 in order to establish: a profi le of the hospital and ICU they work in; their estimate of overall ICU mortality for critically ill cancer patients; the value of six outcome indicators in predicting mortality in two subgroups of oncological candidates for ICU admission; and the local management of acutely deteriorating cancer patients potentially requiring ICU care. Introduction Acute respiratory failure (ARF), a common syndrome, is still poorly clinically appreciated. Literature review reports only a few attempts in neonatology (Silverman score) and in adults (Patrick score [1]) constructed by authors in 1996 for scientifi c research purposes. Both scores have never been validated. Instead, clinicians developed specifi c scores. We constructed a new respiratory failure score, organized in a trimodal manner (Table 1). Items were selected on the basis of pathophysiological and clinical expertise. Particular attention was paid to formulation and scaling to make the score both simple, noninvasive, inexpensive, didactic, and with interesting clinimetric properties. The objective of this study is to validate this score already in use for several years in our ICU. q g Results The ICU mortality rates estimated by survey respondents diff ered from those reported in the literature: for solid tumor 21% (SEM 3) versus 10 to 23%, for metastatic solid tumor 38% (SEM 4) versus 23%, hematological malignancy 45% (SEM 3) versus 33 to 43% with allograft transplant 54.8% (SEM 5) versus 39 to 50% and autograft transplant 56% (SEM 5) versus 44%. Regarding the management of cancer patients, there were confl icts reported between teams (rarely 44%, occasionally 56%, commonly 0.2%). Few units had established triage policies for the acutely ill cancer patient (5%) and it was also not common that plans were made prior to the patient’s deterioration (never 11%, rarely 38%, occasionally 41%, commonly 9%). Figure 1 shows those outcome indicators thought to be important by responders in forecasting ICU prognosis. Table 1 (abstract P415). Characteristics, resource consumption and outcome of cancer patients admitted to ICUs Characteristics, resource consumption and outcome of cancer patients admitted to ICUs R Garcia, L Terceros, I Saez, J Flordelis, L Colino, C Mudarra, S Temprano, J Montejo 12 de Octubre Hospital, Madrid, Spain Critical Care 2012, 16(Suppl 1):P412 (doi: 10.1186/cc11019) p R Garcia, L Terceros, I Saez, J Flordelis, L Colino, C Mudarra, S Temprano, J Montejo 12 de Octubre Hospital, Madrid, Spain Critical Care 2012, 16(Suppl 1):P412 (doi: 10.1186/cc11019) Introduction The development of cancer treatment has improved the prognosis for cancer patients and they need more support measures in the ICU. Our objective is to evaluate the characteristics and evolution of cancer patients admitted to a general ICU of a university hospital. Introduction The development of cancer treatment has improved the prognosis for cancer patients and they need more support measures in the ICU. Our objective is to evaluate the characteristics and evolution of cancer patients admitted to a general ICU of a university hospital. Methods A retrospective study of cancer patients admitted to an ICU from January 2008 to December 2010. We collected demographic and cancer characteristics, reason for admission, complications, resource consumption and mortality. We compared quantitative variables with the Student t test and the qualitative variables with the chi-square test, statistical signifi cance accepted as P <0.05. Methods A retrospective study of cancer patients admitted to an ICU from January 2008 to December 2010. We collected demographic and cancer characteristics, reason for admission, complications, resource consumption and mortality. We compared quantitative variables with the Student t test and the qualitative variables with the chi-square test, statistical signifi cance accepted as P <0.05. Figure 1 (abstract P413). (a) Hematological cancer. (b) Solid tumor. Checked bars, not proven in the literature. Figure 1 (abstract P413). (a) Hematological cancer. (b) Solid tumor. Checked bars, not proven in the literature. i Results A total of 108 patients were admitted with cancer, 23 with cured cancer were excluded, so we selected 85 patients (4.38% of S148 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 this survey, we sought to evaluate the awareness of overall mortality rates in critically ill cancer patients among intensivists in the UK. this survey, we sought to evaluate the awareness of overall mortality rates in critically ill cancer patients among intensivists in the UK. References Results A total of 200 patients (age 70 ± 17 years, 65% males) were admitted to the CCU during the study period; diagnoses included acute coronary syndrome (65%), pulmonary edema (11.5%), congestive heart failure (5.5%) and other (18%). The median length of CCU stay was 5 ± 3 days and the median length of hospital stay 9 ± 7 days. The CCU mortality rate was 20% and in-hospital mortality 24.2%. Both APACHE II and SOFA scores were independently associated with mortality (OR = 1.30; CI: 1.21 to 1.40, P <0.001 and OR = 1.82; CI: 1.53 to 2.16, P <0.001 respectively). The receiver operating characteristic curves confi rmed both scores as equally eff ective predictors of clinical outcome with areas under the curve of 0.92, P <0.001 and 0.91, P <0.001 for APACHE II and SOFA score respectively. 1. Patrick W, Webster K, Ludwig L, Roberts D, Wiebe P, Younes M: Non-invasive positive pressure ventilation in acute respiratory distress without prior chronic respiratory failure. Am J Respir Crit Care Med 1996, 153:1005-1011. p y p 2. Landis JR, Koch GG: The measurement of observer agreement for categorical data. Biometrics 1977, 33:159-174. 2. Minerva Anestesiol 2011, 77:305. P414 Methods A prospective study among 70 patients with ARF on previously healthy lungs. ARF was rated in a randomized blinded manner respect- ively by residents and seniors. An inter-rater reliability analysis using the kappa statistic was performed to determine consistency among raters. P414 Role of illness severity scores in predicting mortality in the coronary care unit G Argyriou, J Terrovitis, G Sainis, V Papas, C Marvaki, S Nanas, C Routsi Medical School, University of Athens, Greece Critical Care 2012, 16(Suppl 1):P414 (doi: 10.1186/cc11021) Introduction Several illness severity scores have been developed in order to predict outcome in multidisciplinary ICUs. However, the role of these scores has not been thoroughly investigated in coronary care units (CCUs) and the results are confl icting [1,2]. The aim of this study was to evaluate the utility of two of the most widely used scores – that is, APACHE II and Sequential Organ Failure Assessment (SOFA) – for the prediction of mortality in patients admitted to CCUs. 2 y Results The inter-rater reliability for the raters was found to be κ = 0.82 (P <0.001), indicating an almost perfect agreement [2]. The area under the ROC curve was revealed very interesting (AUC = 0.88) indicating an excellent prognostic predictive power. Conclusion This new and validated score could drive some advantages in daily practice, allowing accurate assessment of ARF severity, more objective monitoring of patients and easier communication between care providers. It may accurately guide oxygen supplementation and ventilatory support and aff ord accurate monitoring of patho- physiological and etiological treatment of ARF. It could be a valuable tool in randomized clinical trials or physiological studies evaluating treatments in ARF. Finally it could be used as an educational tool. References p y p Methods All patients consecutively admitted to an eight-bed CCU from April 2010 to May 2011 were prospectively studied. Demographic, clinical and laboratory data were recorded. Illness severity on admission was measured using the APACHE II and SOFA scores. For the calculation of the scores, the worst values for each variable on admission day were used. 1. Clin Cardiol 1999, 22:366. Validity of six prognostic scoring systems for septic shock patients admitted to a medical ICU B Khwannimit, R Bhurayanontachai B Khwannimit, R Bhurayanontachai y Prince of Songkla University, Hat Yai, Thailand Critical Care 2012, 16(Suppl 1):P416 (doi: 10.1186/cc11023) Conclusion The APACHE II and SOFA scores on admission are independent predictors of mortality in patients hospitalized in a CCU. Both scores demonstrate excellent performance in discriminating high-risk patients and thus are useful tools to predict clinical outcome in CCUs. Introduction This study aimed to assess the validity of the APACHE II, SAPS II, and SAPS 3, along with each of their customized scores, in predicting hospital mortality in patients with septic shock admitted to our ICU. 1. Azoulay E, et al.: Ann Intensive Care 2011, 1:5. Characteristics, resource consumption and outcome of cancer patients admitted to ICUs Score of respiratory failure Grade Respiratory rate Accessory muscle use Hypoxemia I <30 Intercostal Normal II 30 to 40 Supraclavicular and/or Cyanosis suprasternal III >40 Thoraco-abdominal Circulatory and/or swing/nasal fl aring consciousness disorders IV Gasp Exhaustion/ventilatory Cardio-circulatory arrest arrest Table 1 (abstract P415). Score of respiratory failure Table 1 (abstract P415). Score of respiratory failure G d R i A l H i Table 1 (abstract P415). Score of respiratory failure Grade Respiratory rate Accessory muscle use Conclusion The awareness of improved outcome in critically ill cancer patients diff ers among physicians, and in general estimated mortalities were far higher than those reported in the literature. Conclusion The awareness of improved outcome in critically ill cancer patients diff ers among physicians, and in general estimated mortalities were far higher than those reported in the literature. Reference 1. Azoulay E, et al.: Ann Intensive Care 2011, 1:5. References References Methods A prospective cohort study was conducted over a 6-year period in a medical ICU of a tertiary referral university teaching hospital in Thailand. S149 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Figure 1 (abstract P416). Calibration curves for customized APACHE II, SAPS II and SAPS 3. Figure 1 (abstract P416). Calibration curves for customized APACHE II, SAPS II and SAPS 3. Results A total of 880 patients were enrolled and a hospital mortality rate of 57.4% was found. Community-acquired infections accounted for 57.2% and 32.8% of patients had positive blood culture. The respiratory tract was the most common site of infection (48.7%). The predicted mortality of all the scores was close to the observed mortality, with a standardized mortality ratio (95% confi dence interval) of 0.94 (0.86 to 1.02) for APACHE II, 1.01 (0.92 to 1.1) for customized APACHE II, 0.93 (0.85 to 1.01) for SAPS II, 1.07 (0.98 to 1.17) for customized SAPS II, 0.97 (0.89 to 1.06) for SAPS 3 and 1.02 (0.93 to 1.11) for customized SAPS 3. All six scores were well discriminated, with areas under the receiver operating characteristic curves of 0.82, 0.813, 0.819, 0.815, 0.817 and 0.813, respectively. The Hosmer–Lemeshow goodness-of-fi t showed good calibration in only the customized APACHE II (H-statistic 12.4, P = 0.26). See Figure 1. patients underwent study of endothelial vasodilating function using the method proposed by Celermajer and colleagues [1]. Results A total of 880 patients were enrolled and a hospital mortality rate of 57.4% was found. Community-acquired infections accounted for 57.2% and 32.8% of patients had positive blood culture. The respiratory tract was the most common site of infection (48.7%). The predicted mortality of all the scores was close to the observed mortality, with a standardized mortality ratio (95% confi dence interval) of 0.94 (0.86 to 1.02) for APACHE II, 1.01 (0.92 to 1.1) for customized APACHE II, 0.93 (0.85 to 1.01) for SAPS II, 1.07 (0.98 to 1.17) for customized SAPS II, 0.97 (0.89 to 1.06) for SAPS 3 and 1.02 (0.93 to 1.11) for customized SAPS 3. All six scores were well discriminated, with areas under the receiver operating characteristic curves of 0.82, 0.813, 0.819, 0.815, 0.817 and 0.813, respectively. The Hosmer–Lemeshow goodness-of-fi t showed good calibration in only the customized APACHE II (H-statistic 12.4, P = 0.26). See Figure 1. Reference Scientifi c Clinical Center of Miner’s Health Protection, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P417 (doi: 10.1186/cc11024) Scientifi c Clinical Center of Miner’s Health Protection, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P417 (doi: 10.1186/cc11024) 1. Celermajer DS, et al.: Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992, 340:1111-1115. Introduction The high risk of thromboembolic complications after knee joint prosthetics is conditioned by the series of surgical intervention particularities. The infl uence of intraoperative tourniquet usage on the leg deep venous thrombosis frequency was studied. References Results On 4 to 5 days after surgery, leg deep venous thrombosis was found in 11 patients (8.8% of all patients after prosthetics). For decrease of intraoperative blood loss the tourniquet was applied onto the middle third of the leg in 77 patients (60.6%). In this group DVT was found in 10.4% of cases. In the nontourniquet group (48 patients) DVT was found in 6.25%. The diff erences in the complication frequency were not statistically valid. The data from duplex scanning showed that 43 patients (34.4%) before surgery had changes in the lower leg veins in view of varicose subcutaneous veins and post-thrombophlebitic syndrome combined with disorders of endothelial vasodilating func tion and low venous tone. Tourniquet use in patients with venous pathology resulted in DVT in 30% (fi ve of 15 patients). When a tourniquet was not used in patients with venous disease, DVT was found only in one of 28 patients (3.5%). The test showed a signifi cant diff erence in the frequency of thromboembolic complications in these groups (P <0.001). Conclusion In this study, the customized APACHE II was found to be accurate in predicting hospital mortality in septic shock patients requiring ICU admission. Conclusion Therefore, using a tourniquet in patients with evident base venous pathology in terms of varicose subcutaneous veins or post- thrombophlebitic syndrome in total knee joint endoprosthetics is a risk factor for venous thrombosis development. Reference Risk factors of venous thrombosis in knee joint endoprosthesis SV Vlasov, IV Vlasovai Risk factors of venous thrombosis in knee joint endoprosthesis SV Vlasov, IV Vlasova Scientifi c Clinical Center of Miner’s Health Protection, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P417 (doi: 10.1186/cc11024) P418 P418 Proximal and distal deep venous thrombosis in critically ill patients: incidence and prevalence S Yus Teruel, J Camacho Oviedo, L Cachafeiro Fuciños, M Hernandez Bernal, A Agrifoglio Rotaeche, M Irazábal Jaimes, L Fernandez Rodriguez, B Civantos Martín, J Díez Hospital Universitario La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P418 (doi: 10.1186/cc11025) P418 Proximal and distal deep venous thrombosis in critically ill patients: incidence and prevalence S Yus Teruel, J Camacho Oviedo, L Cachafeiro Fuciños, M Hernandez Bernal, A Agrifoglio Rotaeche, M Irazábal Jaimes, L Fernandez Rodriguez, B Civantos Martín, J Díez Hospital Universitario La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P418 (doi: 10.1186/cc11025) Effi cacy and safety of enoxaparin as deep vein thrombosis prophylaxis in critically ill patients R Al-Hubail, N Hassan King Fahad Specialist Hospital, Dammam, Saudi Arabia Critical Care 2012, 16(Suppl 1):P420 (doi: 10.1186/cc11027) Effi cacy and safety of enoxaparin as deep vein thrombosis prophylaxis in critically ill patients R Al-Hubail, N Hassan King Fahad Specialist Hospital, Dammam, Saudi Arabia Critical Care 2012, 16(Suppl 1):P420 (doi: 10.1186/cc11027) Methods This was a prospective observational study conducted in our medical–surgical and trauma ICU from October 2009 to September 2010. The inclusion criterion was ≥72 hours of ICU stay. Exclusion criteria were admitting diagnosis of pulmonary embolism or DVT, readmission, and patients with support withdrawal orders. The study was approved by the Research Ethics Board of La Paz Hospital. Bilateral lower extremity compression ultrasound was performed within 48 hours of admission to evaluate the prevalence, and twice weekly until discharge to assess the incidence. We collected demographic data, body mass index (BMI), APACHE II score, SOFA score, diagnostic categories, classic risk factors for DVT, femoral catheter and the use of mechanical ventilation and muscle relaxants. For the statistical analysis chi-square and Fisher tests were used, as well as Mann–Whitney and Student tests for data comparison. For the probability of DVT and its relation with the associated factors, the odds ratio and confi dence interval were used. Statistical signifi cance was P <0.05. Introduction Critically ill patients are at high risk of developing deep vein thrombosis (DVT). DVT cannot be detected in most cases, leading to fatal embolic manifestations [1]. The goal of this study was to review the occurrence of DVT in patients receiving enoxaparin during their length of stay in the ICU (ICU LOS). In addition we review the occurrence f j bl di d th b t i d t i of major bleeding and thrombocytopenia secondary to enoxaparin. Methods This was a retrospective database analysis including medical and surgical patients admitted to a tertiary hospital (King Fahad Specialist Hospital Dammam) critical care department from 1 January to 31 December 2010, aged 17 to 70 years, excluding patients with: platelets <50,000/l; evidence of active bleeding; new ischemic or haemorrhagic stroke; spinal or epidural catheter who were already on anticoagulant when admitted to the ICU and who were previously diagnosed with DVT or with pulmonary embolism (PE); DNR (do not resuscitate). The APACHE II score, predicted mortality and ICU LOS were calculated for included patients in the study. Saddle embolism is associated with the major adverse events in patients with nonhigh-risk pulmonary embolism H Kim, W Kim Asan Medical Center, Seoul, South Korea Critical Care 2012, 16(Suppl 1):P419 (doi: 10.1186/cc11026) Saddle embolism is associated with the major adverse events in patients with nonhigh-risk pulmonary embolism H Kim, W Kim Asan Medical Center, Seoul, South Korea Critical Care 2012, 16(Suppl 1):P419 (doi: 10.1186/cc11026) Introduction In some patients with acute pulmonary embolism (PE), thrombi may lodge at the levels of bifurcation of the pulmonary trunk and extend into both main pulmonary arteries, forming so-called saddle embolism (SE). The aim of this study was to assess the incidence of SE and whether it is associated with an increased risk of complicated clinical course in patients with nonhigh-risk PE. p g Methods Between January 2006 and June 2010, 297 consecutive patients with nonhigh-risk PE that was confi rmed with contrast- enhanced spiral computed tomography (CT) in the emergency department were studied. One experienced radiologist evaluated the presence of SE. The clinical information, echocardiographic and CT parameters were reviewed. Patients were divided into SE and non- SE. Multivariate logistic regression was applied to determine factors associated with occurrence of major adverse events (MAE). 1. Greets et al.: Chest 2003, 124:357s-363s. Proximal and distal deep venous thrombosis in critically ill patients: incidence and prevalence Methods The study included 125 patients with gonarthrosis of degree III who received total knee joint endoprosthesis. There were 26 men and 99 women at the age of 36 to 77 (60.7 ± 8.03). For all patients, spinal anesthesia in combination with long-term epidural blockade for postsurgical pain relief was performed. The antithrombotic measures included Klexan 40 mg, 12 to 15 hours before surgery and 8 hours after it. Color mapping of the lower leg vessels with an Acuson 128XP/10c scanner was performed before surgery, on 4 to 5 days after prosthetics and before discharge from the in-patient department. In addition, all p S Yus Teruel, J Camacho Oviedo, L Cachafeiro Fuciños, M Hernandez Bernal, A Agrifoglio Rotaeche, M Irazábal Jaimes, L Fernandez Rodriguez, B Civantos Martín, J Díez Hospital Universitario La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P418 (doi: 10.1186/cc11025) Introduction The aim of this study was to detect deep venous thrombosis (DVT) in patients admitted to a critical care unit (ICU) by S150 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P420fi compression ultrasonography, and to determine the incidence and prevalence of proximal and distal DVT in this setting. Table 1 (abstract P420) Table 1 (abstract P420) Predicted Type of case ICU LOS (days) APACHE II mortality (%) Total cases on enoxaparine 5.29 ± 7.3 16.7 ± 10.5 28.4 ± 23.7 Low platelets cases 11.75 ± 9.7 23 ± 2.3 48.75 ± 11.18 Major bleeding cases 13.5 ± 13.1 22.4 ± 17.4 30 ± 17.5 Effi cacy and safety of enoxaparin as deep vein thrombosis prophylaxis in critically ill patients R Al-Hubail, N Hassan King Fahad Specialist Hospital, Dammam, Saudi Arabia Critical Care 2012, 16(Suppl 1):P420 (doi: 10.1186/cc11027) The hospital electronic system and critical care database were reviewed with the physician order sheet according to the ICU protocol for DVT prophylaxis (enoxaparin 40 mg subcutaneously once daily). i Results We enrolled 182 patients, with male predominance (57.3%), 135 were mechanically ventilated (74.2%) and the mean APACHE II score was 19.3 ( ± 7.8). The mortality in the ICU was 15.4% (28), and 20.9 (38) in hospital. The prevalence of proximal DVT was 29.1% (53/182), and the incidence 24.0% (31/129). Seventy-nine percent of patients received DVT prophylaxis. The localization of incidentally diagnosed DVT was proximal in 29% and distal in 35%; 19 (64%) of these were identifi ed on day 5 of admission. In four patients DVT was clinically suspected and only in one of them was DVT confi rmed. The most frequently involved were soleal veins (67%). Independent risk factors for incidental DVT were: older age (62 ± 15.4 years vs. 54.5 ± 17.1; P = 0.032); BMI (27.7 ± 5.5 kg/m2 vs. 24.9 ± 5.2 kg/m2; P = 0.014); and mechanical ventilation: (OR: 3.3, 95% CI = 1.0 to 10.26). Patients with incidental DVT had a higher hospital mortality (P = 0.03). g y y Results Five hundred and ninety-seven patients were investigated, from which 22 (3.5%) fulfi lled exclusion criteria, 220 (36%) were on a sequential decompression device (SD), and 26 (4%) were not on DVT prophylaxis (protocol violation). This gave a study population of 329 (55%) cases that were on enoxaparin thromboprophylaxis. In this population there were no recorded cases of DVT and two cases (0.6%) of PE. Major bleeding was recorded in seven cases (2.1%), platelets <50,000/l in eight cases (2.4%), and Hb level <1.5 g/dl from baseline without bleeding in 47 cases (14.2%). See Table 1. Conclusion In our study DVT was an early, asymptomatic and frequent event (46% of the ICU patients). In the presence of risk factors, a diagnostic ultrasound test might have a role. Effi cacy and safety of enoxaparin as deep vein thrombosis prophylaxis in critically ill patients R Al-Hubail, N Hassan King Fahad Specialist Hospital, Dammam, Saudi Arabia Critical Care 2012, 16(Suppl 1):P420 (doi: 10.1186/cc11027) Table 1 (abstract P420) Predicted Type of case ICU LOS (days) APACHE II mortality (%) Total cases on enoxaparine 5.29 ± 7.3 16.7 ± 10.5 28.4 ± 23.7 Low platelets cases 11.75 ± 9.7 23 ± 2.3 48.75 ± 11.18 Major bleeding cases 13.5 ± 13.1 22.4 ± 17.4 30 ± 17.5 Conclusion Using the hospital and critical care databases, we observed that the critically ill patients receiving enoxaparin as thromboprophylaxis did not experience DVT, and two (0.6%) had PE during their ICU stay. However, thrombocytopenia and major bleeding were recorded at very low frequencies (2.5%). Reference 1. Greets et al.: Chest 2003, 124:357s-363s. Table 1 (abstract P420) P423 Consequences of suspected heparin-induced thrombocytopenia in the ICU y y Results All patients included in the study before surgery had detected hypercoagulation and inhibition of fi brinolysis: increasing of MA (maximum density of the clot, fi brin-platelet constant of the blood) to 20.7% (P <0.001) and ICD (intensity of coagulation drive (the intensity of clot formation)) to 15.6%; reduction of IRCL (intensity of the retraction and clot lysis) to 13.6% (P <0.05) in both groups compared to normal rates. At the fi rst day after surgery in patients treated with bemiparin (group 1) MA and ICD decline to 12.7 (P <0.05) and 9.6% (P <0.001) respectively, and IRCL increased by 4.6% (P <0.05) compared with preoperatively. In group 2 there was a similar picture: the reduction of MA and ICD to 10.3 (P <0.001) and 6.6% (P <0.05) respectively, and IRCL increased by 4.4% (P <0.001). At the fi fth day the condition of hemostasis in both groups came almost to the same value – a moderate hypocoagulation, normal activity of fi brinolysis. At 7 days of the postoperative period, thrombotic complications developed in one patient of the fi rst group (2.70%). In the second group, complications developed in four (9.52%) patients: in three cases deep venous thrombosis, and in one case coagulopathic bleeding. Conclusion Using a combination of bemiparin and epidural anesthesia reduces the level of postoperative thrombotic complications, such as deep venous thrombosis, and massive bleedings in the patients after total hysterectomy. Using hemoviscoelastography enables quick identifi cation of disorders of hemostasis in patients after hysterectomy before, during and after the surgery. P423 Consequences of suspected heparin-induced thrombocytopenia in the ICU M Ostermann1, L McIntyre2, F Lauzier3, J Alhashemi4, I Qushmaq5, S Langevin3, P Dodek6, M Albert7, K Khwaja8, J Kutsiogiannis9, L Burry10, J Granton10, J Friedrich10, N Ferguson10, J Marshall10, S Finfer11, D Heels-Ansdell12, N Zytaruk12, D Cook12, J Sheppard12, T Warkentin12, M Crowther12 M Ostermann1, L McIntyre2, F Lauzier3, J Alhashemi4, I Qushmaq5, S Langevin3, P Dodek6, M Albert7, K Khwaja8, J Kutsiogiannis9, L Burry10, J Granton10, J Friedrich10, N Ferguson10, J Marshall10, S Finfer11, D Heels-Ansdell12, N Zytaruk12, D Cook12, J Sheppard12, T Warkentin12, M Crowther12 1King’s College London, UK; 2University of Ottawa, Canada; 3CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 4King AbdulAziz University, Jeddah, Saudi Arabia; 5King Faisal Hospital, Jeddah, Saudi Arabia; 6University of British Columbia, Vancouver, Canada; 7Université de Montréal, Canada; 8Université McGill, Montréal, Canada; 9University of Alberta, Edmonton, Canada; 10University of Toronto, Canada; 11The George Institute, Sidney, Australia; 12McMaster University, Hamilton, Canada Critical Care 2012, 16(Suppl 1):P423 (doi: 10.1186/cc11030) Introduction Clinical suspicion of heparin-induced thrombocytopenia (HIT) may prompt changes in drug management and alert clinicians to an increased risk of thrombosis. However, thrombocytopenia in the ICU occurs in about 50% of patients, is multifactorial and is due to HIT in <1%. We aimed to describe the consequences of suspected HIT among medical–surgical critically ill patients in terms of drug and device management, and thrombotic outcomes. g Methods We enrolled 3,746 patients in the PROTECT trial comparing prophylactic dalteparin to unfractionated heparin. We defi ned HIT as occurring in patients with a clinical or laboratory-driven suspicion of HIT and a positive serotonin release assay (SRA). We defi ned suspected HIT as patients whose clinicians were suffi ciently concerned about HIT to withhold heparin. We defi ned consequences of HIT as occurring from 1 day before it was suspected to 30 days thereafter. P422 Cost-eff ectiveness analysis of two thromboprophylactic strategies following major surgery C Ebm, M Cecconi, A Rhodes, M Grounds St George´s Healthcare Trust, London, UK Critical Care 2012, 16(Suppl 1):P422 (doi: 10.1186/cc11029) y p y Results One hundred and thirty patients (3.5%) had heparin held due to clinical suspicion of HIT. Of these, 10 (7.7%) had a positive SRA test. The drugs and devices used for thromboprophylaxis, as well as thrombotic events, are outlined in Table 1. At least one new thrombotic event developed in 23.8% of patients with suspected HIT and 40.0% of patients with HIT. Reference Reference instrumental method – hemoviscoelastography preoperatively, intra- operatively and every day during 10 days after surgery. Prevention of thrombotic complications in group 1 (37 patients), conducted by bemiparin 3,500 units: the fi rst injection 12 hours before surgery, then at 6 hours after the operation in the future once a day for 10 days; group 2 (42 patients) received heparin 5,000 units: the fi rst injection 6 hours before surgery, then 6 hours after the operation, then four times per day for 10 days. 1. Roderick P, Ferris G, Wilson K, Halls H: Towards evidence-based guidelines for the prevention of venous thromboembolism: systematic reviews of mechanical methods, oral anticoagulants, dextran and regional anaesthesia for thromboprophylaxis. Health Technol Assess 2005, 9(49):iii-iv, 1-78. 1. Roderick P, Ferris G, Wilson K, Halls H: Towards evidence-based guidelines for the prevention of venous thromboembolism: systematic reviews of mechanical methods, oral anticoagulants, dextran and regional anaesthesia for thromboprophylaxis. Health Technol Assess 2005, 9(49):iii-iv, 1-78. Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P423 Consequences of suspected heparin-induced thrombocytopenia in the ICU Introduction Patients recovering from major surgery are at high risk of developing life-threatening deep venous thrombosis, which is a key source of postoperative morbidity and mortality. Our objective was to assess the cost-eff ectiveness of two diff erent thromboprophylactic agents for patients admitted to the ICU after high-risk surgery: inter mittent pneumatic compression (IPC) and anti-embolism stockings (AES). P421 P421 Reducing the level of postoperative thrombotic complications by the combination of low molecular weight heparin and epidural anesthesia at the patients after total hysterectomy O Tarabrin, V Dubinina, A Turenko, S Tarasenko, S Shcherbakov, D Gavrychenko, G Mazurenko Odessa National Medical University, Odessa, Ukraine Critical Care 2012, 16(Suppl 1):P421 (doi: 10.1186/cc11028) Results Twenty-seven out of 297 patients (9.1%) were found to have a SE. Overall mortality at 1 month was 12.5% with no diff erence between the groups (11.9% vs. 18.5%, P = 0.32), although SE patients were more likely to receive thrombolytic therapy (8.1% vs. 29.6%, P <0.01). SE patients had s signifi cantly higher rate of MAE (59.3% vs. 25.6%, P <0.01). Presence of SE and the ratio of right ventricular to left ventricular diameter were associated with an odds ratio of MAE within 1 month of 2.48 (95% CI: 1.10 to 6.04, P = 0.03) and 3.34 (95% CI: 1.46 to 7.46, P <0.01). Introduction Each year in the world, cancer of the reproductive system is diagnosed in more than 600,000 women. In 8 to 35% of patients with cancer of the reproductive system, pulmonary embolism was the cause of death – and in 43% the background for other fatal complications. Methods The results of surgical treatment of 79 patients after hysterectomy under prolonged epidural anesthesia during the period from 2008 to 2010 entered the study. The condition of hemostasis was monitored by 12 standard biochemical tests, as well as the new Introduction Each year in the world, cancer of the reproductive system is diagnosed in more than 600,000 women. In 8 to 35% of patients with cancer of the reproductive system, pulmonary embolism was the cause of death – and in 43% the background for other fatal complications. Methods The results of surgical treatment of 79 patients after hysterectomy under prolonged epidural anesthesia during the period from 2008 to 2010 entered the study. The condition of hemostasis was monitored by 12 standard biochemical tests, as well as the new Conclusion SE by CT angiography was associated with PE-related shock, intubation, mortality, thrombolysis, and thrombectomy within 1 month in patients with nonhigh-risk PE and may be a useful method for simple risk stratifi cation. S151 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 R Riessen, M Behmenburg, G Blumenstock, D Guenon, S Enkel, M Haap University Hospital Tübingen, Germany l l (d ) Results Thirty patients were included, with a mean age of 59.6 ± 19.3, APACHE II score 19.1 ± 7.2, SOFA at baseline 8.5 ± 4.0, and most patients had septic shock (63.3%). Baseline iron and transferrin levels were low in 83.3% (14.0 (5.0 to 25.5)) and in 96.7% (94.1 ± 31.6) of the patients, while ferritin was high in 63.3% (954.0 (508.4 to 5,394.0)). In the 19 patients where a day 7 sample was available, variation between baseline and day 7 was statistically signifi cant for transferrin (97.9 ± 37.5 to 132.7 ± 48.3, P = 0.013) and ferritin (478.0 (224.5 to 1,741.0) to 376.0 (187.0 to 886.7), P = 0.018), while iron levels showed a trend towards increasing levels at day 7 (17.0 (6.5 to 44.3) to 29.0 (21.0 to 54.0), P = 0.061). Baseline SOFA score trends to be lower in hypoferrinemic patients (7.7 ± 3.8 vs. 12.4 ± 1.9, P = 0.098). The Spearman test showed a weak correlation only between SOFA and iron levels (P = 0.008; r2 = 0.48). Introduction By introducing a blood-saving-bundle (BSB) consisting of a closed-loop arterial blood sampling system, smaller tubes and an attempt to reduce the number of blood samples, we aimed to reduce blood loss caused by diagnostic blood sampling and to minimize the development of anemia in a high-risk group of mechanically ventilated intensive care patients. p Methods Included were all patients from our medical ICU who were ventilated for more than 72 hours. Exclusion criteria were acute or chronic anemia on admission, a bleeding episode during the ICU stay or end-of-life therapy. The BSB was introduced in 2009 with training and educational support. Patients treated in the year 2008 before the introduction of the BSB served as a control group and were compared to patients treated in 2010 after introduction of the BSB (BSB group). Daily blood loss was calculated on the basis of the documentation of blood samples and laboratory values in the patient data management system and by using data from two representative study periods in which the sample volumes of all diagnostic blood tests were measured. Results The control group comprised of 41 patients (614 observation days), the BSB group of 50 patients (559 observation days). Evaluation of iron, transferrin and ferritin serum levels in patients with severe sepsis and septic shock Evaluation of iron, transferrin and ferritin serum levels in patients with severe sepsis and septic shock M Missano Florido, M Assunção, B Mazza, M Jackiu, F Freitas, A Bafi , F Machado UNIFESP, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P424 (doi: 10.1186/cc11031) M Missano Florido, M Assunção, B Mazza, M Jackiu, F Freitas, A Bafi , F Machado UNIFESP, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P424 (doi: 10.1186/cc11031) Introduction Iron metabolism is altered in critically ill patients leading to hypoferremia. Several studies related it to infl ammatory response [1,2]. The present study aims to evaluate iron, transferrin and ferritin serum levels in patients with severe sepsis and septic shock and its association with severity of organ dysfunction. y g y Methods A prospective observational cohort study, unicentric, in a tertiary teaching hospital. From November 2010 to October 2011 patients over 18 years old with severe sepsis or septic shock with up to 72 hours of organ dysfunction were included. Exclusion criteria were blood transfusion or iron supplementation in the last 90 days, previous inclusion and pregnancy. After obtaining informed consent, blood samples were taken at baseline and on day 7. Demographic and APACHE II and SOFA data were also collected. Patients who were transfused with red blood cells between the two periods were excluded from the day 7 sample. Patients were followed until hospital discharge or death. Conclusion Angiogenic factors and their soluble receptors, particularly sVEGFR1, play pivotal roles in the development of organ dysfunction in DIC associated with severe trauma. The DIC-induced tissue hypoxia and platelet consumption plays crucial roles in inducing sVEGFR1 and Ang2, and in determining the prognosis of the severity of organ dysfunction. P426 P424 g y Results DIC patients showed higher Sequential Organ Failure Assess- ment (SOFA) scores, soluble fi brin and lactate levels. The serum levels of VEGF, Ang1, and the sTie2 levels were lower in the DIC patients than the non-DIC patients. The serum levels of sVEGFR1, Ang2 and the Ang2/Ang1 ratio in the DIC patients were higher than in those without DIC. The sVEGFR2 levels showed no statistically signifi cant diff erence between the patients with and without DIC. The levels of sVEGFR1, Ang2 and the Ang2/Ang1 ratio correlated with the SOFA score. In particular, sVEGFR1 and Ang2 were independent predictors of an increase in the SOFA score. The lactate levels independently predicted increases in the levels of sVEGFR1 and Ang2 and platelet consumption also independently predicted the increase in Ang2 levels in severe trauma patients with DIC. R Riessen, M Behmenburg, G Blumenstock, D Guenon, S Enkel, M Haap University Hospital Tübingen, Germany l l (d ) Mean blood loss per ICU day decreased from 43.3 ml (95% CI 41.2 to 45.3 ml) in the controls to 15.0 ml (14.3 to 15.7 ml) in the BSB group (P <0.001). The introduction of a closed-loop arterial blood sampling system contributed most to this eff ect. Mean hemoglobin values showed a similar decrease in both groups during the ICU stay. However, hemoglobin values <9 g/dl were measured in 21.2% of observation days in the controls versus 15.4% in the BSB group (P = 0.01). In the control group 31.7% (18.1 to 48.1%) of the patients received red blood cell transfusions in contrast to only 8.0% (2.2 to 19.2%) in the BSB group (P = 0.006), while the hemoglobin concentration triggering transfusion was not signifi cantly diff erent (8.2 vs. 7.8 g/dl). The mean number of intubation days was 7.1 days (6.1 to 8.3 days) in the controls and 7.5 days (6.6 to 8.5) in the BSB group (P = NS). However, patients in the BSB group stayed with a mean of 9.8 days (8.6 to 11.3 days) signifi cantly shorter in the ICU than controls with 13.2 days (10.9 to 15.4 days) (P = 0.014). Conclusion Septic patients have low iron and transferrin levels, associated with high ferritin levels, and those levels improved during the course of disease. Low iron levels might be associated with low SOFA scores. References 1 Lagan AL 1. Lagan AL, et al.: Am J Physiol Lung Cell Mol Physiol 2008, 294:L161-L174. 2. Quinlan GJ, et al.: Am J Respir Crit Care Med 1997, 155:479-484. Table 1 (abstract P423) Table 1 (abstract P423) 1 day before to 30 days after heparin held for suspect HIT Intervention Danaparoid 34 (26.2) Lepirudin 8 (6.2) Fondaparinux 11 (8.5) Argatroban 19 (14.6) Any of the above drugs 67 (51.5) Anti-embolic stockings 25 (19.2) Pneumatic compression device 37 (28.5) Anti-embolic stockings or pneumatic 49 (37.7) compression device Any of the above interventions 96 (73.8) Incident thromboses Venous thrombosis (including PE) 30 (23.1) Arterial thrombosis 1 (0.8) Progression of a previous thrombus 2 (1.5) Any of the above 31 (23.8) g Methods A decision model (TreeAge Software 2010) was constructed simulating the impact of AES and IPS on patient outcomes and costs following high-risk surgery in the UK. Probabilities were assessed from published data [1]. ICU and item costs were derived from NHS reference costs tablets. Assessed outcomes were cost per deep vein thrombosis (DVT) and pulmonary embolism (PE) prevented, net monetary benefi t and y yi incremental costs per quality-adjusted life expectancy (QALY) gained. Results Total costs for in-patients receiving AES were £923 and £1,010 for patients treated with IPC. Equipment costs and cost of initial care were higher in patients who received IPC, but this was partly off set by a reduction in costs related to treatment of early (DVT and PE) and late complications (post-thrombotic syndrome and pulmonary hypertension). IPC treatment increased QALY by approximately 0.01 years. The incremental cost-eff ectiveness of the IPC device was £12,650 per QALY gained. One-way sensitivity analysis revealed that the most sensitive variables were probability of developing a DVT resulting from the insignifi cant diff erence in treatment effi cacy.f iffi Conclusion Based on UK cost-eff ectiveness guidelines, our results indicate that IPC stockings should be used for patients at high risk of developing DVT. IPCs decrease the incidence of developing DVT and therefore result in cost savings related to preventive and therapeutic actions. For patients at low risk of developing DVT, AES are favoured due to higher utility and lower maintenance costs associated with AES. Due to the lack of reliable data on the incidence of PE as well as the absence of reliable head-to-head studies between IPC and AES, no generalisable conclusion to favour either strategy can be made. Conclusion Over 90% of patients with suspected HIT did not have HIT. A simple blood-saving bundle reduces diagnostic blood loss in mechanically ventilated patients R Riessen, M Behmenburg, G Blumenstock, D Guenon, S Enkel, M Haap University Hospital Tübingen, Germany Table 1 (abstract P423) One-half of patients with suspected HIT were prescribed another S152 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods Fifty-seven patients with severe trauma were divided into two subgroups: 30 DIC patients and 27 non-DIC patients. The serum levels of angiogenic factors were measured on admission (day 1), day 3, and day 5. We compared serum levels of these angiogenic factors between with and without DIC groups and evaluated their predictive value for organ dysfunction and outcome. anticoagulant and one-third received mechanical prophylaxis. Thrombotic rates are higher in patients with HIT and suspected HIT than other patients. The frequent suspicion of HIT in critically ill patients and initiation of other interventions may create a greater clinical and economic burden than HIT itself. Templating eff ect of clot structure can predict clot development and outcome in diluted blood: a comparison with thromboelastography g p y M Lawrence1, J Kaczynski2, S Stanford1, R Morris3, P Evans2 1Swansea University, Swansea, UK; 2ABMU LHB, Swansea, UK; 3UWIC, Cardiff , UK Critical Care 2012, 16(Suppl 1):P429 (doi: 10.1186/cc11036) g p y M Lawrence1, J Kaczynski2, S Stanford1, R Morris3, P Evans2 1Swansea University, Swansea, UK; 2ABMU LHB, Swansea, UK; 3UWIC, Cardiff , UK Critical Care 2012, 16(Suppl 1):P429 (doi: 10.1186/cc11036) Introduction Safety of patients is possible to increase applying early detection of intraoperative and postoperative hemorrhage using the widening array of monitoring opportunities; not only the hemodynamic parameters, but the detection of total hemoglobin. Continuous noninvasive monitoring of total hemoglobin content is possible due to the Masimo Rainbow SET technology, using multiwave spectrophotometry. Introduction Treatment of major hemorrhage with colloids is known to have an eff ect on clot outcome. However, determining both the rate and extent of these changes is diffi cult. Development of a new biomarker has shown that it can detect structural development earlier and quantifi es these changes to clot outcome accurately when compared to other methods. This study compares the fractal dimension, Df [1], found when the clot fi rst forms to measures of mature clot fi rmness obtained from thromboelastography. p p y Methods Seventy-eight patients aged 15 to 59 (35.9 ± 1.62) with laparoscopic gynecological operations were included in the research after permission of the ethics committee and signing the informing agreement. Total hemoglobin was detected by laboratory method invasively, discretely and delayed. Total hemoglobin was detected by another method oximetrically (SpHb) during the monitoring process on the platform Rainbow SET technology noninvasive, continuous, and promptly. SpHb was compared with total hemoglobin on the following stages of the research: before the operation, during the operation and in the early postoperative period. Statistical analysis was fulfi lled by comparing real and tabular (critical) criteria of reliability – Student test. Results During the detection of total hemoglobin by the laboratory method, the mean value was 121.5 ± 17.28 g/l, while oximetrically it occurred 118.6 ± 17.41 g/l. The real criterion of reliability (tr) was 0.85, the critical criterion of reliability (tcr) was 2.63.if y Methods Forty healthy blood samples were obtained; each sample was allocated a random dilution ratio (10%, 20%, 40%, 60%) and diluted with gelofusine. These were matched with 40 healthy samples that were undiluted. Reference 1. Evans P, et al.: Blood 2010, 116:3341-3346. 8 Use of coagulation screening in the critical care unit A Rice, R Paterson, C Cairns Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P428 (doi: 10.1186/cc11035) Introduction The aim of this audit is to compare the eff ectiveness of indiscriminate coagulation testing versus selective testing based on clinical indications within the HDU setting. Coagulation tests (PT and APTT) are often taken as a matter of routine alongside patient’s daily blood tests in the critical care setting. Abnormal coagulation results rarely alter patient management while repeated testing has signifi cant detrimental fi nancial implications. Reference Templating eff ect of clot structure can predict clot development and outcome in diluted blood: a comparison with thromboelastography An oscillatory shear technique was applied to the blood using an AR-G2 measuring Df (clot structure). Additionally the clot development in terms of fi rmness was measured using a ROTEM analyser measuring at 5, 10, 15 minutes and its maximum (A5, A10, A15, MCF).i Results Df signifi cantly decreases with increasing dilution. The decrease in structural complexity indicates that gelofusine even at 40% dilution is producing poor quality clots. See Table 1. Table 1 (abstract P429). Change in Df with dilution Dilution % Df 0 1.74 (0.05) 10 1.72 (0.04) 20 1.70 (0.06) 40 1.63 (0.05) 60* 1.59 (0.06) *Signifi cant decrease from 0%. Table 1 (abstract P429). Change in Df with dilution Conclusion We did not discover statistically signifi cant diff erences of total hemoglobin determined by two diff erent methods. Thereby, noninvasive monitoring of total hemoglobin contention using multiwave spectrophotometry by Masimo Rainbow SET technology can serve as an appropriate replacement for the laboratory screening of hemoglobin. Conclusion Df that is measured at the incipient clot is found much sooner than the mature clot parameters, between 5 and 30 minutes earlier. Df is signifi cantly correlated (P <0.05) with the mature clot parameters of clot fi rmness (A5, A10, A15 and MCF) and elasticity (G’max). This suggests that in dilution Df can determine the eventual clot outcome very early. Measurement of Df could guide fl uid replacement and component therapy more accurately and earlier than conventional markers. P428 P428 Use of coagulation screening in the critical care unit A Rice, R Paterson, C Cairns Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P428 (doi: 10.1186/cc11035) Using angiogenic factors and their soluble receptors to predict organ dysfunction in patients with disseminated intravascular coagulation associated with severe trauma T Wada1, S Jesmin2, S Gando3, S Zaedi2, H Yokota1 , , , , 1Nippon Medical School, Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan; 3Hokkaido University Graduate School of Medicine, Sapporo, Japan Critical Care 2012, 16(Suppl 1):P425 (doi: 10.1186/cc11032) , , , , 1Nippon Medical School, Tokyo, Japan; 2National Center for Global Health and Medicine, Tokyo, Japan; 3Hokkaido University Graduate School of Medicine, Sapporo, Japan Critical Care 2012, 16(Suppl 1):P425 (doi: 10.1186/cc11032) Introduction Disseminated intravascular coagulation (DIC) is observed after not only sepsis but also trauma. DIC is associated with concomitant activation of coagulofi brinolytic disorder and systemic infl ammation with endothelial dysfunction and microvascular permeability. The angiogenic factors, including vascular endothelial growth factor (VEGF), angiopoietin (Ang), and their receptors, play crucial roles in angiogenesis and microvascular permeability. The aim of the study was to assess: the relationship between angiogenic factors, their soluble receptors and organ dysfunction associated with DIC precipitated by severe trauma; and the eff ects of DIC-induced platelet consumption, thrombin generation and tissue hypoxia on the expression of these factors and receptors. Conclusion Our BSB could easily be implemented and was able to reduce diagnostic blood loss by 65%. After introduction of the BSB we observed less transfusions and a shorter ICU stay in mechanically ventilated patients; this, however, has to be interpreted with caution due to the longitudinal study design. S153 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P427 Comparative assessment of invasive and noninvasive methods for detection of total hemoglobin in gynecological patients’ blood AV Pyregov, SV Petrov Research Center for Obstetrics, Gynecology and Perinatology, Moscow, Russia Critical Care 2012, 16(Suppl 1):P427 (doi: 10.1186/cc11034) P427 P429 Templating eff ect of clot structure can predict clot development and outcome in diluted blood: a comparison with thromboelastography Templating eff ect of clot structure can predict clot development and outcome in diluted blood: a comparison with thromboelastography M Lawrence1, J Kaczynski2, S Stanford1, R Morris3, P Evans2 1Swansea University, Swansea, UK; 2ABMU LHB, Swansea, UK; 3UWIC, Cardiff , UK Critical Care 2012, 16(Suppl 1):P429 (doi: 10.1186/cc11036) P432 3 Thromboelastography (platelet contribution to clot strength) for the assessment of platelet residual function D Haxhiademi1, S Parri1, A Cerillo1, P Del Sarto1, R Paniccia2, D Prisco2 1Fondazione Toscana G. Monasterio, Massa, Italy; 2Thrombosis Centre, University of Florence, Italy Critical Care 2012, 16(Suppl 1):P432 (doi: 10.1186/cc11039) Thromboelastography (platelet contribution to clot strength) for the assessment of platelet residual function D Haxhiademi1, S Parri1, A Cerillo1, P Del Sarto1, R Paniccia2, D Prisco2 1Fondazione Toscana G. Monasterio, Massa, Italy; 2Thrombosis Centre, University of Florence, Italy Critical Care 2012, 16(Suppl 1):P432 (doi: 10.1186/cc11039) P430 Methods Rheometry and confocal laser scanning microscopy (CLSM) were used to monitor and image the formation of fi brin clots. Clotting was initiated by the addition of diff erent levels of thrombin to solutions of a fi xed concentration of fi brinogen. Each sample was divided into two aliquots; one added to the measuring geometry of an AR-G2 rheometer and one to the microscope slide for the spinning disk CLSM (Olympus IX71). Results The micrographs of formed clots (Figure 1) show marked qualitative diff erences in clot architecture. Upon increasing the available thrombin, the clot network (visually) appears more dense. Table 1 shows the value of the structural biomarker, the fractal dimen- sion, that corresponds to the clots formed in Figure 1. Conclusion We demonstrate, for the fi rst time, that the fractal dimen- sion obtained by rheometry is a sensitive measure of visually observed structural diff erences within the fi brin network. Rheometrical detection of incipient clots formed in whole blood provides the clinician with a powerful tool for the diagnosis of thromboembolic disease. Reference 1. Scott et al.: Arterioscler Thromb Vasc Biol 2004, 2:1558-1566. P432 Thromboelastography (platelet contribution to clot strength) for the assessment of platelet residual function D Haxhiademi1, S Parri1, A Cerillo1, P Del Sarto1, R Paniccia2, D Prisco2 1Fondazione Toscana G. Monasterio, Massa, Italy; 2Thrombosis Centre, University of Florence, Italy Critical Care 2012, 16(Suppl 1):P432 (doi: 10.1186/cc11039) Introduction In the early postoperative period after cardiac surgery, platelet dysfunction is one of the main causes of excessive bleeding; there is still controversy regarding the timing of antiplatelet therapy Table 1 (abstract P431). Results of the fractal dimension obtained by rheometry of fi brin clots Thrombin (NIH) Fractal dimension 0.02 1.85 0.1 1.95 0.3 2.13 Table 1 (abstract P430) Mean Df Mean MCF (mm) Pre enoxaparin 1.79 ± 0.08 68.0 ± 8.0 Post enoxaparin 1.64 ± 0.10 64.3 ± 4.2 Figure 1 (abstract P430). Table 1 (abstract P430) Mean Df Mean MCF (mm) Pre enoxaparin 1.79 ± 0.08 68.0 ± 8.0 Post enoxaparin 1.64 ± 0.10 64.3 ± 4.2 Figure 1 (abstract P430). Table 1 (abstract P431). Results of the fractal dimension obtained by rheometry of fi brin clots Thrombin (NIH) Fractal dimension 0.02 1.85 0.1 1.95 0.3 2.13 Table 1 (abstract P431). Results of the fractal dimension obtained by rheometry of fi brin clots Figure 1 (abstract P430). y References 1. Evans PA, et al.: Blood 2010, 116:3341-3346. 2. Levi M, Opal SM: Crit Care 2006, 10:222. P430 architecture with several diseases including sepsis, bleeding or acute thromboembolic disease [1]. We investigate our biomarker by examining the relationship between thrombin generation and clot architecture in an in vitro model. Methods Rheometry and confocal laser scanning microscopy (CLSM) were used to monitor and image the formation of fi brin clots. Clotting was initiated by the addition of diff erent levels of thrombin to solutions of a fi xed concentration of fi brinogen. Each sample was divided into two aliquots; one added to the measuring geometry of an AR-G2 rheometer and one to the microscope slide for the spinning disk CLSM (Olympus IX71). y p Results The micrographs of formed clots (Figure 1) show marked qualitative diff erences in clot architecture. Upon increasing the available thrombin, the clot network (visually) appears more dense. Table 1 shows the value of the structural biomarker, the fractal dimen- sion, that corresponds to the clots formed in Figure 1.i Figure 1 (abstract P430). admission, at 6 hours and 24 hours to assess pathophysiological state and progression. Twelve patients were recruited: nine severe sepsis and three severe DKA with metabolic disorder. Twelve healthy volunteers were recruited as a matched control. Conclusion We demonstrate, for the fi rst time, that the fractal dimen- sion obtained by rheometry is a sensitive measure of visually observed structural diff erences within the fi brin network. Rheometrical detection of incipient clots formed in whole blood provides the clinician with a powerful tool for the diagnosis of thromboembolic disease. Reference Results Mean Df in the control group was 1.73 ± 0.03 whereas mean Df in DKA and sepsis was found to be 1.77 ± 0.07 and 1.65 ± 0.05 respectively. Marked diff erences were observed in Df and maximum clot fi rmness (MCF) in response to treatment intervention (Figure 1). Furthermore, patients saw a dramatic decrease in Df post enoxaparin, but no signifi cant change in MCF was observed (Table 1). 1. Scott et al.: Arterioscler Thromb Vasc Biol 2004, 2:1558-1566. gi g Conclusion Df shows specifi city between severe DKA and sepsis. Df shows sensitivity to treatment intervention and illness progression in the critically ill. P432 P430 i p Methods Over a 14-day period, the blood results of HDU patients were prospectively analysed in order to assess whether or not a coagulation screen was conducted and whether or not this was appropriate based on clinical indications. Following targeted education towards medical and nursing staff , including publicising a list of clinical indications within the unit, the audit cycle was repeated. y p Results Prior to education, only 37% of coagulation screens were clinically indicated. Following implementation of the indications this rose to 50%. Using the guidelines in the second round there was 100% identifi cation of abnormal results compared to only 81% prior to education. On review of all these data we were able to extrapolate that prior to targeted education there was a 2:1 ratio of appropriate to inappropriate coagulation testing, post intervention this rose to 5:1. Conclusion With local targeted education of staff we signifi cantly reduced the number of inappropriate coagulation tests undertaken within our unit from 65% to 27%. Along with this we had a 100% detection rate for abnormal results using our list of clinical indications for testing. In our high turnover critical care unit this would indicate potential savings of around £10,000 per annum; a signifi cant amount in an organisation with longstanding fi nancial constraints. Introduction Recent research [1] has highlighted a novel new biomarker of haemostasis: the fractal dimension (Df). This new biomarker relates the kinetics of clot formation to clot outcome in whole blood and allows us to quantify the complexity of the fi brin network microstructure which is believed to be the template for development of the mature clot. It is well established that abnormalities in haemostasis contribute to the pathogenicity of critical illness [2]. This prospective study aims to assess the eff ect of critical illness on clot structure and monitor the sensitivity of Df to therapeutic intervention. Methods Patients with critical illness inducing SIRS were recruited on admission to the intensive therapy unit in a large teaching hospital in Wales. Blood was taken for routine coagulation testing, ROTEM thromboelastometry and rheological analysis (Df and Tgel) on S154 architecture with several diseases including sepsis, bleeding or acute thromboembolic disease [1]. We investigate our biomarker by examining the relationship between thrombin generation and clot architecture in an in vitro model. References 1. Kauvar DS: Impact of haemorrhage on trauma outcome: an overview of epidemiology clinical presentation and therapeutic considerations. J Trauma 2006, 60:S3-S11. 2. WHO: Injuries and Violence: Facts. Geneva, Switzerland: WHO; 2010. f f 3. Dawn M, Mark D, Pasquale M, Thomas W: Impact of pre-injury warfarin use in elderly trauma patients. J Trauma 2000, 48:3. 3. Dawn M, Mark D, Pasquale M, Thomas W: Impact of pre-injury warfarin use in elderly trauma patients. J Trauma 2000, 48:3. 3. Dawn M, Mark D, Pasquale M, Thomas W: Impact of pre-injury warfarin use in elderly trauma patients. J Trauma 2000, 48:3. P434 Retrospective comparison study of warfarinised trauma patients and an age-matched control group of nonwarfarinised patients M Omar, P Stevens, T Jenkins, K Morris, H Hussain, A Evans Morriston Hospital, Swansea, UK Critical Care 2012, 16(Suppl 1):P434 (doi: 10.1186/cc11041) Introduction There are several studies stating the association of mortality between trauma and anticoagulation; however, it is diffi cult to ascertain a credible conclusion due to the small number of data and inconclusive results. Some studies have showed a signifi cant increased risk of morbidity and mortality. We analysed retrospective data of 45,798 trauma patients, out of which 254 were on warfarin. The incidence of death continues to rise and there are no specifi c strategies to reduce haemodilution and coagulopathy which maybe the underlying cause of mortality. Results There was no signifi cant association between bleeding at 4, 6 and 12 hours and any of the preoperative tests. There was no signifi cant association between bleeding at 4, 6 and 12 hours and any of the standard laboratory tests. Platelet contribution to clot strength (%pltMA) detected by TEG showed a signifi cant association with postoperative blood loss (at 4, 6 and 12 hours, respectively P = 0.02, P = 0.02, P = 0.01). Methods A retrospective analysis of a national database collected in 2009 and 2010, from the Trauma Audit and Research Network (TARN) UK. The data also contain vital information including age, Glasgow Coma Scale (GSC), Injury Severity Score (ISS), INR, blood products given, number of days in hospital and clinical outcome. We evaluated trauma patients who were on warfarin therapy and compared their clinical outcome and mortality to age-matched patients with similar injuries not on warfarin. Conclusion Our data confi rm the utility of perioperative evaluation of platelet contribution to clot strength evaluated by TEG. It helps to understand the mechanism behind the surgical bleeding and reduce empirical transfusions. References 1 Hartmann References 1. Hartmann M, et al.: Transfus Med Rev 2006, 20:230-241. 2. Ferraris V, et al.: Ann Thorac Surg 2011, 91:944-982. 3. Mousa SA, et al.: Thromb Res 2001, 104:49-56. 2. Ferraris V, et al.: Ann Thorac Surg 2011, 91:944-982. Results A total of 45,780 adult patients were analysed. These were subdivided into 32,225 young patients under 65 years with median age 60.5, of which 59 were on warfarin; and 13,555 older patients aged over 65 with median age 80.4, of which 195 were on warfarin. The mortality rate in warfarinised patients was signifi cantly higher than in the nonwarfarinised age-matched group aged <65 (5/59, 8.5% vs. 1,223/32,163, 3.8%; P <0.001; 95% CI). The group age >65 included 13,555, of which 195 were warfarinised (4.7/195, 24.1% vs. 1,501/13,360, 11.3%; P <0.001; 95% CI). 3. Mousa SA, et al.: Thromb Res 2001, 104:49-56. Fractal dimension: a biomarker for detecting acute thromboembolic disease Introduction In the early postoperative period after cardiac surgery, platelet dysfunction is one of the main causes of excessive bleeding; there is still controversy regarding the timing of antiplatelet therapy discontinuation [1]. The Clinical Practice Guidelines of the Society of Thoracic Surgeons recommend that point-of-care (POC) testing may help identify patients who can safely undergo urgent operations [2]. This study was designed to test the relationship between platelet function as revealed by POC tests and postoperative bleeding in patients that undergo cardiac surgery without suspending thienopyridines at least 5 days prior to surgery. K Hawkins1, N Badiei1, J Weisel2, I Chernysh2, PR Williams1, MJ Lawrence1, PA Evans1 1Swansea University, Swansea, UK; 2University of Pennsylvania, Philadelphia, PA, USA Critical Care 2012, 16(Suppl 1):P431 (doi: 10.1186/cc11038) Introduction This study investigates the potential use of rheometry to provide a structural biomarker for acute critical illness. Previous studies have reported an association of altered fi brin clot network Figure 1 (abstract P431). CLSM micrographs of formed fi brin clots at thrombin levels of 0.02, 0.1 and 0.3 NIH. Figure 1 (abstract P431). CLSM micrographs of formed fi brin clots at thrombin levels of 0.02, 0.1 and 0.3 NIH. S155 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods Adult patients scheduled for cardiac operations in which thienopyridines were not discontinued at least 5 days before surgery were included. From November 2010 to February 2011, 20 patients were enrolled in this pilot study. Samples were taken before induction of anesthesia (T0) and 2 hours after arrival in the ICU (T1). Standard laboratory tests and the following POCs were performed: multiple electrode aggregometry (MEA), PFA 100 and thromboelastography (TEG). Functional fi brinogen level (FFL) is a recent modifi cation of TEG used to investigate the function of fi brinogen [3]. We used the combination of TEG and FFL to detect platelet contribution to the clot strength.i Measurement of hemoglobin in the operating room: what methods can we trust? B Giraud, D Frasca, O Mimoz CHU La Milétrie Poitiers, France Critical Care 2012, 16(Suppl 1):P433 (doi: 10.1186/cc11040) Conclusion This data analysis proves that mortality is signifi cantly higher in warfarinised patients compared to the nonwarfarinised age- matched group. Future research needs to focus on both developing a practical procedure reducing risks of morbidity and mortality by exploring coagulopathy and early correction of coagulations. Anticoagulated patients are more likely to receive aggressive i.v. fl uid resuscitations as the result of haemorrhage which leads to haemodilution and further exacerbates coagulopathy. This cascade of events is the underlying mechanism causative to mortality. References Introduction A noninvasive and continuous monitoring of total hemoglobin (Hb) by spectrophotometry was recently marketed (SpHb; Masimo, Irvine, CA, USA). The main objective of this study was to determine the absolute and trend accuracy of SpHb compared to Hb assessment at the laboratory (HbLab) used as the reference method. Methods After obtaining ethics committee approval and informed consent, 51 adult patients (29 men, 22 women, age 18 to 90 years) undergoing major surgery with expected large blood loss were enrolled in the study. Patients wore Rainbow adult resposable sensors (R2–25, Revision E) connected to a Radical-7 Pulse CO-Oximeter, software version 7.6.0.1. HbLab values were obtained by analyzing arterial blood samples at the laboratory using a Sysmex XT-2100i automated hematology analyzer (Roche Diagnostics, Paris, France). The same samples were also analyzed with a satellite laboratory CO- Oximeter (Siemens RapidPoint 405 CO-Oximeter; Siemens, Munich, Germany), HbSat, and a point-of-care hemoglobinometer (HemoCue, Hb201; Ångelholm, Sweden), HcueArt. At the same time, a fourth drop of blood after skin puncture on the ear or fi nger was taken for capillary blood sampling tested also with the HemoCue: HcueCap. Invasive Hb values were compared to Sphb obtained at the time of the blood draw. An initial set was collected before surgery. Then blood samples were taken on approximately an hourly basis or more often if clinically indicated. Bland–Altman method plots were used to compare absolute accuracy of test devices to laboratory values. The ability of the test devices to follow the trend of the changes in Hb values reported by the reference device was assessed by plotting the diff erence between subsequent measurements reported by each device to the diff erence in subsequent measurements reported by the reference device, and a coeffi cient of determination (R2) was calculated. P436 Reducing ICU blood draws with artifi cial intelligence FC Cismondi1, AS Fialho1, SM Vieira2, LA Celi1, SR Reti3, JM Sousa2, SN Finkelstein1 1Massachusetts Institute of Technology, Cambridge, MA, USA; 2IST, Lisbon, Portugal; 3BIDMC, Brookline, MA, USA Critical Care 2012, 16(Suppl 1):P436 (doi: 10.1186/cc11043) Results The analysis of fi ndings showed both sepsis and pneumonia development in an acute period of burn disease to be accompanied by disorders of anticoagulant, fi brinolytic and procoagulant parts of the hemostasis system typical for DIC syndrome. The changes of hemostasis system indices were not only the characteristic of infection in burned patients but they preceded the diagnosis of sepsis and pneumonia in the clinic on average by 2 to 4 days. In patients with pneumonia, relevant and statistically signifi cant were the activity changes of XIIa- dependent fi brinolysis, from the second to sixth days. And on the third to seventh days there was reliable pneumonia development with decreased activity of antithrombin III. In patients with sepsis were revealed changes of XIIa-dependent fi brinolysis activity – from the third to seventh days – and antithrombin III activity – from the third to the sixth days. Introduction Recent studies have demonstrated that frequent laboratory testing does not necessarily relate to better outcomes. Our aim is to reduce unnecessary blood draws for ICU laboratory tests by predicting which tests are likely to return as normal or abnormal and therefore infl uence clinical management around gastrointestinal (GI) bleeding. Methods An artifi cial intelligence tool, namely fuzzy systems, was applied to 1,092 GI bleed patients extracted from a large ICU database with over 32,000 patients. A classifi cation approach for laboratory test outcome was utilized for a total of seven outcome variables shown in Table 1. The outcome for each test was binarized as normal or abnormal. Input variables included 10 physiological variables such as heart rate, temperature and urine output, as well as further data on transfusions for platelets, red blood cells and plasma.i y Conclusion The development of both local and generalized infection in an acute period of burn disease occurs against the background of DIC syndrome induced by a serious heat injury. The indices of hemostasis system can be included into a complex of clinic and laboratory studies aimed at detecting infection and early intensive etiopathological therapy. In medical–surgical ICU patients, major bleeding is common but independent of heparin prophylaxis If one could predict in advance whether a laboratory test would be normal or abnormal then that particular laboratory test may not be ordered, and thereby reducing potential complications and costs. In this work we present an artifi cial intelligence method for the classifying the likelihood of a blood test being normal or abnormal. Our results show acceptable classifi cation accuracy both in terms of sensitivity and specifi city. Conclusion Reducing frequent laboratory testing, and potential phlebotomy complications, is a major concern in critical care medicine. If one could predict in advance whether a laboratory test would be normal or abnormal then that particular laboratory test may not be ordered, and thereby reducing potential complications and costs. In this work we present an artifi cial intelligence method for the classifying the likelihood of a blood test being normal or abnormal. Our results show acceptable classifi cation accuracy both in terms of sensitivity and specifi city. site and severity of each bleeding event, which was reevaluated by two independent blinded adjudicators. Patients with trauma, orthopedic surgery or neurosurgery were excluded. Major bleeding was defi ned as life threatening, occurring in critical sites, requiring ≥2 units of red blood cells or an invasive intervention, or associated with an unexplained decrease in systolic blood pressure (≥20 mmHg) or increase in heart rate (≥20 beats/minute). We used Cox proportional hazard models adjusting for age, APACHE II, reason for ICU admission, end-stage renal disease, drugs aff ecting coagulation, coagulation parameters and life- support interventions to identify predictors of bleeding. P437 y Results Among 3,746 patients, 208 had major bleeding (5.6%, 95% CI 4.9 to 6.3%). The commonest bleeding sites were: gastrointestinal tract (51.9%), surgical site (30.3%), respiratory tract (15.9%), retroperitoneal (8.2%) and intracranial (3.4%). Independent predictors of major bleeding (expressed as hazard ratio with 95% CI) were: prolonged activated partial thromboplastin time (aPTT) (1.10, 1.05 to 1.14 per 10-second increase), thrombocytopenia (1.16, 1.09 to 1.24 per 50×109/l decrease in platelet count), therapeutic heparin (3.26, 1.72 to 6.17), anti- platelet agents (that is, acetylsalicylic acid and/or clopidogrel) (1.38, 1.02 to 1.88), renal replacement therapy (1.75, 1.20 to 2.56) and surgery in the preceding 3 days (1.64, 1.01 to 2.65). Prophylactic dalteparin in the preceding 3 days was not associated with bleeding. In medical–surgical ICU patients, major bleeding is common but independent of heparin prophylaxis F Lauzier1, D Arnold2, C Rabbat2, D Heels-Ansdell2, P Dodek3, B Ashley3, M Albert4, K Khwaja5, M Ostermann6, Y Skrobik4, R Fowler7, L McIntyre8, J Nates9, T Karachi2, R Lopes10, N Zytaruk2, M Crowther2, D Cook2 1CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 2McMaster University, Hamilton, Canada; 3University of British Columbia, Vancouver, Canada; 4Université de Montréal, Canada; 5Université McGill, Montréal, Canada; 6King’s College London, UK; 7University of Toronto, Canada; 8Université d’Ottawa, Canada; 9MD Anderson Hospital, Houston, TX, USA; 10Duke Clinical Research Institute, Durham, NC, USA fi ( ) Results The study included 210 measurements. HbLab ranged between 6.8 and 16.3 g/dl. Compared to the reference method, the average bias was 0.96 ± 2.78 g/dl for SpHb, 0.16 ± 0.45 g/dl for HcueArt, 0.5 ± 1.71 g/dl for HcueCap and 0.81 ± 1.04 g/dl for HbSat. R2 values were 0.39 for SpHb, 0.93 for HcueArt, 0.53 for HcueCap and 0.47 for HbSat. Conclusion This study shows that HcueArt seem the most reliable method of Hb assessment. The SpHb has a lower accuracy, but its ability to monitor Hb continuously and noninvasively remains attractive and development of this method should be encouraged. Critical Care 2012, 16(Suppl 1):P435 (doi: 10.1186/cc11042) Results The study included 210 measurements. HbLab ranged between 6.8 and 16.3 g/dl. Compared to the reference method, the average bias was 0.96 ± 2.78 g/dl for SpHb, 0.16 ± 0.45 g/dl for HcueArt, 0.5 ± 1.71 g/dl for HcueCap and 0.81 ± 1.04 g/dl for HbSat. R2 values were 0.39 for SpHb, 0.93 for HcueArt, 0.53 for HcueCap and 0.47 for HbSat. Introduction Bleeding frequently complicates critical illness. Our objec tives were to describe the incidence, locations and predictors of major bleeding in patients with low risk of bleeding receiving thromboprophylaxis. Conclusion This study shows that HcueArt seem the most reliable method of Hb assessment. The SpHb has a lower accuracy, but its ability to monitor Hb continuously and noninvasively remains attractive and development of this method should be encouraged. p p y Methods In the PROTECT trial comparing dalteparin to unfractionated heparin for thromboprophylaxis in medical–surgical ICU patients, research coordinators used a validated ICU-specifi c tool to describe the S156 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Reducing frequent laboratory testing, and potential phlebotomy complications, is a major concern in critical care medicine. Hemostasis system condition in infectious complication development in severe burned patients Infectious septic complications were diagnosed in the clinic on the basis of clinical and laboratory fi ndings, as well as confi rmed by morphological studies in casualties (44 from 102 patients). Diagnosis of disseminated intravascular coagulation (DIC) syndrome was made based on standard criteria. P436 Results Classifi cation accuracy of greater than 80% was achieved for all of the seven outcome variables (Table 1). Sensitivity and specifi city were satisfactory for all the outcomes. Input variables frequently selected as most predictive of normal or abnormal results include urine output and red blood cell transfusion. Table 1 (abstract P436). Classifi cation results Outcome ACC (%) Sensitivity Specifi city Calcium 85 ± 2.3 0.88 ± 0.3 0.81 ± 0.1 PTT 86 ± 1.2 0.89 ± 0.1 0.82 ± 0.2 Hematocrit 82 ± 1.6 0.84 ± 0.2 0.78 ± 0.1 Fibrinogen 84 ± 2.8 0.87 ± 0.3 0.80 ± 0.4 Lactate 80 ± 2.2 0.82 ± 0.2 0.77 ± 0.4 Platelets 88 ± 1.3 0.90 ± 0.1 0.85 ± 0.2 Hemoglobin 84 ± 3.1 0.85 ± 0.3 0.81 ± 0.2 ACC, accuracy of classifi cation. Table 1 (abstract P436). Classifi cation results Table 1 (abstract P436). Classifi cation results Outcome ACC (%) Sensitivity Specifi city Calcium 85 ± 2.3 0.88 ± 0.3 0.81 ± 0.1 PTT 86 ± 1.2 0.89 ± 0.1 0.82 ± 0.2 Hematocrit 82 ± 1.6 0.84 ± 0.2 0.78 ± 0.1 Fibrinogen 84 ± 2.8 0.87 ± 0.3 0.80 ± 0.4 Lactate 80 ± 2.2 0.82 ± 0.2 0.77 ± 0.4 Platelets 88 ± 1.3 0.90 ± 0.1 0.85 ± 0.2 Hemoglobin 84 ± 3.1 0.85 ± 0.3 0.81 ± 0.2 ACC, accuracy of classifi cation. P438 Randomized comparison of fi brinogen concentrate versus cryoprecipitate for bleeding control in pediatric cardiac surgery (FICCS study) F Galas1, L Hajjar1, B Sorensen2, J Almeida1, M Sundin1, V Guimaraes1, S Zeff erino1, L Camara1, F Maua1, M Moreira1, C Puttini1, M Carmona1, J Auler Jr1, R Nakamura1 1Heart Institute, São Paulo, Brazil; 2Guy’s and St Thomas’ NHS Foundation Trust & King’s College London School of Medicine, London, UK Critical Care 2012, 16(Suppl 1):P438 (doi: 10.1186/cc11045) Introduction We compared hemostatic outcomes after treatment with fi brinogen concentrate or cryoprecipitate in pediatric cardiac surgery patients with intraoperative bleeding. Hemostasis system condition in infectious complication development in severe burned patients Introduction Over the period of the history of combustiology one of the main problems for treatment of patients with burns is infection, both local – bacterial pneumonia – and generalized – sepsis – characterized by extremely severe course, complex diagnostics and high lethality rate. However, the role of hemostasis disorders in infectious complication development in severe burned patients is taken into consideration insuffi ciently. The aim of the study is to reveal the most relevant hemostasis system changes in sepsis and pneumonia in patients with serious heat injury in an acute period of burn disease. Methods Hemostasis and biochemical blood parameters were studied in 169 patients with over 20% of the body burned, from the fi rst to 12th days after burn. Sepsis developed in 33 patients, 69 patients had pneumonia, and in 67 patients there were no complications of sepsis and pneumonia. Infectious septic complications were diagnosed in the clinic on the basis of clinical and laboratory fi ndings, as well as confi rmed by morphological studies in casualties (44 from 102 patients). Diagnosis of disseminated intravascular coagulation (DIC) syndrome was made based on standard criteria.i Introduction Over the period of the history of combustiology one of the main problems for treatment of patients with burns is infection, both local – bacterial pneumonia – and generalized – sepsis – characterized by extremely severe course, complex diagnostics and high lethality rate. However, the role of hemostasis disorders in infectious complication development in severe burned patients is taken into consideration insuffi ciently. The aim of the study is to reveal the most relevant hemostasis system changes in sepsis and pneumonia in patients with serious heat injury in an acute period of burn disease. Conclusion Major bleeding occurred in 5.6% of medical–surgical ICU patients. Prolonged aPTT, thrombocytopenia, therapeutic (but not prophylactic) heparin, anti-platelet agents and recent surgery are potentially modifi able and independent predictors of bleeding. in patients with serious heat injury in an acute period of burn disease. Methods Hemostasis and biochemical blood parameters were studied in 169 patients with over 20% of the body burned, from the fi rst to 12th days after burn. Sepsis developed in 33 patients, 69 patients had pneumonia, and in 67 patients there were no complications of sepsis and pneumonia. P440 Reduced EPO receptor expression may contribute to limited pleiotropic eff ects of EPO during critical illness O McCook, S Matějková, J Matallo, A Scheuerle, P Moeller, M Georgieff , E Calzia, P Radermacher, H Schelzig University Medical School Ulm, Germany Critical Care 2012, 16(Suppl 1):P440 (doi: 10.1186/cc11047) Introduction We showed neuroprotective and renoprotective eff ects of recombinant human erythropoietin (rhEPO) after kidney and spinal cord ischemia/reperfusion (I/R) injury [1,2], but clinical studies using rhEPO to prevent acute kidney injury yielded equivocal results [3,4]. Increased cytokine release and/or oxidative stress can cause EPO resistance due to receptor modifi cation and/or downregulation [5]. Since we recently failed to confi rm rhEPO-related kidney protection in atherosclerotic swine [6], we compared kidney EPO receptor expression in swine strains with and without pre-existing vascular disease and kidney dysfunction. g Results Sixty-three patients (fi brinogen concentrate: 30; cryoprecipitate: 33) completed the study. The median age was 3 years 5 months and the median weight was 6.7 kg. Median fi brinogen doses were 504 mg (fi brinogen concentrate) and 402 mg (cryoprecipitate). Plasma fi brinogen concentrations increased after study medication and were similar in the two groups. No signifi cant between-group diff erences were observed in PT, aPTT or platelet count. In both groups, all ROTEM parameters showed signifi cant improvement after study medication, with no clinically relevant between-group diff erences in any of the EXTEM, INTEM or FIBTEM clotting parameters. Total avoidance of allogeneic blood product transfusion was achieved in 70% of patients in the fi brinogen concentrate group versus 18.2% in the cryoprecipitate group (P <0.001). The mean bleeding mass was signifi cantly lower in the fi brinogen concentrate group than in the cryoprecipitate group after 30 minutes. The thorax was opened after study medication in zero patients (0%) in the fi brinogen concentrate group and in six patients (18.2%) in the cryoprecipitate group (P = 0.025). y y Methods EPO receptor expression was quantifi ed with immunohisto- chemistry (densitometric image analysis) of formalin-fi xed paraffi n sections from pre-injury kidney biopsies taken in young and healthy pigs (German Landrace, up to now n = 4) as well as swine (FBM strain, up to now n = 6) with familial hypercholesteremia (11.1 (7.4; 12.3) vs. 1.4 (1.3; 1.5) mmol/l, P <0.001, n = 20 and 15, respectively, P <0.001) and consecutive, diet-induced atherosclerosis [7]. P441 Recognition and management of haemophagocytic lymphohistiocytosis on the ICU: a case series R Baumber, B Agarwal, M Carrington Royal Free Hospital, London, UK Critical Care 2012, 16(Suppl 1):P441 (doi: 10.1186/cc11048) Recognition and management of haemophagocytic lymphohistiocytosis on the ICU: a case series R Baumber, B Agarwal, M Carrington Royal Free Hospital, London, UK Critical Care 2012, 16(Suppl 1):P441 (doi: 10.1186/cc11048) y p y R Baumber, B Agarwal, M Carrington Methods We performed a randomized, double-blind study in 37 patients who underwent cesarean section. Patients were divided into two groups: the fi rst group (n = 19) received preoperative (30 minutes before operation) tranexamic acid 10 mg/kg; the second group (n = 18) received preoperative placebo. The condition of hemostasis was monitored by haemoviscoelastography. Introduction Haemophagocytic lymphohistiocytosis (HLH) is a rare haematological condition, with a reported incidence of the familial variety of the disease being 1.2 cases per 10,000 children per year. The acquired form of HLH predominantly aff ects adults and is almost always precipitated by infection, with Epstein–Barr virus (EBV) being the commonest trigger. This results in abnormal activation and proliferation of histiocytes and macrophages. Widespread phagocytosis of blood cell components leads cytopenias, a strong proinfl ammatory response and cytokine release leading to tissue necrosis and multiple organ failure. A large majority of these patients will present to the ICU for organ support and the aim of this report is to provide an update on HLH and raise awareness of this rare condition amongst the critical care community. Results All patients included in the study before the surgery had moderate hypercoagulation and normal fi brinolysis: increasing of the intensity of clot formation (ICF) to 11.4% compared to normal rates; the intensity of the retraction and clot lysis (IRCL) was 16.45 ± 1.40 in both groups. At the start of the operation in patients (group 1) – ICF decreased 9.7% (P <0.05), and IRCL decreased 27.6% (P <0.05) compared with preoperatively. In group 2, ICF decreased 8.8% (P <0.05), and IRCL increased 11.4% (P <0.05) compared with preoperatively. At the end of the operation, the condition of hemostasis in both groups came almost to the same value – moderate hypocoagulation, depressed fi brinolysis. In both groups there were no thrombotic complications. P440 Results Atherosclerotic swine presented with reduced glomerular fi ltration rate (creatinine clearance 76 (60; 83) vs. 103 (79; 120) ml/ minute, n = 19 each, P = 0.004) and chronic histological kidney injury (dilatation of Bowman’s space, swelling of Bowman’s capsule, tubular dilatation and necrosis). EPO receptor expression was reduced by nearly two orders of magnitude in this strain (94.6 (8.3; 112.5)×107 vs. 1.7 (0.0; 4.7) ×107 densitometric units, P = 0.010). Conclusion Fibrinogen concentrate raised fi brinogen levels and improved coagulation measures to a similar degree as cryoprecipitate. Bleeding and transfusion of allogeneic blood products were lower in the fi brinogen concentrate group. Fibrinogen concentrate may be a valuable option for controlling bleeding and avoiding transfusion in cardiac surgery. Conclusion Even pretreatment with rhEPO did not infl uence I/R- induced acute kidney in swine with pre-existing impairment of kidney function and histological damage. The lacking benefi cial eff ect of rhEPO was most likely due to the reduced expression of the EPO receptor, which may also explain contradictory results in clinical trials due to the frequent underlying kidney disease in the patients recruited. References 1. Simon F, et al.: Crit Care Med 2008, 36:2143-2150. 2. Simon F, et al.: Intensive Care Med 2011, 37:1525-1533. 3. Song YS, et al.: Am J Nephrol 2009, 30:253-260. 4. Endre ZH, et al.: Kidney Int 2010, 77:1020-1030. 5. van der Putten K, et al.: Nat Clin Pract Nephrol 2008, 4:47-57. 6. Simon F, et al.: Shock 2011, 36(Suppl 1):S24. 7. Thim T: Dan Med Bull 2010, 57:B4161. 1. Simon F, et al.: Crit Care Med 2008, 36:2143-2150. 2. Simon F, et al.: Intensive Care Med 2011, 37:1525-1533. P439 Effi cacy of tranexamic acid in decreasing blood loss during cesarean section O Tarabrin, V Kaminskiy, S Galich, R Tkachenko, A Gulyaev, S Shcherbakov, D Gavrychenko Odessa National Medical University, Odessa, Ukraine Critical Care 2012, 16(Suppl 1):P439 (doi: 10.1186/cc11046) 7. Thim T: Dan Med Bull 2010, 57:B4161. Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unfi nished. Annually in the world 125,000 women die from obstetric hemorrhage. Introduction Despite signifi cant progress in obstetric care, the problem of bleeding during labor remains unfi nished. Annually in the world 125,000 women die from obstetric hemorrhage. P438 S157 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods This was a single-center, randomized, open-label study. Key inclusion criteria were age <15 years, cardiac surgery involving cardiopulmonary bypass, intraoperative bleeding after neutralization of heparin, and hypofi brinogenemia. Patients received fi brinogen concentrate (60 mg/kg body weight; Haemocomplettan® P) or cryo- precipitate (10 ml/kg body weight). After study medication, allogeneic blood products were administered as required. Blood samples taken immediately before randomization and 1, 24 and 48 hours after study medication were subjected to laboratory and thromboelastometry (ROTEM) coagulation tests.i No impact of a massive transfusion protocol on coagulopathy and mortality at a level 1 trauma center: why? C Bourassa-Fulop, J Chauny, J Paquet, R Daoust, E Notebaert Hôpital Sacré-Coeur, Montreal, Canada Critical Care 2012, 16(Suppl 1):P444 (doi: 10.1186/cc11051) Introduction Blood transfusion is associated with worse outcome in critically ill patients. A restrictive strategy of blood transfusion has been advocated in patients undergoing cardiac surgery in order to avoid clinical complications related to exposure to blood components. Nevertheless, the blood transfusion rate remains elevated in clinical practice. Introduction In 2010 we studied the mortality and coagulopathy of all massively transfused patients at our hospital since 2004. We compared those who were transfused before the implementation of our massive transfusion protocol (MTP) (from 2004 to 2006) to those transfused with MTP. We found that our MTP did not lower mortality (35.7%) and our incidence of coagulopathy was high (72.6%). The aim of the present study is to explain those results, while concentrating uniquely on trauma patients. Methods We performed a retrospective study with 750 patients undergoing elective coronary arterial graft bypass (CABG) surgery, valvar surgery or combined procedure under cardiopulmonary bypass (CPB) between October 2010 and October 2011 at a university hospital cardiac surgery referral center in Brazil. We collected baseline characteristics and preoperative laboratory data, EuroSCORE, type of surgical procedure, intraoperative characteristics, blood transfusion exposure, postoperative severe complication as bleeding, low output cardiac syndrome, vasoplegia syndrome, myocardial ischemia, stroke, ventricular or supraventricular tachyarrhythmia, respiratory failure, acute renal failure, infection, ICU length of stay, hospital length of stay and mortality in 30 days. Methods We conducted a retrospective nested case–control study from our trauma registry. We included trauma patients who received 10 packed red blood cells (pRBC) or more in 24 hours and excluded those who died within the very fi rst hours of massive trauma. We extracted supplementary demographic and clinical data from the laboratory database and the hospital fi les. Chi-square tests and multivariate logistic regression were used to compare the eff ect of the two approaches (MTP vs. non-MTP) on mortality and coagulopathy, defi ned as an INR ≥1.8, a PTT ≥54, a fi brinogen <1 g/l or a platelet count <50,000, while controlling for acidosis (defi ned as a pH ≤7.1), hypothermia (defi ned as ≤35°C) and Injury Severity Score (ISS) (critically injured if ISS ≥30). y y Results A total of 512 patients (68.4%) was exposed to blood transfusion components. Red blood transfusion is a predictor of poor outcome in pediatric cardiac surgery Results Twenty-four patients were identifi ed with a diagnosis of HLH, 18 males and six females, with mean age 42.6 years. A history of prior haematological malignancy, HIV infection and immunosuppressive therapy was present in six, fi ve and four patients respectively; no underlying medical condition was found in 5/24 patients. Infective causes were identifi ed in 15/24 patients, EBV in eight out of 15. Other infective causes were Cytomegalovirus, Toxoplasma gonadii, Mycobacterium tuberculosis and Schistosomiasis. All patients were pancytopenic at ICU admission and had signifi cantly elevated serum ferritin (15,771 ± 17,718) and triglyceride (3.8 ± 2.05) levels. Eleven out of 24 patients displayed features of acute liver involvement. Mean APACHE II score was 20.5 ± 5.1 and mean SAPS II was 51.3 ± 12.1. Ten out of 24 survived to ICU discharge, and 6/24 (25%) were alive at the time of hospital discharge. The survivors had lower APACHE and SAPS scores, and were associated with a non-EBV infection and a lower incidence of liver involvement. C Colognesi, R Maia, L Hajjar, F Galas Heart Institute, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P443 (doi: 10.1186/cc11050) Introduction Red blood cell transfusion is associated with morbidity and mortality among adults undergoing cardiac surgery. We aimed to evaluate the association of transfusion with morbidity in pediatric cardiac surgical patients. The purpose of this study was to assess whether red blood cell transfusions result in worse outcomes after cardiac surgery in pediatric patients. g y Methods We studied an observational and prospective cohort of 200 patients undergoing cardiac surgery for congenital heart disease. We recorded baseline characteristics, RACHS-1 score, intraoperative data, cardiopulmonary bypass length, type of surgery, transfusion requirement and postoperative complications as need for reoperation, time of mechanical ventilation, cardiovascular complications, acute renal failure, infection, readmission at ICU and death during 28 days. Results One hundred and twenty-four patients were exposed to blood components. Seventy-seven percent of patients presented at least one major complication. There was no diff erence between transfused and nontransfused patients regarding baseline or intraoperative characteristics. Transfused patients presented a higher incidence of major complications than nontransfused patients (93.5% vs. 54.5%, P = 0.002). In a multivariate analysis, red blood cell transfusion was an independent risk factor for clinical complications including death in 28 days (OR = 2.2 (95% CI 1.4 to 23.4)). P443 variables, HLH disease characteristics, acute physiological derangement (APACHE II and SAPS II), and outcome. 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: Transfusion requirements after cardiac surgery: the TRACS randomized controlled trial. JAMA 2010, 304:1559-1567. Red blood transfusion is a predictor of poor outcome in pediatric cardiac surgery Conclusion HLH is a rare but fatal haematological syndrome that in its acquired form may present to ICU clinicians for organ support. Diagnosis of HLH in the intensive care setting may be diffi cult because sepsis may cause similar clinical and laboratory abnormalities. Presence of more severe acute physiological derangement, EBV aetiology and features of liver failure portend a poor prognosis in HLH. g y Results One hundred and twenty-four patients were exposed to blood components. Seventy-seven percent of patients presented at least one major complication. There was no diff erence between transfused and nontransfused patients regarding baseline or intraoperative characteristics. Transfused patients presented a higher incidence of major complications than nontransfused patients (93.5% vs. 54.5%, P = 0.002). In a multivariate analysis, red blood cell transfusion was an independent risk factor for clinical complications including death in 28 days (OR = 2.2 (95% CI 1.4 to 23.4)). 1. Henter JI, et al.: HLH 2004: diagnostic and therapeutic guidelines for haemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007, 48:124-131. Conclusion Blood transfusion after pediatric cardiac surgery is a risk factor for worse outcome. Avoiding blood transfusion may reduce mortality in this population. Blood transfusion is an independent predicting factor for poor outcome after cardiac surgery J Almeida, S Zeferino, F Galas, J Fukushima, L Camara, M Lima, T Santos, M Ferreira, J Auler Jr, R Kalil Filho, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P442 (doi: 10.1186/cc11049) g y J Almeida, S Zeferino, F Galas, J Fukushima, L Camara, M Lima, T Santos, M Ferreira, J Auler Jr, R Kalil Filho, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P442 (doi: 10.1186/cc11049) P441 Intraoperative blood loss in the fi rst group was 300 ± 40.5 and in the second was 500 ± 60.6.i Methods A single-centre retrospective review of case records of all patients admitted to our ICU, in a tertiary haematology referral centre, in the last 5 years with a confi rmed or suspected diagnosis of HLH, based on HLH-2004 guidelines. Data were collected on demographic Conclusion Using tranexamic acid before surgery signifi cantly reduces intraoperative blood loss by 40%, without thrombotic complications. S158 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P442 Blood transfusion is an independent predicting factor for poor outcome after cardiac surgery J Almeida, S Zeferino, F Galas, J Fukushima, L Camara, M Lima, T Santos, M Ferreira, J Auler Jr, R Kalil Filho, L Hajjar Heart Institute, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P442 (doi: 10.1186/cc11049) P444 No impact of a massive transfusion protocol on coagulopathy and mortality at a level 1 trauma center: why? C Bourassa-Fulop, J Chauny, J Paquet, R Daoust, E Notebaert Hôpital Sacré-Coeur, Montreal, Canada Critical Care 2012, 16(Suppl 1):P444 (doi: 10.1186/cc11051) Massive transfusion practice M C b ll G Y k d l S L y Results We enrolled 37 patients with severe sepsis/septic shock requiring RBC transfusion. After transfusion, the mean arterial pressure increased from 79 ± 9 to 82 ± 10 (T1a vs. T0: P <0.05) and 83 ± 10 mmHg (T1b vs. T0: P <0.001). Besides a nonstatistically signifi cant drop in arterial partial oxygen pressure, we observed no change in arterial blood gases and vital signs. Overall, RBC transfusion did not alter any of the MD-assessed parameters (that is, lactate, pyruvate, glycerol and glucose) or blood lactate, but it decreased the tissue LP ratio from (T0) 18.80 (interquartile range (IQR), 14.85 to 27.45) to (T1a) 17.80 (IQR, 14.35 to 25.20) (P <0.05) and (T1b) 17.90 (IQR, 14.45 to 22.75) (P <0.001). The post-transfusion changes in LP ratio at T1a (r = –0.42; 95% CI, –0.66 to –0.098; P = 0.01) and T1b (r = –0.68; 95% CI, –0.82 to –0.44; P <0.001) were signifi cantly correlated with the pre-transfusion LP ratio but not with baseline demographic characteristics, vital signs, severity scores, hemoglobin level and blood lactate. Finally, 39.0% of the transfused RBC units were leukoreduced and their median storage time was 16 days (IQR, 11 to 24). RBC storage time and leukocyte reduction had no infl uence on the tissue metabolic response to transfusion. Introduction Management of massive blood loss requires a multi- disciplinary team approach. Current guidelines are varied and generic with a lack of adherence when it comes to management of massive haemorrhage. The aim of our survey was to assess the transfusion practice in the management of massive haemorrhage in a busy district general hospital with a tertiary neurosurgical centre and the busiest obstetric unit in London. Methods A retrospective analysis of cases requiring transfusion of more than 6 units of red blood cells (RBC), between January 2009 and January 2010. Sixty-eight cases of massive transfusion were identifi ed, and data collected included causes of the haemorrhage, patient’s demographics and past medical background, investigations (FBC, clotting), use of blood products and patient outcome. Results There were 21 gastrointestinal, 17 vascular, 12 general surgical, seven trauma, six obstetric, and fi ve haematology–oncology patients. Thirty-one per cent of patients were 61 to 80 years old. Overall mortality was 35%, highest mortality among vascular patients. Average blood products per patient: RBC 9 units, fresh frozen plasma (FFP) 4 units, platelets (PLT) 1.2 units, cryoprecipitate 0.67 units. No impact of a massive transfusion protocol on coagulopathy and mortality at a level 1 trauma center: why? Transfused patients presented a higher number of severe clinical complications in the postoperative period compared to non- trans fused patients (74 (34.1%) vs. 312 (61.9%), P <0.0001). Also, the mor tality rate was higher in transfused patients than nontransfused patients (1 (0.5%) vs. 18 (3.6%), P <0.016). In a multivariate analysis, age, obesity, perioperative myocardial ischemia, valve disease, heart failure, blood transfusion and CPB duration are independently associated with mortality. y y y Results Of the 84 trauma patients, 23 were transfused with the MTP and 61 without. The average ISS score was very high (29.2), most were male (73.8%) and the average age was 41 years. The MTP versus non-MTP groups were similar in regards to age, sex, pH, temperature, ISS and Revised Trauma Score, but the MTP group received more transfusions (40% vs. 22% when dichotomized in two groups: above 20 pRBC and between 10 and 20 pRBC). The mortality and coagulopathy were similar in both the MTP and non-MTP group (39% vs. 34% and 65% vs. 75% respectively). PTM did not aff ect mortality or coagulopathy, even when controlling for all other variables. Individually, both hypothermia (OR = 2.6, 95% CI: 1.1 to 6.8) and acidosis (OR = 4.3, 95% CI:1.6 to 13.0) signifi cantly aff ected mortality, while the number of pRBC (OR = 3.8, 95% CI: 1.1 to 14.1) was the main determinate for coagulopathy. Conclusion Blood component exposure is associated with poor outcome and mortality in patients undergoing cardiac surgery. Despite the evidence that blood transfusion is associated with worse outcome, the blood transfusion rates remain unacceptably high in clinical practice. Reference S159 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 http://ccforum.com/supplements/16/S1 data regarding its impact on tissue metabolism. The aim of this study was to explore the eff ect of RBC transfusion on microdialysis-assessed interstitial fl uid metabolic parameters in septic patients. Conclusion In our population of severely injured patients, the MTP was not found to be benefi cial in regards to mortality nor coagulopathy. Hypothermia and acidosis seem to be the main determinants for mortality and should be among the priorities in caring for trauma patients. l Methods We conducted an observational, clinical study in a 25- bed, medical–surgical ICU of a university hospital. Blood transfusion after cardiac surgery increases the hospital length of stay in adult patients Blood transfusion after cardiac surgery increases the hospital length of stay in adult patients L Hajjar1, JL Vincent2, J Almeida1, F Jatene1, A Rodrigues1, J Fukushima1, R Nakamura1, C Silva1, E Osawa1, R Kalil1, F Galas1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Belgium Critical Care 2012, 16(Suppl 1):P447 (doi: 10.1186/cc11054) L Hajjar1, JL Vincent2, J Almeida1, F Jatene1, A Rodrigues1, J Fukushima1, R Nakamura1, C Silva1, E Osawa1, R Kalil1, F Galas1, J Auler Jr1 1Heart Institute, São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Belgium C l C (S l ) P (d / ) Conclusion Blood product use varied widely irrespective of speciality, the dependent factor being individual doctors involved in patient management. Due to diffi culty of accessing and their complexity in emergency situations, it was noted that hospital guidelines were disregarded. FFP was the commonly used blood product while cryoprecipitate and tranexamic acid were underused. Only 56% of patients had FBC and clotting screen to guide transfusion management. In these patients the ratio of cryoprecipitate and PLTs to RBCs was higher. This survey showed the need for revised, easily accessible and user-friendly guidelines for the management of massive haemorrhages. The results of this survey helped to establish point- of-care testing (thromboelastography) to provide a target controlled therapy and make the use of blood and blood products cost-eff ective. References Introduction Transfusion of allogeneic red blood cells (RBC) is a recognized risk factor for adverse outcomes following cardiac surgery. A potential endpoint to assess clinical complications and incremental use of resources is the measurement of hospital length of stay (LOS). The primary objective of this study was to evaluate the relationship between blood transfusion and increased hospital LOS after cardiac surgery. y Methods A prospective observational substudy that analyzed data from the overall 502 patients enrolled in the Transfusion Requirements After Cardiac Surgery (TRACS) study [1]. Patients who received blood transfusion during surgery or ICU stay were further categorized according to the number of prescribed RBC units: nontransfusion group, low transfusion requirement group (3 units or less), and high transfusion requirement group (more than 4 units). 1. CRASH-2 Trial Collaborators et al.: Lancet 2010, 376:23-32. 2. Johansson PI, Stensballe J: Transfusion 2010, 50:701-710. 3. Zink KA, Sambasivan CN, Holcomb JB, Chisholm G, Schreiber MA: Am J Surg 2009, 197:565-570. 4. Blood transfusion after cardiac surgery increases the hospital length of stay in adult patients Enriquez LJ, Shore-Lesserson L: Br J Anaesth 2009, 103(Suppl 1):i14–i22. q g p Results Patients who received any RBC unit had longer median LOS than patients in the nontransfusion group: 15 days (95% CI, 12.66 to 17.34) in high transfusion requirement group versus 10 days (95% CI, 9.1 to 10.9) in low transfusion group versus 8 days (95% CI, 7.4 to 8.6) in nontransfusion group (P <0.001). In a multivariate Cox proportional hazards model the following factors were considered predictive: age older than 65 years (hazard ratio (HR), 1.38 (95% CI, 1.11 to 1.73); P = 0.004), EuroSCORE 3 to 5 (HR, 1.44 (95% CI, 1.12 to 1.86); P = 0.005), EuroSCORE higher than 5 (HR, 1.7 (95% CI, 1.26 to 2.28); P <0.001), valvular surgery (HR, 1.57 (95% CI, 1.26 to 1.95); P <0.001), combined procedure (HR, 1.6 (95% CI, 1.03 to 2.46); P = 0.034), bypass duration higher than 100 minutes (HR, 1.23 (95% CI, 1.01 to 1.51); P = 0.046), LVEF lower than 40% (HR, 1.69 (95% CI, 1.24 to 2.32); P = 0.001), LVEF 40 to 59% (HR, 1.36 (95% CI, 1.1 to 1.69); P = 0.004), RBC transfusion Massive transfusion practice M C b ll G Y k d l S L Tranexamic acid was used in eight cases and factor VII in one case. At the time of haemorrhage, FBC, clotting screen and fi brinogen levels were requested in 56% of patients. In this group, FFP, PLTs and cryoprecipitate were used more frequently with mean use of blood products: RBC 9 units, FFP 5 units, PLT 1.5 units, and cryoprecipitate 1 unit. l p Conclusion Tissue oxygenation is improved by red blood cell transfusion in critically ill septic patients. Monitoring of the tissue LP ratio by microdialysis may represent a useful method for individual clinical management. P447 Reference 1. Cotton BA, et al.: J Trauma 2009, 66:41-49. No impact of a massive transfusion protocol on coagulopathy and mortality at a level 1 trauma center: why? We analyzed the eff ect of transfusion of either 1 or 2 RBC units on interstitial fl uid metabolic activity by means of a microdialysis (MD) catheter inserted in the subcutaneous adipose tissue of the upper thigh. Samples were collected before (T0) and after (T1a and T1b; spaced out by 4 hours) transfusion. Lactate, pyruvate, glycerol and glucose concentrations were measured with a bedside analyzer and the lactate/pyruvate (LP) ratio was calculated automatically. Reference P446 Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients P Kopterides, N Nikitas, M Theodorakopoulou, A Diamantakis, D Vassiliadi, A Kaziani, S Assoti, F Drakopanagiotakis, A Antonopoulou, P Papadopoulos, E Mavrou, C Georgiadou, A Tsantes, A Armaganidis, I Dimopoulou ‘Attiko’ University Hospital, Haidari – Athens, Greece Critical Care 2012, 16(Suppl 1):P446 (doi: 10.1186/cc11053) Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients P Kopterides, N Nikitas, M Theodorakopoulou, A Diamantakis, D Vassiliadi, A Kaziani, S Assoti, F Drakopanagiotakis, A Antonopoulou, P Papadopoulos, E Mavrou, C Georgiadou, A Tsantes, A Armaganidis, I Dimopoulou ‘Attiko’ University Hospital, Haidari – Athens, Greece Critical Care 2012, 16(Suppl 1):P446 (doi: 10.1186/cc11053) Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients P Kopterides, N Nikitas, M Theodorakopoulou, A Diamantakis, D Vassiliadi, A Kaziani, S Assoti, F Drakopanagiotakis, A Antonopoulou, P Papadopoulos, E Mavrou, C Georgiadou, A Tsantes, A Armaganidis, I Dimopoulou ‘Attiko’ University Hospital, Haidari – Athens, Greece Critical Care 2012, 16(Suppl 1):P446 (doi: 10.1186/cc11053) Impact on early trauma mortality of the adoption of the Updated European Guidelines on the management of bleeding C l 1 G d 2 G R ld 2 C S dd 2 S R 2 C l 2 Impact on early trauma mortality of the adoption of the Updated European Guidelines on the management of bleeding E Cingolani1, G Nardi2, G Ranaldi2, C Siddi2, S Rogante2, A Ciarlone2 1Azienda Ospedaliera San Camillo Forlanini, Roma, Italy; 2S.Camillo Hospital, Roma, Italy Critical Care 2012, 16(Suppl 1):P450 (doi: 10.1186/cc11057) Methods We conducted a cross-over randomized interventional study, enrolling 10 healthy adults. Nine volunteers completed the study; one volunteer could not complete the protocol because of anemia. Each volunteer received 1 unit of 40-day and 1 unit of 3-day stored autologous leukoreduced PRBC, on diff erent study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index (RHI) measurements were performed before and 10 minutes, 1 hour, 2 hours, and 4 hours after transfusion. E Cingolani1, G Nardi2, G Ranaldi2, C Siddi2, S Rogante2, A Ciarlone2 1Azienda Ospedaliera San Camillo Forlanini, Roma, Italy; 2S.Camillo Hospital, Roma, Italy E Cingolani1, G Nardi2, G Ranaldi2, C Siddi2, S Rogante2, A Ciarlone2 1Azienda Ospedaliera San Camillo Forlanini, Roma, Italy; 2S.Camillo Hospital, Roma, Italy Introduction Post-traumatic bleeding is the leading cause of potentially preventable death among trauma patients. The Updated European Guidelines (UEG), published at the beginning of 2010, were aimed to provide an evidence-based multidisciplinary approach to improve the management of the critically injured bleeding trauma patients. The aim of this study is to evaluate the impact of the implementation of UEG recommendations on early hospital mortality for severe trauma in a high-fl ow trauma center. Introduction Post-traumatic bleeding is the leading cause of potentially preventable death among trauma patients. The Updated European Guidelines (UEG), published at the beginning of 2010, were aimed to provide an evidence-based multidisciplinary approach to improve the management of the critically injured bleeding trauma patients. The aim of this study is to evaluate the impact of the implementation of UEG recommendations on early hospital mortality for severe trauma in a high-fl ow trauma center. Results The change of RHI after transfusion of 40-day stored PRBC did not diff er as compared to 3-day stored PRBC (P = 0.67). Plasma hemoglobin and bilirubin levels were higher after transfusion of 40-day than after 3-day stored PRBC (P = 0.02 and 0.001, respectively). Plasma levels of potassium, LDH, haptoglobin, cytokines, as well as blood pressure, did not diff er between the two transfusions and remained within the normal range. Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients y y P Kopterides, N Nikitas, M Theodorakopoulou, A Diamantakis, p , , p , , D Vassiliadi, A Kaziani, S Assoti, F Drakopanagiotakis, A Antonopoulou, P Papadopoulos, E Mavrou, C Georgiadou, A Tsantes, A Armaganidis, I Dimopoulou Introduction Even though red blood cell (RBC) transfusion is a common intervention in the critical care setting, there is a paucity of S160 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 of 1 to 3 units (HR, 1.24 (95% CI, 1.01 to 1.53); P <0.001), and RBC transfusion >3 units (HR, 1.96 (95% CI, 1.45 to 2.66); P <0.001). In an adjusted model for age, EuroSCORE, type of surgical procedure, LVEF and cardiopulmonary bypass time, the exposure to RBC transfusion was associated with an elevated LOS. controlled trials (RCTs) and observational studies comparing the eff ect of two or more diff erent PLT:RBC ratios in trauma resuscitation. We excluded studies using whole blood or systematically addressing the use of hemostatic products. Two independent reviewers selected the studies, extracted data using a standardized form, and assessed the risk of bias using the Newcastle–Ottawa scale and a checklist of key methodological elements (for example, use of massive transfusion protocol, survival bias). Disagreements were solved by consensus or a third party. The primary outcome was mortality. Secondary outcomes were multiple organ failure (MOF), lung injury and sepsis. A meta- analysis using random eff ects models was planned. controlled trials (RCTs) and observational studies comparing the eff ect of two or more diff erent PLT:RBC ratios in trauma resuscitation. We excluded studies using whole blood or systematically addressing the use of hemostatic products. Two independent reviewers selected the studies, extracted data using a standardized form, and assessed the risk of bias using the Newcastle–Ottawa scale and a checklist of key methodological elements (for example, use of massive transfusion protocol, survival bias). Disagreements were solved by consensus or a third party. The primary outcome was mortality. Secondary outcomes were multiple organ failure (MOF), lung injury and sepsis. A meta- analysis using random eff ects models was planned. Conclusion Blood transfusion is an independent risk factor for prolonged hospital LOS after cardiac surgery. P449 Liberal use of platelet transfusions in the acute phase of trauma resuscitation: a systematic review J Hallet1, F Lauzier1, O Mailloux2, V Trottier1, P Archambault2, R Zarychanski3, AF Turgeon1 1CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 2Université Laval, Québec, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2012, 16(Suppl 1):P449 (doi: 10.1186/cc11056) Rossaint et al.: Crit Care 2010, 14:R52. Red blood cell transfusion improves microdialysis-assessed interstitial lactate/pyruvate ratio in critically ill septic patients This fi nding can support the development of blood conservation strategies in order to avoid deleterious outcomes of blood exposure. Reference 1. Hajjar LA, Vincent JL, Galas FR, et al.: Transfusion requirements after cardiac surgery: the TRACS randomized controlled trial. JAMA 2010, 304:1559-1567. y gf p Results From 6,123 citations, seven observational studies were included (n = 4,230 patients). No RCT was identifi ed. All studies were considered to be at low risk of bias and addressed confoundings through multivariate regression or propensity scores. Four studies (n = 1,978) reported a decrease in mortality with higher PLT:RBC ratios in patients requiring massive transfusion and one study observed no mortality diff erence (n = 1,181) in nonmassively transfused patients. Two studies reported on the implementation of a massive transfusion protocol with higher PLT:RBC ratios; only one revealed a survival benefi t (n = 211). Of the three studies accounting for survival bias, two demonstrated a survival benefi t (n = 1,300). Among two studies reporting on the secondary outcomes (n = 854), one observed an increase in MOF with higher PLT:RBC ratios. Clinical heterogeneity between studies and methodological limitations precluded the use of a meta-analysis.fi P448 P448 Transfusion of blood stored for longer periods of time does not alter the reactive hyperemia index in healthy volunteers A Coppadoro1, L Berra2, B Yu2, C Lei2, E Spagnolli2, AU Steinbicker2, KD Bloch2, T Lin2, HS Warren2, FY Sammy2, BO Fernandez3, M Feelisch3, WH Dzik2, CP Stowell2, WM Zapol2 1University of Milan-Bicocca, Monza, Italy; 2Massachusetts General Hospital, Boston, MA, USA; 3University of Warwick, Coventry, UK Critical Care 2012, 16(Suppl 1):P448 (doi: 10.1186/cc11055) Introduction The purpose of this study is to investigate the eff ects of transfusing human packed red blood cells (PRBC) after prolonged storage, as compared to short storage. Retrospective data suggest that transfusion of PRBC stored for over 2 weeks is associated with increased mortality and morbidity. During storage, PRBC progressively release hemoglobin, which avidly binds nitric oxide (NO). We hypothesized that the NO-mediated hyperemic response following ischemia would be reduced after transfusion of PRBC stored for 40 days. Conclusion There is insuffi cient evidence to strongly support the use of a specifi c PLT:RBC ratio for acute trauma resuscitation, especially considering survival bias and nonmassively transfused patients. RCTs examining both safety and effi cacy of liberal PLT transfusions are warranted. Impact on early trauma mortality of the adoption of the Updated European Guidelines on the management of bleeding C l 1 G d 2 G R ld 2 C S dd 2 S R 2 C l 2 Plasma nitrite concentrations increased after transfusion of 40-day stored PRBC, but not after transfusion of 3-day stored PRBC (P = 0.01). gl Methods S. Camillo Hospital is a level 1 trauma center based in downtown Rome, with a catchment population of 2.5 million people. UEG recommendations were formally adopted and implemented since 1 April 2010. The pre-existing hospital guidelines were modifi ed as follows: immediate pelvic ring closure for all unstable patients with a suspected pelvic fracture; early administration of plasma with a higher rate of plasma/blood units; early use of thromboelastometry to monitor bleeding patients; and early use of antifi brinolitics for all bleeding patients. Data on trauma admissions and early hospital (6 hours) mortality before (2009) and after the adoption of the UEG were collected using the hospital registry, and were subsequently analysed. Results A total of 1,617 patients met the criteria for full trauma team activation (551 in 2009, 528 in 2010 and 538 during the fi rst 11 months of 2011). There were no diff erences for gender, age, mechanism of injury and average ISS. In 2009 21 patients died within the fi rst 6 hours versus 17 in 2010 and 12 in 2011; P = 0.3, P for trend = 0.1 Hemorrhage was the most important cause of death within this time-span. All early trauma deaths occurred in the operating room or in the emergency room during the initial stabilization. Conclusion Transfusion of 1 unit of autologous PRBC stored for longer periods of time is associated with increased hemolysis, an unchanged RHI and increased levels of plasma nitrite in healthy volunteers. P451 Hemodynamics in the severely injured patient with signifi cant hemorrhage G Nardi, D Piredda, A Cossu, E Cingolani, M Cristofani, I Ghezzi S. Camillo Hospital, Roma, Italy Critical Care 2012, 16(Suppl 1):P451 (doi: 10.1186/cc11058) outcome measures were in-hospital mortality and time to death. The secondary endpoint was to identify the eff ect of chronic medication on mortality. Categorical variables were compared by chi-squared test and continuous variables by Student’s t/Mann–Whitney tests. Multiple logistic regression analysis was used to predict mortality. P <0.05 was considered statistically signifi cant. i Results The inclusion criteria were met by 261 patients. Age average was 75.57 years (SD 5.7). Male gender was more prevalent (58.5%) for all age groups. The median ISS was 17. The most frequent trauma mechanism was low-energy type (58.2%). Patients with chronic ACT numbered 41 (15.7%). The mean ICU stay was 12.8 days (SD 2.8). Global mortality was 34.1%. Age >78 years and ISS >18 were predictive of mortality (P <0.05) with a HR of 6.0 (CI 2.5 to 14.6) and 1.01 (CI 1.01 to 1.05) respectively. Furthermore, the time to death was found to be earlier in both of the latter groups (P <0.05). GCS <4 or bilateral mydriasis was associated with 100% mortality. About 15% of patients with low-energy trauma (LET) underwent ENS compared to 7.8% with high-energy trauma. For the same ISS category, ACT increases the risk with HR 2.7 (CI 1.2 to 6.3) of ENS compared with nonanticoagulated patients. Introduction Very little is known about the hemodynamic impairments induced by trauma and severe hemorrhage. The aim of this study is to contribute to a better understanding of this topic. A recent paper has shown that about 50% of the hemorrhagic patients receive vasopressors [1] together with fl uids, blood and plasma. Fluids and vasopressors are aimed to restore patients’ hemodynamics; however, they might be detrimental. y g Methods The setting was a 10-bed trauma ICU in a level 1 trauma center with a catchment population of over 2.5 million people. This is a retrospective cohort study based on the data of the ICU electronic shift. During a 24-month period (2009 and 2010), 780 patients with major trauma (ISS >15) were admitted to the hospital; 410 of them were subsequently admitted to the shock and trauma ICU. All patients with ISS >15, who had received ≥5 blood units before ICU admission, and who were submitted to semi-invasive hemodynamic monitoring (PICCO), were entered into the study. p g p Conclusion LET accounted for most of the older trauma patients admitted to our ICU and had increased risk of death, especially with ACT. P451 Hemodynamics in the severely injured patient with signifi cant hemorrhage G Nardi, D Piredda, A Cossu, E Cingolani, M Cristofani, I Ghezzi S. Camillo Hospital, Roma, Italy Critical Care 2012, 16(Suppl 1):P451 (doi: 10.1186/cc11058) Although this is not necessarily secondary to alarming mechanisms. Referencei y Results Thirty patients (mean age 42.7 ± 17, mean 37.5 ± 12) met the study criteria. At the time of insertion of the PICCO catheter (T0) the 30 patients had already received an average of 8,760 ml fl uids (3,239 ml blood, plasma and platelets, 4,870 ml crystalloids and 685 ml colloids). Systemic blood pressure, central venous pressure and heart rate at T0 were, as an average, in the normal range. Nevertheless, six patients (20%) had a Cardiac Index lower than 2.5 l/minute, and 76% had a DO2 signifi cantly lower than the normal range. In the subsequent 24 hours following the information of the PICCO, these patients received, as an average, an additional 6,070 ml fl uids, blood and plasma. All vasopressors were discontinued, but 40% of the patients received dobutamine. Within 24 hours (T24), oxygen transport (DO2) and lactate were back to the normal values in all patients but one. ICU mortality and hospital mortality were respectively 13.3% and 16%. 1. Spaniolas et al.: Ground level falls are associated with signifi cant mortality in elderly patient. J Trauma 2010, 69:821-825. Outcomes in older blunt chest wall trauma patients: a retrospective study y C Battle, H Hutchings, PA Evans C Battle, H Hutchings, PA Evans Swansea University, Swansea, UK y Critical Care 2012, 16(Suppl 1):P453 (doi: 10.1186/cc11060) Introduction Blunt chest wall trauma accounts for over 15% of all trauma admissions to emergency departments in the UK and has high morbidity and mortality rates [1]. Reported risk factors for morbidity and mortality in blunt chest trauma patients include patient age, pre-existing disease and three or more rib fractures [2]. No guidelines exist for management of this patient group unless the patient has severe immediate life-threatening injuries. The aim of this study was to investigate whether blunt chest wall trauma patients aged 65 years or more have higher rates of mortality, morbidity (respiratory complications), ICU admissions and hospital length of stay (HLOS) than patients aged less than 65 years. Conclusion A high percentage of the severely injured patients who received ≥5 units of PRC have a low oxygen transport at the time of ICU admission. A high percentage of them is treated with vasopressors. However, as 20% of the patients in our study had a low cardiac index in spite of a normal blood pressure and a highly positive fl uid balance, vasopressors might be harmful. In our experience, hemodynamic monitoring with PICCO allowed the early recognition of inappropriate oxygen transport and a goal-directed treatment. Our data do not support the use of vasopressors to increase blood pressure in trauma patients. Methods A retrospective study was completed in which the notes of 1,056 blunt chest wall trauma patients who presented in 2010 to the emergency department of a large regional trauma centre in Wales were examined. A total of 94 out of the 1,056 (9%) patients were admitted to hospital in 2010 with blunt chest wall trauma. Data were recorded for each of the admitted patients including patient age, severity of injury, morbidity, mortality, ICU admission and HLOS. Patients were grouped according to age; group one included all blunt chest wall trauma patients aged 65 years or more and group two included all patients aged less than 65 years. Pearson’s chi-square analyses were performed to determine whether any diff erences existed between the two groups and signifi cance set at P <0.05. Reference 1. J Trauma 2011, 71:17-19. Liberal use of platelet transfusions in the acute phase of trauma resuscitation: a systematic review y J Hallet1, F Lauzier1, O Mailloux2, V Trottier1, P Archambault2, R Zarychanski3, AF Turgeon1 1CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 2Université Laval, Québec, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2012, 16(Suppl 1):P449 (doi: 10.1186/cc11056) J Hallet1, F Lauzier1, O Mailloux2, V Trottier1, P Archambault2, R Zarychanski3, AF Turgeon1 1CHA-Hôpital de l’Enfant-Jésus, Université Laval, Québec, Canada; 2Université Laval, Québec, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2012, 16(Suppl 1):P449 (doi: 10.1186/cc11056) Introduction With the recognition of early trauma coagulopathy, trauma resuscitation has recently shifted towards early and aggressive transfusion of platelets (PLTs). However, the clinical benefi ts of this strategy remain controversial. This systematic review examined the impact of an aggressive approach (higher PLT:RBC ratios) compared to restrictive PLT transfusions (lower PLT:RBC ratios) in the acute phase of trauma resuscitation. Conclusion This is a retrospective cohort study based on the data of the S. Camillo Hospital registry and the emergency department electronic shift. With the limitations of all retrospective studies, our data suggest that the implementation of the European Guidelines recommendations might contribute to a relevant reduction in early trauma mortality. Reference Methods We systematically searched Medline, Embase, Web of Science, Biosis, Cochrane Central and Scopus to identify relevant randomized Rossaint et al.: Crit Care 2010, 14:R52. S161 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P451 Hemodynamics in the severely injured patient with signifi cant hemorrhage G Nardi, D Piredda, A Cossu, E Cingolani, M Cristofani, I Ghezzi S. Camillo Hospital, Roma, Italy Critical Care 2012, 16(Suppl 1):P451 (doi: 10.1186/cc11058) P451 1. Trauma Audit and Research Network: Blunt Chest Trauma Admissions in the UK in 2010. TARN; 2011. (Kindly provided by Tom Jenks.) 2. Blecher GE, Mitra B, Cameron PA, et al.: Failed emergency department disposition to the ward of patients with thoracic injury. Injury 2008, 39:586-591. P455 Trauma patients and cervical spine protection in critical care: the impact of a spinal checklist on clinical care and documentation A Chick, C Scott, H Ellis, A Tipton Sheffi eld Teaching Hospitals NHS Foundation Trust, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P455 (doi: 10.1186/cc11062) Introduction In October 2010 a specifi c online proforma for cervical spine (C-spine) assessment in the context of trauma was introduced in critical care in a large UK teaching hospital. The aim of this study is to assess the impact of the Metavision Spinal Checklist (MSC) on clinical care and documentation. Prior to October 2010, the documentation of C-spine status on admission to critical care was incomplete or unclear in over 40% of these patients. Conclusion Surgical stabilization of spinal fractures avoids restrictive spinal braces and permits mobilization. Surgical fi xation of spinal fractures appears to decrease mortality and ITU stay and has a benefi cial eff ect on respiratory function, with regards to degree of ventilatory support and development of respiratory failure. Methods Patients were identifi ed from a comprehensive critical care database. Inclusion criteria: age >16; polytrauma or traumatic brain injury; other trauma where mechanism of injury suspicious for C-spine injury; admission date after 1 October 2010, before 30 November 2011. Exclusion criteria: pre-existing spinal injury; mechanism of trauma not consistent with C-spine injury. Clinical and MSC details were recorded, including sequential forms for individual patients where the C-spine status changed (for example, C-spine cleared and hard collar removed). Results A total of 62 patients met the inclusion criteria; 47% of these had been transferred from a district hospital. In patients with an MSC completed, there was 100% documentation of time, date and name of the completing critical care consultant. Seventy-fi ve per cent of initial MSCs indicated the name of the responsible consultant spinal surgeon. Seventy-nine per cent of patients with a completed MSC required their C-spines to be cleared after critical care admission. When completed, the initial MSC allowed clearance of C-spine and immediate removal of hard collar in 67% of those patients. There were clearly documented instructions for C-spine care from a spinal consultant in 92% of patients with a completed MSC. Overall, an MSC was completed for only 39% of patients, despite 53% of patients having sustained a spinal fracture at some level (for example, lumbar, thoracic or cervical). Eff ect of instrumented spinal fi xation on outcome in polytrauma patients in the ICU y j y g Results A total 951 patients were included (427 (30 months) before RRTT and 524 (39 months) after RRTT). Of these, 83 patients (8.8%) were dead after admission and analyzed for characters of mortality. The average age of mortality patients was 38.7 ± 16.3 years. Male was the predominant gender. The most common mechanism of injury was a motorcycle accident. Although there were no diff erences of character and mechanism of injuries between the two periods, patients associated with maxillofacial injury had signifi cant lower mortality after RRTT (28.5% vs. 10.5%; P = 0.04). However, the after RRTT group had signifi cantly higher occurrence of urinary complication and acute renal failure. The average adjusted monthly mortality rate was lower after RRTT (9.0 ± 6.1 vs. 6.9 ± 4.0%). Time series analysis between two periods demonstrated a decrease trend in monthly mortality after RRTT (coeffi cient (95% CI) = –0.61 (–1.13 to –0.23); P <0.01)). Introduction Spinal injuries in polytrauma patients carry high morbidity and mortality often necessitating intensive care admission. A review of polytrauma patients admitted to the ICU at The Royal Liverpool University Hospital was undertaken to investigate the eff ect of spinal instrumentation on outcome in the ITU. Methods A retrospective review of all polytraumatized patients admitted to the RLUH ICU over 3 years with a thoraco-lumbar spinal fracture. Clinical records, laboratory results and radiological records were accessed. Patients were grouped according to the use of instrumented spinal fi xation versus conservative management and outcomes compared. Results Fourteen polytrauma patients with spinal fractures were admitted to the ICU over 3 years, fi ve managed conservatively with a TLSO brace and nine managed operatively with instrumented spinal fi xation. The degree of injury as graded by the Injury Severity Scale (ISS) was lower in the nonoperative group (mean: 27, range: 14 to 59) compared to the operative group (mean: 36.1, range: 14 to 57). Mortality was signifi cantly higher in patients conservatively managed (nonoperative: 60%, operative: 0%) (P <0.01). The intubation time was lower in patients who underwent spinal instrumentation (mean: 12.3 days, range: 1 to 27 days), when compared to conservative management (mean: 16 days, range: 11 to 27 days), and similarly the ITU length of stay was reduced in the operative group (operative: mean 20.6 days, nonoperative: 32.25 days). Reference Reference Introduction The Department of Surgery, Faculty of Medicine, Chiang Mai University established a rapid response trauma team (RRTT) in July 2006. The aims of this study were to verify mortality rate alteration after setting up the RRTT. 1. Morris CG, et al.: BMJ 2004, 329:495-499. P457 P457 P456f Methods We retrospectively collected data between January 2004 and September 2009. The month before July 2006 was defi ned as before RRTT and after July 2006 as after RRTT. The monthly mortality rate, severity injury score (ISS) and demographic data were collected. Mortality trend alteration of thoracic injury after rapid response trauma team establishment K Chittawatanarat, C Ditsatham, K Chandacham, T Jirapongchareonlap, N Chotirosniramit P454 Mortality trend alteration of thoracic injury after rapid response trauma team establishment K Chittawatanarat, C Ditsatham, K Chandacham, T Jirapongchareonlap, N Chotirosniramit Chiang Mai University, Chiang Mai, Thailand Critical Care 2012, 16(Suppl 1):P454 (doi: 10.1186/cc11061) Conclusion The uptake of this checklist has not been optimal, but the MSC provides an excellent tool for clear documentation of C-spine status. During this initial trial phase, October 2010 to December 2011, the MSC has been consultant-only. Further action will involve rolling- out the checklist to critical care trainee doctors to improve the rate of documentation of C-spine status and improve patient safety in this area of signifi cant clinical risk [1]. P455 The median time from critical care admission to MSC completion was 36 hours (range 3 hours to 12 days, mean 48 hours). Eff ect of instrumented spinal fi xation on outcome in polytrauma patients in the ICU Development of respiratory failure was decreased in patients treated with instrumented fi xation (operative 33.3%, nonoperative: 71%). Conclusion Rapid response trauma team establishment could decrease the mortality trend. A protective eff ect was predominant in patients associated with maxillofacial injury. Critical older trauma patients M Irazábal, S Yus, L Fernández M Irazábal, S Yus, L Fernández Introduction The aim of this study was characterize the older injured patient in our setting and identify risk factors that might predict mortality. Trauma is the fi fth leading cause of death over the age of 65. In Spain, it has become a major public health problem as a result of the increase of this population. It represents 30% of the trauma admissions to our ICU. Geriatric patients may have comorbidities, limited physiologic reserve, may be taking chronic medication and the injury pattern is diff erent [1]. i Results There was no signifi cant diff erence in severity of injury between the groups. The mortality rate and HLOS in the patients aged 65 years or more were signifi cantly higher (P <0.05) than in the younger patient group. There were no signifi cant diff erences between the morbidity rates and number of ICU admissions. Conclusion Blunt chest wall trauma patients have a signifi cantly higher rate of mortality and hospital length of stay if aged 65 years or more when compared to those patients aged less than 65 years. Older blunt chest wall trauma patients should be considered for a higher level of care on admission to hospital from the emergency department. References f Methods We retrospectively analyzed trauma patients aged 65 years and older admitted to our ICU from January 2000 through December 2010. Three groups were formed on the basis of age: 65 to 70, 71 to 78 and older than 78 years. The Injury Severity Score (ISS) was categorized into three ranges: >12, 12 to 18 and >18. Variables studied include: age, gender, mechanism of injury, anticoagulant therapy (ACT), ISS, Glasgow Coma Scale (GCS) or presence of pupillary abnormalities and need for emergent neurosurgery (ENS) at admission. Primary 1. Trauma Audit and Research Network: Blunt Chest Trauma Admissions in the UK in 2010. TARN; 2011. (Kindly provided by Tom Jenks.) y y 2. Blecher GE, Mitra B, Cameron PA, et al.: Failed emergency department disposition to the ward of patients with thoracic injury. Injury 2008, 39:586-591. S162 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P454 Mortality trend alteration of thoracic injury after rapid response trauma team establishment K Chittawatanarat, C Ditsatham, K Chandacham, T Jirapongchareonlap, N Chotirosniramit Chiang Mai University, Chiang Mai, Thailand Critical Care 2012, 16(Suppl 1):P454 (doi: 10.1186/cc11061) Whole body computed tomography scanning for severe blunt polytrauma: analysis of Trauma Audit and Research Network database 2005 to 2010 PA Hunt1, F Lecky2, O Bouamra2 1James Cook University Hospital, Middlesbrough, UK; 2Hope Hospital, Salford, UK Critical Care 2012, 16(Suppl 1):P457 (doi: 10.1186/cc11064) Introduction There is growing evidence to recommend the use of whole body computed tomography (WBCT) scanning in the early management of severe blunt polytrauma patients. One recent study reported a survival advantage when using WBCT compared to a conventional imaging approach [1]. A number of UK NHS institutions already utilise WBCT protocols based upon either injury mechanism- related or physiological factors, or a combination of these. However, the UK Royal College of Radiologists is yet to provide recommendations on the use of WBCT in polytrauma. We present the results of our analysis of a large retrospective case series from 2005 to 2010 taken from the Trauma Audit and Research Network (TARN) database. We believe this is the fi rst analysis of its kind involving UK trauma cases and provides S163 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 of maximal CPK levels (15,780 to 52,600 U/l), but more severe acidosis (lowest pH 7.0 to 7.2, maximum lactate: 7.5 to 28 mmol/l, acidosis duration: 72 to 84 hours). This acidosis turned out to be due to intra- abdominal complications: post-traumatic pancreatitis and mesenteric ischemia. The vital prognosis of post-traumatic crush injury was good but the sequelae of the compartment syndrome were major. The need for RRT was not linked to CPK levels but rather to acidosis due to intra- abdominal complications. of maximal CPK levels (15,780 to 52,600 U/l), but more severe acidosis (lowest pH 7.0 to 7.2, maximum lactate: 7.5 to 28 mmol/l, acidosis duration: 72 to 84 hours). This acidosis turned out to be due to intra- abdominal complications: post-traumatic pancreatitis and mesenteric ischemia. The vital prognosis of post-traumatic crush injury was good but the sequelae of the compartment syndrome were major. The need for RRT was not linked to CPK levels but rather to acidosis due to intra- abdominal complications. important evidence to support the use of WBCT and guide best clinical practice. Controlled mechanical ventilation tactics in patients with polytrauma during interhospital transportation to the specialized center A Shatalin1, S Kravtsov2, V Agadzhanyan2, D Skopintsev2 1Federal State Budgetary Medical Prohylactic Institution, Leninsk-Kuznetsky, Russia; 2Federal State Budgetary Medical Prohylactic Institution ‘Scientifi c Clinical Center of the Miners Health Protection’, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P460 (doi: 10.1186/cc11067) A Shatalin1, S Kravtsov2, V Agadzhanyan2, D Skopintsev2 1Federal State Budgetary Medical Prohylactic Institution, Leninsk-Kuznetsky, Russia; 2Federal State Budgetary Medical Prohylactic Institution ‘Scientifi c Clinical Center of the Miners Health Protection’, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P460 (doi: 10.1186/cc11067) Methods During 24 months, seven patients admitted to our surgical intensive care after polytrauma (ISS >15) suff ered severe rhabdomyolysis (CPK >5,000 U/l) treated by intensive fl uid resuscitation, bicarbonate and furosemide. Results The following data are reported in Table 1: renal function (initial creatinine, renal replacement therapy (RRT), rhabdomyolysis (maximal CPK and myoglobin), acidosis (lowest pH, highest lactate (HL), time lactate >5 mmol/l) and complications (mortality, neurological sequelae). Introduction This study is an analysis of the infl uence of con- trolled mechanical ventilation (CMV) with PEEP in conditions of pneumocompression of the Chestnut antishock suit on the hemodynamics and blood oxygenation in patients with polytrauma during interhospital transportation. q Conclusion Survival was 100% but neurological impairment in the limbs is a major complication. The two RRT patients had a wide range Methods Seventy-two patients with polytrauma complicated by II and III stage ARDS were included in the study. The mean age was 33 ± 2 years. All patients were divided into two equal groups. The control group (CG) CMV was carried out with no PEEP. The experimental group (EG) CMV was carried out with PEEP 8 to 10 mbar. Both groups received the CMV regimen with Vt 7 ml/kg, Pmax 30 mbar. The injury severity according to the ISS was 37.6 ± 1 points in the EG and 39.1 ± 1 in the CG. The transportation time was 135 ± 7 minutes, the distance was 136 ± 10 km. Immobilization in the lower extremity fractures and pelvis fractures was carried out using the Chestnut suit with pneumocompression over the damaged parts of the body of 40 mmHg and over the remaining parts of the body of 20 mmHg. Statistical analysis was performed using Statistica 6.1. We used Student’s criterion. Table 1 (abstract P458). P460 P460 Controlled mechanical ventilation tactics in patients with polytrauma during interhospital transportation to the specialized center A Shatalin1, S Kravtsov2, V Agadzhanyan2, D Skopintsev2 1Federal State Budgetary Medical Prohylactic Institution, Leninsk-Kuznetsky, Russia; 2Federal State Budgetary Medical Prohylactic Institution ‘Scientifi c Clinical Center of the Miners Health Protection’, Leninsk-Kuznetsky, Russia Critical Care 2012, 16(Suppl 1):P460 (doi: 10.1186/cc11067) Whole body computed tomography scanning for severe blunt polytrauma: analysis of Trauma Audit and Research Network database 2005 to 2010 Methods We utilised retrospective, multicentre data of severe blunt polytrauma (ISS >15) direct ED admissions aged >15 years recorded in the UK TARN database to compare survival at 30 days between two groups of patients: those who underwent WBCT scans, and those who received a focused CT scan approach as part of their initial management in the emergency department. A total of 12,792 cases were included in the fi nal dataset. Post-traumatic rhabdomyolysis: an observational study in seven patients l Conclusion Exertional rhabdomyolysis is not rare, but rarely do such patients present to the emergency department with acute abdominal pain. Whilst triathlon training is popular among amateur sports people, awareness must be raised to train appropriately under proper conditions. M Alezrah, A Berger, P Bentzinger, C Sassot, L Profumo, B Saumande, O Collange, A Meyer, B Calon réanimation chirurgicale, Strasbourg, France Critical Care 2012, 16(Suppl 1):P458 (doi: 10.1186/cc11065) Introduction In the ICU, post-traumatic rhabdomyolysis is a relatively rare (1/5) cause of crush syndrome [1]. Early aggressive treatment is quintessential to avoid complications such as renal failure and death [2]. This observational study intends to assess the incidence of complications after traumatic crush injury in a tertiary trauma center ICU. Reference 1. Huber-Wagner S, et al.: Lancet 2009, 373:1455-1461. g y Results All three patients presented with abdominal pain after triathlon training. On admission, creatinine kinase levels were over 30,000 in all three cases and all required acute hospital admission for pain relief and intravenous fl uids to prevent renal failure. Exertional rhabdomyolysis in female amateur triathletes V Meighan Conclusion Despite the crude mortality rates appearing to demonstrate a poorer outcome in the WBCT group, correcting for confounding factors revealed an around 30% improvement in survival for the WBCT group. However, when also correcting for the potential eff ect of clustering, the benefi t of WBCT is less clear, with an around 20% improvement in survival and a lower level of signifi cance (P = 0.084). This eff ect may, in part, be due to diff ering trauma systems and logistical organisation between institutions. Overall, the results of our investigation appear to suggest a potential survival benefi t from the use of WBCT in severe blunt polytrauma. Exertional rhabdomyolysis in female amateur triathletes V Meighan Galway University Hospital, Galway, Ireland Critical Care 2012, 16(Suppl 1):P459 (doi: 10.1186/cc11066) y y p , y, Critical Care 2012, 16(Suppl 1):P459 (doi: 10.1186/cc11066 Introduction Multisport endurance events are becoming increasingly popular in Ireland. Overexertion, especially in the heat, of overweight or poorly conditioned athletes increases the risk of rhabdomyolysis. This study presents a case series of three female amateur triathletes presenting with acute abdominal pain caused by rhabdomyolysis. Methods The medical case notes of three female athletes presenting to the emergency department were reviewed. References 1. Bagley WH, Yang H, Shah KH: Rhabdomyolysis. Intern Emerg Med 2007, 2:210-218. 1. Bagley WH, Yang H, Shah KH: Rhabdomyolysis. Intern Emerg Med 2007, 2:210-218. i Results A total 2,822 (22%) of 12,792 cases underwent WBCT from the ED. The median ISS for the WBCT group was 22 (IQR 14 to 33) compared to 16 (IQR 9 to 25) for the focused CT group. The calculated crude mortality rate for the WBCT group was 10.1% compared to 8.7% in the focused CT group (P = 0.0124). Multivariate analysis with adjustments for potential confounding factors demonstrated an OR of 1.313 (95% CI = 1.083 to 1.592, P = 0.006) in favour of survival in the WBCT group. 1. Bagley WH, Yang H, Shah KH: Rhabdomyolysis. Intern Emerg Med 2007, 2:210-218. 2. Bosch X, Poch E, Grau JM: Rhabdomyolysis and acute kidney injury. N Engl J Med 2009, 361:62-72 2. Bosch X, Poch E, Grau JM: Rhabdomyolysis and acute kidney injury. N Engl J Med 2009, 361:62-72 Impact of fl uid resuscitation volume on the severity of organ failures in severely burned patients Impact of fl uid resuscitation volume on the severity of organ failures in severely burned patients N Depaye, G Minguet, A Magnette, D Jacquemin, D Ledoux, P Damas University Hospital of Liege, Belgium Critical Care 2012, 16(Suppl 1):P461 (doi: 10.1186/cc11068) N Depaye, G Minguet, A Magnette, D Jacquemin, D Ledoux, P Damas University Hospital of Liege, Belgium Critical Care 2012, 16(Suppl 1):P461 (doi: 10.1186/cc11068) Introduction Adequacy of fl uid resuscitation remains a cornerstone of early burn management. The Parkland formula – that is, administration of 4 ml/kg/% total of the body surface area (TBSA) burned with Ringer’s lactate for the fi rst 24 hours post injury – has been used for decades. The purpose of this study was to evaluate the eff ect of adherence with the Parkland protocol and its impact on the severity of organ failure during the fi rst week post injury using the Sequential Organ Failure Assessment (SOFA) score. Table 1 (abstract P462). SOFA during the resuscitation phase Day 0 Day 1 Day 2 Day 3 SOFA 3.40 ± 2.48 4.26 ± 2.99 4.95 ± 3.04 5.25 ± 3.25 Respiratory 1.38 ± 1.12 1.32 ± 1.09 1.81 ± 1.09 1.76 ± 1.07 Cardiovascular 1.19 ± 1.76 2.06 ± 1.94 2.10 ± 1.85 2.22 ± 1.89 Conclusion In the resuscitation phase of our critical burn patients the initial dysfunction was respiratory and cardiovascular, progressing later to cardiovascular dysfunction and haematological dysfunction appearing at the third day of admission. Knowing the possible evolution of organ dysfunction may help early detection and treatment. Reference 1 Latenser B: Crit Care Med 2009 37:2819 2826 Table 1 (abstract P462). SOFA during the resuscitation phase Table 1 (abstract P462). SOFA during the resuscitation phase Day 0 Day 1 Day 2 Day 3 SOFA 3.40 ± 2.48 4.26 ± 2.99 4.95 ± 3.04 5.25 ± 3.25 Respiratory 1.38 ± 1.12 1.32 ± 1.09 1.81 ± 1.09 1.76 ± 1.07 Cardiovascular 1.19 ± 1.76 2.06 ± 1.94 2.10 ± 1.85 2.22 ± 1.89 Methods We conducted a retrospective review of burns’ resuscitation data, from 2000 to 2007, on 101 adult patients (aged ≥16 years) admitted within the fi rst 24 hours following injury, with a %TBSA burned of 20 or more. A classifi cation of patients into four groups, according to fl uids administered, was done for comparison between these groups. The SOFA score was calculated daily for the fi rst week after admission. 1. Jeff rey RS: The phenomenon of ‘fl uid creep’ in acute burn resuscitation. J Burn Care Res 2007, 28:382-395. P463 i Results A total of 62 patients with complete data on fl uid administration were included in the analysis. Median age was 41 (28 to 54) years, median TBSA burned was 35.5 (25 to 50); median ICU stay was 38 (12 to 62) days and 13 (21%) patients died. Ten patients suff ering from inhalation injury were excluded from further analysis. Median fl uids administered was 4.9 (4.1 to 6.2) ml/kg/%TBSA at 24 hours. Five patients received <3. 5ml/kg/%TBSA, 15 between 3.5 and 4.5 ml/kg/%TBSA, 17 between 4.5 and 6 ml/kg/%TBSA and 15 patients >6 ml/kg/%TBSA. No diff erences existed between groups concerning the cause and surface of injury, age, sex, and comorbidities. Compared to others, patients who received >6 ml/kg/%TBSA had a signifi cant increase in respiratory failure (P = 0.03). The amount of fl uids administered had no impact on the incidence of cardiovascular (P = 0.89), renal (P = 0.11), liver (P = 0.52) and coagulation (P = 0.86) failure. Organ dysfunction in the resuscitation phase of critical burn patients A Agrifoglio, M Sánchez, M Hernández, J Camacho, L Cachafeiro, M Asensio, E Herrero, A García de Lorenzo, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P462 (doi: 10.1186/cc11069) Introduction Sequential Organ Failure Assessment (SOFA) is useful to assess organ dysfunction in burn patients [1]. The aim of this study was to determine the change in organ dysfunction from admission to day 3. Methods We performed a prospective observational cohort study with critical burn patients (total body surface area (TBSA) >20% and/ or inhalation injury) admitted to our burn ICU from September 2008 to December 2010. Epidemiological data and SOFA score at admission (day 0) and days 1, 2 and 3 were collected. Conclusion Use of CMV with PEEP in patients with polytrauma- complicated ARDS provided more expressed improvement of the blood oxygenation. Improvement of the blood gas exchange was accompanied by lactate decrease in both groups: by 24% in the EG, and by 13% in the CG. Application of the Chestnut allowed one to level the hemodynamic disorders using CMV with PEEP by means of preload maintenance and of the systolic output as a consequence. y y Results Sixty-four patients were enrolled (70% men) with mean age of 48.2 ± 19.0 years; Abbreviated Burn Severity Index (ABSI): 8.78 ± 2.59; APACHE II score: 13.5 ± 6.5. Twenty-three patients (35.9%) had inhalation injury and 19 patients (29.7%) died. The SOFA score was increased from day 0 to day 3. At admission the most frequent dysfunctions were cardiovascular and respiratory. The respiratory was similar in the next days and the cardiovascular dysfunction worsened (Table 1). Haematological dysfunction appeared at day 3 (1.05 ± 1.0) and neurological, renal and hepatic dysfunction were uncommon in the resuscitation phase. P461 Impact of fl uid resuscitation volume on the severity of organ failures in severely burned patients The neurological component of SOFA was left out because of the diffi culty to assess the actual Glasgow Coma Scale in sedated patients. Organ failures were defi ned by partial SOFA ≥3. Data are expressed as median (Q1 to Q3) and are analyzed using the chi- square test (P <0.05 was considered statistically signifi cant).l Conclusion In the resuscitation phase of our critical burn patients the initial dysfunction was respiratory and cardiovascular, progressing later to cardiovascular dysfunction and haematological dysfunction appearing at the third day of admission. Knowing the possible evolution of organ dysfunction may help early detection and treatment. Reference 1. Latenser B: Crit Care Med 2009, 37:2819-2826. Controlled mechanical ventilation tactics in patients with polytrauma during interhospital transportation to the specialized center Results Initial creatinine (μmol/l) 69 to 198 RRT 2/7 Maximal CPK (103 U/l) 11 to 144 Maximal myoglobin (103 U/l) 4 to 159 pH 7 to 7.3 Highest lactate (mmol/l) 2 to 28 Time lactate >5 mmol/l (hours) 0 to 84 Mortality 0 Neurological sequelae 6/7 Table 1 (abstract P458). Results Results In the EG there were the high values of SpO2 during all observation periods and PaO2/FiO2 after completion of the trans- portation in 1 and 12 hours (P <0.05). PaCO2 in the EG was lower after completion of the transportation in 1 and 12 hours compared to the CG (P <0.05). In the EG the value of FiO2 decreased from 0.5 ± 0.01 in S164 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P462 the early examination to 0.4 ± 0.01 in 12 hours after transportation. In the CG, FiO2 did not change. Hemodynamics diff erences between the groups were not documented, except for HR (P >0.05). Tachycardia was less expressed in the EG. The diff erence from the CG according to this index occurred 12 hours after completion of the transportation, 83 ± 1 and 87 ± 0.7 beats/minute respectively (P <0.05). The lactate rate was lower in the EG during all periods of observation (P <0.05). After completion of the transportation, the lactate rate in the EG was 2.2 ± 0.1 mol/l and in the CG was 2.7 ± 0.1 mol/l. Organ dysfunction in the resuscitation phase of critical burn patients A Agrifoglio, M Sánchez, M Hernández, J Camacho, L Cachafeiro, M Asensio, E Herrero, A García de Lorenzo, M Jiménez Hospital Universitario La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P462 (doi: 10.1186/cc11069) Early administration of parenteral estrogen suppresses the deleterious local and systemic infl ammatory response in severe burns Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fl uid resuscitation and 0.5 mg/kg i.p. estrogen (or placebo) 15 minutes post burn. From each group of 80, eight animals were sequentially sacrifi ced (and burn tissue and blood sampled for IL-6, TNFα, IL-1β) at one of 10 time points as follows: 0.5, 1, 2, 4, 6, 8, 18 and 24 hours and 7 days (7 days only for the eight shams). Introduction The purpose of this study was to examine the fl uid resuscitation of severely burned patients admitted to our regional centre and to review whether our practice had changed over the last 5 years in light of concerns of fl uid creep. Fluid creep is the term coined by Pruitt used to describe fl uid resuscitation in excess of that predicted by the Parkland formula and which is associated with abdominal compartment syndrome (ACS) [1]. l Methods Male rats (n = 168) were assigned randomly to one of three groups: (1) sham (no) burn (n = 8); (2) burn given placebo (n = 80); and (3) burn given E2 (estrogen). Groups 2 and 3 had 40% TBSA third-degree dorsal burns, early fl uid resuscitation and 0.5 mg/kg i.p. estrogen (or placebo) 15 minutes post burn. From each group of 80, eight animals were sequentially sacrifi ced (and burn tissue and blood sampled for IL-6, TNFα, IL-1β) at one of 10 time points as follows: 0.5, 1, 2, 4, 6, 8, 18 and 24 hours and 7 days (7 days only for the eight shams). Methods We completed a retrospective review in accordance with clinical governance guidance of patient notes evaluating all admissions in two groups (Group A: 1 May 2005 to 30 April 2006 and Group B: 1 May 2010 to 30 April 2011). The review examined the fi rst 72 hours of fl uid resuscitation in patients with ≥15%TBSA burns who were admitted less than 24 hours post burn injury. p j y Results There were 12 patients in each group. Both groups were comparable in both admission (Table 1) and resuscitation data. The total fl uid (mean ± SD) given in the fi rst 24 hours post burn-centre admission was 5.36 ± 2.22 ml/kg/%TBSA in Group A and 5.72 ± 3.00 ml/kg/%TBSA in Group B (P = 0.817) with three patients in each group receiving in excess of 250 ml/kg. 63 Epidemiological study of critical burn patients in an ICU L Cachafeiro, M Sanchez, E Herrero, J Camacho, M Hernandez, A Agrifoglio, A García de Lorenzo, M Jimenez Hospital La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P463 (doi: 10.1186/cc11070) Introduction Burn injuries remain a signifi cant problem with high associated morbidity and mortality, long average stays and high costs. The aim of our study is to analyze the epidemiology and mortality of critical burn patients admitted to the ICU at a university hospital in Madrid, Spain. Methods We performed a prospective, observational and descriptive study in patients admitted with burns over 20% of the total body surface area (TBSA), from October 2008 to December 2009. Demographic data were collected, TBSA, location and mechanism of burns, severity scores (ABSI, APACHE II, SOFA at admission, and next 3 days) length of stay, complications and mortality. Data are presented as number and percentage or as median and interquartile range, and they were analyzed with the Fisher exact test and Mann–Whitney test. Conclusion This single-centre retrospective study indicates that fl uid resuscitation volumes frequently overcome those previously established by the Parkland protocol. This fl uid over-resuscitation may have deleterious eff ects on patient outcome by increasing the incidence of respiratory failure. Results During this period, 64 patients were admitted to our unit, 45 (70.3%) were men and 19 (29.7%) were women. The mean age was 48 ± 19. SOFA score at admission was 3 ± 2, APACHE II score 15 ± 6 (range 4 to 38) and ABSI 8 (range 5 to 16). The TBSA average was 40 ± 20% and the mechanism of burn was by fl ame in 60 patients (93.8%), scald in four (6.3%), electrical in two (3.1%) and chemical in one (1.6%). The most frequent location was in the upper limbs in 60 patients (93.8%), followed by thorax in 50 (78.19%), head and neck in 43 (67.2%), lower S165 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P464) Patient data Group A Group B P value Number (n) 12 12 Age (years) 49 (18 to 69) 38.5 (21 to 77) 0.260 Weight (kg) 72 (55 to 109) 75 (60 to 99) 0.794 % TBSA 37.5 (16 to 70) 31 (18 to 60) 0.602 Inhalation injury (n) 6/12 3/12 0.206 Trauma (n) 1/12 0/12 0.307 Admission base defi cit –5.95 (–15 to +1) –6.55 (–11.7 to +2.5) 0.931 Admission lactate (mmol/l) 3.03 (0.98 to 5.4) 2.05 (0.5 to 4.1) 0.081 Survival (n) 6/12 9/12 0.206 Data presented as median (range). Figure 1 (abstract P463). Figure 1 (abstract P463). limbs in 43 (67.2%), and back in 29 (45.3%). Reference Reference Early administration of parenteral estrogen suppresses the deleterious local and systemic infl ammatory response in severe burns Conclusion In our study the most common burns were caused by fl ame in the upper limbs, chest, neck and face. Eighty-nine percent of our patients had complications, and they increased signifi cantly the length of stay and mortality. Based on the SOFA score, patients had higher scores for respiratory and cardiovascular systems. However, mortality was lower than expected in severity scores. JG Wigginton1, PE Pepe1, JW Simpkins2, JW Gatson1, KG Wigginton1, KR Kareem1, JP Minei1, D Maass1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2012, 16(Suppl 1):P465 (doi: 10.1186/cc11072) JG Wigginton1, PE Pepe1, JW Simpkins2, JW Gatson1, KG Wigginton1, KR Kareem1, JP Minei1, D Maass1 1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2University of North Texas, Fort Worth, TX, USA Critical Care 2012, 16(Suppl 1):P465 (doi: 10.1186/cc11072) P464 Fluid creep in burn resuscitation: the tide has not yet turned E James, M Hayes, P McCabe, G Williams, M Takata, MP Vizcaychipi Chelsea and Westminster Hospital and Imperial College, London, UK Critical Care 2012, 16(Suppl 1):P464 (doi: 10.1186/cc11071) Introduction Soon after severe burns, deleterious cytokines are produced and found in the burned skin, including dead tissue in third- degree injuries. This is followed by a systemic surge in these markers and correlated with subsequent multiorgan failure (MOF). In animal models, this response can be somewhat blunted by early debridement, but such early intervention is not usually feasible in most clinical settings. As estrogen is a powerful anti-infl ammatory/anti-apoptotic agent, we tested parenteral 17β-estradiol (E2) as a feasible early alternative intervention to dampen the proinfl ammatory response. Introduction Soon after severe burns, deleterious cytokines are produced and found in the burned skin, including dead tissue in third- degree injuries. This is followed by a systemic surge in these markers and correlated with subsequent multiorgan failure (MOF). In animal models, this response can be somewhat blunted by early debridement, but such early intervention is not usually feasible in most clinical settings. As estrogen is a powerful anti-infl ammatory/anti-apoptotic agent, we tested parenteral 17β-estradiol (E2) as a feasible early alternative intervention to dampen the proinfl ammatory response. Methods Male rats (n = 168) were assigned randomly to one of three groups: (1) sham (no) burn (n = 8); (2) burn given placebo (n = 80); and (3) burn given E2 (estrogen). 63 Epidemiological study of critical burn patients in an ICU L Cachafeiro, M Sanchez, E Herrero, J Camacho, M Hernandez, A Agrifoglio, A García de Lorenzo, M Jimenez Hospital La Paz, Madrid, Spain Critical Care 2012, 16(Suppl 1):P463 (doi: 10.1186/cc11070) Six patients had trauma associated and 23 had inhalation injury. Thirty-two patients (50.0%) required escharotomy at admission and 16 (25.0%) had compartment syndrome. Forty-four patients (68.8%) needed mechanical ventilation, and 20 (31.3%) tracheostomy. Fifty-six patients had complications. The most frequent were: shock (70.3%), ARDS (31.3%), sepsis (35.9%) and renal failure (26.6%). All complications increased signifi cantly the mortality (P <0.001). The length of stay was 30 days and global mortality was 29.7% (19 patients). See Figure 1.l titration of fl uid administration to urine output and the specifi c role of colloids in early resuscitation. Reference 1. Pruitt BA Jr: J Trauma 2000, 49:567-568. titration of fl uid administration to urine output and the specifi c role of colloids in early resuscitation. Reducing the indication of ventilatory support in the severely burnt patient and improving outcomes: results of a new protocol approach within a regional burns centref Affi liation to group A correlated with a shorter time of ventilation after admission (P <0.01); 61.1% of these patients were extubated within 6 hours after admission (vs. 14.3% in group B). Group A showed lower mortality rates (1 (1.4%) vs. 8 (14%), P = 0.01), shorter total hospital stay (34.2 ± 23.9 vs. 50 ± 38.4, P = 0.014) and lower incidence of sepsis (24 (39.3%) vs. 39 (68.4%), P <0.01). Methods A clinical observation study, approved by the local ethical committee, was designed and executed. Criteria for early spontaneous breathing were defi ned. These were formulated into a protocol for the management of severely burnt patients and trialled over 2 years in clinical practice on all admitted patients (group A). The ventilation period, complications and fi nal outcomes were recorded and compared with a retrospective control group of patients (group B) collated prior to implementation of the protocol. Initial study analysis revealed high inclusion rates of superfi cial burns in the intervention group. To achieve comparability these were excluded and further analysis was conducted only for patients with an abbreviated burn severity index (ABSI) ≥7. y y Results CPET was performed in 259 patients who subsequently underwent an elective open AAA repair. Outcome data were available for 185 patients from a potential 222 in whom 1-year follow-up was available (83%). Baseline demographics included AT ≤10.9 ml/kg/ minute in 39% and >10.9 ml/kg/minute in 61% of patients with respective median ages in these groups being 73 and 72. Regression analysis demonstrated that AT was the only predictor of survival at 30 days, 90 days and 1 year. Age and AT remained independent predictors of survival at 90 days and 1 year following multivariate analysis. Of note, 87 patients underwent elective endovascular aneurysm repair and CPET, median age 76, during the period analysed. In particular, 26.4% were older than 80 years old, versus 14.7% in the AAA group. See Figure 1. only for patients with an abbreviated burn severity index (ABSI) ≥7. Results In total 118 patients were included. The demographics and injury characteristics of both groups were similar. Patients of group A (n = 61) had fewer ventilator days in the time course of treatment (3.9 ± 11.7 vs. 17.1 ± 19.6 days, P <0.01). Reducing the indication of ventilatory support in the severely burnt patient and improving outcomes: results of a new protocol approach within a regional burns centref AH Raithatha, S Smith, K Chakrabarti, A Tridente, K Kerr Sheffi eld Teaching Hospitals NHS Trust: Northern General Hospital, Sheffi eld, UK Introduction A reduced oxygen uptake at anaerobic threshold (AT) and an elevated ventilatory equivalent for carbon dioxide (VE/VCO2) have been shown to be predictors of outcome after major surgery [1]. We report the demographic and outcome data of patients undergoing elective open abdominal aortic aneurysm (AAA) surgery who underwent cardiopulmonary exercise testing (CPET) testing within our unit and examine the relationship between age, AT and VE/VCO2 on survival outcomes. Introduction Initial management of the severely burnt patient often includes sedation and mechanical ventilatory support as routine. Conversely it is documented in the literature that nonjudiciously applied mechanical ventilatory support can itself lead to poorer patient outcomes [1]. Exploring means to reduce this iatrogenic risk, a standardised in-house fi ve-point protocol off ering clinical guidance on the use and duration of ventilation was introduced, analysed and the impact on outcome assessed. Methods A retrospective observational analysis of our unit’s CPET Excel database was conducted to identify patients who underwent CPET testing for elective open AAA repair over a 6-year period. Demographic data and survival at 30 days, 90 days and 1 year were extracted. Logistic regression analysis was undertaken using STATA statistical software to determine if age, AT or VE/VCO2 were predictors of survival at 30 days, 90 days or 1 year. p Methods A clinical observation study, approved by the local ethical committee, was designed and executed. Criteria for early spontaneous breathing were defi ned. These were formulated into a protocol for the management of severely burnt patients and trialled over 2 years in clinical practice on all admitted patients (group A). The ventilation period, complications and fi nal outcomes were recorded and compared with a retrospective control group of patients (group B) collated prior to implementation of the protocol. Initial study analysis revealed high inclusion rates of superfi cial burns in the intervention group. To achieve comparability these were excluded and further analysis was conducted only for patients with an abbreviated burn severity index (ABSI) ≥7. Results In total 118 patients were included. The demographics and injury characteristics of both groups were similar. Patients of group A (n = 61) had fewer ventilator days in the time course of treatment (3.9 ± 11.7 vs. 17.1 ± 19.6 days, P <0.01). P467 P467 Cardiopulmonary exercise testing and elective open abdominal aortic aneurysm surgery over a 6-year period in a UK teaching hospital AH Raithatha, S Smith, K Chakrabarti, A Tridente, K Kerr Sheffi eld Teaching Hospitals NHS Trust: Northern General Hospital, Sheffi eld, UK Critical Care 2012, 16(Suppl 1):P467 (doi: 10.1186/cc11074) Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results In placebos, very high levels of cytokines appeared almost immediately in the echars and circulation, persisting 7 days post burn. In the estrogen group, cytokines, including tissue and circulating IL-6, the greatest predictor of MOF, remained suppressed at all time points, even day 7 (Figure 1). No patients fulfi lling the inclusion criteria required re-intubation or emergency intubation. Conclusion Extended periods of mechanical ventilatory support are known to be associated with poorer outcomes in the severely burnt patient. Guidance on minimising ventilator dependency through introduction of a protocol has led to improved outcomes of such patients within a regional burns centre. This study suggests that many burns patients are overtreated through routine ventilation. Reference y g Conclusion Early single-dose parenteral estrogen can dramatically suppress both the local and systemic massive proinfl ammatory responses in severe burns. Based on these data, estrogen may not only be an inexpensive, simple, adjunctive therapy in burn management, it may obviate the need for many subsequent interventions altogether. References 1. Mackie D, Spoelder E, Paauw R, et al.: Mechanical ventilation and fl uid retention in burn patients. J Trauma 2009, 67:1233-1238. 1. Mackie D, Spoelder E, Paauw R, et al.: Mechanical ventilation and fl uid retention in burn patients. J Trauma 2009, 67:1233-1238. 1. Crit Care Med 2010, 38:S620-S629. 1. Crit Care Med 2010, 38:S620-S629. 2. J Neuroinfl amm 2009, 6:30-36. Early administration of parenteral estrogen suppresses the deleterious local and systemic infl ammatory response in severe burns Almost one-third of the fl uid administered was colloid in each group. The hourly urine output (mean ± SD) was 1.34 ± 0.72 ml/kg/hour in Group A and 1.53 ± 1.47 ml/kg/hour in Group B (P = 0.817). Inhalational injury was present in six patients in Group A and three in Group B. The inhalational injury group (mean ± SD) received 6.64 ± 2.51 ml/kg/%TBSA whilst the noninhalational injury group received 4.88 ± 2.50 ml/kg/%TBSA (P = 0.101). There was no reported incidence of ACS.l Figure 1 (abstract P465). Burned skin IL-6 levels at day 7. Conclusion Despite our awareness of fl uid creep, our practice has not changed signifi cantly over the last 5 years. Fluid was administered in excess of that predicted by the Parkland formula despite almost one-third being given as colloid and no cases of ACS being reported. A multicentre randomised control trial is required to examine stricter S166 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Perioperative evaluation of elective surgical patients: is it possible to plan ICU admission? p LM Mozzoni, FR Ruggeri, MN Nastasi Ospedale Ceccarini Riccione, Italy Critical Care 2012, 16(Suppl 1):P468 (doi: 10.1186/cc11075) p LM Mozzoni, FR Ruggeri, MN Nastasi Ospedale Ceccarini Riccione, Italy Critical Care 2012, 16(Suppl 1):P468 (doi: 10.1186/cc11075) Results There was no signifi cant diff erence between both groups intraoperatively concerning arrhythmias, blood transfusion, and hemodynamic support. Off -pump patients had a signifi cantly higher mean number of constructed grafts than in the ONCAB group (mean, 3.30 ± 0.88 vs. 2.84 ± 0.80, P = 0.02). There were no signifi cant diff erences between off -pump and on-pump regarding postoperative blood loss, blood transfusion, length of the ICU and the hospital stay, the ventilation time, the use of intraaortic balloon pump, renal complications, respiratory complications, and reopening. However, graft occlusion, MI, raised cardiac enzymes, ventricular tachycardia, cardiogenic shock, and disturbed conscious level were signifi cantly higher in the OPCAB group. The postoperative mortality rate was signifi cantly higher in the OPCAB group than in the ONCAB group (15% vs. 0%, P = 0.046). Follow-up angiograms in 40 patients out of 65 (61.5%) who underwent 124 grafts revealed that no signifi cant diff erence between off -pump and on-pump groups regarding the overall rate of graft patency (83.5% vs. 84.4%, P = 0.84). No mortality was reported in both groups at 6-month follow-up. Introduction The aim of the study is to evaluate the possibility to predict ICU admission in elective surgical patients, studying the perioperative period variables. Introduction The aim of the study is to evaluate the possibility to predict ICU admission in elective surgical patients, studying the perioperative period variables. Methods This is a prospective, nonintervention study concerning 207 patients, who have been operated on under elective conditions from January to October 2011. The group we studied was aff ected by thoracic (n = 78) or abdominal (n = 129) cancer. Mean age was 67.8 (SD 11.3; limits 24 to 91). ASA score III concerned 107 patients (51.7%) and score II 98 patients (47.3%). A senior anesthetist screened all patients before operation, assigning them to one of these three possible groups: G0 (patient who does not need ICU admission), G1 (patients who could need ICU admission), G2 (patients who defi nitely need ICU admission). Scheduling of patients into groups was made considering medical history, laboratory data, physical evaluation and type of surgery. Patients were studied from surgical intervention to discharge. Aortic aneurysm disease versus aortic occlusive disease: diff erences in postoperative ICU requirements after open elective abdominal aortic surgery Aortic aneurysm disease versus aortic occlusive disease: diff erences in postoperative ICU requirements after open elective abdominal aortic surgery Aortic aneurysm disease versus aortic occlusive disease: diff erences in postoperative ICU requirements after open elective abdominal aortic surgery J Bisgaard1, HK Jørgensen1, T Gilsaa1, E Ronholm1, P Toft2 1Littlebaelt Hospital Kolding, Denmark; 2Odense University Hospital, Odense, Denmark Critical Care 2012 16(Suppl 1):P470 (doi: 10 1186/cc11077) J Bisgaard1, HK Jørgensen1, T Gilsaa1, E Ronholm1, P Toft2 1Littlebaelt Hospital Kolding, Denmark; 2Odense University Hospital, Odense, Denmark J Bisgaard1, HK Jørgensen1, T Gilsaa1, E Ronholm1, P Toft2 1Littlebaelt Hospital Kolding, Denmark; 2Odense University Hospital, Odense, Denmark Critical Care 2012, 16(Suppl 1):P470 (doi: 10.1186/cc11077) Conclusion Preoperative evaluation does not appear to be a signifi cant predictor for ICU admission, which is determined by intraoperative or organizational factors. The ICU admission reduces the incidence of postoperative complications; mortality is mainly due to the immediate perioperative period. Introduction Open elective abdominal aortic surgery is a high- risk procedure involving clamping of the aorta. Indications include abdominal aortic aneurysm (AAA) or aortic occlusive disease (AOD) causing lower limb ischaemia. These patients are often regarded as one entity in postoperative study settings. However, previous studies indicate that risk profi les, infl ammatory activity, and haemodynamic capacity may diff er between these groups [1,2]. The aim of this study was to evaluate postoperative ICU requirements after open elective abdominal aortic surgery, hypothesising that AAA patients had longer ICU stays and needed more mechanical ventilation or acute dialysis than did patients with AOD. Reference Reference 1. Rhodes A, et al.: Intensive Care Med 2011, 37:1466-1472. 1. Rhodes A, et al.: Intensive Care Med 2011, 37:1466-1472. Perioperative evaluation of elective surgical patients: is it possible to plan ICU admission? All data were analyzed using IBM SPSS statistics v19 (SPSS Inc.), using adequate test and accepting P <0.05. Conclusion There was a higher incidence in postoperative complications and mortality in the off -pump procedure than the on- pump. At 6-month follow-up, no signifi cant diff erences between both techniques were found in graft patency and mortality. Hence, longer- term mortality from randomized trials of off -pump versus on-pump CABG is needed. q p g Results Sixty-six patients (31.9% of all patients) were in G0, 70 (33.8%) in G1 and 71 (34.3%) in G2. The ASA score can distinguish patients in G0 and G2, but not in G1 (P <0.05). The decision to schedule patients in a group arises mainly from the coexistence of both cardiovascular and respiratory diseases [1]. Ninety patients (43.5%) entered the ICU; 30 (42.8%) of these were in G1 and 34 (47.9%) in G2; 26 (39.4%) were in G0. Distribution in the three groups of ICU-admitted patients was similar (P = NS) and there was no signifi cant relationship between the ASA score (and its distribution in the three groups) and ICU admission (P = NS). Patients admitted had undergone surgery of longer duration or had problems in the theater (low output syndrome, diffi cult weaning at the end of procedure, bleeding) or organizational problems (P <0.05). ICU-admitted patients show a lower number of postoperative complications as arrythmias and wound infections (P <0.05). Four patients died, all had been hospitalized in the ICU. The mortality rate was 1.9% (75% were in G2). Patients with complications requiring further surgery were 15 (7.2%), seven of which had been hospitalized in the ICU.i Reference Reducing the indication of ventilatory support in the severely burnt patient and improving outcomes: results of a new protocol approach within a regional burns centref Affi liation to group A correlated with a shorter time of ventilation after admission (P <0.01); 61.1% of these patients were extubated within 6 hours after admission (vs. 14.3% in group B). Group A showed lower mortality rates (1 (1.4%) vs. 8 (14%), P = 0.01), shorter total hospital stay (34.2 ± 23.9 vs. 50 ± 38.4, P = 0.014) and lower incidence of sepsis (24 (39.3%) vs. 39 (68.4%), P <0.01). Conclusion Our data support existing evidence that AT can be used as a predictor of survival in open elective AAA surgery. In addition, age at Figure 1 (abstract P467). Elective AAA mortality rates by group. Figure 1 (abstract P467). Elective AAA mortality rates by group. S167 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods This nonrandomized single-centre control trial was prospectively conducted on 65 patients who were subjected to coronary artery bypass surgery followed by stay in the Open Heart Intensive Care Center of the Police Authority Hospital, in the period from July 2009 to January 2010. Patients were divided into two groups; group A, 25 patients underwent surgery using cardiopulmonary bypass pump (on coronary artery bypass pump (ONCAB)); and group B, 40 patients underwent surgery without using cardiopulmonary bypass pump (off -pump coronary artery bypass (OPCAB)). All of the demographic, operative and postoperative data were prospectively collected and analyzed statistically. Six months later, the patients underwent coronary angiography. CPET also predicted 90-day and 1-year survival; however, VE/VCO2 was not a predictor of survival in this cohort. Reference CPET also predicted 90-day and 1-year survival; however, VE/VCO2 was not a predictor of survival in this cohort. Reference 1. Carlisle J, et al.: Mid-term survival after abdominal aortic aneurysm surgery predicted by cardiopulmonary exercise testing. Br J Surg 2007, 94:966-969. P469 Cardiac-specifi c biomarkers and life-threatening complications of off -pump versus on-pump coronary bypass surgery in Egyptian patients H Elabd1, A Alsherif2, T El Gohary2, M Hagras2, S Salah Eldin2 1Student Hospital, Cairo University, Giza, Egypt; 2Kasr Alaini Hospitals, Cairo, Egypt Critical Care 2012, 16(Suppl 1):P469 (doi: 10.1186/cc11076) Reference 1. Shroyer AL, Grover FL, Hattler B, et al.: On-pump versus off -pump coronary- artery bypass surgery. N Engl J Med 2009, 361:1827-1837. P471 P471 High postoperative blood levels of macrophage migration inhibitory factor are associated with less organ dysfunction in patients after cardiac surgery C Stoppe1, G Grieb1, D Simons1, R Rossaint1, J Bernhagen1, S Rex2 1University Hospital of the RWTH, Aachen, Germany; 2University Hospital Gasthuisberg, KU Leuven, Belgium Critical Care 2012, 16(Suppl 1):P471 (doi: 10.1186/cc11078) Introduction Macrophage migration inhibitory factor (MIF) is a structurally unique infl ammatory cytokine [1] that exerts protective eff ects during ischemia and reperfusion [2]. We hypothesized that elevated MIF levels in the early postoperative time course might be inversely associated with postoperative organ dysfunction as assessed by SAPS II and SOFA score in patients after cardiac surgery. y p g y Methods Fifty-two cardiac surgical patients (mean age (± SD) 67 ± 10 years; EuroSCORE: 7 (2 to 11)) were enrolled in this monocenter, prospective, observational study. Serum levels of MIF and clinical data were obtained after induction of anesthesia, at admission to the ICU, 4 hours thereafter and at the fi rst and second postoperative day (POD). Patient outcome was assessed using the SAPS II at POD1 and SOFA score for the fi rst 3 days of the eventual ICU stay. Conclusion Prolonged mechanical ventilation occurred in 40% of our patients after heart transplantation. A higher creatinine level during the fi rst 24 hours after the surgery was associated with prolonged mechanical ventilation in this study. P472 and malignant disease (2.7 vs. 0.6%, P = 0.02). In contrast, AOD patients had a higher prevalence of smoking (95 vs. 86%, P <0.001), and diabetes (16 vs. 9%, P <0.001). AAA patients had larger intraoperative blood losses (1,610 (1,000 to 2,500) vs. 1,200 (750 to 1,800) ml, P <0.001), but duration of surgery was shorter (161 (130 to 205) vs. 194 (160 to 240) minutes, P <0.001). Postoperatively, more AAA patients had ICU stays >24 hours (62 vs. 45%, P <0.001), tended to need mechanical ventilation more often (16 vs. 12%, P = 0.08), and more needed acute dialysis (3.8 vs. 0.9%, P <0.03). and malignant disease (2.7 vs. 0.6%, P = 0.02). In contrast, AOD patients had a higher prevalence of smoking (95 vs. 86%, P <0.001), and diabetes (16 vs. 9%, P <0.001). AAA patients had larger intraoperative blood losses (1,610 (1,000 to 2,500) vs. 1,200 (750 to 1,800) ml, P <0.001), but duration of surgery was shorter (161 (130 to 205) vs. 194 (160 to 240) minutes, P <0.001). Postoperatively, more AAA patients had ICU stays >24 hours (62 vs. 45%, P <0.001), tended to need mechanical ventilation more often (16 vs. 12%, P = 0.08), and more needed acute dialysis (3.8 vs. 0.9%, P <0.03). 1. Shteinberg D, et al.: Eur J Vasc Endovasc Surg 2000, 20:462-465. 2. Johnston WE, et al.: Anesthesiology 1987, 66:386-389. f References 1. Shteinberg D, et al.: Eur J Vasc Endovasc Surg 2000, 20:462-465. 2. Johnston WE, et al.: Anesthesiology 1987, 66:386-389. Results The mean age of the patients (71% male) was 31.5 ± 16.8 years and the incidence of prolonged mechanical ventilation was 40%. Compared with patients who did not require prolonged mechanical ventilation, those who did had signifi cantly lower preoperative hemoglobin levels (12.0 ± 1.5 vs. 13.7 ± 2.4 mg/dl, P = 0.03), higher intraoperative lactate levels (7.14 ± 4.13 vs. 3.5 ± 1.82 mmol/l, P = 0.006), higher postoperative day 1 serum creatinine levels (2.2 ± 0.9 vs. 1.2 ± 0.7 mg/dl, P = 0.002), and longer cardiopulmonary bypass times (143.0 ± 24.2 vs. 122.8 ± 29.1 minutes, P = 0.005). Binary logistic regression revealed that the postoperative day 1 serum creatinine level was an independent risk factor for prolonged mechanical ventilation after heart transplantation (OR: 5.109; 95% CI: 1.362 to 19.159, P = 0.016). Length of hospital stay was signifi cantly longer in patients with PMV than those who did not require prolonged mechanical ventilation (36.4 ± 30.4 vs. 21.8 ± 12.7, P = 0.049). The respective mortality rates for patients with prolonged mechanical ventilation and those without prolonged mechanical ventilation were 60% versus 40%, P = 0.15. Predictors of prolonged mechanical ventilation after heart transplantation Introduction Several studies have reported that prolonged mechanical ventilation is associated with high mortality and morbidity rates, length of hospital stay, and costs after coronary artery and valvular surgeries. However, no study has focused on the incidence and risk factors of prolonged mechanical ventilation after heart transplantation. The aim of this study was to determine the incidence and predictors of prolonged mechanical ventilation after heart transplantation. y Conclusion Compared to the AOD group, more AAA patients had ICU stays >24 hours and more often needed acute dialysis. Distinguishing between these two diseases may be useful in planning and distribution of ICU resources. Furthermore, considering these two patient groups as diff erent pathological entities may be advised in future studies. References Methods We retrospectively analyzed the records of 38 out of 45 patients who underwent heart transplantation from February 2003 to November 2010 at our center. Patients under 12 years of age and those who died before extubation were excluded. We defi ned prolonged mechanical ventilation as mechanical ventilation longer than 36 hours. Preoperative, intraoperative, and postoperative variables were collected. Reference 1. Cislaghi F, et al.: Minerva Anestesiol 2007, 73:615-621. 1. Cislaghi F, et al.: Minerva Anestesiol 2007, 73:615-621. i Results MIF_AUC, the computed area under the curve of MIF serum levels from admission until POD1, was inversely correlated with SAPS II and SOFA score on POD1 (Table 1). MIF at admission (r = 0.296; P = 0.041) and MIF at 4 hours (r = 0.367; P = 0.012) correlated inversely with the paO2/FiO2 ratios at POD1. Moreover, postoperative MIF values were inversely correlated with SAPS II (r = 0.528; P = 0.044) and SOFA scores during the early postoperative stay (Table 1). In addition, MIF values on POD1 were related to the calculated Cardiac Power Index (r = 0.420; P = 0.009). Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Cardiac-specifi c biomarkers and life-threatening complications of off -pump versus on-pump coronary bypass surgery in Egyptian patients Cardiac-specifi c biomarkers and life-threatening complications of off -pump versus on-pump coronary bypass surgery in Egyptian patients p Methods This cohort study was based on prospectively registered data from the Danish National Vascular Registry and the Danish ICU Database between 1 January 2007 and 1 May 2010. The study population comprised all patients (n = 1293) undergoing open elective, primary aorto-iliac bypass, or aorto-femoral bypass procedures (n = 363) or abdominal aortic aneurysm repair (n = 930) in the eight hospitals performing these procedures in Denmark. The primary endpoints were: ICU stay >24 hours, mechanical ventilation, and acute dialysis. Results Patients in the AAA group were older (70 ± 7 vs. 62 ± 9 years, P <0.001), predominantly males (80 vs. 49%, P <0.001), with a higher prevalence of preoperative cardiac co-morbidity (34 vs. 24%, P = 0.001), p H Elabd1, A Alsherif2, T El Gohary2, M Hagras2, S Salah Eldin2 1Student Hospital, Cairo University, Giza, Egypt; 2Kasr Alaini Hospitals, Cairo, Egypt Critical Care 2012, 16(Suppl 1):P469 (doi: 10.1186/cc11076) gyp Critical Care 2012, 16(Suppl 1):P469 (doi: 10.1186/cc11076) Introduction Coronary artery bypass grafting (CABG) has traditionally been performed with the use of cardiopulmonary bypass (ONCAB). This study aims to compare between on-pump and off -pump surgery concerning postoperative morbidity and mortality, and also to evaluate 6-month graft patency in Egyptian patients. Results Patients in the AAA group were older (70 ± 7 vs. 62 ± 9 years, P <0.001), predominantly males (80 vs. 49%, P <0.001), with a higher prevalence of preoperative cardiac co-morbidity (34 vs. 24%, P = 0.001), S168 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Oxygen delivery index during goal-directed therapy predicts complications and hospital length of stay in patients undergoing high-risk surgery y M Cecconi, N Arulkumaran, R Suleman, D Shearn, M Geisen, J Mellinghoff , D Dawson, J Ball, M Hamilton, M Grounds, A Rhodes St George’s Hospital, London, UK Critical Care 2012, 16(Suppl 1):P474 (doi: 10.1186/cc11081) Introduction National guidance for patients presenting to the emergency department (ED) with a traumatic head injury advises that head computed tomography (CT) should be performed and reported within 1 hour [1]. The operative intervention or injury to knife time should be within 4 hours [2]. With more than 50% of patients requiring neurosurgical intervention in the UK taken to hospitals without onsite neurosurgical services [3], secondary transfer is necessary prior to defi nitive intervention. Are we achieving timely transfers in rural England? Introduction National guidance for patients presenting to the emergency department (ED) with a traumatic head injury advises that head computed tomography (CT) should be performed and reported within 1 hour [1]. The operative intervention or injury to knife time should be within 4 hours [2]. With more than 50% of patients requiring neurosurgical intervention in the UK taken to hospitals without onsite neurosurgical services [3], secondary transfer is necessary prior to defi nitive intervention. Are we achieving timely transfers in rural England? Introduction The aim of this study was to evaluate the effi cacy of a goal-directed therapy (GDT) protocol designed to augment the oxygen delivery index (DO2I) and to assess the relationship between DO2I measurements and postoperative complications and length of stay. Methods A single-centre retrospective cohort study assessing the data obtained during an 8-hour post-operative GDT protocol in consecutive major surgical patients admitted to the ICU. Introduction The aim of this study was to evaluate the effi cacy of a goal-directed therapy (GDT) protocol designed to augment the oxygen delivery index (DO2I) and to assess the relationship between DO2I measurements and postoperative complications and length of stay. Methods A single-centre retrospective cohort study assessing the data obtained during an 8-hour post-operative GDT protocol in consecutive major surgical patients admitted to the ICU. y Methods The Royal Cornwall Hospital is a district general hospital serving a population of 300,000. The regional neurosurgical unit is 100 km away. All patients undergoing transfer to the neurosurgical unit during 2009 were identifi ed. A notes review was undertaken of all these patients transferred to the care of neurosurgeons. The operative logs were also reviewed. Oxygen delivery index during goal-directed therapy predicts complications and hospital length of stay in patients undergoing high-risk surgery Time lines were created of their care from ambulance call to neurosurgical intervention. j g p Results Thirty-seven patients were included. The median DO2I increased over the 8-hour protocol from a baseline level of 407 ml/minute/m2 to a maximum of 537 ml/minute/m2 (P <0.0001) (Figure 1). Twenty-one (57%) patients developed a postoperative complication. Patients who developed zero or one complication had a higher maximum oxygen delivery index DO2I than patients who had more than one complication (602 vs. 477 ml/minute/m2, P = 0.018) (Table 1). The proportion of patients with a length of stay greater than 2 weeks was less in patients who achieved a DO2I of at least 600 ml/minute/m2 (P = 0.035). Results Ten patients in total were transferred for neurosurgical intervention. Two of these patients required two transfers as they were initially seen in satellite minor injury units. No patient had CT within 1 hour of arriving in the ED. The median time was 2 hours 56 minutes. The CT report was available at a median of 3 hours 17 minutes. None of these patients arrived in the tertiary referral centre within 4 hours of their injury. The fastest time to intervention was 8 hours 29 minutes, median 22 hours 59 minutes after injury. 2 Conclusion Postoperative GDT was able to increase DO2I in the postoperative period. Patients who achieved a DO2I of 600 ml/minute/ m2 were less likely to suff er postoperative complications and have a signifi cantly reduced length of hospital stay. y Conclusion We are not meeting targets for CT head acquisition and transfer for neurosurgical intervention. Prompt transfer of a trauma patient from a rural district general hospital in the UK to a tertiary referral centre for neurosurgical intervention is a multifactorial problem. The introduction of trauma centres and of protocols for direct admission to tertiary centres by paramedics may reduce the delays that our audit has highlighted. Figure 1 (abstract P474). Increase in DO2I from baseline to maximum over the 8-hour protocol. Atrial fi brillation following major noncardiac thoracic surgery: signifi cance and impact on morbidity P475 P475 Transfer delays in patients referred for neurosurgical intervention with traumatic brain injury L Smith1, B Jordan2, J Paddle1 1Royal Cornwall Hospital NHS Trust, Truro, UK; 2Derriford Hospital, Plymouth, UK Critical Care 2012, 16(Suppl 1):P475 (doi: 10.1186/cc11082) P475 Transfer delays in patients referred for neurosurgical intervention with traumatic brain injury L Smith1, B Jordan2, J Paddle1 1Royal Cornwall Hospital NHS Trust, Truro, UK; 2Derriford Hospital, Plymouth, UK Critical Care 2012, 16(Suppl 1):P475 (doi: 10.1186/cc11082) P475 Transfer delays in patients referred for neurosurgical intervention with traumatic brain injury References 1. Head Injury Triage: Assessment, Investigation and Early Management of Head Injury in Infants, Children and Adults, Methods Evidence and Guidance. Commissioned by the National Institute for Health and Clinical Excellence. [http://guidance.nice.org.uk/CG56/NICEGuidance/pdf/English] p g g p g 2. Better Care for the Severely Injured. Joint Report. Royal College of Surgeons of England and British Orthopaedic Association. [http://www.rcseng.ac.uk/ publications/docs/severely_injured.html]i p y j 3. Trauma: Who Cares? Report. National Confi dential Enquiry into Patient Outcome and Death. [http://www.ncepod.org.uk/2007report2/Downloads/ SIP_report.pdf] P474 Oxygen delivery index during goal-directed therapy predicts complications and hospital length of stay in patients undergoing high-risk surgery M Cecconi, N Arulkumaran, R Suleman, D Shearn, M Geisen, J Mellinghoff , D Dawson, J Ball, M Hamilton, M Grounds, A Rhodes St George’s Hospital, London, UK Critical Care 2012, 16(Suppl 1):P474 (doi: 10.1186/cc11081) Atrial fi brillation following major noncardiac thoracic surgery: signifi cance and impact on morbidity Metaxa Hospital, Athens, Greece Critical Care 2012, 16(Suppl 1):P473 (doi: 10.1186/cc11080) Table 1 (abstract P471) MIF level SOFA 1. POD SOFA 2. POD SOFA 3. POD ICU admission r = –0.2; P = 0.18 r = –0.4; P = 0.11 r = –0.6; P = 0.05 4 hours later r = –0.4; P = 0.40 r = –0.5; P = 0.05 r = –0.8; P = 0.01 MIF_AUC r = –0.4; P = 0.01 r = –0.2; P = 0.55 r = –0.4; P = 0.19 1. POD r = –0.3; P = 0.08 r = –0.6; P = 0.03 r = –0.7; P = 0.02 Conclusion Elevated postoperative MIF levels are inversely correlated with organ dysfunction in patients after cardiac surgery. References 1. Calandra T, et al.: Macrophage migration inhibitory factor: a regulator of innate immunity. Nat Rev Immunol 2003, 3:791-800. 2. Koga K, et al.: Macrophage migration inhibitory factor provides cardioprotection during ischemia/reperfusion by reducing oxidative stress. Antioxid Redox Signal 2011, 14:1191-1202. Introduction Atrial fi brillation (AF) is a common complication after noncardiac thoracic surgery. Its impact on overall mortality has not yet been fully assessed and few data are available on the eff ects of the noncardiac post-thoracotomy AF on clinical outcomes. Methods From July 2006 to July 2011, 226 consecutive patients undergoing lung resection for lung cancer were studied retrospectively. Preoperative data and serial electrocardiograms were evaluated. Hypertension, dyslipidaemia, diabetes mellitus, smoking and advanced age (>75 years) were considered as risk factors. Patients (n = 97) who had structural heart disease or ≥2 risk factors were considered a high- risk group whereas those with <2 risk factors constituted the low-risk group. g p Results Thirty-two patients (14.16%) experienced new-onset post- operative AF. The high-risk group had a 58% incidence of AF compared with 23% in the low-risk group (P <0.001). Moreover, following β-blocker administration, more of the high-risk group required antiarrhythmic treatment with amiodarone than did the low-risk group (67% vs. 35% respectively, P = 0.02). Patients who developed AF had 2. Koga K, et al.: Macrophage migration inhibitory factor provides cardioprotection during ischemia/reperfusion by reducing oxidative stress. Antioxid Redox Signal 2011, 14:1191-1202. Atrial fi brillation following major noncardiac thoracic surgery: signifi cance and impact on morbidity S169 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 P475 Transfer delays in patients referred for neurosurgical intervention with traumatic brain injury L Smith1, B Jordan2, J Paddle1 1Royal Cornwall Hospital NHS Trust, Truro, UK; 2Derriford Hospital, Plymouth, UK Critical Care 2012, 16(Suppl 1):P475 (doi: 10.1186/cc11082) Table 1 (abstract P474). Postoperative complications by achievement of an oxygen delivery index of 600 DO2I >600 DO2I <600 P value Number of patients 16 (43%) 21 (57%) – Complications 13 (29%) 32 (71%) P = 0.003 Mortality 0 (0%) 4 (100%) P = 0.12 Table 1 (abstract P474). Postoperative complications by achievement of an oxygen delivery index of 600 Table 1 (abstract P474). Postoperative complications by achievement of an oxygen delivery index of 600 a signifi cantly longer hospital stay (P <0.01). The 30-day mortality rate was signifi cantly higher in the high-risk group (11% vs. 2%; P = 0.03) but AF was not an independent risk factor for death. In the multivariate analysis, major resection (pneumonectomy) and advanced age were identifi ed as independent risk factors for the development of postoperative AF (P = 0.004 and P = 0.008 respectively).i a signifi cantly longer hospital stay (P <0.01). The 30-day mortality rate was signifi cantly higher in the high-risk group (11% vs. 2%; P = 0.03) but AF was not an independent risk factor for death. In the multivariate analysis, major resection (pneumonectomy) and advanced age were identifi ed as independent risk factors for the development of postoperative AF (P = 0.004 and P = 0.008 respectively).i DO2I >600 DO2I <600 P value Number of patients 16 (43%) 21 (57%) – Complications 13 (29%) 32 (71%) P = 0.003 Mortality 0 (0%) 4 (100%) P = 0.12 Conclusion Atrial fi brillation occurrence after lung resection does not independently aff ect the short-term mortality but is associated with a prolonged length of hospital stay. Performances of ventilator at simulated altitude 1 k1 l 1 l h1 Performances of ventilator at simulated altitude E Forsans1, L Franck1, T Leclerc1, M Bensalah1, J Tourtier1, Y Auroy1, C Bourrilhon2 1HIA Val-de-Grâce, Paris, France; 2Institut de Recherche Biomédicale des Armées, Brétigny sur Orges, France Critical Care 2012, 16(Suppl 1):P476 (doi: 10.1186/cc11083) E Forsans1, L Franck1, T Leclerc1, M Bensalah1, J Tourtier1, Y Auroy1, C Bourrilhon2 1HIA Val-de-Grâce, Paris, France; 2Institut de Recherche Biomédicale des Armées, Brétigny sur Orges, France Critical Care 2012, 16(Suppl 1):P476 (doi: 10.1186/cc11083) E Forsans1, L Franck1, T Leclerc1, M Bensalah1, J Tourtier1, Y Auroy1, C Bourrilhon2 C Bourrilhon 1HIA Val-de-Grâce, Paris, France; 2Institut de Recherche Biomédicale des Armées, Brétigny sur Orges, France Critical Care 2012, 16(Suppl 1):P476 (doi: 10.1186/cc11083) Introduction We have assessed the ability of three ventilators to deliver to a normal lung model a set tidal volume (Vt) at diff erent simulated cabin altitudes. We studied the performance of the LTV-1200 (Viasys Healthcare, USA), the Elisée 350 (Resmed, Australia) and the Medumat transport (Weinmann, Germany). Figure 1 (abstract P474). Increase in DO2I from baseline to maximum over the 8-hour protocol. Figure 1 (abstract P474). Increase in DO2I from baseline to maximum over the 8-hour protocol. S170 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 critical care capacity and as a consequence the surge in admissions had a signifi cant impact on both critical care and critical care dependent hospital services. Figure 1 (abstract P476). p Methods Data were collected prospectively through the Critical Care Minimum Data Set: the number of critical care admissions with confi rmed or highly suspected infl uenza, co-morbidities, mortality rate, level 3 bed day occupancy, number and mode of advanced respiratory support days, numbers of nonclinical and clinical transfers, and numbers of cancelled operations requiring critical care. p q g Results In a 10-week period 128 patients in Wales required critical care with infl uenza. A total of 1,692 level 3 bed days were required. There are 95 potential level 3 beds across Wales per day. Therefore >25% of level 3 beds over 10 weeks were occupied by infl uenza patients. Fifty percent of patients had signifi cant comorbidities; pregnancy, COPD, morbid obesity, immunocompromise (Figure 1). The overall mortality rate for all aff ected critical care patients was 23.4%. Mortality was 25% in those with comorbidities and 22% in those without. Performances of ventilator at simulated altitude 1 k1 l 1 l h1 The overall mortality rate for all aff ected patients treated in Wales during the 2009/10 infl uenza pandemic was 9.6%. The UK has fewer critical care beds per head of population than comparable nations, and Wales fewer still so critical care in Wales is more vulnerable to surges in admissions. This was apparent in the peak in nonclinical critical care transfers seen during this period, performed due to units exceeding their capacity, and in an increase in cancellations of elective surgery requiring critical care. Methods We used a decompression chamber to mimic the hypobaric environment at a range of simulated cabin altitudes of 2,438 and 3,657 m (8,000 and 12,000 feet). Ventilators were tested with a set fraction of inspired oxygen of 50% and Vt set at 450. Respiratory rate was 12 breaths/minute. Comparisons of preset to actual measured values were accomplished using a t test for each altitude. The protocol included 36 measurements for each Vt set at each simulated altitude. A signifi cant diff erence was defi ned by P <0.05. Methods We used a decompression chamber to mimic the hypobaric environment at a range of simulated cabin altitudes of 2,438 and 3,657 m (8,000 and 12,000 feet). Ventilators were tested with a set fraction of inspired oxygen of 50% and Vt set at 450. Respiratory rate was 12 breaths/minute. Comparisons of preset to actual measured values were accomplished using a t test for each altitude. The protocol included 36 measurements for each Vt set at each simulated altitude. A signifi cant diff erence was defi ned by P <0.05. Conclusion The shortage of critical care capacity in Wales is made more apparent during times of increased critical care requirement such as the infl uenza in the winter 2010/2011. Hospital services are increasingly dependent on critical care, and government and health boards need to provide targeted increases in critical care bed provision to match those levels in other similar nations to mitigate the eff ect on critical care and dependent services due to surges in demand. Results Figure 1 summarizes the data. Comparisons of actual delivered Vt in altitude and set Vt demonstrated a signifi cant diff erence for the three ventilators. P478f 8 Eff ects of levels of clinical supervision during simulated ICU scenarios on resident learning and patient care: a qualitative study D Piquette1, M Mylopoulos2, VR LeBlanc3 1Sunnybrook Health Sciences Centre, Toronto, Canada; 2SickKids Hospital, Toronto, Canada; 3Wilson Centre, Toronto, Canada Critical Care 2012, 16(Suppl 1):P478 (doi: 10.1186/cc11085) Performances of ventilator at simulated altitude 1 k1 l 1 l h1 Conclusion The LTV-1200 showed a very signifi cant increase in Vt delivered with increasing altitude (suggesting a lack of effi cacy of altimetric correction in hypobaric conditions), whereas the Elisée 350 and Medumat transport delivered respectively a stable and a rather stable Vt. P479 P479 Virtual reality and live scenario simulation: options for training medical students in mass casualty incident triage PL Ingrassia, L Ragazzoni, L Carenzo, FL Barra, D Colombo, G Gugliotta, F Della Corte CRIMEDIM Research Center in Disaster and Emergency Medicine, Novara, Italy Critical Care 2012, 16(Suppl 1):P479 (doi: 10.1186/cc11086) Introduction Multicasualty triage is the process of establishing the priority of care among casualties in disaster management. Recent mass casualty incidents (MCI) revealed that health personnel are unfamiliar with the triage protocols. The objective of this study is to compare the relative impact of two simulation-based methods for training medical students in mass casualty triage using the Simple Triage and Rapid Treatment (START) algorithm. Results The transportation time by ambulance was shorter for the fi rst time since statistics were fi rst kept in 1999, the mean time was 33.7 minutes in 2009 and 33.2 minutes in April 2011. Furthermore, the new system is expected to reduce the operational costs by 40,000,000 yen a year. The data on the transportation time by ambulance are continually stored in the system and analyses are continuing. Conclusion The introduction of iPad to the new 99 Saga Net has three benefi cial points. First, the utilization of information and communication technology is useful for a realistic emergency medical setting. Second, the situation of a realistic emergency medical setting is visualized in real time. Finally, both the emergency personnel and the medical staff in the hospital share the information in an emergency medical setting by eliminating vertically divided administrative functions. Medical personnel will work with local governments in the future to analyze the data from this new system. Methods A prospective randomized controlled longitudinal study. Medical students enrolled in the emergency medicine course were randomized into two groups (A and B). On day 1, group A students were exposed to a virtual reality (VR) scenario and group B students were exposed to a live scenario (LS), both exercises aiming at triaging 10 victims in a limited period of time (30 seconds/victim). On day 2 all students attended a 2-hour lecture about medical disaster management and START. On day 3 group A and B students were exposed to a LS and to a VR scenario respectively. The vital signs and clinical condition of the 10 victims were identical in the two scenarios. Impact of H1N1 infl uenza on critical care and dependent services in Wales during winter 2010/2011 Introduction Closer clinical supervision of residents is often perceived as a double-edged sword, improving patient safety but limiting resident participation in patient care. There has been little empirical research on the educational eff ects of closer supervision. We examined the impact of levels of clinical supervision on clinical learning and patient care during acute simulated resuscitation. Introduction Infl uenza H1N1 admissions to critical care from December 2010 to January 2011 had a signifi cant impact on intensive care bed occupancy across Wales. Wales is relatively underprovided in Methods Fifty-four ICU residents (PGY1 to 4) were randomly assigned to complete a simulated ICU scenario in one of three levels of Figure 1 (abstract P477). Infl uenza admissions into critical care in winter 2010/2011. Figure 1 (abstract P477). Infl uenza admissions into critical care in winter 2010/2011. S171 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 supervision (physical proximity of supervising ICU fellow: distant, immediately available, direct). In-person and telephone interactions between participants were recorded and transcribed. We conducted an inductive thematic analysis of anonymized transcripts using constant comparison within and between scenarios. Distributed cognition theory was used as a framework to guide analysis. supervision (physical proximity of supervising ICU fellow: distant, immediately available, direct). In-person and telephone interactions between participants were recorded and transcribed. We conducted an inductive thematic analysis of anonymized transcripts using constant comparison within and between scenarios. Distributed cognition theory was used as a framework to guide analysis. prompting critical decisions in mass casualty drills. In the beginning the average time to complete the VR scenario was higher than the LS. This could be due to the fact that on day 1 very detailed VR victims created a higher challenge for untaught students. However, the higher triage accuracy recorded at the end of day 3 in VR could be explained by a lower stress level compared to the LS, which could be creating a more stressful environment in taught students. y g y Results Both distant and direct levels of supervision resulted in variable involvement of residents in patient care. A shift of control over patient care from residents to fellows often occurred regardless of the physical distance of the fellow. Direct supervision did not always result in decreased resident contributions. Impact of H1N1 infl uenza on critical care and dependent services in Wales during winter 2010/2011 Fellows were found to facilitate more elaborated cognitive contributions from the residents during direct supervision. In addition, practicing in the presence of a supervisor was more likely to lead to timely feedback. However, a presence at the bedside allowed fellows to infl uence the nature of resident involvement by delegating specifi c tasks such as technical procedures. During distant supervision, fellows had to use residents as proxies to obtain information about patients and to deliver care, with potentially serious consequences: when residents’ interpretations of the clinical information were problematic, the quality of fellows’ clinical decisions was negatively aff ected. Higher cognitive work required of fellows during distant supervision appeared to limit their ability to invest cognitive resources in teaching. P480 P479 Ability of the groups to manage a simulated triage scenario was then compared (times and triage accuracy). Reference 1. Fire and Disaster Management Agency: White Paper on Fire and Disaster (2010) [in Japanese]. [http://www.fdma.go.jp/html/hakusho/h22/h22/html/2-4-5_4.html] p g y Results Groups A and B were composed of 25 and 28 students respectively. During day 1 group A LS triage accuracy was 58%, while the average time to assess all patients was 4 minutes 28 seconds. The group B VR scenario triage accuracy was 52%, while the average time to complete the assessment was 5 minutes 18 seconds. During day 3 the triage accuracy for group A VR simulation was 92%, while the average time was 3 minutes 53 seconds. Group B triage accuracy during the LS was 84%, with an average time of 3 minutes 25 seconds. Triage scores improved signifi cantly during day 3 (P <0.001) in the two groups. The time to complete each scenario decreased signifi cantly from day 1 to day 3. Utilization of iPad in the system of emergency demand and acceptance Utilization of iPad in the system of emergency demand and acceptance K Yamada1, Y Sakamoto1, Y Enjiyouji2 1Saga University, Saga, Japan; 2Saga Prefectural Government, Saga, Japan Critical Care 2012, 16(Suppl 1):P480 (doi: 10.1186/cc11087) Introduction This study reports that the transportation time by ambulance was shorter following the introduction of iPad (Apple, Inc.) to the current system of emergency demand and acceptance in Saga Prefecture, Japan. There were about over 5,000,000 ambulance dispatches in Japan, and the time for transportation is increasing (the national average: 36.1 minutes) [1]. The administration has made various eff orts nationwide that did not achieve any positive results. Although the information system of medical institutions and the emergency medical service (99 Saga Net) was established in 2003 in Saga, it has been underutilized. The Saga prefectural government renewed the previous system as the real-time system of emergency demand and acceptance for the fi rst time in Japan in April 2011. Conclusion Level of clinical supervision was not the main determinant of resident engagement in patient care. Both distantly and directly supervised scenarios presented learning opportunities for residents. Given the observed negative eff ects of distant supervision on patient care, strategies to optimize unique learning opportunities off ered by direct supervision should be investigated. i Methods Cloud computing has provided new system to facilitate Internet access from ambulances. In addition, iPads were put into all ambulances (about 55) and emergency medical technicians can get the picture of acceptable hospitals in real time. Emergency personnel who arrive on the scene select the patient’s symptoms with an iPad, and this new system displays an up-to-date list of acceptable hospitals. The data that the emergency personnel entered into the system from the iPad are uploaded to 99 Saga Net immediately. Therefore, both the emergency personnel and medical staff in the hospital share the information of where the emergency occurred, the transportation and the medical institutes to which patients were transferred in real time. Mass evacuation of victims from emergency areas by medica modules aboard the aircraft of EMERCOM of Russia I Yakirevich, A Popov EMERCOM of Russia, Zhukovsky, Moscow Region, Russia Critical Care 2012, 16(Suppl 1):P481 (doi: 10.1186/cc11088) I Yakirevich, A Popov p EMERCOM of Russia, Zhukovsky, Moscow Region, Russia Critical Care 2012, 16(Suppl 1):P481 (doi: 10.1186/cc11088) Introduction During elimination of medical consequences of various emergencies the issues concerning victims’ mass evacuation to a specialized hospital base are constantly brought up. The physicians of the Central Airmobile Rescue Service of EMERCOM of Russia and the specialists of Kazan Helicopter Plant ‘Zarechye’ developed two types of modules. The Medical Airplane Module (MMS) is used for medical Introduction During elimination of medical consequences of various emergencies the issues concerning victims’ mass evacuation to a specialized hospital base are constantly brought up. The physicians of the Central Airmobile Rescue Service of EMERCOM of Russia and the specialists of Kazan Helicopter Plant ‘Zarechye’ developed two types of modules. The Medical Airplane Module (MMS) is used for medical Conclusion The study demonstrates that the training course generates signifi cant improvement in triage accuracy and speed. It also reveals that VR simulation compared to live exercises has equivalent results in S172 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion Our triage system shows a good interrater reliability and validity in predicting resource consumption. To our knowledge, this is the fi rst prospective Italian study that tests the relationships between the triage category assigned by the nurses (using a four-level triage method) and resource consumption. Conclusion Our triage system shows a good interrater reliability and validity in predicting resource consumption. To our knowledge, this is the fi rst prospective Italian study that tests the relationships between the triage category assigned by the nurses (using a four-level triage method) and resource consumption. evacuation of four victims aboard Ilyushin 76 aircraft. The Medical Helicopter Module (MMV) is used for medical evacuation of two victims aboard an MI 8 helicopter. MMS and MMV advantages are: mobility – the possibility of installation in various aircraft cabins types; and versatility – the possibility of any required equipment installation for the treatment of victims with various trauma severity, safe fi xation of medical equipment straight on the module, equipment operation off - line as well as using the aircraft power supply network. g p pp y Methods From December 2008 until now 28 medical evacuations were carried out using MMS aboard Iluyshin 76 aircraft: traffi c accident victims, terrorism act victims and manmade catastrophes. P483 Coordination of emergency resources after Lorca’s earthquakes L Escobar1, A Ferrández2, J Jimenez1, A Peláez1, A Corbatón3, R Alvaro4 1Hospital Rafael Méndez, Lorca, Spain; 2Gerencia 061, Murcia, Spain; 3Hospital Clínico San Carlos, Madrid, Spain; 4Área II Servicio Cantabro de Salud, Santander, Spain Critical Care 2012, 16(Suppl 1):P483 (doi: 10.1186/cc11090) Introduction This work’s purpose is to describe the coordination of diff erent medical resources after Lorca’s 2011 earthquakes. They caused 11 deaths, including two pregnant women and their babies, many injured, moderate or severe damage to 80% of the buildings, and more than 30,000 people without shelter.i p p Methods A descriptive study of the fi les of Murcia’s Emergency Coordination Center (ECC) on the activation of resources after the earthquakes. Results MMS and MMV application in case of mass evacuation in fl ight ensures spare victims’ transportation, total monitoring and treatment continuity. It enables one to carry out anesthetic and resuscitation treatment, intensive care, monitoring and treatment of all the victims. Conclusion The quality of mass medical evacuation of extremely injured victims has considerably improved and the time of transportation from emergency area to specialized hospitals to render them effi cient medical aid has reduced. Results MMS and MMV application in case of mass evacuation in fl ight ensures spare victims’ transportation, total monitoring and treatment continuity. It enables one to carry out anesthetic and resuscitation treatment, intensive care, monitoring and treatment of all the victims. Results Time 17:06 hours: fi rst call. Local resources and city emergency plan are activated. Four medical teams (UME) are pre-activated. 18:49 hours: incoming calls alert of buildings crumbling, dead among the rubble, and hundreds of injured. 18:55 hours: seven UME from fi ve cities are sent to Lorca. 19:00 hours: telephone communications collapse. The ECC uses its internal network. An Advanced Command Point (ACP) is established with a fi eld hospital. 19:10 hours: Rafael Mendez Hospital (225 patients) has to be evacuated. Medical personnel of the hospital, private ambulances and UMEs begin the evacuation. The emergency service of the hospital continues to be operative in the building until evacuation is completed and in a fi eld hospital later. 19:20 hours: the Military Emergencies Unit is required for activation. 19:30 hours: the military and emergency services fi eld hospitals are sent to Lorca. 19:50 hours: Virgen del Alcázar Hospital has to be evacuated (145 patients). P482 Reliability and validity of an Italian four-level emergency triage system N Parenti1, G Rastelli2, C Ferri2, V Serventi1, R Lazzari3, L Sarli1 1University of Parma, Bologna, Italy; 2Ospedale Fidenza, Italy; 3University of Modena, Italy Critical Care 2012, 16(Suppl 1):P482 (doi: 10.1186/cc11089) Critical Care 2012, 16(Suppl 1):P482 (doi: 10.1186/cc11089) Introduction The goal of this study is to assess the reliability and validity of a four-level emergency triage system (Urgency Category (UC) 1 = immediate response; UC 2, 3 and 4 assessment within 20, 60 and 120 minutes respectively) used in an Italian large urban hospital with 60,000 emergency department (ED) visits annually. Methods Three triage nurses, using our triage system, independently assigned, at the same time, triage scores to each patient admitted to the ED from June to August 2011. We collected demographic and clinical characteristics, nurse triage category, resources used for each triage code (for example, laboratory tests, EKG, radiographs, procedures), admission status and site, nurse triage forms that included presenting complaint, vital signs, and pain score. For each scenario, the most frequent UC (the mode) has been considered as true triage. Weighted kappa (K) was used to calculate inter-rater reliability. Validity was evaluated by studying the relationships between the triage category assigned by the nurses and resource consumption. Conclusion Coordination of the diff erent medical and emergency services by the ECC made possible correct use of resources and fast attention to the population that minimized the eff ects of the catastrophe. Figure 1 (abstract P483). Thirty thousand people need shelter after the earthquakes. g y p Results A total of 315 patients admitted to the ED were included in the study randomly (35 were excluded for incomplete data). Mean age was 47 years. Five patients were admitted to the ICU, 48 to nonintensive units. Trauma was the most frequent symptom at triage (44%). The mean time of rating was 2 minutes. The UCs assigned were: 14% with UC 4, 60% UC 3, 25.7% UC 2, 0.3% UC 1. We found 2/315 (0.6%) cases with a marked discordance (2 or more points), 69/315 (21.9%) cases with partial agreement (2/3) and 244/315 (77.5%) cases with a complete agreement (5/5) among nurses who used the triage method. Interrater reliability among the three nurses was K = 0.71 (CI: 0.58 to 0.84). Hospital admission by our triage system was as follows: 1 (100%), 2 (30%), 3 (12%), 4 (2%). Mass evacuation of victims from emergency areas by medica modules aboard the aircraft of EMERCOM of Russia I Yakirevich, A Popov EMERCOM of Russia, Zhukovsky, Moscow Region, Russia Critical Care 2012, 16(Suppl 1):P481 (doi: 10.1186/cc11088) In total, 198 patients were evacuated (including 12 children), 55 victims with artifi cial lung ventilation (ALV). Medical evacuation of severely injured children and adults from regional hospitals to Moscow specialized hospitals in order to provide effi cient and modern medical aid was carried out using MMV. In total, 27 patients were evacuated (including fi ve children), fi ve patients with ALV. The majority of victims were in severe and extremely severe conditions with associated multisystem trauma. Closed craniocerebral injury was observed in 75% of victims with mass aff ection of locomotor apparatus, mine and explosion trauma, gunshot wounds, burn shock and burn disease. Constant monitoring, oxygen therapy, ALV, analgesia and sedation, intensive and anti-shock care as well as wound dressing were carried out in fl ight. The victims’ general condition was evaluated according to the Glasgow Coma Scale, APACHE II and SOFA scales.l P483 20:25 hours: at the ACP fi eld hospital of the Red Cross, Civil Protection and Emergency Services are being set. 20:30 hours: 11 hospitals in six provinces are contacted to relocate evacuated patients. 20:40 hours: all buildings in Lorca are have been evacuated. Thirty thousand people need shelter. Ten camps with tents are set throughout Lorca by the Red Cross, Emergency Services and Civil Protection to give shelter to 16,000 people. See Figure 1.f g Conclusion The quality of mass medical evacuation of extremely injured victims has considerably improved and the time of transportation from emergency area to specialized hospitals to render them effi cient medical aid has reduced. P484 Lightning injuries in a lightning city: a district hospital experience in Singapore factor analysis to analyze responses with regard to factors such as face wash, toilets, sleep, clothes, and food. p Results The overall response rate was 59.5% (n = 47/79); the response rate was 38.3% (n = 18/47) for medical doctors, 36.2% (n = 17/47) for nurses, and 25.5% (n = 12/47) for logisticians. The mean length of career was 16.5 years (standard deviation, 9.75). Descriptive statistics revealed that the participants reported high satisfaction with regard to the command system and consistent satisfaction with regard to membership. However, some were unsatisfi ed with the deployment length. Almost all participants wanted to be part of a relief team if given an opportunity again. Factor analysis derived one factor (eigenvalue shows 3.48 (one factor), 0.33 (two factors), 0.17 (three factors), and 0.13 (four factors)) as comfort. Face wash (–0.95) contributed the most satisfaction compared to other factors such as toilets (–0.86), sleep (–0.81), clothes (–0.74), and food (–0.69).i Y Mok, P Tan, R Jagadesan Changi General Hospital, Singapore Critical Care 2012, 16(Suppl 1):P484 (doi: 10.1186/cc11091) Introduction Tropical Singapore’s meterological profi le makes it one of the world’s lightning capitals. This study aims to assess the profi le of the at-risk patient, and possibly identify factors predicting the length of hospital stay in patients with lightning injuries over a period of 11 years. Methods This is an 11-year retrospective study of patients who were admitted to Changi General Hospital, the only hospital located in eastern Singapore, from 2000 to 2011 with the diagnosis of lightning injuries. j Results There were a total of 27 subjects, with 25 (95.6%) males and two (7.4%) females in the sample. Their age ranged from 17 to 62 years; 63% of the subjects were between 20 and 40 years old. All except three subjects had no comorbidities, with the latter having only hypertension or hyperlipidemia. Most of the events occurred during two periods, March to April and October to December, which is consistent with previously observed seasonal peaks. The length of hospital stay ranged from 1 to 10 days for all patients, except one who stayed for 78 days and one who was transferred to another hospital. Six patients (22.2%) required admission to the ICU or high dependency. There were three mortalities, all found in asystole at the incident site and also suff ered hypoxic ischemic encephalopathy (HIE). P482 The mean of resources used for each triage code was: 4.5 (SD 2.2) for UC 2; 3.2 (SD = 1.67) for UC 3; 1.89 (SD 0.84) for UC 4. Figure 1 (abstract P483). Thirty thousand people need shelter after the earthquakes. S173 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 References 1. Chao TC, et al.: A study of lightning deaths in Singapore. Singapore Med J 1981, 22:150-157. 1. Chao TC, et al.: A study of lightning deaths in Singapore. Singapore Med J 1981, 22:150-157. 2. Feng Z, et al.: Lightning city. The Straits Times, 22 November 2011, B1. Nuclear disaster and the medical problems during the earthquake in Japan, 2011 Y Haraguchi, Y Tomyasu, H Nishi, M Sakai, E Hoshino, M Hoshino, T Takeda-Nozawa Japanese Compendium Team for Disaster Medicine, Kawasaki, Japan Critical Care 2012, 16(Suppl 1):P486 (doi: 10.1186/cc11093) Introduction The roles of medicine including intensivists against natural mega-disaster followed by artifi cial disaster are discussed. Methods The Higashinihon earthquake caused more than 2,000 deaths or missing, which was followed by the Fukushima Daiichi nuclear plant explosion. This study was mainly studied based upon on the actual experience in and around the nuclear station.fi Results Many medical teams, rescue teams and public offi cials worked hard. However, many serious problems are revealed, even if they are limited to the medical fi elds, which are as follows: inappropriate basic preparedness against the largest degree of mega-disaster; lack of offi cial education for medical teams against special disaster, such as nuclear disaster (that is, most members of the Japan DMAT or disaster medical assistance team seemed to be laypersons); incorrect standard/ rules of Japan DMAT, which were thought to be excessively focused upon the cure of the injured patients and a planned short period or nearly 48 hours; and insuffi cient consideration for the weak people or CWAP: children, (pregnant) women, aged people, and the poor people/ sick patients. Many CWAP seemed not to have survived. Conclusion The results of this study add to the small but increasing literature on lightning injuries and may serve to increase physician awareness in this medical niche. References 1. Singh G, et al.: Psychiatry Clin Neurosci 2003, 57:333-336. 2. Kondo Y, et al.: Jpn J Reanimatol 2011, 30:77-81. Satisfaction survey among medical staff involved in relief operations following the Great East Japan Earthquake and Tsunami Satisfaction survey among medical staff involved in relief operations following the Great East Japan Earthquake and Tsunami Y Kondo1, T Abe2, S Deguchi3, Y Kuba4, H Mitsunaga5, H Sekiguchi1, K Kohshi1, I Kukita1 , 1University of the Ryukyus, Nishihara, Japan; 2Mito Kyodo General Hospital, University of Tsukuba, Mito, Japan; 3Graduate School of Health Sciences, Meio University, Nago, Japan; 4Medical Corporation Kariyushi Heart-Life Hospital, Nishihara, Japan; 5Tokyo Institute of Technology, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P485 (doi: 10.1186/cc11092) 1University of the Ryukyus, Nishihara, Japan; 2Mito Kyodo General Hospital, University of Tsukuba, Mito, Japan; 3Graduate School of Health Sciences, Meio University, Nago, Japan; 4Medical Corporation Kariyushi Heart-Life Hospital, Nishihara, Japan; 5Tokyo Institute of Technology, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P485 (doi: 10.1186/cc11092) P487 P485 p y Conclusion In order to cope with the mega-disaster, it became evident that it is insuffi cient to take makeshift measures or use cheap tricks. Working out the systematization of disaster medicine, based upon the academic viewpoints and philosophy/reliability, is essential to protect the people and the nation too. P485 Satisfaction survey among medical staff involved in relief operations following the Great East Japan Earthquake and Tsunami Y Kondo1, T Abe2, S Deguchi3, Y Kuba4, H Mitsunaga5, H Sekiguchi1, K Kohshi1, I Kukita1 1University of the Ryukyus, Nishihara, Japan; 2Mito Kyodo General Hospital, University of Tsukuba, Mito, Japan; 3Graduate School of Health Sciences, Meio University, Nago, Japan; 4Medical Corporation Kariyushi Heart-Life Hospital, Nishihara, Japan; 5Tokyo Institute of Technology, Tokyo, Japan Critical Care 2012, 16(Suppl 1):P485 (doi: 10.1186/cc11092) P484 Lightning injuries in a lightning city: a district hospital experience in Singapore Seventeen (63%) events were occupation related with all occurring either at the airbase or open construction sites. Although there were reportedly six mechanisms of lightning injuries (direct strike, contact injury, side fl ash, ground current, upward streamer and blast injury) this study only established two types of mechanisms – direct strike and contact injury – amongst our patients. Clinical and biochemical parameters that were studied included cardiovascular morbidity, rhabdomyolysis, otologic injuries, burns, acute kidney injury and neurological complications. The small numbers limited a statistical analysis for any correlations between clinical factors and prognosis as well as hospital length of stay. Nevertheless, it is notable that all three deaths had asystole arrest at presentation, developed HIE, and a trend towards a higher serum creatinine on admission. Conclusion Almost all participants were satisfi ed with their level of comfort, and the infl uence of factors responsible for this comfort in descending order were face wash, toilets, sleep, clothes, and food. References Family meetings and end-of-life decision-making in Thai critically ill patients P Chatrkaw1, R Champunot2, W Riyakul3 1Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Buddhachinaraj Hospital, Phitsanulok, Thailand; 3Chulalongkorn Hospital, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P489 (doi: 10.1186/cc11096) P Chatrkaw1, R Champunot2, W Riyakul3 1Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Buddhachinaraj Hospital, Phitsanulok, Thailand; 3Chulalongkorn Hospital, Bangkok, Thailand Critical Care 2012, 16(Suppl 1):P489 (doi: 10.1186/cc11096) p Results All stressor categories were diff erently reported by the three groups analysed: environmental (S = 17 (15 to 19), R = 15 (13 to 18), P = 10 (8 to 11), P <0.01), relationships (S = 23 (21 to 25), R = 20.5 (17 to 24.5), P = 14 (11 to 17), P <0.01), emotional (S = 25.5 (23 to 28), R = 24 (20 to 26), P = 18 (15 to 22), P <0.01), and physical (S = 35 (31 to 38), R = 33 (26.5 to 37), P = 27 (21 to 30), P <0.01). Among the staff members, nurses overestimated more than attending physicians, while trainees are closer to relatives’ perception (P = 0.03). Staff members used to conscious sedation overestimate less the impact of environmental stressors (P = 0.03). Years of experience (r = 0.24, P = 0.03) and age (r = 0.27, P = 0.01) are related to stressor overestimation among staff members.f Introduction Limitation of life-sustaining therapy after critical illnesses in Thailand is uncommon. The barriers may be uncertain prognosis, wrong sense of doctor duty, guilty feeling, confl icts on the goals of care and fear of liability. Therefore we set a formal healthcare team meeting followed by a family meeting to fi nd the balance between curative and palliative intention. The objective was to determine the nature and eff ects of family meetings in the Thai social context. f y g Methods A descriptive, retrospective analysis of charts and preference forms after family meetings in the surgical ICU during 2003 to 2005. Close family members were invited and encouraged to express their ideas and feelings. Conclusion Members of the staff should reconsider their beliefs on patients’ perception of stressors. We argue that such an overestimation may bring inappropriate administration of analgesic and sedative drugs, particularly for nurses and older members of staff . Relatives might be useful intermediaries to have a better insight of patients’ perception. Results Thirty-one family meetings were analysed. P489 categories; higher scores refer to a higher stressfulness. The median (IQR) was calculated for each category. Twenty-eight high-risk critically ill at discharge, 55 relatives 48 hours after admission of their next of kin, and a total of 125 staff members (55 attending physicians, 40 nurses and 30 medical students/specialist trainees) were interviewed. Fifty-six of the staff members were used to keep patients consciously sedated as for local guidelines; the remaining used deeper levels of sedation. Nonparametric tests were used as needed. Reference Figure 1 (abstract P489). Preferences for care at the end of life after family meetings. 1. Novaes: Intensive Care Med 1997, 23:1282. Infl uence of burnout on attitudes of ICU doctors and nurses towards liberalization of visiting polices Results From the fi rst measurement it occurred that 66.7% of the relatives faced severe symptoms of PTSD (scores >33) and from the second measurement it occurred that 70% of family members were identifi ed as cases of severe stress symptoms too. No correlation was found between these symptoms and APACHE II score (P >0.05), indicating that such symptoms exist in family members during the whole patient’s stay in the ICU, regardless of the seriousness of the patient’s condition.f A Giannini1, G Miccinesi2, E Prandi1, M Audisio3, A Bencivinni4, E Biagioni5, E Castenetto6, I Laganà7, R Oggioni4, V Porta8, R Salcuni9, A Sarti10, MG Visconti11, C Borreani12 A Giannini1, G Miccinesi2, E Prandi1, M Audisio3, A Bencivinni4, E Biagioni5, E Castenetto6, I Laganà7, R Oggioni4, V Porta8, R Salcuni9, A Sarti10, MG Visconti11, C Borreani12 1Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy; 2Istituto per lo Studio e la Prevenzione Oncologica, Florence, Italy; 3Ospedale di Ciriè, Italy; 4Ospedale Nuovo del Mugello, Borgo San Lorenzo, Italy; 5Azienda Ospedaliera Universitaria di Modena, Italy; 6Ospedale Civico, Chivasso, Italy; 7Azienda Ospedaliera ‘G. Salesi’, Ancona, Italy; 8Ospedale Civile, Legnano, Italy; 9Ospedale di Ivrea, Italy; 10Ospedale S. Maria Nuova, Florence, Italy; 11Ospedale ‘A. Uboldo’, Cernusco sul Naviglio, Italy; 12Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy Conclusion Relatives of ICU patients seem to suff er from symptoms of PTSD. Nurses who work in the ICU, and have direct and longer contact with patients and relatives too, need to recognize, evaluate and minimize these symptoms in order that further disorders and damage to the relatives’ mental health be prevented. y Critical Care 2012, 16(Suppl 1):P490 (doi: 10.1186/cc11097) Introduction The staff working in the ICU have a complex and stressful job and are at risk of burnout [1]. We conjectured that the presence of a burnout profi le may also infl uence the views of ICU doctors and nurses regarding the liberalization of visiting policies. We investigated P488 Role of ICU nurses in the confrontation of post-traumatic stress disorder in relatives of ICU patients in a general hospital of Athens, Greece M Kourti1, T Katostaras2, G Kallergis2, E Christofi lou1, I Floros1, G Fildisis2 1Laiko General Hospital, Athens, Greece; 2University of Athens, Faculty of Nursing, Athens, Greece Critical Care 2012, 16(Suppl 1):P488 (doi: 10.1186/cc11095) Figure 1 (abstract P489). Preferences for care at the end of life after family meetings. Introduction This study was planned to assess post-traumatic stress disorder (PTSD) in relatives of ICU patients and to evaluate the role of ICU nurses in the confrontation of these symptoms. Introduction This study was planned to assess post-traumatic stress disorder (PTSD) in relatives of ICU patients and to evaluate the role of ICU nurses in the confrontation of these symptoms. Conclusion Withholding intubation and withdrawal therapy are uncommon in Thai people. However, most Thai families prefer not to escalate therapy including CPR. All of them died peacefully and the families were satisfi ed with the care at the end of life. Reference Methods The Impact of Event Scale – Revised (IES-R) was translated and distributed to the family members of patients that were hospitalized in the ICU from August 2008 to September 2010. Two measurements took place: the fi rst one 7 to 10 days from the admission of the patient to the ICU and the second one (to the same relative) after 15 to 20 days from the admission. The maximum IES-R score is 88 (0 to 4 for each one of the 22 questions that constitute the scale). Scores over 33 were interpreted as severe cases of PTSD. Patients’ health condition was evaluated with the APACHE II score before each measurement.i 1. Truog RD, et al.: Crit Care Med 2001, 29:2332-2348. Family meetings and end-of-life decision-making in Thai critically ill patients The mean age of the patients was 65.5 (± 12.9) with mean SAPS II of 55.6 (± 14.9). Three patients were post CPR. Metastatic cancer was the most common underlying condition (45.2%). Most families requested to have full support, except CPR. Around 20% were not ready to make a decision, but fi nally agreed not to escalate therapy. See Figure 1. P488 P488 Role of ICU nurses in the confrontation of post-traumatic stress disorder in relatives of ICU patients in a general hospital of Athens, Greece M Kourti1, T Katostaras2, G Kallergis2, E Christofi lou1, I Floros1, G Fildisis2 1Laiko General Hospital, Athens, Greece; 2University of Athens, Faculty of Nursing, Athens, Greece Critical Care 2012, 16(Suppl 1):P488 (doi: 10.1186/cc11095) Stressors in the ICU: diff erent perceptions of patients, relatives and staff members Stressors in the ICU: diff erent perceptions of patients, relatives and staff members M Umbrello1, G Mistraletti1, B Cerri1, E Carloni1, V Mariani2, A Di Carlo1, F Martinetti1, L D’Amato1, S Miori1, G Iapichino1 1Università degli Studi di Milano, Milan, Italy; 2AO San Paolo – Polo Universitario, Milan, Italy Critical Care 2012, 16(Suppl 1):P487 (doi: 10.1186/cc11094) Introduction We conducted an attitude survey regarding satisfaction among medical staff involved in relief operations following the Great East Japan Earthquake (magnitude 9.0) and Tsunami, which struck Japan on 11 March 2011. The damage was enormous and a number of medical relief teams visited the aff ected area to rescue victims. Our Okinawa medical relief team visited Otuchi, Iwate, on 15 March and provided medical support to the victims for 2.5 months. Introduction The high-risk critically ill are exposed to signifi cant stressors, along with diffi culties in communicating them to relatives and members of the staff . The aim of this study was to compare the perception of stressors as reported by patients (P), relatives (R) and ICU staff members (S). Methods We conducted an anonymous paper survey using self- developed questionnaires. The 79 participants included medical doctors, nurses, and logisticians from medical relief teams involved in rescuing victims of the 2011 Great East Japan Earthquake and Tsunami. Data were analyzed using descriptive statistics. We also performed f Methods A validated questionnaire [1] was used to quantitatively assess discomforts related to the ICU stay. Items were clustered into S174 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 References 1. Azoulay E, et al.: Am J Respir Crit Care Med 2005, 171:987-994. 2. Azoulay E, et al.: Am J Respir Crit Care Med 2001, 163:135-139. 3 Horowitz MJ et al : Psych Med 1979 41:209 218 y , p , 2. Azoulay E, et al.: Am J Respir Crit Care Med 2001, 163:135-139. 1. Azoulay E, et al.: Am J Respir Crit Care Med 2005, 171:987-994. References S175 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 with the development of severe BOS. These results confi rm previous fi ndings and highlight that strategies to decrease BOS in ICU clinicians are urgently warranted. this issue in the course of a survey about the impact on ICU staff of liberalization of visiting policies. this issue in the course of a survey about the impact on ICU staff of liberalization of visiting policies. Methods We administered an anonymous closed-question question- naire to nurses and doctors at eight ICUs that were about to increase the daily visiting time to at least 8 hours, soliciting their views on policy changes in their unit. The ICU staff were asked to fi ll in the same questionnaire a year after implementation. On both occasions we also administered the Maslach Burnout Inventory (a 22-item self-completed questionnaire) to survey the incidence of burnout.i P492 Reference Methods We administered an anonymous closed-question question- naire to nurses and doctors at eight ICUs that were about to increase daily visiting time to at least 8 hours, soliciting their views on policy changes in their unit. The ICU staff were asked to fi ll in the same questionnaire a year after implementation.i 1. Embriaco N, et al.: Curr Opin Crit Care 2007, 13:482-488. Opening the ICU: views of ICU doctors and nurses before and after liberalization of visiting policies Opening the ICU: views of ICU doctors and nurses before and after liberalization of visiting policies A Giannini1, G Miccinesi2, E Prandi1, M Audisio3, A Bencivinni4, E Biagioni5, E Castenetto6, I Laganà7, R Oggioni4, V Porta8, R Salcuni9, A Sarti10, MG Visconti11, C Borreani12 A Giannini1, G Miccinesi2, E Prandi1, M Audisio3, A Bencivinni4, E Biagioni5, E Castenetto6, I Laganà7, R Oggioni4, V Porta8, R Salcuni9, A Sarti10, MG Visconti11, C Borreani12 q y Results The fi rst response rate was 91% (234/258), the second 76% (197/258). Most doctors and nurses gave a favourable opinion regarding changes to visiting policy in both the fi rst (72%) and the second survey (71%). In both phases of the study, the percentage of respondents presenting a profi le compatible with burnout was 36% and 41% respectively. In subjects without burnout there was a marked predominance of a favourable opinion (80% vs. 61%), and this favourable attitude was also maintained a year after the implementation of policy change (79% vs. 59%).l 1Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy; 2Istituto per lo Studio e la Prevenzione Oncologica, Florence, Italy; 3Ospedale di Ciriè, Italy; 4Ospedale Nuovo del Mugello, Borgo San Lorenzo, Italy; 5Azienda Ospedaliera Universitaria di Modena, Italy; 6Ospedale Civico, Chivasso, Italy; 7Azienda Ospedaliera ‘G. Salesi’, Ancona, Italy; 8Ospedale Civile, Legnano, Italy; 9Ospedale di Ivrea, Italy; 10Ospedale S. Maria Nuova, Florence, Italy; 11Ospedale ‘A. Uboldo’, Cernusco sul Naviglio, Italy; 12Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy y Critical Care 2012, 16(Suppl 1):P492 (doi: 10.1186/cc11099) y g Conclusion The presence of burnout has a strong infl uence on the opinion of doctors and nurses regarding liberalization of visiting policies in the ICU. A favourable opinion predominates among ICU staff members without burnout symptoms. In preparing for and assisting the opening of ICUs it is important also to be aware of this aspect and to off er nurses and physicians appropriate support. Introduction Italian ICUs still impose restrictive visiting policies (with a median visiting time of about 2 hours/day); however, a revision of current policies is underway [1-3]. No data are available on the views of Italian ICU teams following an at least partial liberalization of visiting policies. We investigated this issue in the course of a survey about the impact on ICU teams of the liberalization of visiting policies. References Methods A survey was conducted in 12 Uruguayan adult ICUs. The level of BOS was evaluated on the basis of the Maslach Burnout Inventory (MBI score). ICU, patient, and clinician characteristics were assessed for their association with the prevalence of severe BOS (that is, highest MBI scores). All variables with P <0.2 in univariate analysis were included in a model of ordinal regression. P <0.05 was considered statistically signifi cant. 1. Giannini et al.: Intensive Care Med 2008, 34:1256-1262. 2. Giannini et al.: Intensive Care Med 2011, 37:1890. 3 Giannini et al : Pediatr Crit Care Med 2011 12:e46 e50 3. Giannini et al.: Pediatr Crit Care Med 2011, 12:e46–e50. Prevalence, risk factors and impact of severe burnout syndrome in 12 Uruguayan ICUs q y p Results The fi rst response rate was 91% (234/258), the second 76% (197/258). In the fi rst instance, 83% of doctors and 67% of nurses expressed a favourable opinion regarding the change in visiting policy. After 1 year a positive opinion was expressed by 84% of doctors and 63% of nurses. Both phases of the study show a signifi cant predominance of positive opinions among doctors (P = 0.032 and 0.005).f y G Burghi1, J LAmbert2, M Chaize2, C Quiroga3, G Pittini4, M Cancela5, H Bagnulo1, S Chevret2, E Azoulay2 1Hospital Maciel, Montevideo, Uruguay; 2Saint Louis Hospital, Paris, France; 3Hospital Español, Montevideo, Uruguay; 4CAAMEPA, Pando, Uruguay; 5Hospital de Clínicas, Montevideo, Uruguay Critical Care 2012, 16(Suppl 1):P491 (doi: 10.1186/cc11098) Conclusion Most ICU staff members view the opening of the unit positively, and on the whole maintain this opinion 1 year after the policy change. Overall, the attitude of doctors is more favourable than that of nurses. It is essential to build up a picture of the diffi culties that liberalizing visiting could create for ICU staff (and particularly for nurses), and to explore the causes and extent of such diffi culties, in order to identify possible solutions and off er nurses and doctors appropriate support. Introduction Burnout syndrome (BOS) is defi ned as a state of emotional fatigue that leads to a loss of motivation, usually progressing towards feelings of inadequacy and failure. Severe BOS is relevant as it leads to loss of psychological well-being, increased absenteeism and turnover, and deterioration in the quality of care provided to patients. The objective was to determine the prevalence of BOS among Uruguayan ICU clinicians. To evaluate personal or organization characteristics associated with the development of severe BOS. Acknowledgments The study was supported by Associazione per il Bambino Nefropatico (Milan, Italy). Opening the ICU: views of ICU doctors and nurses before and after liberalization of visiting policies Introduction Italian ICUs still impose restrictive visiting policies (with a median visiting time of about 2 hours/day); however, a revision of current policies is underway [1-3]. No data are available on the views of Italian ICU teams following an at least partial liberalization of visiting policies. We investigated this issue in the course of a survey about the impact on ICU teams of the liberalization of visiting policies. Acknowledgments The study was supported by Associazione per il Bambino Nefropatico (Milan, Italy). A family-based satisfaction survey on the ICU D Moult, R Breeze, A Molokhia S176 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results We received 29 completed questionnaires. Relatives of 24 of the respondents had survived to ICU discharge. Responses were linearly transformed to give percentage scores. Higher values represented a greater degree of satisfaction. Overall care in the ICU, 88.8%. Courtesy, respect and compassion to the patient (93.8%), to relatives (92.2%); assessment and treatment of pain (94.4%), breathlessness (92.9%), agitation (88.9%); emotional support (89.4%); care from nurses (92.0%), doctors (95.5%); frequency of communication nurses (92.9%), doctors (89.7%). Overall decision-making, 91.3%. Willingness of staff to answer questions (90.5%); honesty (90.5%), completeness (91.4%), consistency of information (90.5%); inclusion in decision-making, 78.7%; support during decision-making, 78.7%; time to think about information given, 96.2%. to provide some history as a surrogate to patient interview. We think the care we provide should encompass both the patient and their family. This is already accepted practice in the paediatric ICU setting [2]. Communication between family and clinical staff , ideally on a daily basis, is clearly imperative and a systematic approach to improve this is good practice. Increasing insight into relatives’ perceptions and expectations will aid the delivery of high-quality care. We believe that involving relatives in the ward round will be of benefi t for us in our professional relationships with them and improve their understanding during an extremely diffi cult time. to provide some history as a surrogate to patient interview. We think the care we provide should encompass both the patient and their family. This is already accepted practice in the paediatric ICU setting [2]. Communication between family and clinical staff , ideally on a daily basis, is clearly imperative and a systematic approach to improve this is good practice. Increasing insight into relatives’ perceptions and expectations will aid the delivery of high-quality care. We believe that involving relatives in the ward round will be of benefi t for us in our professional relationships with them and improve their understanding during an extremely diffi cult time. fi Methods This was a prospective study over 2 months formally inviting up to four families per day to be present for that part of the ward round involving their relative. Subsequently they were asked to complete a questionnaire anonymously on the experience.l Conclusion Family satisfaction with our ICU is high, with satisfaction high in both care and decision-making domains. Family satisfaction in an interdisciplinary ICU: a quality audit UM S h d R Al T R CK H f Introduction Meeting the needs of family members of patients in the ICU is an important criterion in assessment of quality of care in the ICU. Therefore this study was conducted to determine the immediate needs and level of anxiety of families with traumatic brain injury patients admitted to ICUs in Shiraz, Iran in 2008. Introduction The quality of intensive care medicine depends on multiple indicators [1,2]. Meeting relatives’ needs in the challenging situation of ICU visits is crucial. The aim of this audit was to assess next of kin’s satisfaction and infl uencing factors. Methods In this descriptive cross-sectional study, a convenience sample of 60 family members was recruited over a period of 4 months. On the second day of ICU admission, one family member for each patient who met the study criteria were asked to complete three questionnaires, consisting of the Critical Care Family Need Inventory (CCFNI), the State- trait Anxiety Inventory (STAI) and a demographic data sheet. Methods With institutional approval, questionnaires were distributed to family members of ICU patients. The survey included two visual analogue scale ratings (VAS 1: patient care, VAS 2: decision-making) and 24 questions with four dimensions D1 to D4 (general impression, treatment and patient care quality, professional quality) on a fi ve-point Likert scale, transformed into values 1 to 100. Patient-specifi c and relatives’ sociodemographic data were recorded. Data are presented as the mean ± SD, median (Q.5), interquartile range (IQR) and range (minimum/maximum). Subgroup analysis (relative’s and patient’s age, sex, education, marital status, length of stay, visit frequency and mortality) was performed using the Mann–Whitney U test. y y g Results The mean ages of the subjects were 32.2 years. A total of 10 needs statements in the CCFNI were rated to be important or very important needs by 50 of the 60 families (83.3%); seven were needs for assurance, two were needs for information, and one of them was needs for proximity. The mean of CCFNI satisfaction scores were low (16.5 ± 1.5) for needs to comfort, and high for needs to support (38.1 ± 4.7). Also the mean score of state anxiety (56.75 ± 5.7) and trait anxiety score (52 ± 6.2) was higher than previous studies. Family satisfaction in an interdisciplinary ICU: a quality audit UM S h d R Al T R CK H f y p g y Results Questionnaires of 159 patients were analyzed (patients: age = 66.1 ± 13.0 years, 64% female, SAPS = 38.8 ± 17.5, LOS = 13.5 ± 11.8 Conclusion A needs-based education program can decrease the level of family anxiety and increase the level of satisfaction. Table 1 (abstract P496) Mean ± SD Q.5/IQR Minimum/maximum VAS1 9.1 ± 0.9 9.3/0.9 5.5/10 VAS2 8.6 ± 1.5 9.0/1.5 3.1/10 ∑Satisf 87 ± 15.1 80/20 20/100 D1 91.1 ± 15 100/20 20/100 D2 89.2 ± 13 100/20 40/100 D3 86 ± 15.4 80/20 20/100 D4 85.5 ± 15 80/40 20/100 i References 1. Dodek et al.: Crit Care Med 2004, 32:1922-1927. 1. Dodek et al.: Crit Care Med 2004, 32:1922-1927. Conclusion In this single-centre survey we have demonstrated that inviting families to ICU ward rounds is feasible and we believe that this intervention could improve family satisfaction on the ICU. We are investigating the impact of this intervention with a detailed comparative survey, which we will present in the future. References 1. Dodek et al.: Crit Care Med 2004, 32:1922-1927. 2. Wall et al.: Crit Care Med 2007, 35:271-279. 1. Dodek et al.: Crit Care Med 2004, 32:1922-1927. 2. Wall et al.: Crit Care Med 2007, 35:271-279. 2. Wall et al.: Crit Care Med 2007, 35:271-279. A family-based satisfaction survey on the ICU D Moult, R Breeze, A Molokhia y y D Moult, R Breeze, A Molokhia D Moult, R Breeze, A Molokhia D Moult, R Breeze, A Molokhia Results A total of 364 questionnaires were evaluated, including 282 nurses and 82 ICU physicians. The prevalence of severe BOS was 51% among ICU physicians and 42% in nursing staff . For ICU nurses, factors independently associated with lower MBI scores were the following: work on fi xed days (OR 0.6; 95% CI 0.3 to 0.9; P = 0.01), integrated in-ICU working groups (OR 0.6; 95% CI 0.3 to 0.9; P = 0.02), good relationships with physicians (OR 0.8; 95% CI 0.7 to 0.9; P = 0.008) and good relationships with supervisors (OR 0.8; 95% CI 0.7 to 1; P = 0.05). In contrast, at least one death over the last week was associated with higher MBI score (OR 2; 95% CI 1.2 to 3.2; P = 0.008). For ICU physicians, not being partnered was independently associated with higher MBI scores. Conversely, good relationships with colleagues was associated with lower MBI scores (OR 0,5; 95% CI 0.3 to 0.8; P = 0.004). Interestingly, this study confi rms that clinicians with severe BOS had increased burden such as sleep disorders, libido troubles, lack of memory, inadequate money management as well as the wish to leave the ICU. Conclusion The prevalence of severe BOS is very high among ICU workers in Uruguay. We have identifi ed diff erent risk factors associated University Hospital Lewisham, London, UK University Hospital Lewisham, London, UK Critical Care 2012, 16(Suppl 1):P493 (doi: 10.1186/cc11100) y p Critical Care 2012, 16(Suppl 1):P493 (doi: 10.1186/cc11100) Introduction We conducted a prospective survey to determine satisfaction amongst relatives of patients on our ICU. Patient-reported outcome measures have become widely used and accepted in the pursuit of improved quality of care [1]. However, assessing patient satisfaction is diffi cult on the ICU, an environment where we more commonly communicate with the family of patients regarding the care of their relative. Therefore, a more family-centred approach is indicated, for which family satisfaction questionnaires have already been validated [2]. Methods We utilised a 35-point questionnaire-based survey of relatives of patients in our ICU over 10 weeks. Questionnaires were distributed to family members when the decision to discharge from the ICU was made. We limited this to two family members per patient who were in the ICU for more than 48 hours. Immediate needs and level of anxiety of families with traumatic brain injury patients admitted to ICUsf 1. Patient Satisfaction [www.patientsatisfaction.co.uk] j y S Gholamzadeh, R Abdoli, F Shariff , R Gholamzadeh, 1. Patient Satisfaction [www.patientsatisfaction.co.uk] 2. Aronson et al.: Paediatrics 2009, 124:1120-1125. 1. Patient Satisfaction [www.patientsatisfaction.co.uk] 2. Aronson et al.: Paediatrics 2009, 124:1120-1125. 1. Patient Satisfaction [www.patientsatisfaction.co.uk] 2. Aronson et al.: Paediatrics 2009, 124:1120-1125. S Gholamzadeh, R Abdoli, F Shariff , R Gholamzadeh, A Maraghian Mohammadi Shiraz University of Medical Sciences, Shiraz, Iran Critical Care 2012, 16(Suppl 1):P494 (doi: 10.1186/cc11101) 2. Aronson et al.: Paediatrics 2009, 124:1120-1125. A family-based satisfaction survey on the ICU D Moult, R Breeze, A Molokhia Appropriate inclusion with and support during the decision-making process were areas with lower satisfaction scores. The structuring of options for answering these questions may have been a confounding factor in this fi nding. However, this may represent genuine lower levels of satisfaction and steps should be taken to improve this. In response to these fi ndings we have invited families to join their relatives’ part of the consultant ward round to improve inclusion and support in decision-making. We are currently repeating the survey with these changes in place and will present our fi ndings in the future. Results The results that refl ected 31 ward round attendances were unanimous: every family agreed that their attendance had a positive impact, alleviating misconceptions about the intensive care environment and clarifying the processes involved in the care of their relative. The survey also revealed that attendance at the ward round provided an excellent opportunity to have their questions answered by consultants. All those invited wished to attend and all respondents said the experience was valuable and they would like to attend again. Comments included: ‘Explanations very helpful to deal with the stress of the situation’ and ‘Reassuring to have information delivered professionally and compassionately’. P495 Families: the newest members of the ICU multidisciplinary team R Santhirapala, J Lipton, T Hall, R Breeze, A Molokhia University Hospital Lewisham, London, UK Critical Care 2012, 16(Suppl 1):P495 (doi: 10.1186/cc11102) Families: the newest members of the ICU multidisciplinary team R Santhirapala, J Lipton, T Hall, R Breeze, A Molokhia University Hospital Lewisham, London, UK Critical Care 2012, 16(Suppl 1):P495 (doi: 10.1186/cc11102) Introduction We have started inviting the relatives of our patients to remain present during our multidisciplinary team ICU ward round. The aim is to improve their understanding of the complex activity on an ICU and reduce inconsistencies in communication. In the UK it is becoming expected practice that patient satisfaction is an endpoint we should be measuring and improving [1]. Assessing this on the ICU is often very diffi cult due to the confounding factors inherent to critical illness. We often seek assent from families for procedures and S177 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Methods A retrospective analysis of all patients that were treated in the medical ICU of a large German university hospital in 2009 and 2010 and died during their hospital stay. Methods A retrospective analysis of all patients that were treated in the medical ICU of a large German university hospital in 2009 and 2010 and died during their hospital stay. days, mortality = 16.5%; relatives: age = 44.5 ± 26.9 years, 63.7% female, 13% medical/25.5% higher education). High satisfaction (VAS 1/2, D1 to D4) was observed (Table 1). Signifi cant diff erences within subgroups were found: relatives with healthcare education showed higher D1 to D4 satisfaction than the ones with a graduate degree only. Higher VAS scorings were observed from next of kin with high visit frequency (≥5×/ week). g p y Results During the observation period 3,401 patients were treated in our ICU. The ICU mortality was 15% (n = 501), hospital mortality was 19% (n = 658). The mean predictive mortality derived from the SAPS 2 score was 29% for all patients (standardized mortality ratio 0.67), deceased patients had a predictive mortality of 56%. Of all deceased, 232 (35%) had received CPR, 170 of those (73%) outside the ICU. Of all patients who died in the hospital, 126 (19%) had received unlimited therapy. Incidence of post-traumatic stress, anxiety and depression symptoms in patients and relatives during the ICU stay and after discharge Lovell Federal Healthcare Center, North Chicago, IL, USA; 2Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Critical Care 2012, 16(Suppl 1):P499 (doi: 10.1186/cc11106) Introduction The eff ect of the full moon (the lunar eff ect) on human behaviour has occupied researchers for centuries. We aim to determine such a lunar eff ect on mortality among patients admitted to the ICU. Methods We analyze the electronic medical records of patients admitted to the ICU. The subjects were divided into two groups: patients who died on full moon days (14th,15th, and 16th days of the lunar month) and the patients who died on other days of the lunar month. The mortality rates were calculated for patients in both groups. Parameters including age, gender, acute physiology and chronic health evaluation (APACHE) III scores, predicted mortality, type of ICU, and actual mortality were compared between the two groups. Student’s t test was performed to determine whether there were any diff erences between the groups. Introduction The eff ect of the full moon (the lunar eff ect) on human behaviour has occupied researchers for centuries. We aim to determine such a lunar eff ect on mortality among patients admitted to the ICU. Introduction The eff ect of the full moon (the lunar eff ect) on human behaviour has occupied researchers for centuries. We aim to determine such a lunar eff ect on mortality among patients admitted to the ICU. Methods We analyze the electronic medical records of patients admitted to the ICU. The subjects were divided into two groups: patients who died on full moon days (14th,15th, and 16th days of the lunar month) and the patients who died on other days of the lunar month. The mortality rates were calculated for patients in both groups. Parameters including age, gender, acute physiology and chronic health evaluation (APACHE) III scores, predicted mortality, type of ICU, and actual mortality were compared between the two groups. Student’s t test was performed to determine whether there were any diff erences between the groups. y Conclusion At least one-third of family members visiting patients in the ICU suff er from symptoms of anxiety, depression or both. The level of post-traumatic stress symptoms in family members was high after ICU discharge. Depression, anxiety and post-traumatic stress symptoms were higher among family members compared to patients. Female gender and oncologic diagnosis were strongly associated with depression and post-traumatic stress. p References 1. Myhren H, et al.: Crit Care 2010, 14:R14. 2. Pochard F, et al.: Crit Care Med 2001, 29:1893-1897. 3. Fumis R, et al.: Intensive Care Med 2009, 35:899-902. References 1. Intensive Care Med 2007, 33:1913-1920. 2. Intensive Care Med 2009, 35:2051-2059. 1. Intensive Care Med 2007, 33:1913-1920. 2. Intensive Care Med 2009, 35:2051-2059. 1. Intensive Care Med 2007, 33:1913-1920. 2. Intensive Care Med 2009, 35:2051-2059. P498 Conclusion The full moon does not seem to aff ect the mortality of patients admitted to the ICU. P495 Life support was withdrawn in 245 patients (37%) and life support was withheld in 241 patients (36%). In 46 patients (7%) palliative care was instituted right from the beginning of the ICU stay. In 104 cases (16%) the patients themselves made the EOL decision, in 78 cases (12%) an advance directive was present. A legally designated healthcare proxy was involved in 8%. In 541 cases (82%) the relatives were integrated in EOL decisions with the objective of fi nding a broad consensus; however, in these cases the assessment of the medical indication and the prognosis by the medical team was of particular importance. Cases in which relatives were not involved in EOL decisions were in 76% cases with short unsuccessful maximal therapy, for example CPR (median ICU stay 5 hours). The rate of life support withdrawal was highest (60%) in patients with CNS diseases. We did not experience any serious or unsolvable confl icts with relatives. Involvement of a law court was necessary in none of the cases. Conclusion Relatives of ICU patients were in general highly satisfi ed. The educational status and ICU visit frequency of the next of kin were revealed to be infl uencing factors. Incidence of post-traumatic stress, anxiety and depression symptoms in patients and relatives during the ICU stay and after discharge Further actions might be adopted to diminish the incidence of these disorders. Results Data from 4,387 patients who were followed for 23 months were analyzed. Overall, 297 patients died during this period, including 31 patients on full moon days and 266 patients on the other days of the month. Both groups were similar in terms of age (73 vs. 71 years, P = 0.39), APACHE III scores (82.06 vs. 76.52, P = 0.28), and predicted mortality (0.405 vs. 0.370, P = 0.48). There was no diff erence in the frequency of death between the full moon days and the other days (10.33 vs. 9.85, P = 0.81). See Table 1.f Incidence of post-traumatic stress, anxiety and depression symptoms in patients and relatives during the ICU stay and after discharge R Fumis, P Martins, G Schettino R Fumis, P Martins, G Schettino Hospital Sirio-Libanes, São Paulo, Brazil Hospital Sirio-Libanes, São Paulo, Brazi Critical Care 2012, 16(Suppl 1):P497 (doi: 10.1186/cc11104) Critical Care 2012, 16(Suppl 1):P497 (doi: 10.1186/cc11104) Introduction To study the incidence and predictors of post-traumatic stress, anxiety and depression symptoms in medical and surgical patients and relatives during the ICU stay and at 30 and 90 days post ICU discharge. Methods A prospective study of 72 patients and 99 family members that completed the Hospital Anxiety and Depression Scale during the ICU stay and at 30 and 90 days after discharge. The Impact of Event Scale at 30 and 90 days after ICU discharge was used to evaluate post- traumatic stress disorder (PTSD). Conclusion EOL policies were applied in 81% of our intensive care patients who died during their hospital stay. The new German law regulations served as a practical and realizable basis for EOL policies in our medical ICU. Results The prevalence of symptoms of anxiety, depression or both in patients during the ICU stay was 10%, 2.8% and 6.9% respectively. Among family members prevalence was 17.3%, 6.5% and 14.4% respectively, and was signifi cantly higher compared to patients (P = 0.034). PTSD symptoms were present in 39.8% and 32.7% of family members respectively at 30 and 90 days after discharge. Among patients symptoms were signifi cantly lower (P <0.001). Factors associated with symptoms of anxiety and depression during the ICU stay in a multivariate model included patient-related factors as SAPS 3 (OR 1.1, 95% CI 1.01 to 1.24) and length of family member stay in the ICU (OR 1.39, 95% CI 0.89 to 2.16) and family-related factors as female gender (OR 5.43, 95% CI 0.67 to 43.8) and oncologic diagnosis (OR 0.25, 95% CI 0.05 to 1.31). The multivariate model also identifi ed patient age (OR 0.97, 95% CI 0.93 to 1) and oncologic diagnosis (OR 0.27, 95% CI 0.09 to 0.79) associated with symptoms of post-traumatic stress after discharge among family members. P499 Eff ect of a full moon on mortality of patients admitted to the ICU R Nadeem1, A Nadeem1, E Madbouly1, J Molnar2, J Morrison2 1Captain James A. Growing a positive culture in an ICU antimicrobial stewardship program p p y yi g Results Among 720 patients, 165 were women (22.9%), 506 (70.3%) aged <55 years old and 214 (29.7%) ≥55 years old. Overall mortality was 31.7% and 70.2% of deaths occurring following the WLST [2]. Unadjusted mortality was 41.2% in women versus 28.8% in men (P = 0.003). We observed similar fi ndings in patients <55 years old (30.5 vs. 21.4%, P = 0.06), but not among men and women aged ≥55 years old (55.7 vs. 55.0%, P = 0.43). Adjusted hazard ratios (HRs) showed a nonsignifi cantly increased risk of death in women aged <55 years old as compared to men (1.51 (0.92 to 2.47)), and in women aged ≥55 years old (1.53 (0.94 to 2.50)). We observed no diff erence both in the overall unadjusted incidence of WLST between women and men (73.5 vs. 68.8%, P = 0.47) and in women and men aged <55 years old, while there was a nonsignifi cantly increased rate of WLST in women ≥55 years old (HR 1.53 (0.94 to 2.50)). K Walker, J Litynsky, J Powis K Walker, J Litynsky, J Powis y y Toronto East General Hospital, Toronto, Canada Toronto East General Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P502 (doi: 10.1186/cc11109) Toronto East General Hospital, Toronto, Canada Critical Care 2012, 16(Suppl 1):P502 (doi: 10.1186/cc11109) p Critical Care 2012, 16(Suppl 1):P502 (doi: 10.1186/cc11109) Introduction A 3-month pilot antimicrobial stewardship program (ASP) was initiated in a 490-bed urban community hospital medical/surgical ICU. The ASP continued post pilot. ASP goals are to optimize/reduce antimicrobial (AM) usage, improve clinical outcomes and reduce nosocomial Clostridium diffi cile rates [1,2]. Methods The pilot had one pharmacist (Ph) providing ICU clinical service and one AMPh, both working as ICUPhs. The AMPh collected standardized data on patients and were reviewed with the ID physician; then the AMPh and ID physician discussed with the ICU care team for optimal AM use. Post pilot, the ICUPh assumed the AM stewardship role. The ASP reduced from 5 to 3 days/week. Data collection included the ASP time required and interventions. The same metrics were collected pre/post pilot. Conclusion There may be gender-based diff erences in outcome among patients with severe TBI. Overall, mortality for women tended to be higher, as were decisions for WLST. These diff erences may be due to unmeasured confounders, biologic responses to TBI, or diff erences in level of care decision-making. P500 P500 Potential association of gender with mortality and withdrawal of life-sustaining therapies in patients with severe TBI: a Canadian multicentre cohort study AF Turgeon1, F Lauzier1, A Boutin1, N Côte1, R Zarychanski2, R Fowler3, D Scales3, M Meade4, K Burns3, F Bernard5, D Zygun6, L Moore1, D Fergusson7 1Laval University, Quebec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Toronto, Canada; 4McMaster University, Hamilton, Canada; 5Université de Montréal, Canada; 6University of Calgary, Canada; 7Ottawa Hospital Research Institute, Ottawa, Canada Critical Care 2012, 16(Suppl 1):P500 (doi: 10.1186/cc11107) Application of a new German law as a basis for end-of life decisions in a medical ICU Table 1 (abstract P499). Characteristics of patients who died on full moon days versus other days Full moon Other days P value Age 73.6 ± 14.59 71.07 ± 16.1 0.39 Male/female 15/16 133/133 0.86 APACHE III 82.06 ± 24.1 76.52 ± 27.4 0.28 Mortality 0.405 ± 0.249 0.370 ± 0.268 0.48 Table 1 (abstract P499). Characteristics of patients who died on full moon days versus other days R Riessen, C Bantlin, M Haap University Hospital Tübingen, Germany University Hospital Tübingen, Germany y y Critical Care 2012, 16(Suppl 1):P498 (doi: 10.1186/cc11105) Introduction In 2009 a new German law came into eff ect that clarifi ed issues regarding end-of-life decisions, especially the role of patient autonomy and the importance of a medical indication in the course of treating patients with terminal illness. In this study we analyzed the end-of-life (EOL) policies in our medical ICU with a focus on the practicability of this law. S178 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 References was to introduce advanced computerised infusion pumps with in-built drug safety software, so-called smart pumps, in the ICU to facilitate safer drug administration. 1. Wolbank S, Praus G, Smolle-Juettner F, et al.: The infl uence of lunar phenomena on the incidence of emergency cases. Resuscitation 2003, 58:97-102.f Methods The working group consisted of an ICU pharmacist, clinical nurse specialist and consultant intensivist. A drug library was constructed by the ICU pharmacist and consultant intensivist and loaded onto the infusion pumps. The selection of drugs and dose limits were carefully considered to ensure that they were within the boundaries of normal usage so as not to impede patient management whilst maximising patient safety. Super users then provided individualised training to 85 ICU nurses. In the UK there have been 50 system implementations to date. The national average for compliance with the use of the software is 50%. 2. Alves DW, Allegra JR, Cochrane DG, Cable G: Eff ect of lunar cycle on temporal association in cardiopulmonary arrest in seven emergency departments during eleven years. Eur J Emerg Med 2003, 10:225-228. 2. Alves DW, Allegra JR, Cochrane DG, Cable G: Eff ect of lunar cycle on temporal association in cardiopulmonary arrest in seven emergency departments during eleven years. Eur J Emerg Med 2003, 10:225-228. References 1. Fowler RA, Sabur N, Li P, et al.: CMAJ 2007, 177:1513-1519. 2. Turgeon AF, Lauzier F, Simard JF, et al.; Canadian Critical Care Trials Group: CMAJ 2011, 183:1581-1588. References 1. Upton D: 2011 Why are so few infusions smart? Hosp Pharm Eur 57:39-42. 2 Camire E, et al.: Medication errors in critical care: risk factors, prevention and disclosure. CMAJ 2009, 180:936-943. Potential association of gender with mortality and withdrawal of life-sustaining therapies in patients with severe TBI: a Canadian multicentre cohort study Results Feedback following training with the new system was very positive. In our ICU, utilisation of the drug safety software during drug administration was 94% within the fi rst 6 months. Of 18,000 drug infusions, only 1,000 were used outside the drug safety software. There were seven over-rides from the high limit. Of these, two were for furosemide where there was a genuine clinical need for a higher dose. On two other occasions the software prevented insulin being administered at 30 units/hour and potassium at 100 mmol/hour. The number of drug errors reduced from three to zero during the study period. This demonstrated that the design of our package was sensitive enough to ensure safe drug administration and suffi ciently practical to enable consistent use of the system. 1Laval University, Quebec, Canada; 2University of Manitoba, Winnipeg, Canada; 3University of Toronto, Canada; 4McMaster University, Hamilton, Canada; 5Université de Montréal, Canada; 6University of Calgary, Canada; 7Ottawa Hospital Research Institute, Ottawa, Canada Critical Care 2012, 16(Suppl 1):P500 (doi: 10.1186/cc11107) Introduction Diff erences in admission patterns, delivery of care and outcomes between women and men admitted to the ICU have been previously identifi ed [1]. However, these observations have not been well described in patients with traumatic brain injury (TBI). Our objective was to identify diff erences in outcomes between women and men with severe TBI. y Conclusion We have demonstrated that the introduction of an advanced computerised infusion pump system in the ICU can provide a safer drug administration environment if the appropriate health professionals are selected to implement the system, the drug library is constructed carefully and a comprehensive training package is applied. References Methods We used data from a large retrospectively cohort study in which adults with severe TBI (GCS ≤8) admitted to six Canadian level I trauma centres (2005 to 2006) were identifi ed through health records using ICD-10 codes [2]. Demographic, severity of illness, and outcome data were collected by trained abstractors. The primary outcome was the diff erence in mortality and withdrawal of life-sustaining therapies (WLST) between women and men; secondary outcome was the impact of age (<55 vs. ≥55 years old) among genders. Analyses included chi- square tests and Cox regression analyses adjusted for GCS motor and pupillary reactivity, with stratifi cation for age. Growing a positive culture in an ICU antimicrobial stewardship program Results The ASP total patient recommendations/100 patient-days were 5-day mean 9.3, 3-day mean 13.5 (P = 0.030) with an increased ICU physician acceptance (5 days = 95.9%, 3 days = 99.7%). Statistically signifi cant was an increase in recommendations to broaden therapy (Table 1) and nonstatistically signifi cant was a reduction in recommendations to de-escalate therapy (5-day mean 1.4 recommendations/100 patient-days, 3-day mean 1.2 recom mendations/100 patient-days; P = 0.601). Also, there was an increase in recommendations for duration optimization (5-day mean 4.0 recommendations/100 patient-days, 3-day mean 6.0 recommendations/100 patient-days; P = 0.055) and discontinue AMs (5-day mean 2.7 recommendations/100 patient-days; 3-day mean 3.7 recommendations/100 patient-days; P = 0.181). The ASP mean time required (minutes/month) was reduced (5 days 864, 3 days 771; P = 0.267). 1. Fowler RA, Sabur N, Li P, et al.: CMAJ 2007, 177:1513-1519. 2 Turgeon AF Lauzier F Simard JF et al ; Canadian Critical Care Trials Group: 2. Alves DW, Allegra JR, Cochrane DG, Cable G: Eff ect of lunar cycle on temporal association in cardiopulmonary arrest in seven emergency departments during eleven years. Eur J Emerg Med 2003, 10:225-228. P503 Injectional anthrax: the infl ammatory response M Booth, A Hart, L Donaldson Royal Infi rmary, Glasgow, UK Critical Care 2012, 16(Suppl 1):P503 (doi: 10.1186/cc11110) Methods This study employed a Delphi selection process involving 38 intensivist participants using a web-based survey tool. In Round 1, participants were presented with 15 interventions proposed by investigators. Using a fi ve-item Likert scale, they responded to the question: ‘In your opinion as an expert, how suffi cient is the evidence that this intervention reduces the risk of ALI in eligible patients?’ Participants were also prompted to comment and submit additional items for consideration. In Round 2, participants followed the same approach to rate and comment on items submitted by the group. Finally, in Round 3, participants reviewed aggregated ratings and comments for all items, and voted for or against inclusion in the draft checklist. Inclusion was limited a priori to items with at least 70% agreement among participants. Introduction From December 2009 to July 2010 there were 47 cases of anthrax amongst injecting drug users in Scotland with 13 fatalities. The majority presented as severe soft tissue infection following i.v. injection or muscle popping as described by Ringertz and colleagues [1]. At fi rst they were diagnosed as necrotising fasciitis (NF) until the diagnosis of anthrax was made. With experience they appeared to have a milder infl ammatory response to their infection compared to other soft tissue infections such as NF. To investigate this the anthrax group was compared to a cohort of confi rmed NF cases. pi Methods Patients admitted to the ICU with NF or injectional anthrax from 1 January 2008 to 30 June 2011 were identifi ed. The white blood count (WBC) and C-reactive protein (CRP) at presentation were recorded. Demographic data (sex, age, ICU and hospital LOS, APACHE II score, predicted and actual hospital mortality and drug-injecting history) were retrieved. All data were collected prospectively for routine ICU management. g g p p Results Following Round 1, items submitted by participants were aggregated with minimal change into six additional items for Round 2. In Round 3, of the 21 total items, nine were endorsed by 70% of participants for inclusion in a draft checklist. These items were grouped conceptually into two domains: respiratory support and resuscitation. Conclusion The Delphi process of expert consensus can be employed to develop a checklist of time-sensitive interventions, in a manner that combines available evidence with the perspective of expert clinicians. Results There were six patients with injectional anthrax and 16 with NF. References 1. Dellit TH, et al.: Clin Infect Dis 2007, 44:159-177. 2. Polk RE, et al.: Clin Infect Dis 2007, 44:664-670. Multicenter consensus development of a checklist for lung injury prevention p JM Litell1, O Gajic1, J Sevransky2, M Gong3, DJ Murphy2 1Mayo Clinic, Rochester, MN, USA; 2Emory University School of Medicine, Atlanta, GA, USA; 3Montefi ore Medical Center, Bronx, NY, USA Critical Care 2012, 16(Suppl 1):P504 (doi: 10.1186/cc11111) JM Litell1, O Gajic1, J Sevransky2, M Gong3, DJ Murphy2 1Mayo Clinic, Rochester, MN, USA; 2Emory University School of Medicine, Atlanta, GA, USA; 3Montefi ore Medical Center, Bronx, NY, USA Critical Care 2012, 16(Suppl 1):P504 (doi: 10.1186/cc11111) Introduction Acute lung injury (ALI) is linked to almost 75,000 US deaths annually. The syndrome is defi ned clinically by criteria that identify only patients with established ALI, at which point treatment options are limited and largely supportive. After 40 years and more than 25 NIH- funded trials of ALI interventions, only supportive therapy with lung protective ventilation has been associated with a mortality benefi t. The US Critical Illness and Injuries Trials Group lung injury prevention subgroup seeks to standardize best practices for patients at risk of ALI. The recently validated lung injury prediction score (LIPS) identifi es patients at risk of ALI, and can prompt the early use of preventative interventions. This may attenuate the progression to ALI. This study seeks expert consensus about best practices in patients at risk of ALI, as determined by their LIPS. These practices will be incorporated into a checklist for lung injury prevention. Standardization of care may protect patients against ALI development and provide a uniform background for enrollment in other ALI trials. P501 P501 Making drug delivery in the ICU safer: the implementation of advanced computerised intravenous infusion pumps A Dimech, A Le Page, P Gruber, T Wigmore The Royal Marsden, London, UK Critical Care 2012, 16(Suppl 1):P501 (doi: 10.1186/cc11108) Introduction Drug administration errors account for approximately 78% of all medical errors occurring in ICUs [1,2]. The aim of this project Conclusion ASP reduction from 5 to 3 days/week was successful. Necessary skills were developed by the ICUPh. ASP reduction increased S179 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 recommendations for discontinuation and prospective duration optimization goals of AMs. A reduction in recommendations to de- escalate therapy and an increase in broadening therapy may refl ect an increased acceptance goals. The 3-day ASP also demonstrated an increase in total recommendations/100 patient-days and a reduction in the total time required which enhanced use of resources, both fi nancial and human. References Table 1 (abstract P502) ASP days Recommendations/ 100 patient-days 5-day mean 3-day mean P value Broaden 0.4 1.6 0.003 P504 Multicenter consensus development of a checklist for lung injury prevention JM Litell1, O Gajic1, J Sevransky2, M Gong3, DJ Murphy2 1Mayo Clinic, Rochester, MN, USA; 2Emory University School of Medicine, Atlanta, GA, USA; 3Montefi ore Medical Center, Bronx, NY, USA Critical Care 2012, 16(Suppl 1):P504 (doi: 10.1186/cc11111) P503 Injectional anthrax: the infl ammatory response M Booth, A Hart, L Donaldson Royal Infi rmary, Glasgow, UK Critical Care 2012, 16(Suppl 1):P503 (doi: 10.1186/cc11110) The results are presented in Table 1. There was a marked diff erence in the infl ammatory response between the two groups with the CRP being highly statistically signifi cant. The anthrax group was also younger (35.5 vs. 43.2) with a lower severity of illness, lower predicted mortality (18.6% vs. 31.7%) but much higher actual mortality. P506 Introduction The purpose of the medical emergency team (MET) is to fi nd and treat deteriorating ward patients. Suboptimal care and delays on general wards before admission to intensive care have an eff ect on mortality [1] and patients admitted from general wards have a worse outcome than from the operating room (OR) or emergency department (ED) [2]. MET patients have a high rate of ICU admissions but whether their outcome diff ers from other patients admitted from the wards has not been studied before. We evaluated characteristics and outcome of ICU patients based on mode of admittance, via the MET versus the conventional way. Introduction The implementation of an in-hospital rapid response system (RRS) could improve the outcome of a deteriorating patient but could increase the medical emergency team (MET) and ICU staff workload [1,2]. y Methods An observational prospective study of patients admitted from general wards to the central ICU at Karolinska University hospital, Stockholm, Sweden in 2007 to 2009. Two groups were identifi ed: admissions directly following a MET call or the conventional way, usually on request from the ward physician. Patients were analyzed for age, gender, co-morbidities, length of stay, severity scoring system (APACHE II) and mortality. Methods A retrospective analysis of the years pre, during and post implementation of a RRS in a 480-bed hospital with a mean of 17,500 admissions/year. Figure 2 (abstract P506). Hospital mortality predicted and observed before, during and after the RRS implementation. Figure 1 (abstract P506). MET calls and ICU admission before, during and after the RRS implementation. Figure 1 (abstract P506). MET calls and ICU admission before, during and after the RRS implementation. Results Of 2,571 ICU admissions, 694 admissions in 643 patients came from the wards. In total, 355 were admitted by the MET and 339 were conventional admissions. Median age was 65 years in the MET group versus 58 years in the conventional group, hospital LOS prior to ICU admission was median 3 days versus 1 day and APACHE II score was a mean of 26 versus 21. They did not diff er as to proportion of invasive ventilator treatment or dialysis but MET patients more often received noninvasive ventilation, 57.2% versus 29.2% (P <0.01). ICU mortality was 14.5% versus 8.9% (P = 0.04) and 30-day mortality 27.0% versus 19.1% (P = 0.02). P505 Most of the calls were from the emergency department and less from medical and surgical wards. The number of ICU admissions did not increase (Figure 1). During the period of study there was a reduction of observed mortality compared to that predicted from SAPS II score, especially in surgical patients (Figure 2). Finally, there was an increase of ICU length of stay (LOS) from 11.5 to 13.7 days and a reduction of hospital LOS from 24 to 23.1 days. Results A total of 3,629 admissions during a 4-year period pre ICON (August 2004 to August 2008) and 1,446 admissions during 18 months post ICON (August 2009 to February 2011) were audited. Following the introduction of ICON the percentage of unplanned admissions fell from 36.68% to 22.9%. These patients also had a lower mortality rate (14.57% vs. 9.36%) and the SMR decreased from 1.55 to 1.35. Results The number of MET calls initially increased from 34 to 56 and then decreased to 39 calls/1,000 admissions/year. Most of the calls were from the emergency department and less from medical and surgical wards. The number of ICU admissions did not increase (Figure 1). During the period of study there was a reduction of observed mortality compared to that predicted from SAPS II score, especially in surgical patients (Figure 2). Finally, there was an increase of ICU length of stay (LOS) from 11.5 to 13.7 days and a reduction of hospital LOS from 24 to 23.1 days. the emergency department and less from medical and surgical wards. The number of ICU admissions did not increase (Figure 1). During the period of study there was a reduction of observed mortality compared to that predicted from SAPS II score, especially in surgical patients (Figure 2). Finally, there was an increase of ICU length of stay (LOS) from 11.5 to 13.7 days and a reduction of hospital LOS from 24 to 23.1 days. Conclusion Our data show that the mortality rate has decreased since the introduction of ICON although a confounding factor could be a concurrent decreased crude mortality rate (5.5% in 2003 to 2004 vs. 4.2% 2008 to 2010) in all paediatric intensive care patients in the UK [2]. Despite this we believe that ICON is a signifi cant contributing factor in identifying and rescuing patients on the wards before further signifi cant deterioration requiring intensive care. Further ongoing audit is required. References 1. Acutely Ill Patients in Hospital: Full Guideline [http://guidance.nice.org.uk/ CG50/Guidance] 1. Acutely Ill Patients in Hospital: Full Guideline [http://guidance.nice.org.uk/ CG50/Guidance] P505 P505 Impact of the Paediatric Intensive Care Outreach Network service on mortality within intensive care K Sadasivam, S Skellett Great Ormond Street Hospital for Children, London, UK Critical Care 2012, 16(Suppl 1):P505 (doi: 10.1186/cc11112) Conclusion Anthrax releases three factors: lethal factor (LF), edema factor (EF) and protective antigen (PA). PA and LF form lethal toxin which kills macrophages and inhibits B-cell and T-cell function so minimising the immune response to anthrax. This is refl ected in the inappropriately low CRP levels at presentation. Severe soft tissue infection in an injecting drug user associated with subjectively poor infl ammatory response should raise the possibility of anthrax infection. Reference Introduction We audited the mortality rate by admission source in our paediatric ICU, a paediatric tertiary referral centre, from 2004 to 2008 and found that the group of emergency unplanned internal admissions had a higher Standardised Mortality Ratio (SMR) of 1.55 compared to a SMR of 1.00 overall for patients admitted to the paediatric ICU. This was in keeping with data from other large paediatric centres [1]. The reasons for the increased mortality for this internal group were not clear and possibly multifactorial. To help address this, a Paediatric Intensive Care Outreach Network (ICON) team was developed and introduced in September 2009. 1. Ringertz SH, et al.: Injectional anthrax in a heroin skin popper. Lancet 2000, 356:1574-1575. 1. Ringertz SH, et al.: Injectional anthrax in a heroin skin popper. Lancet 2000, 356:1574-1575. 1. Ringertz SH, et al.: Injectional anthrax in a heroin skin popper. Lancet 2000, 356:1574-1575. Table 1 (abstract P503) Anthrax NF Number 6 16 APACHE II score 12.2 19.4 P <0.05 Died (%) 66.6 18.8 P <0.05 WBC 11.6 16.0 NS CRP 71.2 287.3 P <0.001 p Methods A before-and-after study design was used to determine diff erences in percentage of admissions, mortality rate and SMR. Data were collected using the PICANet database for emergency unplanned internal admissions before (August 2004 to August 2008) and after implementation of the ICON team (August 2009 to February 2011). PICANet is a national database that audits all paediatric intensive care admissions in the UK [2]. S180 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Results The number of MET calls initially increased from 34 to 56 and then decreased to 39 calls/1,000 admissions/year. References 1. McQuillan et al.: BMJ 1998, 316:1853-1858. 2. Goldhill et al.: Crit Care Med 1998, 26:1337-1345. 1. McQuillan et al.: BMJ 1998, 316:1853-1858. 2. Goldhill et al.: Crit Care Med 1998, 26:1337-1345. 1. McQuillan et al.: BMJ 1998, 316:1853-1858. 2. Goldhill et al.: Crit Care Med 1998, 26:1337-1345. P507 Medical emergency team admittance to intensive care versus conventional admittance: characteristics and outcome G Jäderling, M Bell, CR Martling, A Ekbom, M Bottai, D Konrad Karolinska Institutet, Stockholm, Sweden Critical Care 2012, 16(Suppl 1):P507 (doi: 10.1186/cc11114) 2. Paediatric Intensive Care Audit Network [http://www.picanet.org.uk/] Medical emergency team admittance to intensive care versus conventional admittance: characteristics and outcome q References 2. DeVita MA, et al.: Crit Care Med 2006, 34:2463-2478. 2. DeVita MA, et al.: Crit Care Med 2006, 34:2463-2478. 1. FO Odetola, et al.: Do outcomes vary according to the source of admission to the PICU? Pediatr Crit Care Med 2008, 9:20-25. P505 y p y Conclusion The implementation of RRS could result in a temporary increase of MET calls but not of ICU admissions; moreover, it could lead to a reduction of mortality and hospital LOS, but not of ICU LOS. References P506 MET patients also had a higher proportion of co- morbidities, with a prevalence of heart failure in 17.3% versus 11.7% (P = 0.0.4) and malignancy in 45.3% versus 35.1% (P <0.01) as well as a higher proportion of limitation of medical treatment (LOMT), 23.0% versus 15.7% (P = 0.02). When LOMT patients were excluded, mortality rates were no longer signifi cantly diff erent, ICU mortality then being 5.7% versus 3.3% (P = 0.2). Figure 1 (abstract P506). MET calls and ICU admission before, during and after the RRS implementation. Figure 2 (abstract P506). Hospital mortality predicted and observed before, during and after the RRS implementation. Conclusion Two distinct groups of patients with intensive care needs are found in general wards. Those admitted by the MET are older, have more severe co-morbidities and have been in hospital longer. We fi nd the MET to be an important tool to identify patients with multiple problems and at high risk of an adverse outcome. R f Intensive care services in Hungary 2000 to 2010: an analysis of bed numbers, occupancy rates, case mix and economics gold standard value. The frequency of these diff erences was analysed. Results Both computer and manual systems returned all the required data, giving a total of 700 data variables. Diff erent values were returned for 183 (26%) variables. The systems had good concordance in the demographic variables, with only 4/300 (1.3%) discrepancies between the computer and manual systems. In the organ support variables, there were 179/400 (45%) discrepancies. Days of renal support had most concordance, with discrepancies in 3/50 patients (6%). Days of level 2 support had least concordance, with discrepancies in 37/50 patients (76%). Overall, the computer method returned the correct variable for 544 (78%) variables, where the manual system returned the correct variable on 591 (84%) variables. p y A Csomos1, B Fulesdi2, M Gresz3 , , 1Semmelweis University, Budapest, Hungary; 2University of Debrecen, Hungary; 3National Institute for Quality and Organisational Development in Healthcare, Budapest, Hungary , , 1Semmelweis University, Budapest, Hungary; 2University of Debrecen, Hungary; 3National Institute for Quality and Organisational Development in Healthcare, Budapest, Hungary Critical Care 2012, 16(Suppl 1):P509 (doi: 10.1186/cc11116) Introduction The purpose of this study is to describe the changes in pattern of intensive care (ICU) use over a 10-year period in Hungary. We attempt to analyze national data in order to improve resource use. Methods A retrospective analysis of national data provided by the hospitals for reimbursement of care to the National Healthcare Fund of Hungary between 2000 and 2010. Conclusion This study shows that both computer and manual data collection methods could be improved, but at present both have similar accuracy. This may be because the criteria for some organ support can be subjective (for example, risk of deterioration), which can be interpreted in diff erent ways between manual data collectors but not by a computer. We plan to rewrite the computer program, aiming for >95% concordance with the gold standard. g y Results The total number of active hospital beds decreased by 33.4% (from 65,532 to 44,300); however, the number of ICU beds increased by 9.8% (from 1,189 to 1,306) between 2000 and 2010. As a result, the percentage of ICU beds to hospital beds increased from 1.89% in 2000 to 2.95% in 2010. The ICU bed occupancy rate ranged between 58.43% and 63.78%; it showed no correlation with the case mix index (r2 = 0.2799). P510 P510 Data acquisition for the UK Critical Care Minimum Data Set: validation of a computer model for automatic calculation from an electronic patient record A Clarke, M Thomas, T Gould, C Bourdeaux Bristol Royal Infi rmary, Bristol, UK Critical Care 2012, 16(Suppl 1):P510 (doi: 10.1186/cc11117) P510 Data acquisition for the UK Critical Care Minimum Data Set: validation of a computer model for automatic calculation from an electronic patient record A Clarke, M Thomas, T Gould, C Bourdeaux Bristol Royal Infi rmary, Bristol, UK Critical Care 2012, 16(Suppl 1):P510 (doi: 10.1186/cc11117) P510 Data acquisition for the UK Critical Care Minimum Data Set: validation of a computer model for automatic calculation from an electronic patient record validation of a computer model for automatic calculation from an electronic patient record A Clarke, M Thomas, T Gould, C Bourdeaux Bristol Royal Infi rmary, Bristol, UK Critical Care 2012, 16(Suppl 1):P510 (doi: 10.1186/cc11117) Introduction This study reports the accuracy of a computer and a manual system at collecting data for the UK Critical Care Minimum Data Set (CCMDS). This is required by the Department of Health to compare performance, to facilitate funding and to plan future resource provision. There are 14 data fi elds in the mandatory dataset, and the full compliment extends to 34 fi elds. At present this is collected manually, which is laborious and subjective. We use an electronic patient record (Innovian, Draeger, Germany) to store all the measured patient observations and laboratory results. We have written a program to interrogate Innovian for the CCMDS data, thereby reducing the administrative time. Conclusion Our data suggest that critical care admission decisions are made based mainly on the assessment of patients’ pre-morbid state and functional capacity, rather than on the extent of acute physiological derangement. This behaviour is more consistent with the application of a prioritization model, defi ning those patients who will benefi t most from critical care admission (Priority 1) to those who will not benefi t at all (Priority 4) and consistent with pressured resources, rather than an objective parameters model or a diagnostic model [1]. References Methods A stratifi ed sample of 50 patients’ data (elective and emergency surgical and medical patients) was analysed. Both manual and computer systems collected the mandatory 14 items of the CCMDS. P508 Factors aff ecting critical care admission to a UK university hospital A Tridente1, A Chick1, S Keep1, S Furmanova2, S Webber1, DC Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2University Hospital Wales, Cardiff , UK Critical Care 2012, 16(Suppl 1):P508 (doi: 10.1186/cc11115) Factors aff ecting critical care admission to a UK university hospital A Tridente1, A Chick1, S Keep1, S Furmanova2, S Webber1, DC Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2University Hospital Wales, Cardiff , UK Critical Care 2012, 16(Suppl 1):P508 (doi: 10.1186/cc11115) Factors aff ecting critical care admission to a UK university hospital A Tridente1, A Chick1, S Keep1, S Furmanova2, S Webber1, DC Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2University Hospital Wales, Cardiff , UK Critical Care 2012, 16(Suppl 1):P508 (doi: 10.1186/cc11115) Figure 2 (abstract P506). Hospital mortality predicted and observed before, during and after the RRS implementation. Introduction Access to critical care is limited, with disparity existing between availability and demand. Guidance to inform triage decisions S181 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Table 1 (abstract P509). Distribution of intensive care services in 2010 Total Total Case mix National data, number number index 2010 of units of beds (mean ± SD) P value University hospitals (level III) 10 412 7.67 (± 4.06) 0.204 County hospitals (level II) 30 584 8.08 (± 2.89) 0.376 City hospitals (level I) 39 280 6.05 (± 1.97) 0.093 Table 1 (abstract P509). Distribution of intensive care services in 2010 has been published but may no longer refl ect current pressures [1,2]. We aimed to identify a set of criteria able to reliably predict likelihood of admission to a critical care unit in a large UK tertiary care centre. We aimed to identify a set of criteria able to reliably predict likelihood of admission to a critical care unit in a large UK tertiary care centre. Methods Consecutive patient referrals were prospectively enrolled in a review cohort. Data were collected using a predefi ned case report form (CRF). The CRF included information on the referral, acute physiological parameters, hospital length of stay (LOS), demographic and functional status, dependency and comorbidities. Logistic regression was performed to identify factors predicting admission, employing STATA [3]. Intensive care services in Hungary 2000 to 2010: an analysis of bed numbers, occupancy rates, case mix and economics A Csomos1, B Fulesdi2, M Gresz3 1Semmelweis University, Budapest, Hungary; 2University of Debrecen, Hungary; 3National Institute for Quality and Organisational Development in Healthcare, Budapest, Hungary Critical Care 2012, 16(Suppl 1):P509 (doi: 10.1186/cc11116) Intensive care services in Hungary 2000 to 2010: an analysis of bed numbers, occupancy rates, case mix and economics A Csomos1, B Fulesdi2, M Gresz3 1Semmelweis University, Budapest, Hungary; 2University of Debrecen, Hungary; 3National Institute for Quality and Organisational Development in Healthcare, Budapest, Hungary Critical Care 2012, 16(Suppl 1):P509 (doi: 10.1186/cc11116) P508 Results Between 17 July and 27 November 2011, 201 patients were referred to critical care, of whom 85 (42.7%) were declined. Median age (interquartile range) was 67 (54 to 79) years, 121 (60.8%) were male, median LOS (interquartile range) was 1 (1 to 3) day. Age, gender, ethnic origin, LOS, referral reason, and markers of acute physiological derangement did not impact on likelihood of admission to critical care. Odds ratios (95% CIs) for admission were 3.1 (1.72 to 5.56) for exercise tolerance >100 yards (P <0.001), 3.03 (1.56 to 5.89) for self-caring status (P = 0.001), 0.38 (0.2 to 0.71) for house-bound status (P = 0.003), 0.28 (0.1 to 0.76) for wheelchair-bound status (P = 0.013), 0.41 (0.23 to 0.74) for cardiovascular (P = 0.003), 0.36 (0.18 to 0.72) for renal system (P = 0.004), 0.34 (0.14 to 0.85) for malignant (P = 0.021), and 0.49 (0.25 to 0.94) for neurological (P = 0.033) comorbidities, respectively. P510 This consists of six demographic variables (for example, admission date, discharge date, date of birth) and eight organ support variables (for example, duration of either advanced or basic cardiovascular, respiratory, renal or neurological support or duration of level 2 or 3 support). Where the computer and manual systems returned diff erent values, a blinded physician analysed the patient records and created a gold standard value. The frequency of these diff erences was analysed. Results Both computer and manual systems returned all the required data, giving a total of 700 data variables. Diff erent values were returned for 183 (26%) variables. The systems had good concordance in the demographic variables, with only 4/300 (1.3%) discrepancies between the computer and manual systems. In the organ support variables, there were 179/400 (45%) discrepancies. Days of renal support had most concordance, with discrepancies in 3/50 patients (6%). Days of level 2 support had least concordance, with discrepancies in 37/50 patients (76%). Overall, the computer method returned the correct variable for 544 (78%) variables, where the manual system returned the correct variable on 591 (84%) variables. 1. Guidelines for intensive care unit admission, discharge, and triage. ACCCM, SCCM. Crit Care Med 1999, 27:633-638. 2. Fair allocation of intensive care unit resources. ATS. Am J Respir Crit Care Med 1997, 156:1282-1301. 2. Fair allocation of intensive care unit resources. ATS. Am J Respir Crit Care Med 1997, 156:1282-1301. 3. STATA 10.1. College Station, TX: StataCorp. 3. STATA 10.1. College Station, TX: StataCorp. P511 P511 To admit or not to admit? The suitability of critical care admission criteria D Marriott, Z Turner, N Robin, S Singh Countess of Chester Hospital, Chester, UK Critical Care 2012, 16(Suppl 1):P511 (doi: 10.1186/cc11118) P509 Intensive care services in Hungary 2000 to 2010: an analysis of bed numbers, occupancy rates, case mix and economics A Csomos1, B Fulesdi2, M Gresz3 1Semmelweis University, Budapest, Hungary; 2University of Debrecen, Hungary; 3National Institute for Quality and Organisational Development in Healthcare, Budapest, Hungary Critical Care 2012, 16(Suppl 1):P509 (doi: 10.1186/cc11116) Intensive care services in Hungary 2000 to 2010: an analysis of bed numbers, occupancy rates, case mix and economics The number of ventilator days increased from 28.9% to 66.1%; it showed good correlation with the case mix index (r2 = 0.9125). Analysing 2010 data, we found signifi cantly lower mortality in level III units (30 ± 18%) compared to level II (51 ± 20%) and level I (56 ± 19%) care (P = 0.001 and 0.003), without signifi cant diff erences in case mix index (Table 1). The mean ICU bed occupancy rate was 59.5% (SD ±12%), and length of hospital stay was 12.3 (SD ±3.0) in 2010. Geographic distribution of ICU beds per 100,000 population ranged between 7.3 and 27.4 (nationwide 12.9/100,000); it showed no correlation with regional gross domestic product values (r2 = 0.4593). P511 Assessing demand for intensive care services: the role of readmission rates Introduction Out-of-hours discharge from the ICU is associated with increased mortality. In Scotland, approximately 15% of discharges occur out of hours [1]. The aim of this study was to determine the reasons behind out-of-hours discharges in our hospital and the eff ect this has on mortality. RA O’Leary, B O’Brien Methods We carried out a retrospective analysis of all patients admitted to our ICU over a 3-year period. Patients who died during their ICU stay, patients <16 years, patients transferred to another ICU, and those with missing data were excluded. Data collected: patient demographics, APACHE II score, time of discharge from the ICU, reason for out-of-hours discharge, and hospital mortality. The out-of-hours period was defi ned as per the Scottish Intensive Care Society (SICS) as 20:00 to 07:59 hours, then later re-defi ned as 17:00 to 07:59 hours. Introduction Irish ICUs typically have bed occupancy rates approaching 100%, with 75 to 80% being the recommended level [1]. Detection of excessive demand from simple databases can thus be diffi cult: expedited turnover and cancellations of elective surgery often ensue, leaving occupancy rates unchanged. We hypothesised that excessive demand would produce higher readmission rates, thus illustrating the strain imposed on ICU resources during the H1N1 infl uenza pandemic. Methods The GICU database was examined from 1 March 2010 to 1 March 2011. The H1N1 pandemic was recognised as a period of strain on the ICU and this period was estimated as 24 December 2010 to 21 January 2011. All ICU readmissions during the same hospital stay were noted. Transfers between GICU, cardiac ICU and theatre recovery were excluded as patients were still being treated by the intensive care team. Patients readmitted after transfer for extracorporeal membrane oxygenation (ECMO) were also excluded. i Results A total of 766 patients were included: 607 discharged between 08:00 and 19:59 hours, 159 discharged between 20:00 and 07:59 hours. Data are expressed as mean values (SD) or percentages, ‘in hours’ versus ‘out of hours’. Both groups were similar: age 51.9 (18.1) versus 54.0 (17.7) years, males 48.9% versus 50.9%, APACHE II score 15.8 (8.7) versus 17.4 (8.0). Hospital mortality following ICU discharge was 9.9% (55/607 deaths) versus 10.0% (16/159 deaths), RR 1.11 (95% CI 0.66 to 1.88). Discharge was delayed due to a shortage of ward beds in 28.5% versus 43.4% of cases. No early discharges were recorded. P511 To admit or not to admit? The suitability of critical care admission criteria Introduction During the 2010/2011 winter the H1N1 infl uenza pandemic placed increased demand on critical care services, prompting our department to devise a modifi ed triage tool for the ICU to be implemented at a time of exceptional bed crisis [1]. Scoring systems such as APACHE or Sequential Organ Failure Assessment Introduction During the 2010/2011 winter the H1N1 infl uenza pandemic placed increased demand on critical care services, prompting our department to devise a modifi ed triage tool for the ICU to be implemented at a time of exceptional bed crisis [1]. Scoring systems such as APACHE or Sequential Organ Failure Assessment Conclusion Our data suggest that intensive care beds are not utilized; a progressive level of care does not function and also there are unnecessary regional diff erences in intensive care provision in Hungary. S182 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 (SOFA) have been used to predict mortality and optimize critical care service utilization [2]. This audit aimed to validate our triage tool for patients admitted to the ICU. when medical staffi ng levels on the wards are highest. The SICS defi ne the out-of-hours period based on the time of handover to nightshift. For discharges at this time, there was no increase in mortality. In our hospital, evening ward cover is the same as overnight. For an out- of-hours period of 17:00 to 07:59, there was a signifi cant increase in mortality following out-of-hours discharge. Methods We retrospectively examined patient notes for all admissions to our adult ICU during December 2010 and January 2011. Patient admission criteria (SpO2 <90% on FiO2 >85%, respiratory acidosis pH ≤7.2, respiratory failure or airway compromise, systolic pressure <90 mmHg, SOFA score ≥7) or refusal criteria (SOFA score ≥12, severe trauma, unwitnessed or non-VF arrest, severe life-limiting condition) were recorded with outcome data. y Conclusion Our data show increased mortality following ICU step- down in the evening as well as at night. Discharge was most often delayed due to a lack of ward beds. To reduce mortality, eff orts must therefore be made to improve bed management and ensure discharge from the ICU before 17:00. Results We analysed 27 sets of notes. Twenty-two patients (81%) fulfi lled at least one admission and no refusal criteria. Two patients (7%) had documented refusal criteria. Delayed discharges revisited: impact of a liaison post on patients’ transition from ICU to ward caref J Mellinghoff , P O’Shea, D Dawson, J Ball, A Rhodes, M Grounds St George’s Healthcare NHS Trust, London, UK Critical Care 2012, 16(Suppl 1):P513 (doi: 10.1186/cc11120) Conclusion The proposed admission criteria concurred with clinical decision-making in 81% of admissions. The patients that met refusal criteria required either prolonged hospital stay or had short survival times and may not represent optimal utilization of critical care facilities during a time of increased demand. Those patients not meeting the admission criteria had short critical care stays illustrating that rigid admission requirements may exclude patients who could benefi t from critical care. A standardized set of admission criteria may supplement decision-making during times of increased critical care demand and strengthen documentation of those decisions. However, no set of criteria can replace clinical judgement in critical care admission. References Introduction This audit reviewed the discharge process of patients from an adult general ICU to the general wards before and after the introduction of a liaison nurse post over a 3-year, 3-month time period. Methods The audit utilised routinely collected retrospective data from a 17-bed ICU. We examined the impact of a liaison post on the length of delays on discharge of patients from the ICU to the general wards. Introduction This audit reviewed the discharge process of patients from an adult general ICU to the general wards before and after the introduction of a liaison nurse post over a 3-year, 3-month time period. Methods The audit utilised routinely collected retrospective data from a 17-bed ICU. We examined the impact of a liaison post on the length of delays on discharge of patients from the ICU to the general wards. Results The study period was from April 2008 until June 2011 with the start date of the liaison nurse post in January 2010. Overall, there were 4,327 patient discharges to hospital wards (before group = 2,063, after group = 2,264). The odds of experiencing a delay in discharge >4 hours were 3.2-fold higher in the before group compared to the after group (95% CI = 2.808 to 3.717, P <0.0001). Accumulated discharge delays decreased by 23% from 1,116 (before group) to 864 days (after group) despite an increase in patient turnover of 10% (n = 201). The median delay time was 7.2 hours (IQR 5.0 hours, 10.4 hours) in the before group and 5.3 hours in the after group (IQR 2.7 hours, 9.0 hours). Reference 1. Scottish Intensive Care Society Audit Group: Audit of Critical Care in Scotland 2011, Reporting on 2010; 2011. Edinburgh: ISD Scotland. Out-of-hours discharge from the ICU: defi ning the out-of-hours period and its eff ect on mortality YL Bramma, R Allan, R Sundaram Royal Alexandra Hospital, Paisley, UK Critical Care 2012, 16(Suppl 1):P512 (doi: 10.1186/cc11119) YL Bramma, R Allan, R Sundaram Royal Alexandra Hospital, Paisley, UK Royal Alexandra Hospital, Paisley, UK y y Critical Care 2012, 16(Suppl 1):P512 (doi: 10.1186/cc11119) Delayed discharges revisited: impact of a liaison post on patients’ transition from ICU to ward caref See Figure 1. Conclusion Our analysis suggests that the introduction of a liaison nurse post within intensive care signifi cantly reduced the length of delays in the discharge process despite an increase in patient turnover. y g p g Results The study period was from April 2008 until June 2011 with the start date of the liaison nurse post in January 2010. Overall, there were 4,327 patient discharges to hospital wards (before group = 2,063, after group = 2,264). The odds of experiencing a delay in discharge >4 hours were 3.2-fold higher in the before group compared to the after group (95% CI = 2.808 to 3.717, P <0.0001). Accumulated discharge delays decreased by 23% from 1,116 (before group) to 864 days (after group) despite an increase in patient turnover of 10% (n = 201). The median delay time was 7.2 hours (IQR 5.0 hours, 10.4 hours) in the before group and 5.3 hours in the after group (IQR 2.7 hours, 9.0 hours). See Figure 1. Conclusion Our analysis suggests that the introduction of a liaison nurse post within intensive care signifi cantly reduced the length of delays in the discharge process despite an increase in patient turnover. 1. Christian MD, et al.: Development of a triage protocol for critical care during an infl uenza pandemic. CMAJ 2006, 175:1377-1381. 1. Christian MD, et al.: Development of a triage protocol for critical care during an infl uenza pandemic. CMAJ 2006, 175:1377-1381. 2. Ling CY, et al.: Outcome scoring systems for acute respiratory distress syndrome. Shock 2010, 34:352-357. P511 To admit or not to admit? The suitability of critical care admission criteria The fi rst of these had a severe life-limiting condition, staying 29 days in the ICU and a further 65 days in hospital. The second was admitted post non-VF arrest, dying after 2 days in the ICU. Three patients (11%) met no admission criteria. These patients stayed between 4 and 6 days in critical care with total hospital stays of 18 to 98 days, one requiring 30 days of rehabilitation. Assessing demand for intensive care services: the role of readmission rates With the out-of- hours period re-defi ned: 393 patients were discharged between 08:00 and 16:59 hours, 373 between 17:00 and 07:59 hours. Both groups were similar: age 51.0 (18.4) versus 53.8 (17.5) years, males 49.9% versus 48.8%, APACHE II 14.9 (8.7) versus 17.4 (8.2). Hospital mortality was 7.7% (28/393 deaths) versus 11.5% (43/373 deaths), RR 1.62 (95% CI 1.03 to 2.55). Discharge was delayed due to a shortage of ward beds in 22.7% versus 41.0% of cases. ICU step-down is most safely performed y Results The number of GICU admissions during the period was 422. There were 19 readmissions (readmission rate of 4.6%). However, this rate increased to 8.6% during the period of high activity encompassing the H1N1 pandemic (Figure 1). Hospital mortality was 36.8% in the readmission group, higher than the average, 24.6%, for the whole GICU population. This is in keeping with previous research showing up to an 11-fold increase in relative risk of mortality in patients readmitted to the ICU [2]. S183 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Conclusion The annual readmission rate for our unit was acceptable [3]. A clear spike was noted during the period of the H1N1 pandemic. Whilst this is a pattern we hope to address, it is a useful indicator of increased demand. Our study suggests that readmission trends in a single institution may be helpful when analysing the severity of epidemics, planning staffi ng needs, and comparing periods of P515 Management of cardiac drugs in a critical care setting M Mallick1, J Walkington1, A Gratrix1, R Pretorius2 1Hull Royal Infi rmary, Hull, UK; 2York Teaching Hospital, York, UK Critical Care 2012, 16(Suppl 1):P515 (doi: 10.1186/cc11122) Figure 1 (abstract P513). Number of ward discharges and accumulated delay. Figure 1 (abstract P514). Readmissions over time. Figure 1 (abstract P513). Number of ward discharges and accumulated delay. Figure 1 (abstract P513). Number of ward discharges and accumulated delay. Figure 1 (abstract P513). Number of ward discharges and accumulated delay. Figure 1 (abstract P514). Readmissions over time. Figure 1 (abstract P514). Readmissions over time. Conclusion The annual readmission rate for our unit was acceptable [3]. A clear spike was noted during the period of the H1N1 pandemic. Whilst this is a pattern we hope to address, it is a useful indicator of increased demand. 2. Rosenberg AL, et al.: Crit Care Med 2001, 2 1. Intensive Care Society: Standards for Intensive Care Units. London: ICS; 1997. 3. Rosenberg AL, et al.: Chest 2000, 118:492-502. 2. Rosenberg AL, et al.: Crit Care Med 2001, 29:511-551. 1. Intensive Care Society: Standards for Intensive Care Units. London: ICS; 1997. 2. Rosenberg AL, et al.: Crit Care Med 2001, 29:511-551. Assessing demand for intensive care services: the role of readmission rates Our study suggests that readmission trends in a single institution may be helpful when analysing the severity of epidemics, planning staffi ng needs, and comparing periods of heightened demand. References Introduction ICU admissions may lead to discontinuation of longstanding evidence-based therapies. A recent study demonstrated how such medications have been discontinued for patients even after their ICU stay [1]. Evidence has shown the benefi cial role of β-blockers in the perioperative period [2], and roles for other drugs such as S184 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 angiotensin-converting enzyme inhibitors (ACE-I) and statins have been demonstrated. The aim of the current study was to examine 30- day mortality and complication rates in the critical care population who were on cardiac medications and did not receive these medications during their ICU stay. angiotensin-converting enzyme inhibitors (ACE-I) and statins have been demonstrated. The aim of the current study was to examine 30- day mortality and complication rates in the critical care population who were on cardiac medications and did not receive these medications during their ICU stay. unindicated SUP use; and reduction in inappropriate i.v. administration (23.1% vs. 0%, P = 0.0024). Conclusion Emphasis on the guidelines for SUP to all members of the team, especially the pharmacist, improves compliance. Inclusion in SUP prescriptions of the intended discontinuation date may further reduce excessive duration of treatment. Re-audit will occur after implementation of new guidelines which acknowledge the diminishing benefi t from SUP and the not-insignifi cant risks associated with its use. Reference g y Methods We looked retrospectively at the last 80 patients admitted to the ICU or HDU in York, 2011. The patients’ case notes were examined to assess if they were on cardiac medications and if those drugs were omitted during their admission. The cardiac medications assessed were β-blockers, ACE-I and statins. We also reviewed any cardiac complications incurred during their stay, alongside 30-day mortality. 1. Cook DJ, et al.: Crit Care 2001, 5:368-375. Results A total of 29.6% of patients on β-blockers received them, whilst 67.8% did not. Complication and mortality rates for medications given versus not given were 12.5% versus 68.4% and 0% versus 42.1% (P = 0.003 and P = 0.007) respectively. A total of 17.6% of patients on ACE-I received them, whilst 82.3% did not. Pharmacists and fastidiousness improve compliance with guidelines for stress ulcer prophylaxis S Sanders, KC Shelley, AJ Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P516 (doi: 10.1186/cc11123) Frenchay Hospital, Bristol, UK Conclusion Although most of the ICU staff believe that the CPOE and bar-code has the potential to improve medication safety and the quality of care for critically ill patients, our survey showed a low level of satisfaction 6 months after implementing the system, particularly for physicians who consider the system unfriendly. Reference Introduction This audit assessed compliance with guidelines for the use of stress ulcer prophylaxis (SUP) in our mixed general/neurosurgical ICU. These patients are at increased risk of gastrointestinal bleeding with clinically important bleeding occurring in about 3.5% of patients ventilated for 48 hours or more [1]. SUP guidelines: all patients at risk of stress ulceration (coagulopathy/IPPV >48 hours/nasogastric (n.g.) feed not absorbed) or already on ant acids should receive ranitidine, enterally where possible. Exceptions are patients on a proton pump inhibitor (PPI) prior to ICU admission. PPIs should continue enterally if possible as lanzoprazole, or as omeprazole i.v. Reference 1. Poon EG, et al.: N Engl J Med 2010, 362:1698-1707. 1. Poon EG, et al.: N Engl J Med 2010, 362:1698-1707. P518 Safer ICU trainee handover: a service improvement project E Godfrey1, I Hassan1, A Carson-Stevens2, AG Saayman1 1University Hospital of Wales, Cardiff , UK; 2Cardiff University, Cardiff , UK Critical Care 2012, 16(Suppl 1):P518 (doi: 10.1186/cc11125) p p p Methods Data were collected from May to August 2010 (Period 1). Results from this were discussed and the following interventions adopted prior to further data collection (Period 2: August to November 2011): prescription of SUP in all ventilated patients on admission to the ICU; discontinuation of SUP after 48 hours if n.g. feeding tolerated; documented daily review of SUP including consideration of discontinuation, drug, route and dose used; and the presence of the ICU pharmacist on ward rounds, briefed specifi cally to prompt correct SUP use. Introduction Quality handover between team members within the ICU is vital for patient safety. Critically ill patients are at high risk of medical errors; these complex patients are exposed to high-risk interventions, medical and procedural [1]. Distractions are known to be particularly prevalent within critical care [2]. This can compromise handover effi ciency, interrupt information-giving and may ultimately lead to poorer patient outcomes [3]. We sought to demonstrate the capability of junior physicians to lead change to their practices that benefi t the quality of patient care in a large critical care unit. P517 P517 Healthcare workers’ experience when using an electronic medical order entry and bar-code technology in an ICU R Fumis, I Souza, V Pizzo, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P517 (doi: 10.1186/cc11124) P517 Healthcare workers’ experience when using an electronic medical order entry and bar-code technology in an ICU R Fumis, I Souza, V Pizzo, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P517 (doi: 10.1186/cc11124) Pharmacists and fastidiousness improve compliance with guidelines for stress ulcer prophylaxis S Sanders, KC Shelley, AJ Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P516 (doi: 10.1186/cc11123) P517 Healthcare workers’ experience when using an electronic medical order entry and bar-code technology in an ICU R Fumis, I Souza, V Pizzo, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2012, 16(Suppl 1):P517 (doi: 10.1186/cc11124) Introduction Medication errors are frequent in the ICU and may occur during medical ordering, transcription or administration of drugs. A system consisting of a computerized physician order entry (CPOE) with bar-code verifi cation of medications (TASY; Web Sistemas, Brazil) has been described as a tool to improve medication safety [1], but few data are available about the satisfaction of healthcare workers with the use of this new technology in the ICU. Methods We conducted a survey to evaluate the satisfaction of healthcare workers when using a CPOE with bar-code verifi cation of medications in a tertiary 40-bed adult ICU in Sao Paolo, Brazil 6 months after implementing the system. A satisfaction questionnaire which consisted of items in a numeric scale type from 1 (low satisfaction) to 10 (high satisfaction) was fi lled out by physicians (n = 42), nurses (n = 58), nurses technicians (n = 84) and other professionals (n = 66). gi y g y Conclusion The study does highlight a trend associated with patients who are on medications who do not receive them to either develop higher complication rates or higher mortality rates or both. Further research involving larger numbers is required to produce validated opinions. f p Results Most subjects were female (66%), below 36 years of age (69%) and used the computer daily at home (81%). On average, respondents were satisfi ed with the CPOE system (score 5.74 ± 2.14) and believed it improved safety (score 7.64 ± 2.42). Satisfaction was lower among physicians (score 4.62 ± 1.79) when compared to other professionals (score 5.97 ± 2.14; P <0.0001). The ease to place the fi rst medical order and to copy the order form the previous day scored 5.41 ± 2.05 and 6.39 ± 1.93. The visualization of the medical order with the bar-code verifi cation of drugs administration scored 5.95 ± 2.51 by the nurses. On average, physicians found the system less user-friendly (score 3.88 ± 1.85) than other professionals (6.40 ± 2.29; P <0.0001). p References References 1. Bell CM: JAMA 2011, 306:840-847. 1. Bell CM: JAMA 2011, 306:840-847. 2. 2009 ACCF/AHA focused update on preoperative beta blockade: a report of the American College of Cardiology Foundation/American Heart Association task force on practice guidelines. Circulation 2009, 120:2123-2151. Assessing demand for intensive care services: the role of readmission rates Complication and mortality rates for medications given versus not given were 0% versus 9.0% and 0% versus 35.7% (P = 0.004 and P = 0.055) respectively. A total of 31.6% of patients on statins received them, whilst 68.4% did not. Complication and mortality rates for medications given versus not given were 25.0% versus 42.3% and 8.3% versus 38.5% (P = 0.256 and P = 0.02 respectively). The global complication and mortality rates for medications given versus not given were 28% versus 55.2% and 11.5% versus 51.7% (P = 0.0648 and P = 0.0039) respectively. Omission of β-blockers resulted in signifi cantly higher complication and mortality rates. Omission of ACE-I resulted in higher complication rates and of statins in higher mortality rates. Omission of cardiac medications resulted in a signifi cantly higher mortality rate. Pharmacists and fastidiousness improve compliance with guidelines for stress ulcer prophylaxis S Sanders, KC Shelley, AJ Marsh Frenchay Hospital, Bristol, UK Critical Care 2012, 16(Suppl 1):P516 (doi: 10.1186/cc11123) We present an improvement project that has transformed handover quality in our ICU. Methods Participant observation of handover practices took place within a high-occupancy 33-bed adult ICU. Quantitative assessment of handover criteria as per Royal College of Anaesthetists guidelines Results Period 1 (n = 86) revealed excess use of SUP, excess use of PPIs when ranitidine was indicated, unnecessary i.v. administration and failure to discontinue prophylaxis appropriately. Period 2 (n = 71) demonstrated: no fall in SUP use in those with indications (93% vs. 97%, P = 0.65); increased prescription accuracy in terms of drug, dose and administration route (40% vs. 84%, P = 0.0001); no increased S185 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 [4] was performed at baseline (handovers: n = 6, patients: n = 119) and 3 months post-intervention (handovers: n = 4, patients: n = 108). Interventions included presentation of data at multiprofessional departmental meetings, education of team members regarding frequency of handover interruptions and development and utilisation of an electronic handover tool. P519 ICU handover: are we forgetting something? A preliminary study T Aslanidis, IL Chytas, A Kontos, I Soultati, A Efthmiou, E Geka, V Ourailoglou, E Anastasiou, M Giannakou-Peftoulidou G.H. AHEPA, Thessaloniki, Greece Critical Care 2012, 16(Suppl 1):P519 (doi: 10.1186/cc11126) ICU handover: are we forgetting something? A preliminary study T Aslanidis, IL Chytas, A Kontos, I Soultati, A Efthmiou, E Geka, V Ourailoglou, E Anastasiou, M Giannakou-Peftoulidou G.H. AHEPA, Thessaloniki, Greece Critical Care 2012, 16(Suppl 1):P519 (doi: 10.1186/cc11126) Introduction The aim of this ongoing study is to review the process of handover in a university teaching hospital ICU, highlight areas of special interest and defi ciency during the process, and improve current practice. Clinical handover, defi ned as a process of transferring authority and responsibility for providing care of patients from departing caregiver to named recipient, is a basic part of clinical practice. Failure to exchange essential information and focus on the important may have disastrous consequences for the patient. y Conclusion Although only 28% of discharge summaries achieved an acceptable or higher rating from the ICU team, GPs valued the majority of discharge summaries issued by our ICU. Further research is needed to explain the diff erence between ICU doctors’ perception of discharge summary quality and the value provided by them to GPs. Reference Methods A prospective observational study was undertaken over a 22-day period to examine the quality and content of clinical handover by nightshift doctor to the medical team. Key aspects expected to be handed over included patient details, diagnosis, system – treatment domains and communication with relatives. Additional data collected also included duration of handover and frequency of interruptions. 1. National Institute for Health and Clinical Excellence: Clinical Guideline 83: Rehabilitation after Critical Illness. London: National Institute for Health and Clinical Excellence; 2009 [www.nice.org.uk/CG83]. P520 Quality and value of intensive care discharge summaries for general practitioners Results Provision of patient details during handover was substandard. Utilisation of a structured handover sheet signifi cantly improved the number of patient details provided; in particular, patient age (18% vs. 100%), duration of stay (29% vs. 79%) and medical management plan (53% vs. 93%). Frequent handover interruptions seen on initial observation signifi cantly improved (100% vs. 25% of handover periods interrupted) following our collaboration with the senior nurse, physiotherapist and other team leads regarding the number and nonurgent nature of interruptions; at re-audit, interruptions occurred for clinically urgent requests only. p F Daruwalla, FJ Lamb, CA Mearns Surrey and Sussex Healthcare NHS Trust, Redhill, UK Critical Care 2012, 16(Suppl 1):P520 (doi: 10.1186/cc11127) Introduction Good communication between healthcare professionals is required to provide continuity of care for patients being discharged from the ICU [1]. It is our unit’s practice to send a copy of a patient’s computerized ICU discharge summary to both the hospital team with ongoing responsibility and to their general practitioner (GP). The aim of this study was to establish and compare the quality and value of the summaries as judged by ICU doctors and GPs. Conclusion Simple measures instituted by junior doctors, such as team education and use of a structured handover tool, can aid high-quality handover within critical care. Evidence suggests that high-quality handover within critical care will translate into improved clinical care for patients. j g y Methods Discharge summaries for patients admitted in July 2011 were obtained from the ICU WardWatcher® database. These were scored independently by two ICU consultants and a trainee doctor using a predefi ned rating scale. The GPs were sent postal questionnaires regarding their perceptions of the quality and value of the summaries. A comparison was made between the ratings made by the ICU team and the responses to the GP questionnaires. P521 Results A total of 207 sets of patients were collected during the study period. All handovers were supervised by a consultant intensivist. Clinical information handed over verbally covered reason for admission in 12% of cases, working diagnosis in 13% and current management plan in 29% (100% in these three in new admissions). Medical comorbidities where also poorly covered (8%). The handover was rather focused on special aspects of clinical information like the respiratory system (86%), fl uid balance and laboratory fi ndings (68%), infections status (67%), CNS (56%) and hemodynamics (54%), while nutrition and GI was poorly covered (20%). Only 26% of handovers covered signifi cant changes in the last shift, 21% commented on the interventions made and 32% had a proposed plan for the forthcoming day discussed. Of the allocated 30 minutes, the duration of the handover varied from 20 to 50 minutes (average 28 minutes). There was a total of 34 interruptions over 22 days of the audited period. Reasons for interruption included telephone calls and requests from visiting teams and nurses.i Volume–outcome relationship in critical care: a systematic review DJ Wallace1, YL Nguyen2, L Trinquart2, DC Angus1, P Ravaud2, JM Kahn1 1CRISMA Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 2Centre d’épidémiologie clinique, CHU Hôtel Dieu, Paris, France Critical Care 2012, 16(Suppl 1):P521 (doi: 10.1186/cc11128) Introduction The relationship between provider volume and patient outcome has been demonstrated for many medical and surgical services, including critical care. This relationship is used as one rationale for regionalization of adult intensive care. However, the volume– outcome relationship is not always consistent across studies, and it has not been explicitly evaluated in a heterogeneous population. We performed a systematic review of studies that assessed the association between volume and outcome among critically ill adult patients. Introduction The relationship between provider volume and patient outcome has been demonstrated for many medical and surgical services, including critical care. This relationship is used as one rationale for regionalization of adult intensive care. However, the volume– outcome relationship is not always consistent across studies, and it has not been explicitly evaluated in a heterogeneous population. We performed a systematic review of studies that assessed the association between volume and outcome among critically ill adult patients. References 1. Reader TW, et al.: Curr Opin Crit Care 2007, 13:732-736. 2. Horn J, et al.: Anaesthesia 2004, 59:658-663. 3. Nimmo G, et al.: JICS 2008, 9:240-242. 4. McQuillan P, et al.: In The Royal College of Anaesthetists. Raising the Standard: A Compendium of Audit Recipes. 2nd edition; 2006:218-219 [http://www.rcoa. ac.uk/docs/ARB-section10.pdf]. 4. McQuillan P, et al.: In The Royal College of Anaesthetists. Raising the Standard: A Compendium of Audit Recipes. 2nd edition; 2006:218-219 [http://www.rcoa. ac.uk/docs/ARB-section10.pdf]. Results Sixty patients were admitted during the study period. All 60 summaries were independently rated by three ICU doctors and good inter-rater reliability was demonstrated (Cronbach’s α = 0.89). There was a strong correlation between the ratings given by the ICU consultants and the trainee doctor (Spearman’s = 0.91). Twenty-eight per cent achieved an acceptable score of 6 out of 10 or greater (median score 5, interquartile range 3 to 6). Fifty-four postal questionnaires were sent to GPs and 36 were returned (response rate 67%). Seventy-six per cent achieved an acceptable score of 16 out of 25 or greater (median score 18, interquartile range 16 to 25). Sixty-nine per cent of GPs found the discharge summary helpful and 86% wanted to be sent this type of summary in future. Correlation between the ICU team rating and the GP score for the summaries was weakly positive (Spearman’s = 0.15). References References 1. Patterson ES, et al.: Jt Comm J Qual Patient Saf 2010, 36:52-61. 2. Brenier G, et al.: Crit Care 2011, 15:491. P523 Accuracy of height and weight estimation by critical care staff K Dunne, S Hickey Accuracy of height and weight estimation by critical care sta K Dunne, S Hickey Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P523 (doi: 10.1186/cc11130) y Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P523 (doi: 10.1186/cc11130) y y Results We reviewed 80 studies, of which 27 (34%) met all inclusion criteria. Studies were excluded most commonly when the majority of the patients did not require critical care (n = 46), the study was presented only in abstract form (n = 4), data were duplicative (n = 2) or an outcome measure was not assessed (n = 1). One publication included three diff erent patient populations; these were counted as separate studies. The fi nal 29 studies represented seven clinical categories: respiratory (n = 9), postoperative (n = 7), cardiovascular (n = 4), general admissions (n = 3), sepsis (n = 2), neurological (n = 2) and gastrointestinal (n = 2). Eighteen studies (62%) demonstrated a statistically signifi cant association between higher patient volume and better health outcomes, although the magnitude of the relationship varied across diagnoses. No study showed a statistically signifi cant association between higher volume and poorer outcomes. Forth Valley Royal Hospital, Larbert, UK Critical Care 2012, 16(Suppl 1):P523 (doi: 10.1186/cc11130) Introduction Patient’s height and weight measurements are used regularly within the critical care setting whether for calculation of drug doses, nutritional intake, ventilator settings or calibration of cardiac output monitoring [1]. In sedated patients these parameters are often obtained via estimation by critical care staff . Errors in these estimations have the potential to cause harm either from errors in drug calculations [2], inappropriate ventilatory settings or underfeeding or overfeeding. Methods We asked members of the critical care team (medical, nursing staff , physiotherapists and dieticians) to anonymously estimate the heights and weights of patients within the unit at that time. Following this we obtained accurate measurements by measuring height with a measuring tape and patients’ weight with the Scotweigh weighing machine. The results were then collated and the percentage inaccuracy of estimate compared to actual measurement was calculated.f Conclusion The majority of studies evaluating the volume–outcome relationship in critically ill patients demonstrated better outcomes with higher clinical volumes. P521 Methods We searched the MEDLINE and EMBASE databases for articles published between January 2001 and December 2011 using medical subject heading terms and text words for conditions related to critical illness in adults. Trauma studies were excluded. Two study investigators independently reviewed titles, abstracts and articles identifi ed from the search algorithm and abstracted study-specifi c data using a standardized abstraction form. Variables of interest included study g y p Methods We searched the MEDLINE and EMBASE databases for articles published between January 2001 and December 2011 using medical subject heading terms and text words for conditions related to critical illness in adults. Trauma studies were excluded. Two study investigators independently reviewed titles, abstracts and articles identifi ed from the search algorithm and abstracted study-specifi c data using a standardized abstraction form. Variables of interest included study Conclusion Our study identifi ed that the structure of the handover was rather focused on a system-based approach. Diffi culty in concentration due to fatigue or frequent interruptions prolongs its duration and disturbs the right fl ow of information. The senior clinician must ensure that handover should be a focused but educational experience for the trainee with appropriate feedback. S186 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 characteristics, patient characteristics, study period, volume defi nition, primary and secondary outcomes, risk-adjustment methodology, statistical analyses, results, risk of bias and funding body. characteristics, patient characteristics, study period, volume defi nition, primary and secondary outcomes, risk-adjustment methodology, statistical analyses, results, risk of bias and funding body. P522 Radiation doses in young ICU patients: a cause for concern? RA O’Leary, C Houlihane, P McLaughlin, M Maher, D Breen Cork University Hospital, Cork, Ireland Critical Care 2012, 16(Suppl 1):P522 (doi: 10.1186/cc11129) Conclusion Although height estimation was measured to within 10% accuracy in the majority of cases, staff were considerably less reliable at estimating an accurate patient weight and on more than one-half of all estimates underestimated the weight by greater than 10%. We therefore strongly discourage the practice of weight estimation in situations where clinical decisions are being based on an often unreliable value, and alternative means of obtaining an accurate weight measurement should be sought. Introduction The aim of this study was to quantify the radiation dose in young ICU patients to determine if it approached a clinically signifi cant level. Ionising radiation is a well-recognised risk factor for development of cancer. The risk is dose-related and there is no lower threshold at which the dose can be considered clinically irrelevant. The availability of computed tomography (CT) scanning has led to a signifi cant increase in exposure to ionising radiation of patients over the last decade. Children and young adults are particularly at risk. This is partly because there is a longer lifetime in which radiation eff ects may be manifest but also because children are up to 10 times more sensitive to radiation than adults. In view of these issues it is important to quantify the risk to young ICU patients. References 1. Wigfull J, et al.: Critical assessment of haemodynamic data. Contin Educ Anaesth Crit Care Pain 2005, 5:84-88. 2. Mahajan RP: Medication errors: can we prevent them? Br J Anaesth 2011, 107:3-5. Figure 1 (abstract P523). Accuracy of weight estimation by critical care staff . q y y g p Methods The general ICU database was examined from 1 March 2010 to 1 March 2011. The overall radiation exposure was quantifi ed using the cumulative eff ective radiation dosage (CED) in millisieverts (mSv). The CED was calculated for all of the procedures performed during the stay in the ICU using average procedure-specifi c eff ective doses published by the UK National Radiation Protection Board. A cohort of patients <30 years of age were selected for subanalysis. y g y Results There were 403 patients admitted to the general ICU during the period of interest. The number of patients <30 years of age was 75 with a mean age of 19 (range 0.5 to 30 years). The mean CED was 10.84 mSv (SD = 15.08) with 10 patients receiving >30 mSv. The mean CED for patients who did not undergo CT examination was 0.063 mSv (n = 31, SD = 0.062). Trauma patients received a far higher dose (21.86 mSv) than either medical (3.1 mSv) or postoperative surgical (3.96 mSv) admissions. Conclusion CT is a useful and necessary tool in our diagnostic and therapeutic armoury. However, our results show that young patients can potentially be exposed to signifi cant doses of ionising radiation in an ICU setting mainly due to CT. In view of the lifetime risk of cancer to these patients we should try to minimise radiation exposure by more judicious utilisation of CT and by use of other imaging modalities. References Figure 1 (abstract P523). Accuracy of weight estimation by critical care staff . P523 Accuracy of height and weight estimation by critical care staff K Dunne, S Hickey There was variability in the association across diagnostic categories, indicating that quality improvement eff orts based on the volume–outcome relationship such as regionalization of care may be more successful in specifi c patient subsets. p Results There were 330 estimations made by 30 members of staff . Height estimation was accurate ±10% for 291 patients (88.4%). Inaccuracy in height estimation ranged from –9.5% to +25% with a mean inaccuracy of 4.75%. Weight estimation was accurate ±10% for 123 patients (38.4%). Inaccuracy of weight estimation ranged from –48.9% to +40.3% with a mean inaccuracy of 16.4%. There was a tendency to underestimate weight with only 33 estimates (10%) greater than 10% of actual weight and 174 estimates (52.7%) less than 10% of actual weight. See Figure 1. 1. Hart D, Wall B: Radiation Exposure of the UK Population from Medical and Dental X-ray Examinations. Chilton: National Radiological Protection Board; 2002. 2. Kinsella SM, et al.: Kidney Int 2010, 78:789-793. 3. Dawson P: Br J Radiol 2004, 77(Spec No 1):S10-S13. 4. Slovis T: Radiology 2002, 223:5-6. 5. Cardis E, et al.: Radiat Res 2007, 167:396-416. 6. Cascade PN, et al.: AJR Am J Roentgenol 1998, 1770:561-564. P524 P524 Implementation of evidence-based care bundles in the ICU G Juknevicius, E Balakumar, A Gratrix Hull Royal Infi rmary, Hull, UK Critical Care 2012, 16(Suppl 1):P524 (doi: 10.1186/cc11131) P524 Implementation of evidence-based care bundles in the ICU G Juknevicius, E Balakumar, A Gratrix Hull Royal Infi rmary, Hull, UK Critical Care 2012, 16(Suppl 1):P524 (doi: 10.1186/cc11131) 1. Hart D, Wall B: Radiation Exposure of the UK Population from Medical and Dental X-ray Examinations. Chilton: National Radiological Protection Board; 2002. 2. Kinsella SM, et al.: Kidney Int 2010, 78:789-793. 3. Dawson P: Br J Radiol 2004, 77(Spec No 1):S10-S13. 4. Slovis T: Radiology 2002, 223:5-6. 5. Cardis E, et al.: Radiat Res 2007, 167:396-416. 6. Cascade PN, et al.: AJR Am J Roentgenol 1998, 1770:561-564. 1. Hart D, Wall B: Radiation Exposure of the UK Population from Medical and Dental X-ray Examinations. Chilton: National Radiological Protection Board; 2002. 2. Kinsella SM, et al.: Kidney Int 2010, 78:789-793. 3. Dawson P: Br J Radiol 2004, 77(Spec No 1):S10-S13. 4. Slovis T: Radiology 2002, 223:5-6. 5. Cardis E, et al.: Radiat Res 2007, 167:396-416. 6. Cascade PN, et al.: AJR Am J Roentgenol 1998, 1770:561-564. 2. Kinsella SM, et al.: Kidney Int 2010, 78:789-793. Introduction Implementation of an evidence-based care bundle in critically ill patients has been shown to improve outcome. Use of care bundles to reduce ventilator-associated pneumonia and other ICU complications has been increasing in critical care practice. 6. Cascade PN, et al.: AJR Am J Roentgenol 1998, 1770:561-564. S187 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 confi dence in its use, a similar proportion to the original audit. Eighty per cent would have an ODP for unplanned intubations. One hundred per cent were airway trained. Outcomes A designated consultant was assigned to teach diffi cult airway management at quarterly departmental induction sessions which included equipment location and algorithms. Trainees and consultants underwent simulation and mannequin training, including tracheostomy and surgical airway management. Regular updates and case-based teaching sessions were implemented. Airway profi ciency assessments were conducted at induction. confi dence in its use, a similar proportion to the original audit. Eighty per cent would have an ODP for unplanned intubations. One hundred per cent were airway trained. Outcomes A designated consultant was assigned to teach diffi cult airway management at quarterly departmental induction sessions which included equipment location and algorithms. P526 A new patient mobilization scoring system in the ICU: what is the degree of similarity in scores between assessors in daily use? M Vogel, CW Casteleijn, P Bruins, AJ Meinders St Antonius Ziekenhuis, Nieuwegein, the Netherlands Critical Care 2012, 16(Suppl 1):P526 (doi: 10.1186/cc11133) P526 A new patient mobilization scoring system in the ICU: what is the degree of similarity in scores between assessors in daily use? M Vogel, CW Casteleijn, P Bruins, AJ Meinders St Antonius Ziekenhuis, Nieuwegein, the Netherlands Critical Care 2012, 16(Suppl 1):P526 (doi: 10.1186/cc11133) Introduction Inactivity and immobility in ICU patients have signifi cant deleterious physiologic eff ects, including atelectasis, pressure ulcers, and increased susceptibility to aspiration and pneumonia. A new trend on the ICU is early mobilization of critically ill adult patients. However, evidence of when to start mobilization is missing. Casteleijn developed a new scoring system, the Patient Mobilization Frame (PMF), to improve early mobilization in the ICU. The framework is based on a multidisciplinary agreement. The aim of this study was to evaluate interobserver agreement using the PMF. Conclusion It is very challenging to implement care bundles despite evidence showing that they improve outcome. A recent study suggests that doing a daily quality rounds checklist (QRC) will improve long-term compliance, thereby reducing potential complications for intensive care patients [1]. We have implemented QRC in our practice and will be re-auditing in 6 months to ensure continued adherence. 1. DuBose et al.: Measurable outcomes of quality improvement in the trauma intensive care unit: the impact of a daily quality rounding checklist. J Trauma 2008, 64:22-29. Methods A prospective observational study in 47 critically ill patients in the ICU was performed. The PMF categorizes patients into one of three stages of possible training using a scoring system based on 14 items. Various factors infl uencing individual stage are used including circulation, respiration, infection, kidney function, wounds and neurology. Stage A (critically ill) permits only passive physical examination. Whereas stage B (stable) and stage C (nearly recovered) permit (guided) active mobilization and functional training, respectively. Two staff members and one resident obtained 47 independent observation series of the PMF. All observations were at the same date and time and were compared. Awareness of diffi cult airway equipment on the ICU A W i k A I Royal Liverpool University Hospital, Liverpool, UK Critical Care 2012, 16(Suppl 1):P525 (doi: 10.1186/cc11132) Critical Care 2012, 16(Suppl 1):P525 (doi: 10.1186/cc11132) Introduction It is widely recognised that critically ill patients can be diffi cult to intubate, requiring the use of advanced airway skills and equipment. The range of airway equipment necessary for patients on the ICU has recently been recommended [1]. Our ICU has a comprehensive diffi cult airway trolley (DAT) which is regularly maintained. With a high turnover of trainees, we were keen to determine if there was a training need to be met regarding airway management in ICU patients. The objectives were to determine awareness of the DAT, assess knowledge of its contents and ascertain confi dence in its use. Results Interobserver reliability of observers 1, 2 and 3 proved to be adequate. Kappa for observers 1 and 2 was 0.9. Kappa for observers 1 and 3 was 0.6. Kappa for observers 2 and 3 was 0.6. The value of kappa can range from 0 (disagreement) to 1 (perfect agreement). Kappa larger than 0.6 was regarded as substantial agreement. Conclusion Casteleijn’s PMF proved to be a reliable scoring system as both resident and staff members had comparable results for staging the physical abilities of the critically ill patient in the ICU. i Methods We audited against previously described standards [1] using a short questionnaire, disseminated to trainees and consultants working on the ICU in November 2010: 100% of clinicians should be aware of the location and contents of the DAT; 100% of anaesthetists should have had diffi cult airway equipment training. P524 Trainees and consultants underwent simulation and mannequin training, including tracheostomy and surgical airway management. Regular updates and case-based teaching sessions were implemented. Airway profi ciency assessments were conducted at induction. Methods We conducted a prospective audit on implementation of a care bundle after audit approval. We collected data for 101 patient days from all patients admitted to Hull Royal Infi rmary ICU during the month of November 2011. We collected information regarding stress ulcer prophylaxis, deep vein thrombosis (DVT) prophylaxis, ventilator care bundle, blood glucose control, daily assessment of need for a central line, sedation score assessment and delirium score assessment at least twice a day. y Results All patients received stress ulcer prophylaxis. At least 95% of patients received DVT prophylaxis, adequate blood glucose control and appropriate sedation need assessment. There was further scope for improvement in areas of sedation hold practice and assessing daily need for a central line. Poor clinical practice was identifi ed in delirium score assessment and head elevation to reduce VAP. See Table 1. Conclusion This audit highlights our variable workforce. The presence of junior, nonairway-trained staff on the ICU calls for regular, compulsory airway teaching sessions for all, regardless of grade. Airway competency must be formally assessed at the start of an ICU attachment. Airway instructions for challenging patients should be clearly documented with advice on access to senior assistance for emergencies. Reference Table 1 (abstract P524) Intervention in eligible patients Adherence, n (%) Stress ulcer prophylaxis 101/101 (100) DVT/PE prophylaxis 94/97 (97) Head elevation 30% in ventilated patients 62/75 (83) Daily sedation hold 28/32 (88) Blood glucose control 96/101 (95) Need for central line assessed 73/85 (86) Sedation score assessment 98/101 (97) CAM-ICU score at least twice a day 29/101 (28) Reference 1. Jeanrenaud P, et al.: Diffi cult airway trolleys for the critical care unit. JICS 2010, 11:98-103. P529 Methods A cross-sectional retrospective descriptive and observational study of rehabilitation of bedridden patients in hospital from January 2010 to June 2011. The programme is implemented in Section 30 (21, 9, and 20 rooms). The inclusion criteria for the rehabilitation programme were patients of both sexes, without age limit, inpatient of Hospital F.J. Muñiz coming from the ICU, in bedridden condition (limitation or motor disability in which the patient cannot move or perform activities of daily living and must depend on the care of others), with Barthel scale value 0 to 35 with total or severe dependence and stability hemodynamics. P529 Muscle strength assessment of critically ill patients is associated with functional ability and quality of life at hospital discharge G Sidiras, I Patsaki, M Dakoutrou, E Karatzanos, V Gerovasili, A Kouvarakos, A Kardara, K Apostolou, S Dimopoulos, V Markaki, S Nanas University of Athens, Greece Critical Care 2012, 16(Suppl 1):P529 (doi: 10.1186/cc11136) Introduction Patients with critical illness after hospital discharge often exhibit poor functional ability and quality of life as a consequence of acquired muscle weakness. The Medical Research Council (MRC) strength score and hand-grip dynamometry (HGD) are reliable and valid methods to detect clinically signifi cant muscle weakness. The objective of this study is to examine the correlation of these instruments to functional ability and quality-of-life questionnaires at hospital discharge. Introduction Patients with critical illness after hospital discharge often exhibit poor functional ability and quality of life as a consequence of acquired muscle weakness. The Medical Research Council (MRC) strength score and hand-grip dynamometry (HGD) are reliable and valid methods to detect clinically signifi cant muscle weakness. The objective of this study is to examine the correlation of these instruments to functional ability and quality-of-life questionnaires at hospital discharge. Results We included patients who met the inclusion criteria. The program presented an intensive character in terms of the frequency of weekly sessions as the number of exercises implemented in the form was specifi ed according to the pathology of the patient. Ninety percent of patients were male. The median age was 41 years. The predominant infectious pathology was pulmonary tuberculosis (90%), cerebral toxoplasmosis (50%), spastic paraplegia (6%), bilateral pneumonia (6%), and fumigares aspergillosis (6%). Motor and respiratory intensive rehabilitation in bedridden patients Motor and respiratory intensive rehabilitation in bedridde patients E Canedo, V Nunes Velloso, L Calejman, N Leidi Hospital de Infecciosas F.J. Muñiz, Buenos Aires, Argentina Critical Care 2012, 16(Suppl 1):P527 (doi: 10.1186/cc11134) E Canedo, V Nunes Velloso, L Calejman, N Leidi Hospital de Infecciosas F.J. Muñiz, Buenos Aires, Argentina Critical Care 2012, 16(Suppl 1):P527 (doi: 10.1186/cc11134) fi A re-audit was conducted in June 2011 to complete the audit cycle. Results One hundred per cent of clinicians were aware of the DAT. Only 35% had read the folder detailing its contents with instructions. Ninety per cent could confi dently name the equipment which should be readily available for diffi cult intubations but only 70% were confi dent to use it unaided. Fifty per cent would request the presence of an operating department practitioner (ODP) for an unplanned intubation on the ICU. Twenty-eight per cent were not airway trained. Re-audit showed 100% of respondents were aware of the equipment. Sixty per cent had Introduction Inability to play signifi cant social roles due to a pattern of motor disability aff ects the quality of a person’s life, and is where the motor and respiratory rehabilitation process takes fundamental importance. This disability prevents one to function independently in basic tasks such as dressing and feeding and in more complex tasks such as handling in public and/or work. It can also be a constraint for the Introduction Inability to play signifi cant social roles due to a pattern of motor disability aff ects the quality of a person’s life, and is where the motor and respiratory rehabilitation process takes fundamental importance. This disability prevents one to function independently in basic tasks such as dressing and feeding and in more complex tasks such as handling in public and/or work. It can also be a constraint for the S188 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 4. Mohammadi B, et al.: J Neurol 2008, 255:265-272. 5. Latronico N, et al.: Lancet Neurol 2011, 10:931-941. 6. Bolton CF, et al.: Crit Care Med 1996, 24:1408-1416. dependent patient in personal care activities. The objective of a motor rehabilitation plan is to reduce the impact caused by this alteration of motor ability, facilitating the restoration of functional patient capacity so they can eff ectively engage in occupations, reaching the highest level of functional independence possible. dependent patient in personal care activities. 1. Tennilä A, et al.: Intensive Care Med 2000, 26:1360-1363. 2. Khan J, et al.: Neurology 2006, 67:1421-1425. 3. Latronico N, et al.: Crit Care 2007, 11:R11. P528 Severity of electrophysiological alterations correlates with severity of illness in the early phase of critical illness polyneuropathy R Nemes, Z Fülep, B Fülesdi University of Debrecen, Hungary Critical Care 2012, 16(Suppl 1):P528 (doi: 10.1186/cc11135) Conclusion The signifi cantly reduced muscle strength of critically ill survivors could have detrimental eff ects on their mobility and quality of life. By this study it was shown that muscle strength assessment was well associated with functional ability. We assume that this might be a possible signifi cant prognostic role. Introduction We aimed to investigate the early characteristics of critical illness polyneuropathy in surgical patients in a 5-day follow-up setting. Methods Twenty critically ill patients were enrolled showing signs of systemic infl ammatory response, sepsis or multiorgan failure featuring APACHE II score ≥12 on admittance aged 26 to 86 years. Routine noninvasive nerve conduction study of bilateral median and ulnar nerves was performed on a two-channel portable Keypoint Medtronic apparatus. Nerve conduction studies were performed on fi ve consecutive days starting within at most 2 days after admittance, then weekly follow-up was carried out. Electrophysiological fi ndings were compared to age-matched control group parameters.i P530 Functional dependency in the direct post-ICU phase in patients with prolonged mechanical ventilation S Vossenberg1, I Drogt2, N Bruins2, C De Jager2, EC Boerma2, M Tijkotte1 1Zorggroep Noorderbreedte, Leeuwarden, the Netherlands; 2Medical Centre Leeuwarden, the Netherlands Critical Care 2012, 16(Suppl 1):P530 (doi: 10.1186/cc11137) Functional dependency in the direct post-ICU phase in patients with prolonged mechanical ventilation S Vossenberg1, I Drogt2, N Bruins2, C De Jager2, EC Boerma2, M Tijkotte1 1Zorggroep Noorderbreedte, Leeuwarden, the Netherlands; 2Medical Centre Leeuwarden, the Netherlands Critical Care 2012, 16(Suppl 1):P530 (doi: 10.1186/cc11137) Motor and respiratory intensive rehabilitation in bedridden patients The objective of a motor rehabilitation plan is to reduce the impact caused by this alteration of motor ability, facilitating the restoration of functional patient capacity so they can eff ectively engage in occupations, reaching the highest level of functional independence possible. P529 The profi t was 100% of kinesic treatment adherence, 94% of cases won full independence valued on the Barthel scale with a value of 100, and a single case achieved independence moderated by the presence of spastic paraplegia. Methods Two hundred and sixty-six consecutive patients who had been discharged from the ICU were evaluated and 37 of them were eligible (inclusion criteria: in mechanical ventilation >72 hours, a cognitive status that allows assessment) for the study (mean ± SD: age 55 ± 15; APACHE 14 ± 5; SOFA 8 ± 3; length of ICU stay 22 ± 22 days; duration of mechanical ventilation 17 ± 19 days). Muscle strength was evaluated with the MRC score and HGD every 7 days until discharge from the hospital. The Functional Independence Measure (FIM) was used to evaluate the functional ability while health-related quality of life was assessed by the Nottingham Health Profi le (NHP).i Conclusion The intensive rehabilitation programme presented a great benefi t for hospitalized patients; taking them from being bedridden to total independence in the AVD, the outpatient had better social and labor conditions. y gi Results At hospital discharge the MRC scale and HGD were signifi cantly correlated with FIM (r = 0.69, P <0.001 and r = 0.58, P <0.001, respectively). There seems to be a good correlation of the MRC scale (r = –0.57, P <0.001) with the section of mobility of the NHP. There is also certain association among the domain of mobility and energy of the NHP with the FIM (r = –0.88, P <0.001 and r = –0.61, P <0.05, respectively). 3. Latronico N, et al.: Crit Care 2007, 11:R11. . Mahoney FI, Barthel DW: Functional evaluation: the Barthel Index. Md Med J 1965, 2:61-65. Functional dependency in the direct post-ICU phase in patients with prolonged mechanical ventilation p g g p p Results On fi rst examination, within 2 days following admission 17 of 20 (85%) patients showed signs of axonal type sensory-motor polyneuropathy. Medians of compound muscle action potential (CMAP) and sensory nerve action potential (SNAP) amplitudes of all nerves showed a signifi cant decrease compared to control values (P <0.001). During the 5-day study period four patients showed improvement. Sensory nerve fi bres were less severely aff ected than motor fi bers. The consecutive measurements revealed negative correlation with the severity of peripheral interstitial oedema determined by circumference of the elbow. Changes in CMAP and SNAP amplitudes also showed a negative correlation with daily rated APACHE II and SAPS II severity scores, and thus with patients’ general condition. Introduction Prolonged mechanical ventilation and length of stay (LOS) in the ICU is associated with long-term impaired functional capacity. However, little is known about functional dependency in the direct post-ICU phase. Therefore the timing and location for optimal post-ICU rehabilitation programs remain to be established. Methods In this single-centre observational study we aimed to quantify functional dependency at three diff erent time points: discharge from ICU (DI), discharge from hospital (DH) and discharge from nursing home rehabilitation unit (DR). To this end we retrospectively assed Barthel scores (BS) for individual patients [1], with a duration of mechanical ventilation >48 hours. Data are presented as median (IQR). Comparison between time points was performed with nonparametric tests for paired data and repeated measurements. P <0.05 was considered signifi cant. p g Conclusion Electrophysiological alterations appear early after the development of critical illness [1-4]. Early electrophysiological investigations are advisory although results should be evaluated cautiously, as it is hard to diff erentiate between defi nitive lesions and temporary disorder caused by bioenergetic failure [3,5-6] of the nerve which tend to improve with normalisation of patients’ condition. References i Results Thirty-four patients were included. Baseline characteristics: APACHE II score 20 (17 to 25), age 68 (55 to 73) years, LOS ICU 22 (8 to 36) days, mechanical ventilation 8 (2 to 17) days, LOS hospital 21 (14 to 30) days, LOS rehabilitation unit 53 (31 to 85) days. Median BS at DI was 2 (1 to 3), indicating total functional dependency. In comparison 1. Tennilä A, et al.: Intensive Care Med 2000, 26:1360-1363. 2. Khan J, et al.: Neurology 2006, 67:1421-1425. 3. Latronico N, et al.: Crit Care 2007, 11:R11. Functional dependency in the direct post-ICU phase in patients with prolonged mechanical ventilation S189 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 Critical Care 2012, Volume 16 Suppl 1 http://ccforum.com/supplements/16/S1 to baseline, BS increased to 8 (2 to 12) at DH (P <0.001), indicating severe dependency, and fi nally to 16 (11 to 18) at DR, indicating independency with some disabilities (P <0.001). The absolute increase in BS was signifi cantly greater during the stay in the rehabilitation unit, as compared to the general hospital ward (P <0.001). p g p Conclusion ICU patients with prolonged mechanical ventilation remain severely functionally dependent after ICU discharge, but dependency reduces signifi cantly during rehabilitation in hospital and in a nursing home rehabilitation unit. . Mahoney FI, Barthel DW: Functional evaluation: the Barthel Index. Md Med J 1965, 2:61-65.
https://openalex.org/W4394813296	https://bmcnephrol.biomedcentral.com/counter/pdf/10.1186/s12882-024-03571-5	English	null	Atypical anti-glomerular basement membrane disease with membranous hyperplasia: diagnostic challenges and treatment variability	BMC nephrology	2,024	cc-by	6,022	†Ruoyu Tong and Zhengmao Luo contributed equally to this work. Correspondence: Yuanhang Huang h.yuanhang@163.com 1Nephrology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China 2Pathology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China Correspondence: Yuanhang Huang h.yuanhang@163.com 1Nephrology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China 2Pathology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China Correspondence: Yuanhang Huang h.yuanhang@163.com 1Nephrology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China 2Pathology Department, Chinese PLA Southern Theater Command General Hospital, 510010 Guangzhou, China Atypical anti-glomerular basement membrane disease with membranous hyperplasia: diagnostic challenges and treatment variability Ruoyu Tong1†, Zhengmao Luo1†, Xianyang Zhong1, Liming Fan1, Huangwen Lai2, Meng Shen1 and Yuanhang Huang1 BMC Nephrology BMC Nephrology BMC Nephrology Tong et al. BMC Nephrology (2024) 25:132 https://doi.org/10.1186/s12882-024-03571-5 Open Access Abstract This case report presents a detailed analysis of a 31-year-old male patient who presented with a complex array of clinical symptoms, including proteinuria, hematuria, edema, and kidney insufficiency. Despite undergoing multiple tests, the results for anti-glomerular basement membrane antibodies yielded negative findings. Subsequently, kidney biopsy pathology revealed a distinct diagnosis of atypical anti-glomerular basement membrane (anti-GBM) disease with membrane hyperplasia. Treatment was initiated with a comprehensive approach involving high doses of corticosteroids therapy and cyclophosphamide (CTX). However, contrary to expectations, the patient’s kidney function exhibited rapid deterioration following this therapeutic regimen. The culmination of these complications necessitated a pivotal transition to maintenance hemodialysis. This case underscores the intricate challenges associated with diagnosing and managing rare and atypical presentations of kidney disorders. The negative anti-GBM antibody results and subsequent identification of atypical anti-GBM nephropathy highlight the need for tailored diagnostic strategies to discern subtle nuances within complex clinical scenarios. Additionally, the unexpected response to the treatment regimen emphasizes the potential variability in individual patient responses, underlining the necessity for vigilant monitoring and adaptable treatment strategies. This case report contributes to the evolving understanding of atypical kidney pathologies and the complexities involved in their management. Keywords Atypical anti glomerular basement membrane disease Membranous hyperplasia Kidney Keywords Atypical anti-glomerular basement membrane disease, Membranous hyperplasia, Kidney †Ruoyu Tong and Zhengmao Luo contributed equally to this work. Introduction disease presented with pulmonary hemorrhage [8]. Sec ondly, kidney damage in atypical anti-GBM disease tends to be less severe, primarily characterized by hematuria, proteinuria, and mild kidney insufficiency. This stands in contrast to the acute progressive nephritis syndrome observed in typical anti-GBM disease [5, 6]. The serum creatinine for patients with atypical anti-GBM disease was 2.2 mg/dL, and none of them required dialysis [8]. This suggests that kidney damage is notably milder in comparison to patients with typical anti-GBM disease. Lastly, individuals with atypical anti-GBM disease com monly exhibit negative or low titers of anti-GBM anti bodies circulating in their blood, in contrast to patients with typical anti-GBM disease who demonstrate a posi tive rate ranging from 65–100% [6, 8, 9].h Anti-glomerular basement membrane disease (anti-GBM disease) is a form of small-vessel vasculitis character ized by the deposition of circulating anti-GBM antibod ies within kidney and/or lung tissue. This condition is characterized by the deposition of circulating anti-GBM antibodies within the basement membranes of glom eruli and, at times, the pulmonary alveoli, leading to rapidly progressive glomerulonephritis and, occasion ally, pulmonary hemorrhage [1, 2]. However, the clinical and pathological manifestations of anti-GBM disease are not uniform across all cases. Classic anti-GBM disease, often referred to as Goodpasture’s syndrome, primar ily involves the kidneys and lungs [3]. The autoimmune response targets the alpha-3 chain of type IV collagen, a major component of glomerular and alveolar basement membranes. The ensuing immune complex deposition triggers a cascade of inflammation, resulting in crescen tic glomerulonephritis and, in severe cases, pulmonary hemorrhage [4]. Rapid diagnosis and intervention are essential for favorable outcomes, typically relying on the detection of circulating anti-GBM antibodies and subse quent kidney biopsy to assess disease extent. The varying clinical manifestations and atypical pre sentation can obscure accurate diagnosis, necessitating a comprehensive understanding of the disease’s mecha nisms, diagnostic criteria, and treatment approaches. This case report outlines a patient presenting with edema and kidney dysfunction, later diagnosed with atypical anti-GBM disease. As we embark on an in-depth explo ration of this enigmatic case, we endeavor to unravel the intricate connections between the diverse clinical features, laboratory findings, and potential underly ing pathophysiology. By delving into the complexities of this presentation, we hope to contribute to the evolv ing understanding of atypical clinical scenarios, thereby enriching the realm of medical knowledge and clinical practice. © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 8 Page 2 of 8 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology Introduction Atypical anti-GBM disease is a rare and intriguing kid ney disorder that presents with unique clinical and diag nostic challenges [5]. Unlike classical anti-GBM disease, atypical cases deviate from the conventional presenta tion, making timely diagnosis and effective management paramount [6]. Atypical anti-GBM disease typically exhibits milder clinical manifestations. Firstly, unlike the 34–62% incidence of pulmonary hemorrhage observed in typical anti-GBM disease, atypical cases often lack pul monary hemorrhage [2, 6, 7]. Notably, Nasr et al. have reported none of the 20 patients with atypical anti-GBM Case presentation G: IgG, A: IgA, ALB: albumin, K: kappa Fig. 2 Morphologic analysis of renal biopsy. H&E stain shows general tissue architecture and cell morphology. Periodic Acid-Schiff (PAS) stain highlights basement membranes and mesangial matrix. Jones’ Methena mine Silver (PASM) stain reveals the details of the glomerular basement membranes and mesangial regions. Masson’s Trichrome stain distinguish es between collagen (green) and cellular components (red) loops. Anti-glomerular basement membrane antibod ies (chemiluminescence) returned negative results on multiple occasions. Additionally, immune and other tests, tumor indicators, as well as infection indicators, all yielded negative results. As showed in Fig. 2, we observed 16 glomeruli with no glomerulosclerosis or segmental sclerosis. The glo merular mesangial cells and stroma were moderately to severely hyperplastic with nodular changes, the glo merular capillary loops were lobulated, the capillary lumen was narrowed and occluded, the basement mem brane was thickened, and the mesangial insertion and double-track formation could be seen, and there were no angiomatous dilatation and “eyelash-like” structures, no nail-like structures, and no mesangial areas, There was no obvious complex red protein deposition in the mesan gial, subepithelial and subendothelial areas. Segmental fibrinoid necrosis was seen in individual glomerular cap illary loops, with 2 cellular fibrous, 3 small cellular, and 5 cellular fibrous crescentic formations. Kidney tubu lar epithelial granular and vacuolar degeneration, a few tubular lumen dilatation, epithelial cell detachment, bris tle edge disappearance, multifocal and patchy atrophy (atrophy area of about 55%), kidney interstitium multi focal and patchy inflammatory cell infiltration accompa nied by fibrosis, small arteriolar wall thickening, luminal narrowing. Fig. 2 Morphologic analysis of renal biopsy. H&E stain shows general tissue architecture and cell morphology. Periodic Acid-Schiff (PAS) stain highlights basement membranes and mesangial matrix. Jones’ Methena mine Silver (PASM) stain reveals the details of the glomerular basement membranes and mesangial regions. Masson’s Trichrome stain distinguish es between collagen (green) and cellular components (red) 3 antibody (C-PR3), and total anti-cardiolipin antibody were also within normal ranges. The preliminary diag nosis included acute kidney failure, nephrotic syndrome, hyperuricemia, and hypertension. Additionally, there is consideration of membranoproliferative glomeru lonephritis combined with anti-basement membrane nephropathy, or anti-basement membrane nephropathy. As shown in Fig. 1, kidney biopsy pathology revealed the presence of IgG (++++); IgA (+/-); IgM (+); C3 (negative); C1q (negative); Fib (negative); ALB (negative); kappa (+); and lambda (+). Case presentation A 31-year-old male was admitted to our hospital with facial and double lower limb edema of unknown origin on April 10, 2023. The patient is experiencing symmetri cal pitting edema without fever or malaise, and no symp toms of urinary frequency, urgency, or pain. There has been no decrease in urine output or reduction in urine volume. Urinary analysis conducted in the outpatient set ting revealed occult blood at 3 + and protein at 2+. After admission, the two-dimensional ultrasound and Doppler flow study of both kidneys revealed no significant abnor malities. The lung CT scan indicated the presence of a small nodule in the anterior segment of the upper lobe of the right lung. As shown in Table 1, the urine routine analysis revealed elevations in erythrocytes (71.2/HPF), leukocytes (5.3/HPF), and urinary protein (4 + g/L). Lab oratory results indicated elevated serum levels of creati nine (2.21 mg/dL), cystatin C (3.43 mg/L), uric acid (489 µmol/L), and urea nitrogen (17.8 mmol/L), along with decreased albumin (22.4 g/L). Furthermore, ELISA tests revealed an increased IgG level of 10.90 AU/ml and nor mal levels of complement C3 (1.2 g/L) and C4 (0.35 g/L). The levels of total anti-β2 glycoprotein 1, anti-protease Table 1 Laboratory test result Test Item Result Reference Range Urinary Erythrocytes 71.2/HPF 0–2/HPF Urinary Leukocytes 5.3/HPF 0–5/HPF Urinary Protein 4 + g/L -g/L(negative) Serum Creatinine 2.21 mg/dL 0.74-1.35 mg/dL Cystatin C 3.43 mg/L 0.51-1.09 mg/L Uric Acid 489 µmol/L 210–430µmol/L Urea Nitrogen 17.8 mmol/L 3.1-8.0mmol/L Albumin 22.4 g/L 40–55 g/L IgG Level (ELISA) 10.90 g/L 7–16 g/L Anti-Myeloperoxidase Antibody (P-MPO) 1.81 AU/ml 0-16AU/ml Total Anti-β2 Glycoprotein 1 < 2.00 AU/ml 0-16AU/ml Anti-Protease 3 Antibody (C-PR3) < 2.00 AU/ml 0-16AU/ml Total Anti-Cardiolipin Antibody < 5.00 AU/ml 0-16AU/ml Anti glomerular base ment membrane antibody (chemiluminescence) negative negative Page 3 of 8 Tong et al. BMC Nephrology (2024) 25:132 (2024) 25:132 Tong et al. BMC Nephrology Fig. 1 Immunofluorescence analysis of renal biopsy. Immunofluorescence shows diffuse and globular IgG deposits along capillary loops, less pro nounced, scattered granular IgA deposits, albumin was barely present within the capillary, and Kappa light-chain deposits. G: IgG, A: IgA, ALB: albumin, K: kappa Fig. 1 Immunofluorescence analysis of renal biopsy. Immunofluorescence shows diffuse and globular IgG deposits along capillary loops, less pro nounced, scattered granular IgA deposits, albumin was barely present within the capillary, and Kappa light-chain deposits. Case presentation Within individual capillary lumens, interstitial erythrocyte aggregates were observed. potentially resulting in kidney damage and/or pulmo nary hemorrhage. The target antigen in this context is the non-collagenous domains of the α3(IV)NC1 and α5(IV) NC1 in the GBM [2, 4]. In the development of anti-GBM disease, the immu nological characteristics of anti-GBM antibodies play a crucial role in its pathogenicity. This involves a gradual increase in titer, the progressive emergence and ampli fication of IgG1 subtypes, and a shift in target antigens recognized by the antibodies. Initially limited to α3(IV) NC1 and α5(IV)NC1, the antibodies eventually come to recognize all five target antigens from α1 to α5(IV)NC1 [9, 10]. Plasma exchange has proven effective in remov ing circulating antibodies, leading to the recovery of pul monary hemorrhage and kidney function. Recently, there has been the identification of atypical anti-GBM disease, which presents some unique features. Despite revealing typical immunoglobulin deposition along the GBM lin eage in kidney immunofluorescence, the serum tests neg ative for anti-GBM antibodies. Furthermore, the clinical manifestations, serologic and pathologic features, and prognosis of atypical anti-GBM disease differ from those of the typical variant, suggesting the possibility of distinct underlying pathogenic mechanisms [5, 6, 11]. The diagnosis was atypical anti-GBM nephropathy with membranous hyperplasia. Methylprednisolone was administered at a dose of 500 mg for 3 days, which was then adjusted to 60 mg/day, in combination with two doses of cyclophosphamide (1.0 g each) administered on May 21 and June 20. The patient commenced dialysis on May 11 due to worsened edema, inadequate diuretic response (furosemide 80 mg, intravenous injection, every other day), and an elevated serum creatinine of 3.47 mg/ dL. From July 2 onwards, the patient exhibited symptoms including fever, reduced blood oxygen levels, and tested positive for cytomegalovirus IgM antibodies. A chest CT indicated interstitial pneumonitis. Cytomegalovirus infection was suspected, leading to a reduction in cor ticosteroid dosage, as well as administration of gamma globulin and ganciclovir as part of anti-viral treatment. The lung infection improved after implementing anti- infection measures. The patient declined further cyclo phosphamide treatment. The corticosteroid dosage was reduced to 30 mg/day and combined with 500 mg Myco phenolate Mofetil (MMF) taken twice daily. On July 24th, the patient was discharged from dialysis, and subsequent tests showed blood creatinine levels at 2.28 mg/dL and albumin levels at 26 g/L. Case presentation The IgG deposits were found to exhibit a diffuse, globular, and linear pattern along the capillary Electron microscopy analysis in Fig. 3 revealed two glomeruli. One displayed ischemic change with a crum pled appearance, while the other exhibited crescent formation. The latter showed lobulated capillary loops within relatively preserved areas. Marked vacuolar Fig. 3 TEM analysis of renal biopsy. (A) The left panel shows extensive podocyte effacement and irregular thickening of the glomerular basement mem brane (GBM). (B) The middle panel reveals subepithelial deposits characteristic of membranous nephropathy, with spikes and dome appearance of the GBM. (C) The right panel highlights areas of GBM disruption and cellular interposition, suggesting active glomerular injury. Scale bars represent 2 μm Fig. 3 TEM analysis of renal biopsy. (A) The left panel shows extensive podocyte effacement and irregular thickening of the glomerular basement mem brane (GBM). (B) The middle panel reveals subepithelial deposits characteristic of membranous nephropathy, with spikes and dome appearance of the GBM. (C) The right panel highlights areas of GBM disruption and cellular interposition, suggesting active glomerular injury. Scale bars represent 2 μm Fig. 3 TEM analysis of renal biopsy. (A) The left panel shows extensive podocyte effacement and irregular thickening of the glomerular basement mem brane (GBM). (B) The middle panel reveals subepithelial deposits characteristic of membranous nephropathy, with spikes and dome appearance of the GBM. (C) The right panel highlights areas of GBM disruption and cellular interposition, suggesting active glomerular injury. Scale bars represent 2 μm Page 4 of 8 Page 4 of 8 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology Tong et al. BMC Nephrology degeneration of endothelial cells, focal endothelial cell hyperplasia, and capillary loop compression were also observed. The mural layer of the kidney capsule exhibited thickening and stratification, alongside mural cell hyper plasia featuring vacuolar degeneration. The basement membrane displayed diffuse, homogeneous thickening up to 1000 nm, with a few tethered insertions. Epithelial cells of the visceral layer exhibited swelling with vacu olar degeneration. The presence of foot process fusion was also noted. Prominent hyperplasia of the mesangial cells was observed, without any electron-dense mate rial deposits. Additionally, vacuolar degeneration of kid ney tubular epithelial cells, thickening of the basement membrane in a few tubules, and epithelial cell detach ment were noted. The kidney interstitium displayed mild edema along with a minor infiltration of inflamma tory cells. Case presentation After cessation of dialysis, the patient’s corticosteroid dosage was tapered to 15 mg/day of oral prednisone acetate and MMF at a dose of 500 mg twice daily. Diuretic therapy consisted of 20 mg of oral furosemide every other day. One month following this, the patient chose to discontinue steroids and immu nosuppressants at an outpatient clinic due to personal preferences. Our patient exhibited pathological features such as dif fuse and globular deposition of IgG along capillary loops, diffuse and homogeneously thickened basement mem branes, crescent formation (62.5%), moderate-to-severe hyperplasia of mesangial cells, and the visible insertion of mesangial membranes. Additionally, nodular changes were evident in the form of double-track formation. Con sidering the clinical presentation alongside multiple neg ative laboratory indicators for anti-GBM antibodies, we concluded that the case involved atypical anti-GBM dis ease combined with membrane hyperplasia. The absence of electron-dense material in the ultrastructure excluded the possibility of membranoproliferative nephropathy in conjunction with crescent formation. The linear IgG deposition in atypical anti-GBM disease may be due to an abnormal GBM or non-traditional char acteristics of the antibody-binding sites [8, 12]. The phe nomenon of “serum negativity” for anti-GBM antibodies could be explained by an “immunological sink,” wherein high-affinity antibodies are selectively sequestered from the circulation, leaving behind low-affinity antibodies undetectable by standard ELISA [13]. This hypothesis is indirectly supported by the fact that when anti-GBM disease was first identified in 1967, it was found that cir culating anti-GBM antibodies increased rapidly when patients underwent bilateral nephrectomy [14]. Discussion Unlike the clinical presen tation of classical anti-GBM disease, it is instead similar to a diabetic nephropathy grade III condition, where the blood creatinine is not high, but the urine output is not at normal levels [15]. Furthermore, the pathology of this case was com pounded by membranous hyperplasia. Previous reports have shown that anti-GBM disease can also be associ ated with or secondary to other glomerulopathies, the most common of which is membranous nephropathy. It is hypothesized that these pre-existing glomerulonephri tis may cause damage to the glomerular basement mem brane structure, exposing the antigen which is originally in a masked state, inducing autoimmune reaction to pro duce autoantibodies, leading to the development of anti- GBM disease. In this case, the pathology happened to be combined with a manifestation of membranous hyper plasia as well. Previous reports have shown that anti- GBM disease can also be associated with or secondary to other glomerulopathies, the most common of which is membranous nephropathy. It is hypothesized that these pre-existing glomerulonephritides may cause damage to the glomerular basement membrane structure, exposing the antigen that is originally in a masked state, inducing an autoimmune reaction that produces autoantibod ies, ultimately leading to the development of anti-GBM disease. In this case, we favored the pathological mani festation of membranous hyperplasia secondary to atypi cal anti-GBM nephropathy because no electron-dense material was observed in the ultrastructure, and this case is exceptionally rare. This case was similar to diabetic nephropathy grade III in both clinical presentation and pathology, and it was hypothesized that the two shared a common pathogenesis. p g In the management of anti-GBM disease, timely and effective treatment is crucial for improving outcomes. The standard therapeutic strategy includes a combina tion of immunosuppression (usually with corticosteroids and cyclophosphamide) and plasmapheresis [1, 16]. The goal of immunosuppression is to dampen the immune response and reduce antibody production, while plasma pheresis aims to remove circulating anti-GBM antibod ies from the plasma, thereby reducing their pathogenic impact on the kidneys and lungs [17]. For patients with atypical anti-GBM disease, as in our case, treatment deci sions can be challenging due to the absence of circulat ing antibodies detectable by standard assays. However, the presence of organ involvement necessitates a similar approach to that of the typical form. Clinical symptoms and pathology of atypical anti-GBM disease differ from those of typical anti-GBM nephropathy with specificity. Discussion Compared with typical anti-GBM disease, atypical anti-GBM disease develops more slowly. In this case, the patient’s kidney failure progressed slower than typi cal anti-GBM disease. This was primarily due to acute Anti-GBM diseases are rare spectrum of autoim mune disorders characterized by circulating anti-GBM antibodies that deposit in kidney and/or lung tissues, Tong et al. BMC Nephrology (2024) 25:132 Page 5 of 8 Page 5 of 8 Tong et al. BMC Nephrology for the appropriate management of acute and chronic lesions, guiding treatment decisions that may involve massive corticosteroid therapy and cyclophosphamide. Here, we reviewed a total of 20 case reports of pathol ogy of atypical anti-GBM disease. Atypical anti-GBM disease shares similarities with typical anti-GBM disease in linear deposition of IgG. As shown in Table 2, a defin ing characteristic of atypical anti-GBM disease is nega tive serum anti-GBM antibodies. Cellular glomerular crescent, a histomorphological indicator of a rupture of glomerular capillaries, is the common pathological find ing in both atypical anti-GBM disease and anti-GBM disease. As shows in Tables 2 and 75% (15/20) of atypi cal anti-GBM disease cases exhibit glomerular crescents. This phenomenon is usually a consequence of an intense immune attack involving cytotoxic elements, such as the complement membrane attack complex and extracellular histones, known as necroinflammation. Mesangial hyper plasia is more common in diabetic nephropathy [18], and it can be observed in 40% (8/20) of cases with atypical anti-GBM disease. If the blood glucose levels and screen ing of microalbuminuria are normal, it’s not considered diabetic nephropathy [15]. Membranoproliferative glo merulonephritis (MPGN) is identified by specific pat terns of glomerular injury seen on a kidney biopsy. These patterns are characterized by distinct light microscopic changes, which include hypercellularity and thickening of the glomerular basement membrane (GBM) [19]. In this case, we observed the thickening of the glomerular basement membrane. However, since we didn’t observe the dense deposits by electron microcopy, MPGN was not considered. Thickening of the glomerular basement membrane is not a common phenomenon in atypical anti-GBM disease, with only 15% (3/20) observed. This highlights the importance of a comprehensive diagnostic approach that considers multiple factors. water and sodium retention from corticosteroids therapy, which was later withdrawn after fluid hemodialysis was restored with oral diuretics. Discussion An accurate diagnosis based on pathology is essential In summary, although atypical anti-GBM disease shares similarities with typical anti-GBM disease in terms of immunofluorescence, where immunoglobulin is deposited linearly along the GBM, its clinicopathologic manifestations, response to treatment, and prognosis dif fer from those of typical anti-GBM disease. This suggests that it has a pathogenesis distinct from that of typical anti-GBM disease. Therefore, it is recommended to eval uate active and chronic lesions through pathology before deciding whether to pursue treatment involving massive corticosteroid therapy and cyclophosphamide. Page 6 of 8 Page 6 of 8 Tong et al. BMC Nephrology (2024) 25:132 Table 2 Literature review Case (age/sex) Pathology Immunofluorescence IgG anti-GBM antibodies Electron microcopy 31/M Proliferative basement membranes, focal cres cents, and mesangial proliferation. IgG (+); IgA (+); IgM (+); C3 (-); C1q (-); Fib (-); ALB (-); kappa (+); and lambda (+). linear deposition Negative No electron-dense deposits and pres ence of foot process fusion. 57/F [20] Mesangial proliferation, and focal crescents. IgG (+); IgA (+); IgM (-); C3 (+); C1q (-); kappa (+); and lambda (+). linear deposition Negative No electron-dense deposits and exten sive FPE. 53/M [21] Proliferative basement membranes, focal cres cents, and segmental nodular glomerulosclerosis IgG1 (+); IgG4 (+). linear deposition Negative Fibrillar deposition. 74/M [22] Mesangial proliferation, no crescents, and thin ning basement membranes IgG1 (+);IgG4 (+);IgG2 (+); kappa (+); and lambda (+). linear deposition Negative Accumulation of flocculent debris. 43/F [23] Focal crescents and endocapillary hypercellularity IgG (+); IgA (-); IgM (-); C3 (-); C4 (-); C1q (-); IgG1 (+);IgG4 (+); IgG2 (-);IgG3 (-); kappa (+); and lambda (+). linear deposition Negative Some FPE 13/F [24] Mesangial proliferation, focal crescents, some globally or segmentally sclerosed glomeruli IgG (+); IgA (+); IgM (-); C3 (+);C1q (-); IgG1 (+);IgG4 (+);kappa (+); and lambda (+). linear deposition Negative NA 4/F [25] Subtle segmental scars, segmental endocapillary hypercellularity, and cellular crescent IgG (+);IgA (-); IgM (-); C3 (-); C1q (-). linear deposition Negative No electron-dense deposits 30/M [26] Normal IgG (+);C3 (+);kappa (+); and lambda (+). linear deposition Negative NA 58/M [27] Focal crescents and proliferative, and sclerosing glomerulonephritis IgG (+); IgA (+); kappa (+); and lambda (+). linear deposition Negative NA 79/M [28] NA IgG (+); IgA (+); C3 (+); and lambda (+); fibrinogen (+). Discussion linear deposition Negative NA 48/F [11] Some globally sclerotic glomeruli, cellular and fi bric crescents, Bowman’s capsules and segmental sclerotic glomeruli, tubular atrophy accompanied by interstitial fibrosis, interstitial infiltration of lymphocytes, monocytes, and plasmocytes IgG (+) linear deposition Negative No electron-dense deposits and pres ence of foot process fusion. 37/M [29] diffuse proliferation in the glomeruli, with in creased mesangial proliferation; crescents IgG (+);C3 (+). linear deposition Negative No electron-dense deposits. 64/M [30] Necrotizing glomerulonephritis, cellular crescent, and diffuse segmental endocapillary proliferation. IgG1-κ (+). linear deposition Negative NA 69/M [31] Mesangial proliferation, diffuse endocapillary hypercellularity, mesangiolysis, and extracapillary proliferation IgG (+); IgG1 (+); IgG2 (+);IgG3 (-); IgG4 (-); linear deposition Negative No electron-dense deposits, diffuse foot process effacement and subendothelial space widening 70/M [32] Cellular crescents and necrotic lesions IgG (+);IgM (-); C3 (-);kappa (+); and lambda (+); fibrinogen (+). linear deposition Negative Proliferative base ment membranes 46/F [12] Cellular crescents and fibrinoid necrosis. IgG (+); IgG1 (+); IgG2 (+);IgG3 (-); IgG4 (+). linear deposition Negative Normal 53/F [33] Interstitial fibrosis, tubular atrophy, moderate arteriosclerosis, and moderate arteriolar hyalinosis of moderate chronicity IgG1-kappa (+); IgG1 (+); IgG2 (+);IgG3 (-); IgG4 (+). linear deposition Negative No electron-dense deposits and exten sive FPE 24/M [34] Cellular crescents and mesangial proliferation, IgG (+); IgA (+); IgM (-); C3 (-); C1q (-);IgG2 (+); IgG4 (+). linear deposition Negative No electron-dense deposits 33/M [35] Mesangial proliferation, focal interstitial fibrosis, and tubular atrophy IgG (+);C3 (+); C4d (-). linear deposition Negative NA 37/M [36] Cellular crescents IgG (-); IgA (+). linear IgA deposition Negative NA +: positive; -: negative. FPE: podocyte foot process effacement. NA: not available Page 7 of 8 Page 7 of 8 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology Abbreviations anti-GBM anti-glomerular basement membrane MMF Mycophenolate Mofetil CTX cyclophosphamide MPGN Membranoproliferative glomerulonephritis GBM glomerular basement membrane 9. Cui Z, Zhao MH, Jia XY, Wang M, Hu SY, Wang SX, et al. Antibodies to alpha5 chain of collagen IV are pathogenic in Goodpasture’s disease. J Autoimmun. 2016;70:1–11. Abbreviations anti-GBM anti-glomerular basement membrane MMF Mycophenolate Mofetil CTX cyclophosphamide MPGN Membranoproliferative glomerulonephritis GBM glomerular basement membrane 10. Zhao J, Cui Z, Yang R, Jia XY, Zhang Y, Zhao MH. Anti-glomerular basement membrane autoantibodies against different target antigens are associated with disease severity. Kidney Int. 2009;76:1108–15. with disease severity. Kidney Int. 2009;76:1108–15. 11. Acknowledgements None declared. 12. Adapa S, Konala VM, Hou J, Naramala S, Agrawal N, Dhingra H, et al. Sero negative atypical anti-glomerular basement membrane crescentic glomeru lonephritis. Ann Transl Med. 2019;7:246–246. Data availability Th d d 17. Prendecki M, Pusey C. Plasma exchange in anti-glomerular basement mem brane disease. Presse Med. 2019;48(11 Pt 2):328–37. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 18. Thomas HY, Ford Versypt AN. Pathophysiology of mesangial expansion in diabetic nephropathy: mesangial structure, glomerular biomechanics, and biochemical signaling and regulation. J Biol Eng. 2022;16:19. Received: 10 September 2023 / Accepted: 4 April 2024 Received: 10 September 2023 / Accepted: 4 April 2024 23. Tamura R, Doi T, Hirashio S, Sasaki K, Masuda Y, Shimizu A, et al. A case report of atypical anti-glomerular basement membrane disease. BMC Nephrol. 2022;23:373. 24. Bajaj V, Thakur S, Barwad A, Sinha A, Bagga A, Singh G. IgA nephropathy and atypical Anti-GBM disease: a Rare Dual Pathology in a Pediatric Rapidly Progressive Glomerulonephritis. Glomerular Dis. 2021;2:54–8. Consent for publication 21. Qin J, Zhu T, Zhang H, Ou S. An atypical anti-GBM disease complicated by idiopathic nodular glomerulosclerosis: Case report. Clin Nephrol. 2023;99:98–104. Informed consent was obtained from the patient for the publication of this study. 22. Roy S, Hou J, Chourasia P, Yalamanchili A, Basuli D, Errabelli PK, et al. Sero negative atypical anti-glomerular basement membrane Glomerulonephritis Associated with thrombotic Microangiopathy: Case Report and Literature Analysis. J Investig Med high Impact case Rep. 2023;11:23247096231184760. Ethics approval and consent to participate 20. Hoi S, Ogawa M, Munemura C, Takata T, Isomoto H. Atypical anti-glomerular basement membrane Nephritis after the first dose of the severe Acute Respiratory Syndrome Coronavirus 2 mRNA vaccine. Yonago Acta Med. 2023;66:300–5. The IRB approval is not required due to the retrospective nature of this study in this section. Inform consent was obtained from the patient. Declarations 19. Alchi B, Jayne D. Membranoproliferative glomerulonephritis. Pediatr Nephrol. 2010;25:1409–18. Author contributions We declare that all the listed authors have participated actively in the study and all meet the requirements of the authorship. Dr. Yuanhang Huang designed the study and wrote the protocol, Drs. Ruoyu Tong, Zhengmao Luo, Xianyang Zhong and Liming Fan acquired the manuscript, Drs. Ruoyu Tong, Zhengmao Luo, Huangwen Lai, and Meng Shen analyze the data, Drs. Ruoyu Tong and Zhengmao Luo wrote the first draft of the manuscript and mainly revised the manuscript. All authors approved the final version of the manuscript. p 13. Glassock RJ. Atypical anti-glomerular basement membrane disease: lessons learned. Clin Kidney J. 2016;9:653–6. 13. Glassock RJ. Atypical anti-glomerular learned. Clin Kidney J. 2016;9:653–6. 14. 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J Med Case Rep. 2021;15:186. 3. DeVrieze BW, Hurley JA. Goodpasture syndrome, in StatPearls. 2023: Treasure Island (FL) ineligible companies. Disclosure: John Hurley declares no relevant financial relationships with ineligible companies. 28. Bae JY, Hussein KI, Leibert E, Archer HM. Seronegative Goodpasture’s syndrome associated with organising pneumonia. BMJ Case Rep. 2021;14:e239390. 4. Funding None decla 16. Yamashita M, Takayasu M, Maruyama H, Hirayama K. The Immunobiological agents for treatment of Antiglomerular Basement membrane disease. Med (Kaunas). 2023;59(11):2014. Discussion Guo N, Yin Q, Lei S, He Y, Fu P. Atypical anti-glomerular basement membrane disease with anti-GBM antibody negativity and ANCA positivity: a case report BMC Nephrol. 2021;22:53. References Reynolds J, Preston GA, Pressler BM, Hewins P, Brown M, Roth A, et al. Autoim munity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by autoantigen complementarity. J Autoimmun. 2015;59:8–18. 4. Reynolds J, Preston GA, Pressler BM, Hewins P, Brown M, Roth A, et al. Autoim munity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by autoantigen complementarity. J Autoimmun. 2015;59:8–18. 5. Bharati J, Yang Y, Sharma P, Jhaveri KD. Atypical anti-glomerular basement membrane disease. Kidney Int Rep. 2023;8:1151–61. 29. Elshirbeny M, Alkadi MM, Mujeeb I, Fituri O. Atypical anti-glomerular basement membrane disease with diffuse crescentic membranoprolifera tive glomerulonephritis: case report and review of literature. Qatar Med J. 2020;2020:16. 6. Shen CR, Jia XY, Cui Z, Yu XJ, Zhao MH. Clinical-pathological features and outcome of atypical anti-glomerular basement membrane disease in a large single cohort. Front Immunol. 2020;11:2035. 30. Tsuji T, Ohashi N, Sato T, Goto D, Nagata S, Matsuyama T, et al. Monoclonal immunoglobulin G1 kappa-type atypical antiglomerular basement mem brane disease accompanied by necrotizing glomerulonephritis. Clin Nephrol 2020;93(3):152–7. 7. Cui Z, Zhao J, Jia XY, Zhu SN, Jin QZ, Cheng XY, et al. Anti-glomerular base ment membrane disease: outcomes of different therapeutic regimens in a large single-center Chinese cohort study. Med (Baltim). 2011;90:303–11. 31. Isobe S, Tomosugi T, Futamura K, Okada M, Hiramitsu T, Tsujita M, et al. A case of recurrent atypical anti-glomerular basement membrane nephritis suspicion after renal transplantation. Nephron. 2020;144(Suppl 1):49–53. 8. Nasr SH, Collins AB, Alexander MP, Schraith DF, Herrera Hernandez L, Fidler ME, et al. The clinicopathologic characteristics and outcome of atypical anti- glomerular basement membrane nephritis. Kidney Int. 2016;89:897–908. Page 8 of 8 Page 8 of 8 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology (2024) 25:132 Tong et al. BMC Nephrology 36. Antonelou M, Henderson SR, Bhangal G, Heptinstall L, Oliveira B, Hamour S, et al. Binding truths: atypical anti-glomerular basement membrane disease mediated by IgA anti-glomerular basement membrane antibodies targeting the alpha1 chain of type IV collagen. Kidney Int Rep. 2018;4(1):163–7. 33. Olivier M, Watson H, Lee D, Mohanlal V, Madruga M, Carlan S. Monotypic IgG1-kappa atypical anti-glomerular basement membrane nephritis: a Case Report. Case Rep Nephrol Dial. 2019;9:8–14. 32. Hanna A, Ross J, Heitor F. Rare case of atypical crescentic glomerulonephritis and interstitial lung disease with negative anti-GBM antibody and positive anti-MPO antibody. BMJ Case Rep. 2019;12:e229256. 33. Olivier M, Watson H, Lee D, Mohanlal V, Madruga M, Carlan S. Monotypic IgG1-kappa atypical anti-glomerular basement membrane nephritis: a Case Report. Case Rep Nephrol Dial. 2019;9:8–14. 35. Olewicz-Gawlik A, Żeromski J, Świerczewska M, Idasiak-Piechocka I, Pawlik M, Sikorska D, et al. A case of bizarre posttransplant anti-glomerular basement membrane disease. Cent Eur J Immunol. 2019;44:210–3. 32. Hanna A, Ross J, Heitor F. Rare case of atypical crescentic glomerulonephritis and interstitial lung disease with negative anti-GBM antibody and positive anti-MPO antibody. BMJ Case Rep. 2019;12:e229256. 33. Olivier M, Watson H, Lee D, Mohanlal V, Madruga M, Carlan S. Monotypic IgG1-kappa atypical anti-glomerular basement membrane nephritis: a Case Report. Case Rep Nephrol Dial. 2019;9:8–14. 34. Sporinova B, McRae SA, Muruve DA, Fritzler MJ, Nasr SH, Chin AC, et al. A case of aggressive atypical anti-GBM disease complicated by CMV pneumonitis. BMC Nephrol. 2019;20:29. 35. Olewicz-Gawlik A, Żeromski J, Świerczewska M, Idasiak-Piechocka I, Pawlik M, Sikorska D, et al. A case of bizarre posttransplant anti-glomerular basement membrane disease. Cent Eur J Immunol. 2019;44:210–3. 34. Sporinova B, McRae SA, Muruve DA, Fritzler MJ, Nasr SH, Chin AC, et al. A case of aggressive atypical anti-GBM disease complicated by CMV pneumonitis. BMC Nephrol. 2019;20:29. Publisher’s Note 34. Sporinova B, McRae SA, Muruve DA, Fritzler MJ, Nasr SH, Chin AC, et al. A case of aggressive atypical anti-GBM disease complicated by CMV pneumonitis. BMC Nephrol. 2019;20:29. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 35. Olewicz-Gawlik A, Żeromski J, Świerczewska M, Idasiak-Piechocka I, Pawlik M, Sikorska D, et al. A case of bizarre posttransplant anti-glomerular basement membrane disease. Cent Eur J Immunol. 2019;44:210–3.
https://openalex.org/W2351317484	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0155338&type=printable	English	null	Composing a Tumor Specific Bacterial Promoter	PloS one	2,016	cc-by	9,266	Igor V. Deyneko1, Nadine Kasnitz1, Sara Leschner1, Siegfried Weiss1,2 1 Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany, 2 Institute of Immunology, Medical School Hannover, Hannover, Germany Igor V. Deyneko1, Nadine Kasnitz1, Sara Leschner1, Siegfried Weiss1,2 1 Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany, 2 Institute of Immunology, Medical School Hannover, Hannover, Germany 1 Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany, 2 Institute of Immunology, Medical School Hannover, Hannover, Germany * Igor.Deyneko@helmholtz-hzi.de Editor: Irving Coy Allen, Virginia Tech University, UNITED STATES Editor: Irving Coy Allen, Virginia Tech University, UNITED STATES Received: February 22, 2016 Accepted: April 27, 2016 Published: May 12, 2016 Received: February 22, 2016 Accepted: April 27, 2016 Published: May 12, 2016 Received: February 22, 2016 Accepted: April 27, 2016 Published: May 12, 2016 Copyright: © 2016 Deyneko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Deyneko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract a1111 Systemically applied Salmonella enterica spp. have been shown to invade and colonize neoplastic tissues where it retards the growth of many tumors. This offers the possibility to use the bacteria as a vehicle for the tumor specific delivery of therapeutic molecules. Speci- ficity of such delivery is solely depending on promoter sequences that control the production of a target molecule. We have established the functional structure of bacterial promoters that are transcriptionally active exclusively in tumor tissues after systemic application. We observed that the specific transcriptional activation is accomplished by a combination of a weak basal promoter and a strong FNR binding site. This represents a minimal set of control elements required for such activation. In natural promoters, additional DNA remodeling ele- ments are found that alter the level of transcription quantitatively. Inefficiency of the basal promoter ensures the absence of transcription outside tumors. As a proof of concept, we compiled an artificial promoter sequence from individual motifs representing FNR and basal promoter and showed specific activation in a tumor microenvironment. Our results open possibilities for the generation of promoters with an adjusted level of expression of target proteins in particular for applications in bacterial tumor therapy. RESEARCH ARTICLE OPEN ACCESS OPEN ACCESS Citation: Deyneko IV, Kasnitz N, Leschner S, Weiss S (2016) Composing a Tumor Specific Bacterial Promoter. PLoS ONE 11(5): e0155338. doi:10.1371/ journal.pone.0155338 Citation: Deyneko IV, Kasnitz N, Leschner S, Weiss S (2016) Composing a Tumor Specific Bacterial Promoter. PLoS ONE 11(5): e0155338. doi:10.1371/ journal.pone.0155338 Introduction Cancer is one of the most frequent cause of death and its incidence is rising [1]. This renders the development of powerful therapeutic strategies of high demand. Besides the improvement of established treatment schedules, alternative therapies need to be exploited to eventually win the fight against this disease. One of such non-conventional strategies that is presently inten- sively followed, is bacteria-mediated tumor therapy [2]. Several preclinical and clinical trials have been initiated along this line [3–6]. The approach is based on an observation that cancer patients with bacterial infections sometimes experience spontaneous regression of their tumor [7]. In the meantime, it was shown for several kinds of bacteria that they are able to target and colonize solid tumors after systemic administration [2]. Apart from obligate anaerobic bacteria like Clostridia spp. that are able to grow exclusively in necrotic tumor areas without oxygen supply, facultative anaerobes like Salmonella enterica spp. have been shown to target tumors and spread throughout the entire neoplastic tissue. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Basal promoter elements The strategy employed here is illustrated in Fig 1. The DNA fragments isolated from the S. Typhimurium genome containing promoters with high expression in tumors and absence of expression in spleen and liver were identified using promoter trap library strategies [8]. Based on the fact that Salmonella mainly targets these two organs and only traces of the bacteria could be detected in the rest of the body we have defined these fragments as tumor specific pro- moters or TSP. Fragments were fused with the bare GFP coding DNA sequence preceded only by a ribosomal binding site (Shine-Dalgarno box). Therefore, TSPs should contain basal pro- moter structures like -10 and -35 elements as a prerequisite for gene transcription. The kernel method identified basal promoters in 12 out of 13 TSPs giving a frequency of BasalPTSP = 0.92. In the NP set the frequency is BasalPNP = 0.27. For HMM, the values are BasalPTSP = 0.83 and BasalPNP = 0.43, respectively. It is clear, that the specificity of predictions by the kernel method is much higher than by HMM. Thus, the kernel method was assumed to be more reliable and results obtained by it considered further. To identify exact positions of TATA-box and Inr element, program BROM [13] was applied. Tumor Specific Bacterial Promoter Besides their inherent anti-cancer effect, these bacteria offer the possibility to act as trans- port vehicles for therapeutic agents. Such molecules are usually toxic and should exclusively be expressed directly in the tumor. On the other hand, to be most effective, sufficient concentra- tions of such therapeutic molecules should be reached throughout the entire cancerous tissue. Facultative anaerobic bacteria like Salmonella would be perfect candidates to be used as such transporters. However, the normal target organs of Salmonella, spleen and liver, are also colo- nized in tumor bearing hosts. Thus, to prevent the destruction of these healthy tissues the expression of the therapeutic agent must be exclusively restricted to the tumor mass. In our previous work [8] we could isolate from the genome of S. Typhimurium several DNA fragments containing promoters that specifically respond to the physiological conditions of cancerous tissue (promoters and associated genes are listed in S1 Table). These fragments were classified into groups depending on the level of differential expression in tumor tissue and spleen. The latter served as an example of a normal target organ. First bioinformatics analysis of promoters showing high expression in tumors revealed a so-called tusp motif apparently responsible for tumor specific activation [8]. However, further experimental data and advanced bioinformatics analysis of other groups of fragments with lower expression in tumors or with limited expression in spleen revealed that it was an oversimplification [9, 10]. Therefore, it was required to thoroughly investigate the principles of the tumor specific transcriptional regula- tion and reveal contributing functional elements. Understanding such principles will not only allow the optimization of existing promoters but possibly also the creation of new promoters with the required expression profile and high transcriptional activity. In addition, these pro- moters can serve as probes to understand specific activating conditions provided by the micro- environment of a solid tumor. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by the Deutsche Krebshife and the Ministry for Education and Research (BMBF). NK was supported by the Helmholtz Graduate School for Infection Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 1 / 17 Tumor specific regulatory elements Two programs for basal promoter recognition, one based on sequence alignment kernel [11] and another on Hidden Markov Model (HMM) [12] were applied to the TSP set (DNA sequences are given in the S1 Text). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see Methods). This will ensure the generality and specificity of the recognized feature. doi:10.1371/journal.pone.0155338.g001 The remainder of this subsection will be organized as follows: i) identification of known DNA motifs, ii) identification of novel DNA motifs, iii) identification of other possible features of the promoters and finally iv) combinatorial analysis of identified motifs and other features. The remainder of this subsection will be organized as follows: i) identification of known DNA motifs, ii) identification of novel DNA motifs, iii) identification of other possible features of the promoters and finally iv) combinatorial analysis of identified motifs and other features. Recognition of known transcription factor binding motifs on DNA is usually carried out with the help of position weight matrixes (PWMs) that are collected in databases like DPInter- act [14], TRANSFAC [15] and JASPAR [16]. Although the latter two describe PWMs of eukaryotic transcription factors, they still can be applied to our data, given that the following is kept in mind: a “eukaryotic motif” identified in a prokaryotic genome can be bound by a completely different protein factor. Thereby, the eukaryotic PWM libraries should be regarded, in our case, solely as a library of DNA motifs. Potential binding motifs were identified using all three databases. Thresholds for each PWM were varied to maximize the discrimination between TSP and NP as described in Meth- ods. Following this strategy, motifs for a number of prokaryotic (DnaA, FNR, NagC and RscAB) and eukaryotic (BRSZ4, HNF1, MEF2, SOX9, TGIF and TEF) transcription factors were identified as specific for the TSP dataset. Top scoring motifs are shown in Fig 2. Along with library based searches, programs for DNA motif detection exist that do not require databases of PWMs. Tumor specific regulatory elements Tumor specific transcriptional activation is apparently achieved by features encoded in the DNA sequence of TSP promoters. Such features could be, for example, transcription factor binding motifs or specific conformations of the DNA. Therefore, it was hypothesized that TSP promoters should contain a motif or motifs that either activate transcription exclusively in tumors and/or suppress basal promoter activity in tissues other than tumors such that the observed specificity of transcription would be achieved. 2 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Tumor Specific Bacterial Promoter Fig 1. Identification and analysis steps of tumor specific promoters. A schematic overview. Two programs for basal promoter recognition, one based on sequence alignment kernel [11] and another on Hidden Markov Model (HMM) [12] were applied to the TSP set (DNA sequences are given in the S1 Text). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see Methods). This will ensure the generality and specificity of the recognized feature. doi:10.1371/journal.pone.0155338.g001 Fig 1. Identification and analysis steps of tumor specific promoters. A schematic overview. Two Fig 1. Identification and analysis steps of tumor specific promoters. A schematic overview. Two programs for basal promoter recognition, one based on sequence alignment kernel [11] and another on Hidden Markov Model (HMM) [12] were applied to the TSP set (DNA sequences are given in the S1 Text). As a negative control, a set of DNA fragments that do not initiate expression either in tumor or in spleen was selected (negative promoters, NP). Both programs recognize potential promoters in either dataset and the number of predictions greatly depends on user-defined threshold parameters. To evaluate the specificity of predictions, it was assumed that a basal promoter should be recognized in at least 75% of TSP and at most 50% of NPs (see Methods). This will ensure the generality and specificity of the recognized feature. doi 10 1371/jo rnal pone 0155338 g001 Fig 1. Identification and analysis steps of tumor specific promoters. A schematic overview. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Tumor specific regulatory elements Such programs evaluate the statistical occurrence of DNA motifs and usually do not differentiate between pro- and eukaryotic genomes. Several programs were applied to our data and the resulting PWMs were examined for specificity to the TSP set as above. Only a few programs identified specific motifs, namely: 6 motifs by Meme [17], 2 by DME [18], 2 by CMF [19] and 5 by MDScan [20]. Given that these programs do not suggest any bio- logical function of the recognized motifs, we named these motifs by program name followed by a number. Most significant motifs are included in Fig 2 and a full list is given in S1 Fig. Features like nucleotide composition are also known to affect gene expression. It was found that TSP promoters are in general AT-rich (ATTSP = 0.512, ATNP = 0.468) and can be specifi- cally characterized by the presence of AT-rich regions (ATregionTSP = 0.77, ATregionNP = 0.22, see Methods). In particular, an (A)8 repeat is often found in the TSP set (A8 TSP = 0.77), but not 3 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Tumor Specific Bacterial Promoter Fig 2. Top motifs identified in the set of tumor specific promoters and its distribution within different groups of promoters. Values are normalized numbers of promoters in a set containing at least one motif. P-values were calculated as a binomial probability to observe the actual number of promoters with a motif in the TSP set compared to RP set. A potential overfitting effect of prediction methods can be estimated using an independent lowTSP set (see Methods). Fig 2. Top motifs identified in the set of tumor specific promoters and its distribution within different groups of promoters. Values are normalized numbers of promoters in a set containing at least one motif. P-values were calculated as a binomial probability to observe the actual number of promoters with a motif in the TSP set compared to RP set. A potential overfitting effect of prediction methods can be estimated using an independent lowTSP set (see Methods) doi:10.1371/journal.pone.0155338.g002 in the set of NP (A8 NP = 0.47). This feature may represent a general transcriptional activity of the promoters not connected to tumor activation and therefore may represent a general pro- moter feature. It is clear from the above that many motifs in the TSP set could be identified. Each might explain to some extent tumor specific transcriptional activity. However, before proceeding to experimental testing of each motif it appeared more efficacious first to search for specific groups of motifs and to test such groups rather than every single motif. Clearly, it would be beneficial if potential groups of motifs would localize densely in the promoters, such that a cut- and-test strategy could be applied. In general, promoters of genes vary greatly in length. Therefore, no reasonable window size parameter as required for many programs could be suggested for our particular dataset. There- fore, we have developed a bioinformatics method that identifies combinations of heterogeneous features, like, DNA motifs, CpG islands, repeats, that are co-localized on a DNA sequence [10]. This method is based on a genetic algorithm and searches for a collection of motif combinations that exhibit high specificity for the positive dataset and localize separately on DNA sequences. 4 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Tumor Specific Bacterial Promoter Using this method several highly specific combinations of DNA motifs were identified (listed in S2 Table). Further it was decided to perform the experiments in two steps. First, pro- moters P0.48, P0.156, P0.271, P0.272 and P0.301 were split into three fragments such that the 5' and 3' fragments preferably contain a single combination. Here, we denote these promoters as "P0." (round 0) followed by a number corresponding to the number used in [8]. For further identification, names of promoter fragments will be supplemented with an underscore and a consecutive number. By testing such fragments, it became possible to exclude many non-func- tional motif combinations. Second, on the basis of localization the remaining motifs, promoters P0.212, P0.134 and sev- eral functional fragments from the first step were split into shorter fragments of 50-100bp. As in the previous step, the rationale in selection of fragments is to efficiently separate testable motifs. By experimental analysis the shortest promoter P0.212_1 comprising one functional module was identified (schematically shown in Fig 3). This module consists of three DNA motifs for factors TGIF, FNR and NagC, respectively, complemented by basal promoter elements. Functional role of each regulatory element The promoter model identified above consists of three regulatory DNA elements and accord- ing to the literature all of them may regulate transcription both positively and negatively [21– 23]. To test the functionality of each element and to establish its contribution to the overall effect, a knock out strategy was implemented. Each motif in P0.212_1 was mutated at positions designated as critical in studies where the motif had been discovered (Fig 3, sequences are given in S2 Fig). In addition, to evaluate the prediction of the basal promoter we mutated the TATA-box by introducing 'G' and 'C' nucleotides. Finally, to verify that there are no other ele- ments which had not been discovered in the previous step, intra-motif spacers were also mutated. All variants of P0.212_1 were synthesized de novo (promoters P1.1 –P1.7, here and further named as round 1 promoters "P1."), cloned and confirmed by sequencing. Results of the analysis by flow cytometry are presented in Fig 3. From this analysis it became clear that the motif for transcription factor NagC is not func- tional. Mutation of this motif did not change differential tumor specific expression. On the other hand, modification of the FNR motif completely disrupted transcription (clone P1.2, FNR knockout). The same effect was found for the TATA-box (clone P1.4, TATA knockout). Deletion in the TGIF motif reduced transcription to approx. 75% of the level of the original promoter (clone P1.5, TGIF knockout). Mutations at insignificant positions in motifs FNR and NagC (clone P1.6) did not influence specificity of transcription, but led to increase in expres- sion in tumors by approximately 60% (Fig 3). Changes of nucleotides in intra-motif regions did not influence the transcription specificity, but reduced its level down to 40% (clone P1.7). In all the experiments a unified threshold to separate signal from cellular debris was used. In case of promoter 1.6, this led to detection of a weak GFP signal in spleen (Fig 3). Additional tests including liver as another target organ of Salmonella yielded the same results (Fig 4). However, taking into account the enhanced expression in the tumor, the expression in liver and spleen can be considered as negligible. Taken together: two elements–FNR motif and the basal promoter–form a backbone of a tumor specific bacterial promoter. Other elements, like TGIF and a general nucleotide context, exhibit a minor role and may intensify or downgrade the transcription. Artificial promoter constructs Having identified the principle components of a tumor specific promoter, it was challenging to develop a synthetic promoter with potentially improved characteristics. This would 5 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Tumor Specific Bacterial Promoter additionally prove the concept of two specific elements that are necessary and sufficient for the tumor specific expression. As a basis for such a promoter, a DNA fragment that cannot initiate any expression was taken from the negative promoters (NP) set. A randomly selected region of 100bp from this fragment was used as template. The idea here was to create a minimal set of promoters comprising all discovered functional elements for FNR and TSS including their con- sensus sequences from the literature. This should confirm the validity of the proposed principle and the functionality of each element. Five promoters were constructed by implanting the FNR motif together with basal promoter elements into the template. We denote these as round 2 promoters "P2." (schematically shown in Fig 5). Basal promoters were constructed from short motifs representing -35 and -10 ele- ments and a region of transcription initiation taken from P0.212, P0.134 or P1.6. One promoter was compiled using consensus sequences for the TATA box and Inr element (P2.4) and another one was complemented by an additional FNR consensus sequence (P2.5). It is known that the region of transcription initiation is characterized by a low melting tem- perature that is achieved by a high AT content [24]. As was established above, tumor specific Fig 4. Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver. Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific. doi:10 1371/journal pone 0155338 g004 Fig 4. Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver. Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific. Fig 4. Expression of promoters P1.6 and P0.212_1 in tumor, spleen and liver. Homogenates were analyzed via two color flow cytometry and plating to allow normalization. Given are mean and SD. Expression of P1.6 in spleen and liver can be considered negligible compared to expression in tumor. Therefore expression of promoter P1.6 was accepted as tumor specific. Tumor Specific Bacterial Promoter Fig 3. Schematic representation of promoter structure. Left: binding motifs for factors TGIF, FNR and NagC are shown in green, yellow and brown, respectively. Basal promoter is shown as a directed arrow. Knockout of essential nucleotides within motifs were according to the literature and are represented by crossing lines. Mutation of non-essential nucleotides within motifs was random and is represented by dashed crossing lines. Nucleotides outside motifs were mutated randomly. Right: representative flow-cytometric analyses of GFP-expression in tumor and spleen. Each green point on the blots represents GFP expression levels of an individual bacterial cell. Displayed values of expression are relative to the expression values of the original promoter P0.212_1. All nucleotide substitutions are presented in the S2 Fig. doi:10 1371/journal pone 0155338 g003 Fig 3. Schematic representation of promoter structure. Left: binding motifs for factors TGIF, FNR and NagC are shown in green, yellow and brown, respectively. Basal promoter is shown as a directed arrow. Knockout of essential nucleotides within motifs were according to the literature and are represented by crossing lines. Mutation of non-essential nucleotides within motifs was random and is represented by dashed crossing lines. Nucleotides outside motifs were mutated randomly. Right: representative flow-cytometric analyses of GFP-expression in tumor and spleen. Each green point on the blots represents GFP expression levels of an individual bacterial cell. Displayed values of expression are relative to the expression values of the original promoter P0.212_1. All nucleotide substitutions are presented in the S2 Fig. doi:10.1371/journal.pone.0155338.g003 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 6 / 17 doi:10.1371/journal.pone.0155338.g004 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 7 / 17 Tumor Specific Bacterial Promoter Fig 5. Schematic representation of artificial promoters and their expression in tumor environment. Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions around TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. Only promoter P2.3 showed expression in tumor. No expression in tumor or spleen could be observed for the other promoters. Nucleotide sequences are presented in the S3 Fig Foot note: I, II, III—corresponding sequences were taken from promoters P0.212, P1.6, P0.134 respectively. cons—consensus sequences for the corresponding elements. a, b—modification of loci around putative start of transcription to facilitate DNA melting by random substitution of "C/G"s by "A/T"s. Fig 5. Schematic representation of artificial promoters and their expression in tumor environment. Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions aroun TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. Only promoter P2 Fig 5. Schematic representation of artificial promoters and their expression in tumor environment. Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions around TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. Only promoter P2.3 showed expression in tumor. No expression in tumor or spleen could be observed for the other promoters. Nucleotide sequences are presented in the S3 Fig. Foot note: I, II, III—corresponding sequences were taken from promoters P0.212, P1.6, P0.134 respectively. cons—consensus sequences for the corresponding elements. a, b—modification of loci around putative start of transcription to facilitate DNA melting by random substitution of "C/G"s by "A/T"s. Fig 5. Schematic representation of artificial promoters and their expression in tumor environment. Promoters were compiled by introduction of the respective elements taken from promoters P0.212, P1.6, P0.134 as well as consensus sequences into the template sequence. Additionally, regions around TSS were enriched for A/T to facilitate DNA melting. Expression values are relative to the expression of P0.212_1. Given are mean±SD. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 In vitro experiments After tumor colonization, the bacteria are believed to reside in areas of low oxygen supply. To test the hypothesis that tumor specific promoters might be regulated exclusively by hypoxic conditions, we tested 14 selected constructs in vitro under aerobic and anaerobic culture condi- tions. We extended such tests also by using acidic induction medium as tumors might also present a microenvironment of low pH. Results were compared with the established in vivo sit- uation in tumor and spleen and are shown in Fig 6A. Five promoters, namely P1.2, P2.1, P2.2, P2.4, P2.5, did not show any expression either under aerobic or anaerobic in vitro conditions and are not shown. All other promoters could be divided into three functional groups. The first group of pro- moters showed high expression under anaerobic conditions and in tumors and low under aero- bic conditions and in spleen (Fig 6A group A). In the second group, expression levels were similar under both in vitro conditions, but still strong differential expression was observed in tumors compared to spleen (group B). The third set of promoters showed even an increased level of expression under aerobic in vitro conditions. In vivo expression of such promoters was Fig 6. Activation of bacterial tumor-specific promoters under various conditions. (A) Expression ratios of tumor specific promoters in anaerobic and aerobic environments. For comparison, ratios for tumor and spleen are given. Three groups can be identified: group A–promoters that have similar expression (tumor—high; spleen—low; anaerobic -high; aerobic -low); group B–promoters that lost expression under anaerobic conditions and group C– promoters showing higher expression under aerobic conditions compared to anaerobic. Data were acquired 4 hrs after initiation of the cultures. (B) Promoter activation under acidic induction medium conditions. Only promoter P0.134 and its fragment P0.134_1 show expression. Promoter grouping is the same as in Fig 6A. Data were acquired 3 hrs after initiation of the cultures. The experiments were carried out twice under similar conditions. Results were essentially the same. d i 10 1371/j l 0155338 006 Fig 6. Activation of bacterial tumor-specific promoters under various conditions. (A) Expression ratios of tumor specific promoters in anaerobic and aerobic environments. For comparison, ratios for tumor and spleen are given. Tumor Specific Bacterial Promoter P0.134. The DNA sequence of this promoter is AGACCAATGG ACATCCACGG CGATTAT- TAC GTTGATCATG ATCAAGCAGT TTTAAGACTA TACCAACTTG ATTTAATTCT TGTAATAAAC GAATGCC. Expression under control of this promoter is highly restricted to the tumor tissue. Absolute level of expression is approx. 75% compared to promoter P0.212 and 86% compared to P0.134. This demonstrates that the elements identified in the previous stage are necessary and sufficient for the specific transcriptional response in the tumor micro- environment. It opens the possibility for further development of specific promoters with highly individual expression profiles. doi:10.1371/journal.pone.0155338.g006 Only promoter P2.3 showed expression in tumor. No expression in tumor or spleen could be observed for the other promoters. Nucleotide sequences are presented in the S3 Fig. Foot note: I, II, III—corresponding sequences were taken from promoters P0.212, P1.6, P0.134 respectively. cons—consensus sequences for the corresponding elements. a, b—modification of loci around putative start of transcription to facilitate DNA melting by random substitution of "C/G"s by "A/T"s. doi:10.1371/journal.pone.0155338.g005 promoters indeed display higher AT content and contain many AT-rich regions. However, the template we selected exhibits a GC content of 0.55 and particularly a motif "GGTGGG" around the prospected start of transcription. We therefore randomly changed several nucleotides to A and in one case introduced a motif "AATAAAC" taken from the promoter P0.134 (Fig 5, sequences are given in S3 Fig). All fragments underwent the same cloning and testing proce- dure as before. Results of the expression analysis are shown in Fig 5. All fragments that were constructed from basal promoter elements taken from the promoter P0.212 or from consensus sequences could not initiate any transcription. Such functional deficiency under all tested conditions (see also the next section) can only be explained by lack of functionality of basal promoters. The obvious explanation of this is a low prediction accuracy of the bioinformatic methods and the insufficient knowledge on basal promoter elements. The only promoter which showed tran- scriptional activity was P2.3, that was combined using elements from promoters P1.6 and 8 / 17 Histological analysis These heterogeneous results prompted us to investigate in which tumor region the Salmonella are precisely localized. Therefore, colonized tumor tissue was analyzed by histology. An accu- mulation of immune cells mainly consisting of neutrophils was visualized by hematoxylin and eosin (H&E) staining between necrotic and viable tumor zones (Fig 7A). Partially overlapping with this zone, a large hypoxic region could be detected with a similar shape as the leukocyte zone (Fig 7B). Additionally, this hypoxic region bordered on the necrotic tumor zone where no viable cells were present and which is most likely anoxic (Fig 7A and 7B). Salmonella appar- ently colonize the hypoxic region of the tumor as well as the anoxic necrotic zone (Fig 7C). Thus, the bacteria colonize a very heterogeneous environment which is consistent with our finding that the promoters are activated by heterogeneous factors, only some of which are evident. The presented results indicate that we have identified critical elements of tumor specific promoters. We also show that apparently other DNA features are present in particular TSP promoters that render some of them responsive to hypoxic or induction media conditions. Our data also suggest that there are features, probably distributed along the promoter sequences that quantitatively influence the level of expression. The artificial promoter that lacks these fea- tures responds exclusively to the tumor microenvironment that was proved in all experiments. Understanding these features may shed light on attributes of the tumor microenvironment that may distinguish solid tumors from other tissues. Tumor Specific Bacterial Promoter still restricted to the cancerous tissue (Fig 6A group C). This is somewhat surprising, since in the spleen aerobic conditions should be dominating. We also tested all promoters for activation in induction and minimal medium which might mimic the low nutrient supply and the low pH encountered in a tumor. Only promoter P0.134 and its fragment P0.134_1 were activated when cultivated in induction and minimal medium (Fig 6B, data for minimal medium are similar and not shown). These experiments indicate that many of our promoters specifically respond to additional, presently unknown, factors encountered in tumor environments. Interestingly, the artificial promoter P2.3 that is fully functional in tumors but not in spleen is not sensitive to low oxygen conditions nor is it responding to induction medium. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 In vitro experiments Three groups can be identified: group A–promoters that have similar expression (tumor—high; spleen—low; anaerobic -high; aerobic -low); group B–promoters that lost expression under anaerobic conditions and group C– promoters showing higher expression under aerobic conditions compared to anaerobic. Data were acquired 4 hrs after initiation of the cultures. (B) Promoter activation under acidic induction medium conditions. Only promoter P0.134 and its fragment P0.134_1 show expression. Promoter grouping is the same as in Fig 6A. Data were acquired 3 hrs after initiation of the cultures. The experiments were carried out twice under similar conditions. Results were essentially the same. doi:10.1371/journal.pone.0155338.g006 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 9 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Probable mechanism of tumor specific activation According to the bioinformatic and experimental results we may speculate on how tumor spe- cific activation is achieved. In normal tissues, a level of the active dimerized form of FNR pro- tein is low and the promoter receives no activation signals. To avoid leakage, the basal promoter should be inefficient enough such that it is not able to initiate transcription by itself, since no repressor element is found in the promoters. The "extent of inefficiency" is presumably very vague and cannot be defined as a number of mismatches from consensus Inr or TATA- box sequences. Once, a boost signal from FNR is received, for example, under anaerobic condi- tions, some promoters already show pronounced transcription (Fig 6A group A). For other promoters, activation only by FNR is still not sufficient (Fig 6A group B). However, they are transcriptionally active in the tumor microenvironment where additional factors play a role and the overall signal is sufficient enough to initiate transcription. The mechanism of addi- tional factors also agrees with our data on mutation of "insignificant" nucleotides (P1.6 and P1.7, Fig 3) that led to significant changes in transcriptional activity. One of the reasons for this could be a change in overall physico-chemical properties of a DNA stretch that is shown to PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 10 / 17 Tumor Specific Bacterial Promoter 11 / 17 Tumor Specific Bacterial Promoter Fig 7. Localization of Salmonella expressing GFP under control of P0.212_1 within various regions of solid murine tumors. Consecutive tumor sections are shown. (A) Hematoxylin and eosin (H&E) staining showing infiltration of live immune cells (closed purple nuclei) between viable (V) and necrotic (N) tumor zones. (B) Immunochemical detection of hypoxic tissue (light brown staining) by a rabbit-anti-pimonidazole antibody and (C) S. Typhimurium strain SL7207 cells (dark brown) by a rabbit-anti-salmonella antibody. Here, arrow heads indicate vessles, long arrows indicate sebaceous glands. doi:10.1371/journal.pone.0155338.g007 Fig 7. Localization of Salmonella expressing GFP under control of P0.212_1 within various regions of solid murine tumors. Consecutive tumor sections are shown. (A) Hematoxylin and eosin (H&E) staining showing infiltration of live immune cells (closed purple nuclei) between viable (V) and necrotic (N) tumor zones. (B) Immunochemical detection of hypoxic tissue (light brown staining) by a rabbit-anti-pimonidazole antibody and (C) S. Typhimurium strain SL7207 cells (dark brown) by a rabbit-anti-salmonella antibody. Here, arrow heads indicate vessles, long arrows indicate sebaceous glands. Efficient binding of FNR In the absence of oxygen FNR protein forms dimers and only this active state promotes gene expression [21]. Modifications of nucleotides in the middle of the FNR binding motif led to an increased level of expression (Fig 3, P1.6). The modified fully symmetrical FNR motif is sup- posed to bind the FNR dimer more efficiently [21] and this might explain the intensified transcription. Probable mechanism of tumor specific activation doi:10.1371/journal.pone.0155338.g007 significantly influence transcription [25, 26]. But could such factors initiate transcription by themselves? The absence of expression of promoter P1.2 (FNR knock out) in tumor, spleen and under an- and aerobic conditions shows that FNR is a compulsory prerequisite for tran- scription. Altogether: DNA tertiary structure and nucleotide context within and around the basal promoter serve as a trigger under the special conditions realized solely in tumors. Further we discuss some of the factors in more detail. DNA remodeling A single nucleotide deletion in TGIF motif (P1.5, Fig 3) led to the reduction of expression by 25%. TGIF is a eukaryotic transcription factor and most probably is not relevant in this context. But the motif itself "CTTTGTCAGAA" contains a conserved triplet "TGT" which is known to significantly bend DNA [27]. A specifically bended DNA of a promoter region can initiate transcription more effectively by more efficient binding the CAP protein [27]. Besides TGT, there are other regions not covered by the identified motifs that contribute to the rate of tran- scription, but not to the specificity. This can be concluded from the mutations of "insignificant" nucleotides in promoter P1.7 which led to a significant reduction in the level of expression. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Construction of insert fragments To construct plasmids that contain fragments of the original library inserts [30], oligonucleo- tides of the desired sequence were either directly ordered (Eurofins MWG Operon, Germany) and cloned into the vector (pMW82), or for longer sequences, primers were designed accord- ingly to amplify the fragment from the original plasmid. SL7207 was transformed with plas- mids containing the amplification products and plasmid DNA was sequenced to confirm correct sequence of the amplification products. Ethics statement Procedures involving animals and their care were fully in compliance with the German Animal Welfare Act (Tierschutzgesetz, 1998) and with the permission number 33.9.42502-04-050/09 of LAVES (Niedersaechsisches Landesamt fur Verbraucherschutz und Lebensmittelsicherheit). Tumor Specific Bacterial Promoter other conditions exist which make promoters active. Osmolaritiy and pH are known to be dis- tinct between normal tissue and neoplasias and could be a reason for the activation via specific DNA remodeling. In addition, the insufficient nutrient supply as mimicked by minimal medium might trigger some regulatory mechanisms. We could also show that tumors colo- nized by bacteria strongly attract neutrophilic granulocytes [29]. Signals from such cells might also induce transcription via anti-microbial peptides or other secreted molecules. Thus, molec- ular definition of such additional transcriptional inducers will lead to a more complete picture of tumor microenvironment. Obviously, promoters of Salmonella are not evolutionary selected for the microenvironment of a solid tumor. Rather, the tumor mimics natural habitats of the bacteria. Hypoxia and anoxia, as can be found in the central necrotic or its neighboring regions, was a first apparent suggestion by us and others. Such conditions might not prevail in systemic organs but are most likely excessive in the large intestine. This idea was only partly confirmed. Apparently, the tumor microenvironment represents a highly complex environment for which a natural equiv- alent cannot be envisioned yet. It will be important to unravel such conditions further as it may provide new targets for therapy by bacteria or other means. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Weak basal promoter TSP promoters exhibit relatively low promoter recognition score of 82.7±5.5 (SEM) as identi- fied by the BROM program [13]. So for example, core promoter elements of the well studied P0.212 are TAGCTT (-35) and TTTAAT (-10) and appeared to be not optimal compared to the known consensus sequences TTGTCA and TATAAT. To compare: promoter recognition score for the Salmonella housekeeping genes is 95.3±3.4 (genes: aroC, dnaN, hemD, hisD, purE, sucA and thrA [28], for NP promoters is 81.1±3.7 and for RP promoters is 78.2±7.9. From another side, the overall score revealed by the kernel method [11] of the TSP promot- ers is significantly higher compared to NP or RP promoters (see results section). The latter pro- gram additionally accounts for nucleotides between and around of -35 and -10 elements. Therefore, we may suggest that the specificity of expression of TSP promoters is achieved by very fine tuning of the basal promoter (in-)efficacy. This also explains why a quite frequent combination of FNR and normal promoter do not provide required tumor specificity. The deviation from the well known assumption that promoters should mainly respond to anaerobic conditions, known as Warburg effect, is interesting. Promoters of group A in Fig 6 respond to anaerobic conditions as predicted, promoters in groups B and C do not, but all pro- moters respond to tumor conditions. It demonstrates that in the tumor microenvironment 12 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Animal experiments Eight weeks old female BALB/c mice were purchased from Janvier (France) and subcutane- ously injected with 5x105 CT26 colon carcinoma cells (ATCC CRL-2638). When tumors reached volumes of approximately 200 mm3, mice were infected intravenously with 5x106 bac- teria (Salmonella Typhimurium strain SL7207) in 100 μl PBS. One, three, and five days after infection, mice were sacrificed by exposure to CO2, respective tissues were removed and homogenized in 2 ml PBS. The homogenates were diluted 1:10 (spleen, liver) or 1:100 (tumors) in 0.1% TritonX-100/PBS containing 2 mM EDTA, filtered through a 30 μm CellTrics filter (Partec, Germany) and sorted. Samples were analyzed via two color flow cytometry on a FAC- SAria or LSRII, respectively (Becton Dickinson, USA) and plated on LB plates containing 50 μg/ml ampicillin to allow normalization. No plasmid loss was confirmed via plating on ampicillin. The two color flow cytometry is a method that allows to discriminate GFP express- ing bacteria from autofluorescent cellular debris since GFP expressing Salmonella have a sub- stantially lower orange/green emission ratio [31]. Additionally, forward and side scatter were used to discriminate Salmonella from larger particles by setting an appropriate scatter gate. For more detailed information see [8]. For histological analyses, mice received 1d p.i. 1.5 mg of the PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 13 / 17 Tumor Specific Bacterial Promoter hypoxia marker pimonidazole hydrochloride (Hydroxyprobe, Inc) dissolved in 100 μl saline. The tumors were harvested 45 min after administration, fixed in 4% neutrally buffered formal- dehyde for 24 to 48 hours, embedded in paraffin and consecutive 3 μm sections were stained with the affinity purified rabbit-anti-pimonidazole antibody (PAb2627AP 0.5mg/ml IgG), rab- bit-anti-salmonella sp. antibody or hematoxylin-eosin. Sections were analyzed by light micros- copy with an Olympus BX51 microscope and cellSens software. Bacterial growth under aerobic and anaerobic conditions Respective bacterial strains were streaked out from glycerol stocks onto LB agar plates contain- ing the appropriate antibiotics. After overnight growth at 37°C, the cultures were used to inoc- ulate (i) 4 ml LB medium with antibiotics and grown at 37°C overnight with shaking at 180 rpm or (ii) 15 ml of induction medium (IM) and minimal medium (MM). Both are M9 medium based [32] without CaCl2, supplemented with the appropriate antibiotics, 100 μM MgSO4, 40 μg/ml histidine, 40 μg/ml phenylalanine, 40 μg/ml tryptophane, 40 μg/ml tyrosine, 10 μg/ml para-aminobenzoic acid, 10 μg/ml 2,3-dihydroxybenzoate and 0.2% glucose. The salt concentration was decreased to 0.05% NaCl and the pH was adjusted to 5.5 (IM) or to 7.4 (MM). From the 4 ml liquid cultures (i) 200 μl were used to start two new cultures of 20 ml LB medium each. One was grown under aerobic and the other under anaerobic conditions. Before 1:100 inoculation of the 15 ml liquid cultures for condition (ii), the cultured bacterial cells were washed twice in PBS and adjusted to OD600 of 1.0. Cultures were analyzed at different time points by flow cytometry and parallel by OD600 measurements or plating to allow normaliza- tion. Data presented were derived from 3 hrs (minimal medium) or 4 hrs (aerobic/anaerobic) cultures, respectively. PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 Bioinformatics analysis Datasets of promoter sequences were compiled on the basis of our previous research. Accord- ing to that, tumor specific promoters (TSP) are 13 promoters from class 1, 115 negative pro- moters (NP) are from class 5 and lowTSPs are 12 promoters from class 2 [8]. LowTSP promoters show lower expression in tumors than TSP and may additionally have some low but non-zero expression in spleen. A random promoter dataset (RP) was compiled by splitting ran- domly the entire Salmonella genome into fragments following the same length distribution as in the TSP set, resulting in 7682 sequences. Negative promoters (NP) are DNA fragments from the Salmonella genome that are proved not to initiate any transcription either in tumors or spleen [8]. Promoter nomenclature will be as follows. Promoters from [8] will be denoted as "P0." (round 0) followed by a number that corresponds to the number used in [8]. Fragments of P0. promoters will be supplemented with a consecutive number (for example, P0.212_1). Promot- ers in knockout experiments will be denoted as P1., artificial promoters as P2. both followed by a consecutive number. Parameters of the methods for recognition of basal promoters, regulatory motifs and other elements were selected such that they maximize discrimination between tumor specific pro- moters and negative promoters. As boundary condition, it was set that at least 75% (10 out of 13) of TSPs must have a minimum of one recognized element and at most 50% of NPs may contain such an element. We will denote a portion of TSPs that have an element as ElementNa- meTSP and for negative promoters as ElementNameNP. Recognition was performed for a range of values for each parameter required by a method and those values that maximize the ratio ElementNameTSP/ElementNameNP were selected as optimal, provided that the boundary PLOS ONE \| DOI:10.1371/journal.pone.0155338 May 12, 2016 14 / 17 Tumor Specific Bacterial Promoter conditions are met (i.e. ElementNameTSP 0.75 and ElementNameNP0.50). The higher the ratio the more specific is an element to the promoters. This principle was applied for recognition of basal promoters using Kernel [11] and HMM [12] methods, for the identification of DNA binding motifs using position weight matrixes (PWMs) and for the evaluation of AT-rich regions. AT-rich regions were defined as 100bp regions with an overall A+T content over 0.6. When searching for the repeat AAAAAAAA (we denote A8), one mismatch is allowed. Acknowledgments This work was supported in part by the Deutsche Krebshilfe and the Ministry for Education and Research (BMBF). NK was supported by the Helmholtz Graduate School for Infection Research. Special thanks goes to Dr. S. Lienenklaus for lending important electronic hardware without which this manuscript would not exist in its present form and to Regina Lesch for technical assistance. Bioinformatics analysis To identify exact positions of TATA-box and Inr element program BROM [13] was applied, which is developed by the same authors as the sequence alignment kernel [11]. Due to limitations of the program it could not be applied to batch pro- cessing, but only to single promoters. S1 Table. Genes associated with tumor specific promoters. (DOC) S2 Table. Combinatorial modules found in tumor specific promoters. Each module consists of two or more elements indicated by "+". P–values are calculated as a binomial probability to observe the actual number of promoters with a module in the TSP set compared to RP set. (DOC) Supporting Information S1 Fig. Motifs identified in the set of tumor specific promoters. Values are normalized num- bers of promoters in a set containing at least one motif. P–values are calculated as a binomial probability to observe the actual number of promoters with a motif in the TSP set compared to RP set. (DOC) S2 Fig. DNA sequence and nucleotide substitutions introduced into the minimal promoter and results of expression analysis. DNA motifs for FNR are shown in yellow, NagC in light brown, TGIF in green, TATA-box and Inr element in grey. Mutated nucleotides are shown in red. (DOC) S2 Fig. DNA sequence and nucleotide substitutions introduced into the minimal promoter and results of expression analysis. DNA motifs for FNR are shown in yellow, NagC in light brown, TGIF in green, TATA-box and Inr element in grey. Mutated nucleotides are shown in red. (DOC) S3 Fig. DNA sequence of artificial promoter constructs. DNA motifs for FNR are shown in yellow, -35 and -10 elements are in grey. Nucleotides introduced into the template sequence are in capital and marked red. 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https://openalex.org/W3215693740	https://diposit.ub.edu/dspace/bitstream/2445/181783/1/716191.pdf	Latin	null	Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain	International journal of molecular sciences	2,021	cc-by	12,136	Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain Esther García‐García 1,2,3, Nerea Chaparro‐Cabanillas 1, Albert Coll‐Manzano 1,2, Maria Carreras‐Caballé 1, Albert Giralt 1,2, Daniel Del Toro 1,2,3, Jordi Alberch 1,2,3,4, Mercè Masana 1,2,3,,† and Manuel J. Rodríguez 1,2,3,,† 1 Department of Biomedical Sciences, Institute of Neurosciences, School of Medicine and Health Sciences, Universitat de Barcelona, E‐08036 Barcelona, Spain; egarcia92@ub.edu (E.G.‐G.); nereachc01@gmail.com (N.C.‐C.); albert.coll@ub.edu (A.C.‐M.); carrerasc.maria@gmail.com (M.C.‐C.); albertgiralt@ub.edu (A.G.); danieldeltoro@ub.edu (D.D.T.); alberch@ub.edu (J.A.) 1 Department of Biomedical Sciences, Institute of Neurosciences, School of Medicine and Health Sciences, Universitat de Barcelona, E‐08036 Barcelona, Spain; egarcia92@ub.edu (E.G.‐G.); nereachc01@gmail.com (N.C.‐C.); albert.coll@ub.edu (A.C.‐M.); carrerasc.maria@gmail.com (M.C.‐C.); albertgiralt@ub.edu (A.G.); danieldeltoro@ub.edu (D.D.T.); alberch@ub.edu (J.A.) g ( ) ( ) (J ) 2 August Pi i Sunyer Biomedical Research Institute (IDIBAPS), E‐08036 Barcelona, Spain 3 Networked Biomedical Research Centre for Neurodegenerative Disorders (CIBERNED), E‐08036 Barcelona, Spain 4 Production and Validation Center of Advanced Therapies (Creatio), Faculty of Medicine and Health Science, 4 Production and Validation Center of Advanced Therapies (Creatio), Faculty of Medicine and Health Science, University of Barcelona, E‐08036 Barcelona, Spain * Correspondence: mmasana@ub.edu (M.M.); marodriguez@ub.edu (M.J.R.); Tel.: +34‐93‐4035287 (M.M.); +34‐93‐4024525 (M.J.R.) † These authors contributed equally to this work. Abstract: Loss‐of‐function mutations in the human vacuolar protein sorting the 13 homolog A (VPS13A) gene cause Chorea‐acanthocytosis (ChAc), with selective degeneration of the striatum as the main neuropathologic feature. Very little is known about the VPS13A expression in the brain. The main objective of this work was to assess, for the first time, the spatiotemporal distribution of VPS13A in the mouse brain. We found VPS13A expression present in neurons already in the em‐ bryonic stage, with stable levels until adulthood. VPS13A mRNA and protein distributions were similar in the adult mouse brain. We found a widespread VPS13A distribution, with the strongest expression profiles in the pons, hippocampus, and cerebellum. Interestingly, expression was weak in the basal ganglia. VPS13A staining was positive in glutamatergic, GABAergic, and cholinergic neurons, but rarely in glial cells. At the cellular level, VPS13A was mainly located in the soma and neurites, co‐localizing with both the endoplasmic reticulum and mitochondria. However, it was not enriched in dendritic spines or the synaptosomal fraction of cortical neurons. In vivo pharma‐ cological modulation of the glutamatergic, dopaminergic or cholinergic systems did not modulate VPS13A concentration in the hippocampus, cerebral cortex, or striatum. These results indicate that VPS13A has remarkable stability in neuronal cells. Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain Understanding the distinct expression pattern of VPS13A can provide relevant information to unravel pathophysiological hallmarks of ChAc. Citation: García‐García, E.; Chaparro‐Cabanillas, N.; Coll‐Manzano, A.; Carreras‐Caballé, M.; Giralt, A.; Del Toro, D.; Alberch, J.; Masana, M.; Rodríguez, M.J. Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain. Int. J. Mol. Sci. 2021, 22, 13018. https://doi.org/10.3390/ ijms222313018 Received: 28 October 2021 Accepted: 25 November 2021 Published: 1 December 2021 Received: 28 October 2021 Accepted: 25 November 2021 Published: 1 December 2021 Keywords: Chorea‐acanthocytosis; VPS13A; brain distribution; movement disorders; basal gan‐ glia; neurodegeneration Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Citation: García‐García, E.; Chaparro‐Cabanillas, N.; Coll‐Manzano, A.; Carreras‐Caballé, M.; Giralt, A.; Del Toro, D.; Alberch, J.; Masana, M.; Rodríguez, M.J. Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain. Int. J. Mol. Sci. 2021, 22, 13018. https://doi.org/10.3390/ ijms222313018 1. Introduction The human vacuolar protein sorting the 13 homolog A (VPS13A) gene encodes a large protein of 3174 amino acids named VPS13A or chorein. Human VPS13A protein is of great interest because loss‐of‐function mutations in its coding gene lead to Cho‐ rea‐acanthocytosis (ChAc; MIM 200150), a very rare and complex autosomal recessive adult‐onset neurodegenerative disorder [1,2]. In accordance with this etiology, ChAc has recently proposed to be renamed as VPS13A disease [3]. The main neuropathologic fea‐ ture in VPS13A disease is a selective degeneration of the caudate and putamen nuclei [4– 6], due to massive cell death of medium spiny neurons (MSNs) and striatal interneurons [7,8]. Moreover, many other neuronal subtypes, such as dopaminergic neurons or mo‐ Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 13018. https://doi.org/10.3390/ijms222313018 Int. J. Mol. Sci. 2021, 22, 13018 2 of 23 2 of 23 toneurons, are affected as well, contributing to the explanation of a plethora of patho‐ logical symptoms that include chorea, dystonia, involuntary oral biting, and orofacial dyskinesia, among others [9]. However, the study of structure, activity, and cell function of VPS13A has been poorly addressed. Pioneering functional studies were conducted in the only VPS13 pro‐ tein in Saccharomyces cerevisiae [10]. These studies determined that yeast VPS13 is a pro‐ tein located at membrane contact sites between the endoplasmic reticulum (ER) and other membranous organelles [11–14]. In cell models, VPS13A has been proposed to be necessary for stabilization of ER‐mitochondria contact sites, which enables transfer of li‐ pids between these two organelles [15]. VPS13A has been involved in several cellular functions and its loss of function has been associated with a wide range of cellular defects in eukaryotic cell models. These cellular defects include impaired autophagic degrada‐ tion, defective protein homeostasis [16–18], endocytic trafficking and lysosomal degra‐ dation impairment [19], and abnormal calcium homeostasis [20–22]. VPS13A is widely distributed in the body. According to the human Genotype‐Tissue Expression (GTEx) project (Accession number: ENSG00000197969.11; October 2021), VPS13A is expressed by many tissues including in the testis and kidney, as well as car‐ diovascular and digestive tissues. A similar distribution pattern was found in the mouse [23]. Nevertheless, little is known about the VPS13A distribution in neural cells and the brain. 1. Introduction A preliminary study showed that VPS13A is present in microsomal and synapto‐ somal fractions in the mouse brain [23]. In the same study, cell‐specific patterns of VPS13A‐like immunoreactivity were detected in the striatum, cerebral cortex, and hip‐ pocampus [23]. Despite that general overview, a time course of VPS13A expression and an in‐depth, detailed protein brain localization are yet to be unraveled. Moreover, it is interesting to know that when VPS13A expression starts, it allows for understanding a putative role of the protein in development. Therefore, a time course and regional ex‐ pression analysis of VPS13A in the mouse brain would be valuable in assessing the ear‐ liest possible influence of the lack of VPS13A in ChAc pathogenesis. In this study, we sought to determine, by molecular and histological methods, the presence and the re‐ gional distribution of VPS13A mRNA and protein in the embryonic, early postnatal, and adult mouse brain. Furthermore, we tested its potential dynamic changes upon several different types of challenges to the glutamatergic, dopaminergic, and cholinergic sys‐ tems. 2. Results 2.1. VPS13A Expression Starts from Embryonic Stage and Is Stable over Time To assess the time course of VPS13A expression in the mouse brain, we performed fluorescent in situ hybridization (FISH) and quantitative real‐time PCR (qRT‐PCR) of the cerebral cortex, striatum, hippocampus, and cerebellum at different ages (E15.5, P0, P7, P34 and 16 weeks). At the mid‐stages of mouse brain development (E15.5), we found that VPS13A is predominantly expressed in neuron‐enriched areas compared with germinal zones in both the cortex and hippocampus (Figure 1A,C). This finding is consistent with single‐cell RNA profiling data from E15.5 mouse cortex [24], which also showed that VPS13A is highly expressed in neurons compared with apical progenitors (t = 2.841, p = 0.0295; Student t‐test) (Figure 1B). We then analyzed the VPS13A expression in P0, P7, and P34 postnatal stages to evaluate putative changes of expression and/or distribution over time (Figure 1E). We found VPS13A expression in the cerebral cortex, striatum, hippocampus, and cerebellum for all ages analyzed. That labeling was homogeneous throughout all layers of the cerebral cortex, the striatum, and the pyramidal layer of all hippocampal subfields as well as the granular layer of dentate gyrus. 3 of 23 Int. J. Mol. Sci. 2021, 22, 13018 Figure 1. Vacuolar protein sorting the 13 homolog A (VPS13A) is mainly expressed by neurons and its expression is stable over time. (A) Drawing showing the location of the cortex (CTX), striatum (STR), and hippocampus (HIP) of a E15.5 mouse brain. (B) VPS13A expression in neurons and apical progenitors (AP) was quantified using single‐cell RNA pro‐ filing data for the cortex, as published in Florio et al. (2015) (GSE65000) [24]. n = 15–20; , p < 0.05, two‐tailed Student’s t‐test. Data are presented as whisker plots. (C) VPS13A mRNA staining in representative sagittal sections of the mouse brain. Scale bar 250 μm; n = 3. (D) Drawings showing the location of the CTX, STR, HIP and cerebellum (CB) of a P0, P7, and P34 mouse brain. (E) Expression of VPS13A mRNA in representative sagittal sections of the mouse brain at the dif‐ ferent stages. Scale bar 250 μm; n = 3. VPS13A mRNA levels were analyzed at different stages in (F) frontal cortex, (G) Figure 1. Vacuolar protein sorting the 13 homolog A (VPS13A) is mainly expressed by neurons and its expression is stable over time. 2. Results (A) Drawing showing the location of the cortex (CTX), striatum (STR), and hippocampus (HIP) of a E15.5 mouse brain. (B) VPS13A expression in neurons and apical progenitors (AP) was quantified using single‐cell RNA pro‐ filing data for the cortex, as published in Florio et al. (2015) (GSE65000) [24]. n = 15–20; , p < 0.05, two‐tailed Student’s t‐test. Data are presented as whisker plots. (C) VPS13A mRNA staining in representative sagittal sections of the mouse brain. Scale bar 250 μm; n = 3. (D) Drawings showing the location of the CTX, STR, HIP and cerebellum (CB) of a P0, P7, and P34 mouse brain. (E) Expression of VPS13A mRNA in representative sagittal sections of the mouse brain at the dif‐ ferent stages. Scale bar 250 μm; n = 3. VPS13A mRNA levels were analyzed at different stages in (F) frontal cortex, (G) 4 of 23 Int. J. Mol. Sci. 2021, 22, 13018 striatum, (H) hippocampus, and (I) cerebellum. n = 8; * p < 0.05, the Bonferroni post‐hoc test for CTX, STR, and CB and the Dunn test for HIP. striatum, (H) hippocampus, and (I) cerebellum. n = 8; * p < 0.05, the Bonferroni post‐hoc test for CTX, STR, and CB and the Dunn test for HIP. VPS13A expression was found in the external area of P0 and P7 cerebellum. In P34 cerebellum, this expression was mainly localized in Purkinje cell and granular layers. To better quantify changes in expression over time, we quantified VPS13A mRNA by qRT‐PCR (Figure 1F–I). We found an increase in VPS13A expression in the striatum (Figure 1G), with a mean 86.97% increase from E15.5 to P7 ages to then reach a plateau (F(4,34) = 5.080, p = 0.0026; one‐way ANOVA). In the hippocampus (Figure 1H), the ex‐ pression level was similar in all ages except in P34 samples, which showed a mean 71.5% VPS13A mRNA level increase (F(4,30) = 5.415, p = 0.0021; one‐way ANOVA). In the cerebral cortex (Figure 1F) and cerebellum (Figure 1I), we found stable VPS13A expression levels over all stages analyzed (F(4,33) = 1.519, p = 0.2193; one‐way ANOVA and F(3,26) = 1.291, p = 0.2986; one‐way ANOVA, respectively). 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain To further assess the distribution of VPS13A mRNA and protein in the adult mouse brain, we performed FISH and immunohistochemistry procedures, respectively, and evaluated the pattern of expression throughout the brain (Figures 2–4). Fluorescent in‐ tensity of VPS13A mRNA and protein staining was color‐coded using a standard 8‐bit 16‐color lookup table with ImageJ 1.51a (National Institutes of Health, Bethesda, MD, USA), and the semi‐quantitative visual analysis is summarized in Table 1. We found a wide distribution of mRNA and protein labeling throughout the mouse brain, with dis‐ tinct staining intensity profiles between nuclei. In general, the mRNA localization re‐ sembled that of the protein one (Table 1), with minor changes in intensity profile. 5 of 23 Int. J. Mol. Sci. 2021, 22, 13018 Figure 2. VPS13A mRNA and protein are widely localized in the cortex and basal ganglia‐related nuclei of the adult mouse brain. (A) Drawings with the location of the different brain regions shown in (B–S). Specific labeling of VPS13A mRNA (red) and protein (green) in the representative sagittal section of the mouse brain. Specific brain regions with de‐ tected expression were (B–D) frontal cortex, (E–H) somatosensorial cortex, (I,J) striatum, (K,L) globus pallidus, (M,N) substantia nigra, (O–Q) thalamus, and (R,S) subthalamic nucleus. CTX, cortex; cc, corpus callosum; CP, caudoputamen; GP, globus pallidus; I, II, III, IV, V and VI are cortical layers; SN, substantia nigra; PVT, paraventricular nucleus of the thalamus; TH, thalamus; RT, reticular nucleus of the thalamus; STN, subthalamic nucleus; and ZI, zona incerta. n = 3. Scale bar 250 μm. Figure 2. VPS13A mRNA and protein are widely localized in the cortex and basal ganglia‐related nuclei of the adult mouse brain. (A) Drawings with the location of the different brain regions shown in (B–S). Specific labeling of VPS13A mRNA (red) and protein (green) in the representative sagittal section of the mouse brain. Specific brain regions with de‐ tected expression were (B–D) frontal cortex, (E–H) somatosensorial cortex, (I,J) striatum, (K,L) globus pallidus, (M,N) substantia nigra, (O–Q) thalamus, and (R,S) subthalamic nucleus. CTX, cortex; cc, corpus callosum; CP, caudoputamen; GP, globus pallidus; I, II, III, IV, V and VI are cortical layers; SN, substantia nigra; PVT, paraventricular nucleus of the thalamus; TH, thalamus; RT, reticular nucleus of the thalamus; STN, subthalamic nucleus; and ZI, zona incerta. n = 3. Scale bar 250 μm. 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain 6 of 23 Int. J. Mol. Sci. 2021, 22, 13018 Figure 3. VPS13A mRNA and protein are widely localized in hippocampal‐related nuclei of the adult mouse brain. (A) Drawings with the location of the different brain regions shown in (B–J). Specific labeling of VPS13A mRNA (red) and protein (green) in representative sagittal section of the mouse brain. Specific brain regions with detected expression were (B–C) entorhinal cortex, (D–H) hippocampus, (I–J) induseum grisum. ENT, entorhinal cortex; SUB, subiculum; DG, dentate gyrus; I, IIa, IIb, III, IV, V and VI are cortical layers; IG, induseum grisum; and cc, corpus callosum. n = 3. Scale bar 250 μm. Figure 3. VPS13A mRNA and protein are widely localized in hippocampal‐related nuclei of the adult mouse brain. (A) Drawings with the location of the different brain regions shown in (B–J). Specific labeling of VPS13A mRNA (red) and protein (green) in representative sagittal section of the mouse brain. Specific brain regions with detected expression were (B–C) entorhinal cortex, (D–H) hippocampus, (I–J) induseum grisum. ENT, entorhinal cortex; SUB, subiculum; DG, dentate gyrus; I, IIa, IIb, III, IV, V and VI are cortical layers; IG, induseum grisum; and cc, corpus callosum. n = 3. Scale bar 250 μm. 7 of 23 Int. J. Mol. Sci. 2021, 22, 13018 J. Mol. Sci. 2021, 22, 13018 7 Figure 4. VPS13A mRNA and protein are widely localized in subcortical and non‐basal ganglia nuclei of the adult mouse brain. (A) Drawing with the location of the different brain regions shown in (B–M). Specific labeling of VPS13A mRNA (red) and protein (green) in the representative sagittal section of the mouse brain. Specific brain regions with detected expression were (B–C) hypothalamus, (D–E) lateral septum, (F–G) medulla, (H–J) pons, and (K–M) cerebellum. PVH, paraventricular hypothalamic nucleus; HY, hypothalamus; VMH, ventromedial hypothalamic nucleus; PMd, ventral premammillary nucleus; MM, medial mammillary nucleus; cc, corpus callosum; LS, lateral septum; RM, nucleus raphe magnus; GRN, gigantocellular reticular nucleus; IO, inferior olivary complex; PG, pontine gray; TRN, tegmental reticular nucleus; and SOC, superior olivary complex. n = 3. Scale bar 250 μm. Figure 4. VPS13A mRNA and protein are widely localized in subcortical and non‐basal ganglia nuclei of the adult mouse brain. (A) Drawing with the location of the different brain regions shown in (B–M). Specific labeling of VPS13A mRNA (red) and protein (green) in the representative sagittal section of the mouse brain. 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain Specific brain regions with detected expression were (B–C) hypothalamus, (D–E) lateral septum, (F–G) medulla, (H–J) pons, and (K–M) cerebellum. PVH, paraventricular hypothalamic nucleus; HY, hypothalamus; VMH, ventromedial hypothalamic nucleus; PMd, ventral premammillary nucleus; MM, medial mammillary nucleus; cc, corpus callosum; LS, lateral septum; RM, nucleus raphe magnus; GRN, gigantocellular reticular nucleus; IO, inferior olivary complex; PG, pontine gray; TRN, tegmental reticular nucleus; and SOC, superior olivary complex. n = 3. Scale bar 250 μm. 8 of 23 Int. J. Mol. Sci. 2021, 22, 13018 Table 1. VPS13A expression in the mouse brain. Brain Structure mRNA Protein Brain Structure mRNA Protein Motor Cortex Basal ganglia Layer I − − Caudate putamen + + Layer II/III ++ ++ Fundus of striatum + + Layer V ++ ++ Globus pallidus + + Layer VI ++ ++ Bed nucleus of stria terminalis ++ + Somatosensory Cortex Nucleus accumbens + + Layer I − − Substantia nigra + + Layer II/III ++ + Subthalamic nucleus +++ ++ Layer IV ++ + Amygdaloid complex Layer V +++ ++ Basolateral amygdalar nucleus ++ ++ Layer VI ++ + Basomedial amygdalar nucleus ++ ++ Visual Cortex Central amygdalar nucleus ++ ++ Layer I − − Thalamus Layer II/III ++ + Reticular nucleus ++ ++ Layer IV ++ + Lateral dorsal nucleus + + Layer V ++ ++ Posterior complex + + Layer VI ++ + Ventral medial nucleus + + Entorhinal Area Ventral/Dorsal geniculate n. + + Layer I − − Paraventricular nucleus ++ ++ Layer II +++ +++ Medial habenula ++ ++ Layer III ++ + Nucleus of reuniens + + Layer IV ++ + Hypothalamus Layer V/VI ++ + Paraventricular hypothalamic n. ++ ++ Hippocampal Region Ventromedial hypothalamic n. 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain ++ ++ CA3 +++ ++ Ventral premammillary nucleus ++ ++ CA2 +++ ++ Lateral mammillary nucleus ++ n/a CA1 ++ + Medial mammillary nucleus ++ n/a Granular layer of the DG +++ + Medial preoptic area + + Polymorphic layer of the DG + + Arcuate hypothalamic nucleus + + Molecular layer of the DG − − Suprachiasmatic nucleus + + Hilus ++ ++ Zona incerta + + Postsubiculum ++ ++ Septal region Presubiculum ++ ++ Lateral septal nucleus + + Subiculum ++ +++ Medial septal nucleus + + Induseum griseum ++ ++ Septohippocampal nucleus + + Mid‐Brain Cerebellum Superior colliculus + + Purkinje cell layer +++ +++ Inferior colliculus + + Molecular layer + + Edinger–Westphal nucleus ++ ++ Granular layer ++ ++ Trochelar nucleus ++ n/a White matter structures Oculomotor nucleus ++ n/a Corpus callosum + − Pons Anterior commissure − − Pontine gray +++ +++ Fornix system + + Tegmental reticular nucleus +++ +++ Optic tract + − Pontine reticular nucleus ++ n/a Ventral hippocampal commissure + − Motor nucleus trigeminal +++ n/a Non‐neuronal tissue Table 1. VPS13A expression in the mouse brain. 9 of 23 9 of 23 Int. J. Mol. Sci. 2021, 22, 13018 Medulla Choroid plexus +++ +++ Gigantocellular reticular n. ++ ++ Nucleus raphe magnus ++ ++ Facial motor nucleus +++ n/a Relative expression levels of VPS13A mRNA and protein in adult mouse brain are expressed in the following four cate‐ gories: ‐ not detectable, + weak signal, ++ moderate signal, and +++ strong signal. CA, Cornu Ammonis; DG, dentate gy‐ rus; n., nucleus. We observed VPS13A mRNA and protein throughout Layers II to VI of the cerebral cortex, with distinct intensity profiles between different cortical regions (Figure 2B–H, Table 1). Thus, the motor cortex presented the strongest VPS13A labeling, which was homogeneous within Layers II to VI (Figure 2B–D). Conversely, the somatosensory cor‐ tex had moderate staining (Figure 2E–H), except for Layer V, which displayed higher VPS13A staining, compared to the other cortical layers (Figure 2G,H). At the cellular level, we found VPS13A immunostaining mainly located in the perinuclear zone. We also observed protein labeling in the apical dendrite of pyramidal neurons (Figure 2D,G,H). In the basal ganglia nuclei, cells from the caudate putamen, globus pallidus, and sub‐ stantia nigra had the weakest VPS13A labeling (Figure 2I–N). 2.2. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain Within the thalamic nuclei, the reticular and paraventricular nuclei presented the higher VPS13A expression, com‐ pared with the other thalamic nuclei, which presented moderate labeling (Figure 2O–Q). Finally, the subthalamic nucleus presented high VPS13A mRNA staining and moderate protein labeling (Figure 2R,S). We also evaluated the VPS13A expression in hippocampal related structures, in‐ cluding input and output nuclei (Figure 3). We observed moderate staining in the ento‐ rhinal cortex, although in this cortical region Layer II displayed higher VPS13A staining compared to the other layers (Figure 3B,C). The hippocampal formation presented high VPS13A labeling in the pyramidal layer of all hippocampal subfields and the granular layer of dentate gyrus, with a more intense mRNA labeling in CA3 and CA2 subfields and dentate gyrus (Figure 3D). However, there were differences between FISH and im‐ munohistochemical labelings (Figure 3E). Thus, VPS13A protein staining was moderate in the CA1 pyramidal layer (Figure 3F) and high in the CA3 and CA2 pyramidal layers (Figure 3G), whereas the staining in the granular dentate gyrus was considerably weaker (Figure 3H). The induseum grisum presented moderate labeling (Figure 3I,J). ( g ) g p g ( g J) We also found VPS13A expression in hypothalamic regions (Figure 4B,C). Particu‐ larly, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the ventral premammillary nucleus, which presented high staining (Figure 4B,C), while the rest of the hypothalamic nuclei presented moderate VPS13A expression. The septal nucleus presented moderate VPS13A expression (Figure 4D,E). The gigantocellular reticular nucleus and the nucleus raphe magnus also presented high VPS13A mRNA and protein staining, compared with the other medullar nuclei (Figure 4F,G). Finally, the pons was one of the structures that presented the highest VPS13A expression (Figure 4H– J). The pontine gray, tegmental reticular, and pontine reticular nuclei were the subnuclei of the pons with the highest mRNA and protein labeling. The cerebellum was another of the nuclei with high VPS13A expression (Figure 4K,M). Particularly, the Purkinje cells displayed the highest labeling in this brain region, while cells in the granular layer pre‐ sented moderate VPS13A expression and the molecular layer presented the weakest la‐ beling (Figure 4M). 2.3. VPS13A is Enriched in Glutamatergic, GABAergic, and Cholinergic Neurons The expression pattern of VPS13A suggests that it is expressed in glutamatergic neurons, as observed in the cerebral cortex (Figure 2D,G,H) and hippocampus (Figure 3E–G). It is also expressed by GABAergic neurons, as observed in Purkinje cells in the cerebellum (Figure 4M), indicating that VPS13A could be expressed in different neuronal types. To analyze the expression of VPS13A in different neuronal subpopulations, we Int. J. Mol. Sci. 2021, 22, 13018 10 of 23 10 of 23 performed double immunostaining using antibodies against VPS13A; either calbindin or parvalbumin were used as specific markers for GABAergic neuronal subpopulations or ChAT as a marker of cholinergic neurons (Figure 5). We found VPS13A immunostaining in calbindin‐positive GABAergic neurons (Figure 5A,B), in parvalbumin‐positive GA‐ BAergic neurons (Figure 5C), and in ChAT‐positive cholinergic neurons (Figure 5D). performed double immunostaining using antibodies against VPS13A; either calbindin or parvalbumin were used as specific markers for GABAergic neuronal subpopulations or ChAT as a marker of cholinergic neurons (Figure 5). We found VPS13A immunostaining in calbindin‐positive GABAergic neurons (Figure 5A,B), in parvalbumin‐positive GA‐ BAergic neurons (Figure 5C), and in ChAT‐positive cholinergic neurons (Figure 5D). re 5. VPS13A is present in GABAergic, cholinergic neurons, and a few astrocytes, as well as to a lesser extend in ol‐ endrocytes or microglia. Orthogonal view from different planes of representative confocal images of (A,B) 13A/Calbindin‐positive GABAergic neuron, (C) VPS13A/Parvalbumin‐positive GABAergic neuron, (D) Figure 5. VPS13A is present in GABAergic, cholinergic neurons, and a few astrocytes, as well as to a lesser extend in ol‐ igodendrocytes or microglia. Orthogonal view from different planes of representative confocal images of (A,B) VPS13A/Calbindin‐positive GABAergic neuron, (C) VPS13A/Parvalbumin‐positive GABAergic neuron, (D) 11 of 23 11 of 23 Int. J. Mol. Sci. 2021, 22, 13018 VPS13A/ChAT‐positive cholinergic neurons, (E,F) VPS13A/GFAP‐positive astrocytes, (G) VPS13A/CNPase‐positive oli‐ godendrocytes, and (H) VPS13A/Iba1‐positive microglia in sagittal sections of the mouse brain. n = 3. Scale bar 10 μm. VPS13A/ChAT‐positive cholinergic neurons, (E,F) VPS13A/GFAP‐positive astrocytes, (G) VPS13A/CNPase‐positive oli‐ godendrocytes, and (H) VPS13A/Iba1‐positive microglia in sagittal sections of the mouse brain. n = 3. Scale bar 10 μm. VPS13A/ChAT‐positive cholinergic neurons, (E,F) VPS13A/GFAP‐positive astrocytes, (G) VPS13A/CNPase‐positive oli‐ godendrocytes, and (H) VPS13A/Iba1‐positive microglia in sagittal sections of the mouse brain. n = 3. Scale bar 10 μm. 2.3. VPS13A is Enriched in Glutamatergic, GABAergic, and Cholinergic Neurons To further evaluate whether glial cells also express VPS13A, we carried out double immunostaining using antibodies against this protein and either GFAP, CNPase, or Iba1, as specific markers for astrocytes, oligodendrocytes, and microglia, respectively (Figure 5). We found VPS13A staining in some, but not all, GFAP‐positive cells (Figure 5E). Thus, we found VPS13A‐positive cells in the corpus callosum, the cerebral cortex, and both the oriens and radiatum strata of the hippocampus. However, most GFAP‐positive cells from all other brain regions, such as the striatum, cerebellum, and thalamic nuclei, did not express VPS13A (Figure 5F). VPS13A/S100B double immunostaining reached similar results (data not shown). Finally, we found VPS13A staining in neither CNPase‐positive oligodendrocytes nor Iba1‐positive microglia (Figure 5G,H). 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria Our brain tissue observations indicate that the VPS13A protein is expressed in the soma of neurons. We also observed VPS13A immunolabeling in neuronal processes, such as pyramidal apical dendrites (Figures 2–5). To better understand the subcellular VPS13A distribution, we determined the VPS13A localization in mouse cortical primary cultured neurons by immunocytochemistry (Figure 6). Cultured neurons presented a strong punctate VPS13A labeling in the perinuclear zone, followed by punctate staining of lower intensity in neuronal processes, whereas the nucleus was devoid of specific staining (Figure 6A). Since VPS13A has been described to interact with ER and mitochondria in yeast, we evaluated if these interactions were also present in neuronal cells. Thus, we carried out double immunostaining in cortical primary cultures using antibodies against VPS13A, plus either calnexin as a specific marker for ER membrane or TOMM20 as a marker for the external mitochondrial membrane. VPS13A labeling co‐localized with calnexin (Figure 6B) and also with TOMM20 (Figure 6C). Co‐immunolabeling quantifi‐ cation is evidenced by higher VPS13A co‐localization with calnexin (Manders’ tM1 = 0.915 and Manders’ tM2 = 0.928) than with TOMM20 (Manders ’tM1 = 0.634 and Man‐ ders’ tM2 = 0.572), suggesting stronger enrichment in ER compartments in neurons. To investigate whether VPS13A is present, not only in the dendritic shaft but also in the synaptic terminals, we assessed its presence in synaptic spines of cultured cortical neurons by double staining using phalloidin to label dendritic F‐actin puncta. We found weak VPS13A immunolabeling within dendritic spines of cortical primary cultures (Figure 6D). To delve into this result, we also isolated crude synaptosomes of cerebral cortex tissue and quantified VPS13A by Western blot. We found VPS13A present but not significantly enriched in the crude synaptosome fraction of cerebral cortex tissue (t = 0.9179, p= 0.3754; student t‐test) (Figure 6E). 12 of 23 Int. J. Mol. Sci. 2021, 22, 13018 gure 6. VPS13A is mainly present in the soma of the neurons, colocalizes with endoplasmic reticulum and mitocho ia, and is found in synaptosomal fraction lysates. (A) Representative confocal images of VPS13A immunostaining rtical primary neurons. (B) Orthogonal view from different planes of representative confocal images PS13A/Calnexin colocalization in cortical primary neurons. 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria (C) Colocalization scatter plot showing the relationshi tween the signals in each pixel for VPS13A and Calnexin. (D) Orthogonal view from different planes of representati nfocal images of VPS13A/TOMM20 colocalization in cortical primary neurons. (E) Colocalization scatter plot showin e relationships between the signals in each pixel for VPS13A and TOMM20. Scale bar, A,B,D, 10 μm ; detail in A, 5 μm Orthogonal view from different planes of representative confocal images of VPS13A/F‐Actin localization in cortic imary neurons. Scale bar 5 μm. (G) Homogenate (H) and synaptosomal fractions (S) of mouse cortex were analyzed b estern blot. SV2A (pre‐synaptic protein) and PSD‐95 (post‐synaptic protein) were used as the control for synaptosom ction isolation. Actin was used as a loading control. Data representing mean ± SEM and differences were analyzed b udent’s t‐test; * p < 0.001. n = 8. Figure 6. VPS13A is mainly present in the soma of the neurons, colocalizes with endoplasmic reticulum and mitochon‐ dria, and is found in synaptosomal fraction lysates. (A) Representative confocal images of VPS13A immunostaining in cortical primary neurons. (B) Orthogonal view from different planes of representative confocal images of VPS13A/Calnexin colocalization in cortical primary neurons. (C) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and Calnexin. (D) Orthogonal view from different planes of representative confocal images of VPS13A/TOMM20 colocalization in cortical primary neurons. (E) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and TOMM20. Scale bar, A,B,D, 10 μm ; detail in A, 5 μm. (F) Orthogonal view from different planes of representative confocal images of VPS13A/F‐Actin localization in cortical primary neurons. Scale bar 5 μm. (G) Homogenate (H) and synaptosomal fractions (S) of mouse cortex were analyzed by Western blot. SV2A (pre‐synaptic protein) and PSD‐95 (post‐synaptic protein) were used as the control for synaptosomal fraction isolation. Actin was used as a loading control. Data representing mean ± SEM and differences were analyzed by Student’s t‐test; * p < 0.001. n = 8. Figure 6. VPS13A is mainly present in the soma of the neurons, colocalizes with endoplasmic reticulum and mitochon‐ dria, and is found in synaptosomal fraction lysates. (A) Representative confocal images of VPS13A immunostaining in cortical primary neurons. (B) Orthogonal view from different planes of representative confocal images of VPS13A/Calnexin colocalization in cortical primary neurons. 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria (C) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and Calnexin. (D) Orthogonal view from different planes of representative confocal images of VPS13A/TOMM20 colocalization in cortical primary neurons. (E) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and TOMM20. Scale bar, A,B,D, 10 μm ; detail in A, 5 μm. (F) Orthogonal view from different planes of representative confocal images of VPS13A/F‐Actin localization in cortical primary neurons. Scale bar 5 μm. (G) Homogenate (H) and synaptosomal fractions (S) of mouse cortex were analyzed by Western blot. SV2A (pre‐synaptic protein) and PSD‐95 (post‐synaptic protein) were used as the control for synaptosomal fraction isolation. Actin was used as a loading control. Data representing mean ± SEM and differences were analyzed by Student’s t‐test; * p < 0.001. n = 8. Figure 6. VPS13A is mainly present in the soma of the neurons, colocalizes with endoplasmic reticulum and mitochon‐ dria, and is found in synaptosomal fraction lysates. (A) Representative confocal images of VPS13A immunostaining in cortical primary neurons. (B) Orthogonal view from different planes of representative confocal images of VPS13A/Calnexin colocalization in cortical primary neurons. (C) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and Calnexin. (D) Orthogonal view from different planes of representative confocal images of VPS13A/TOMM20 colocalization in cortical primary neurons. (E) Colocalization scatter plot showing the relationships between the signals in each pixel for VPS13A and TOMM20. Scale bar, A,B,D, 10 μm ; detail in A, 5 μm. (F) Orthogonal view from different planes of representative confocal images of VPS13A/F‐Actin localization in cortical primary neurons. Scale bar 5 μm. (G) Homogenate (H) and synaptosomal fractions (S) of mouse cortex were analyzed by Western blot. SV2A (pre‐synaptic protein) and PSD‐95 (post‐synaptic protein) were used as the control for synaptosomal fraction isolation. Actin was used as a loading control. Data representing mean ± SEM and differences were analyzed by Student’s t‐test; * p < 0.001. n = 8. Int. J. Mol. Sci. 2021, 22, 13018 13 of 23 13 of 23 2.5. Manipulation of the Dopaminergic, Glutamatergic, and Cholinergic Circuits Does Not Modify VPS13A Levels 2.5. Manipulation of the Dopaminergic, Glutamatergic, and Cholinergic Circuits Does Not Modify VPS13A Levels While VPS13A seems not to be a core synaptic protein, previous studies suggested that VPS13A was modulated by the dopaminergic system [25]. 3. Discussion Characterizing the detailed VPS13A mRNA and protein neuroanatomical distribu‐ tion should help to unravel its function in the brain and provide novel insights toward the knowledge of ChAc pathophysiology. We report, for the first time, wide stable VPS13A expression by mature neurons in the embryonic stage and throughout the adult mouse brain. We also found that VPS13A mRNA localization and expression levels re‐ semble those of the protein one, with a distinct distribution in the brain between nuclei. Particularly, we detected an enrichment of VPS13A in the pons, cerebellum, and hippo‐ campus, moderate staining in the cortex as well as in the thalamic and hypothalamic nu‐ clei, and weak staining in the basal ganglia nuclei. Only pyramidal cells, in the cerebral cortex as well as the hippocampal CA and granular cells in the DG, presented some dis‐ crepancies between mRNA and protein contents, suggesting differential mechanisms of translational control in these regions. The cerebral distribution of VPS13A expression in the mouse brain is consistent with that of the human brain reported in the Genotype‐Tissue Expression (GTEx) project (Accession number: ENSG00000197969.11; October 2021). However, a thorough, in‐depth analysis of the VPS13A distribution in the human brain is necessary to determine the degree of expression similarity between the two species. Nevertheless, the widespread VPS13A stable expression during development and adult ages suggests an essential role of this protein in brain function. This is especially important for those nuclei where VPS13A is highly expressed, such as the pons, cerebellum, and hippocampus. Since these particular brain nuclei are involved in autonomic and sensory functions [26], motor co‐ ordination and execution [27], and acquisition and storage of memories [28], respectively, a relevant role of VPS13A in those functions may be expected and ought to be confirmed. y p g Indeed, the distinct VPS13A brain distribution contributes to explaining the ChAc neuropathology. For example, severe atrophy, neuronal loss, and gliosis have been found in the hippocampus, temporal, and frontal lobes, or prefrontal cortex, of some patients [5,29,30]. Other authors report cerebellar atrophy [31,32], impairment of the hypotha‐ lamic endocrine function [33], and oculomotor abnormalities due to brainstem dysfunc‐ tion [34]. Thus, the high‐to‐moderate VPS13A expression levels in these brain areas evi‐ dences the important role of this protein in neuronal functioning and survival through‐ out the nervous system. 2.4. VPS13A is Present Mainly in the Soma of Neurons and Co‐Localizes with Both ER and Mitochondria Thus, we explored if VPS13A expression was modulated by the manipulation of specific neurotransmitter systems. To modulate the dopaminergic, glutamatergic, and cholinergic systems, we subjected mice to amphetamine, ketamine, or lithium‐scopolamine‐pilocarpine treat‐ ments, respectively, and evaluated VPS13A levels in the cortex, striatum, and hippo‐ campus by Western blot. VPS13A protein levels remained similar between control and treated mice in all of the tested treatments (Figure 7A–C). As these results could be re‐ lated to VPS13A being a highly stable protein for a long period of time, we assessed VPS13A protein stability using a cycloheximide (CHX) chasing assay in the STHdhQ7/Q7 striatal cell line. The protein synthesis inhibitor revealed that VPS13A is a very stable protein with a relative half‐life of 18.04 h in our conditions (Figure 7D). Figure 7. VPS13A protein concentration is not modulated by dopaminergic, glutamatergic, or cho‐ linergic systems. VPS13A levels were analyzed by Western blot in the prefrontal cortex, striatum, and hippocampus of an adult mouse treated with (A) D‐amphetamine (3 mg/kg, i.p., 8 days, n = 12– Figure 7. VPS13A protein concentration is not modulated by dopaminergic, glutamatergic, or cho‐ linergic systems. VPS13A levels were analyzed by Western blot in the prefrontal cortex, striatum, and hippocampus of an adult mouse treated with (A) D‐amphetamine (3 mg/kg, i.p., 8 days, n = 12– Int. J. Mol. Sci. 2021, 22, 13018 14 of 23 14 of 23 13), (B) ketamine (30 mg/kg, i.p., 8 days, n = 12–14) or (C) pilocarpine (45 mg/kg, i.p., single dose, n = 10–12). Tubulin was used as a loading control. Data represent mean ± SEM and differences were analyzed by Student’s t‐test; * p < 0.05. (D) Cultured cells of the STHdhQ7/Q7 cell line were treated with cycloheximide (CHX, 50 μg/mL) for 6, 24, and 48 h. Levels of VPS13A were then analyzed by Western blot. Tubulin was used as a loading control. Data represent mean ± SEM. The VPS13A half‐life was calculated by fitting the curve to a one‐phase decay type exponential equation. R2 = 0.8678. 3. Discussion y However, the main neuropathological feature in ChAc patients is the selective de‐ generation of the caudate nucleus and putamen [6,35] and, to a lesser extent, other basal ganglia nuclei [4,8,36]. Interestingly, we found weak VPS13A staining in these basal ganglia nuclei. Thus, the vulnerability of striatal neurons to VPS13A disease seems not to be related to the amount of protein present in the cell, but to specific striatal functional properties and MSN cell processes specifically affected by the lack of VPS13A. As a con‐ sequence, to define the VPS13A function in MSN activity and its involvement in basal ganglia circuitry functionality is a peremptory need to understand the ChAc neuropa‐ thology. With respect to its expression by different cell types, VPS13A is a neuronal protein present in all the glutamatergic, GABAergic, and cholinergic neuronal subpopulations analyzed. By contrast, astrocytes in most brain regions, oligodendrocytes, and microglia lacked specific VPS13A staining. In accordance, the VPS13A mRNA level obtained by RNA sequencing of glial cells of the mouse cerebral cortex was very low in myelinating Int. J. Mol. Sci. 2021, 22, 13018 15 of 23 15 of 23 oligodendrocytes and microglia, compared with neurons and astrocytes [37]. Interest‐ ingly, we found astrocytic VPS13A in the corpus callosum and, to some extent, in the hippocampus. Some studies support that astrocytes have heterogeneous phenotypes according to both their origin and environment [38]. Indeed, these cells are proposed to exert different effects on neuronal populations depending on their localization within brain circuits [39] and, thus, contribute to a selective vulnerability to injury [40]. Whether VPS13A has a role in such brain circuitry‐specific astrocyte function and vulnerability needs to be further explored. At the subcellular level, we found VPS13A in the soma, mainly in the perinuclear region, yet also in the neuronal processes. These results are also observed in dopaminer‐ gic PC12 cells, which present VPS13A localized in the same cell regions [41]. This peri‐ nuclear localization and high co‐localization with ER and mitochondria markers may be related with a role of VPS13A in the interplay between neuronal membranous organelle. Indeed, VPS13A is located at ER and the mitochondria contact sites of HeLa cells, where it is proposed to enable a lipid transfer required for mitochondria function [42]. VPS13A‐depleted HeLa cells have a decreased number of ER‐mitochondria contact sites, leading to mitochondria fragmentation and decreased mitophagy [15]. 3. Discussion However, since mitochondria and ER co‐localize with each other, in our approach TOMM20/VPS13A co‐localization can be coincidental, and a role of VPS13A outside mitochondria‐ER con‐ tacts should not be discarded. Nevertheless, the fact that VPS13A in neurons show simi‐ lar subcellular localization suggests that VPS13A may have similar functions, at least in rodents. Of note, other members of the VPS13 family, such as VPS13C or VPS13D, have been involved in the interaction of ER and their loss‐of‐function mutations also induce motor dysfunction [43,44]. Thus, further in‐depth analysis of the role of VPS13A in the neuronal ER‐mitochondria interplay may also help to understand the specific vulnera‐ bility and degeneration of striatal cells in the VPS13A disease and the involvement of ER function in motor impairment. Further, clarification of the putative interactions between different VPS13 family members may help to understand differences in the responses to VPS13A loss of function and, therefore, explain tissue‐specific differential responses. p p p At the synaptic level, we confirmed the presence of VPS13A in the synaptosomal fractions of the cerebral cortex, as already described [23]. However, we found neither VPS13A enrichment in these fractions nor specific labeling within the dendritic spines of cortical primary cultures. These results suggest that VPS13A is not a core synaptic pro‐ tein. Indeed, the fact that VPS13A is not enriched in the synapse, together with the unal‐ tered VPS13A expression levels after pharmacological manipulation of dopaminergic, glutamatergic, or cholinergic systems, is in line with a neuronal role not directly involved in the synaptic transmission. Actually, the stable expression during development and adult ages, even after manipulation of different neurotransmitter systems, is consistent with an essential homeostatic function of VPS13A in neurons [45] that could be related with the control of mitochondrial function [15]. Despite this stable concentration, possible changes in VPS13A subcellular location or expression by different cell types associated with neuronal stimuli cannot be discarded. To summarize, VPS13A is a stable protein expressed heterogeneously throughout distinct mouse brain nuclei; its expression pattern can provide the basis for future studies aiming to further understand the pathophysiological hallmarks of the VPS13A disease. While VPS13A subcellular localization suggests that it is not directly involved in the core molecular mechanisms of synaptic transmission, VPS13A may have a role in maintaining neuronal homeostasis and function. 4.2. Tissue Sampling E15.5, P0, and P7 mice were decapitated and the brains were removed and immer‐ sion‐fixed with 4% paraformaldehyde (PFA) at 4 °C overnight. At the appropriated age, postnatal and adult mice were anesthetized with pentobarbital and transcardially per‐ fused with ice‐cooled 0.1 M phosphate buffer saline (PBS, pH 7.4), followed by 4% PFA. Then, brains were removed and immersion fixed with 4% PFA at 4 °C overnight. All PFA‐fixed brains were cryoprotected with 30% sucrose in 0.1 M PBS‐0.02% sodium azide and frozen in dry ice‐cooled isopentane. Specimens were stored at −80 °C until section‐ ing. Either sagittal or coronal serial sections were collected onto SuperFrost Plus slides (Sigma Aldrich, Burlington, MA, USA) with a cryotome. A total of 200 sagittal or 240 coronal serial sections of 14 μm were collected. For histological analysis, consecutive sections were used for Nissl standard staining, FISH, and immunohistochemistry. 4.1. Animals Male B6CBA and C57BL6J wild‐type mice were housed under a standard 12:12 h light/dark cycle with access to food and water ad libitum in a colony room kept at 19–22 °C and 40–60% humidity. All animal procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ap‐ proved by the local animal care committee of the Universitat de Barcelona (226/17) and Generalitat de Catalunya (17/9837), in accordance with the Spanish RD 53/2013 and the 2010/63/EU Directive of the European Commission. 3. Discussion Thus, detailed VPS13A brain distribution maps of mRNA and protein is the first step to unravel the function of VPS13A in neurons and should help to characterize its role in the basal ganglia brain circuitry to finally under‐ stand the ChAc neuropathology. Int. J. Mol. Sci. 2021, 22, 13018 16 of 23 16 of 23 4.4. Immunohistochemistry The procedure was carried out as previously described [46]. Briefly, sections were first washed in 0.1 M PBS containing 0.3% Triton X‐100, treated with 50 mM NH4Cl, and incubated with a blocking solution of 0.1 M PBS containing 0.3% Triton‐X100 and 10% normal donkey serum (NDS; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. Sections were then incubated overnight with an anti‐VPS13A antibody (1:500, Cat: PA5‐54483, Invitrogen, Waltham, MA, USA, USA). After washing with 0.1 M PBS, sec‐ tions were then incubated for 2 h with AF488 donkey anti‐rabbit IgG (H + L) (1:200, Thermo Fisher Scientific, Waltham, MA, USA). Antibodies were diluted in 0.1 M PBS, containing 0.1% Triton X‐100 and 5% NDS. Incubations with normal rabbit IgG (Sig‐ ma‐Aldrich, St. Louis, MA, USA) as primary antibodies were used for negative controls (Supplementary Figure S1). Incubations with a mixture of anti‐VPS13A antibody and its antigen (PrEST Antigen VPS13A, Sigma‐Aldrich, St. Louis, MA, USA) were performed to test the specificity of the VPS13A staining (Supplementary Figure S1). Moreover, to ana‐ lyze the reliability of the protein staining pattern, a comparison of immunostaining using anti‐VPS13A antibodies from Invitrogen (Cat: PA5‐54483) and from Sigma (Cat: HPA021662) was performed (Supplementary Figure S1). All washes and incubations were completed at RT, except for the primary antibody incubation, which was completed at 4 °C. After secondary antibody incubation, slices were washed, incubated with Hoechst (1:10,000), mounted with Prolong, and kept in the dark. ( ) g p Double immunofluorescence labeling was carried out using specifics markers for GABAergic neurons (anti‐calbindin IgG antibody (1:500, Cat: CB300, Swant, Burgdorf, Switzerland) and anti‐parvalbumin antibody (1:500, Cat: P‐3088, Sigma‐Aldrich, St. Louis, MA, USA)), cholinergic neurons (anti‐ChAT antibody; 1:500, Cat: AB144P, Sig‐ ma‐Aldrich, St. Louis, MA, USA), astrocytes (anti‐GFAP antibody; 1:1000, Cat: G3893, Sigma‐Aldrich, St. Louis, MA, USA), oligodendrocytes (anti‐CNPase antibody; 1:500, Cat: MAB326, Merck Millipore, Burlington, MA, USA), and microglia (anti‐Iba1 anti‐ body; 1:500, Cat: ab5076, Abcam, Cambridge, UK). Antibodies against the IgG of appro‐ priated species and conjugated with the fluorescent dye Cy3 were used as secondary an‐ tibodies (Cy3‐conjugated donkey anti‐goat IgG (H + L) (1:200; Jackson ImmunoResearch, St. Thomas’ Place, UK) or Cy3‐conjugated donkey anti‐mouse IgG (H + L) (1:200, Jackson ImmunoResearch, St. Thomas’ Place, UK)). Confocal images were acquired by a confocal laser scanning microscope (ZEISS LSM880, Zeiss, Oberkochen, Germany). 4.3. Fluorescence In Situ Hybridization A FISH procedure was performed using the RNAscope® 2.5 High Definition–Red Assay kit (Advanced Cell Diagnostics, Newark, CA, USA), in accordance with the in‐ structions of the manufacturer. Briefly, slices were washed with 0.1 M PBS, baked 30 min at 60 °C, and post‐fixed with 4% PFA for 5 min at 4 °C. After dehydration, brain slices were air dried and treated first with RNAscope® Hydrogen Peroxide solution (Advanced Cell Diagnostics, Newark, CA, USA) for 10 min at room temperature (RT), then with RNAscope® Target Retrieval solution (Advanced Cell Diagnostics, Newark, CA, USA) for 5 min at 98‐102 °C, and, finally, with RNAscope® Protease Plus (Advanced Cell Diagnos‐ tics, Newark, CA, USA) for 30 min at 40 °C. The target probe for VPS13A gene (Probe‐Mm‐Vps13a‐E61‐E71‐C2, Advanced Cell Diagnostics, Newark, CA, USA) was hybridized for 2 h at 40 °C, followed by a series of signal amplification and washing steps. Hybridizations were performed in a HybEZTM Hybridization System (Advanced Cell Diagnostics, Newark, CA, USA). Negative controls were performed with a negative control probe (targeting the DapB gene from the Bacillus subtilis strain SMY) provided by the kit. Specific hybridization signals were detected by fluorescence, and RNA stain‐ ing was identified as red dots. Images of the VPS13A expression staining were obtained with an inverted micro‐ scope (Leica DMI6000 B, Thermo Fisher Scientific, Waltham, MA, USA). A mosaic con‐ taining 21 × 35 images (8022.87 × 13,683.67 μm) with 10% overlapping between images was obtained from the whole brain slice. Mosaics from three different brains were pro‐ cessed. For qualitative visual analysis of the intensity of VPS13A mRNA labeling in the slices, digital images were processed using an 8‐bit 16‐color lookup table with ImageJ 1.51a (National Institutes of Health, Bethesda, MD, USA). For semiquantitative analyses, the intensity of staining was evaluated based on the scales of pseudocolor images (Sup‐ plementary Figure S2). According to the color scale of this intensity analysis, VPS13A expression was classified as low, medium, or high. Int. J. Mol. Sci. 2021, 22, 13018 17 of 23 17 of 23 4.4. Immunohistochemistry Orthogonal views were obtained with ImageJ 1.51a (National Institutes of Health, Bethesda, MD, USA) to assess the presence of VPS13A in the different cell types. For semiquantitative analyses, the intensity of staining was evaluated based on the scale of pseudocolor im‐ ages, as explained for the FISH experiments (Supplementary Figure S2). 4.7. Primary Cell Cultures Cortical primary mouse cultures were performed, as previously described [47]. Brains from E18.5 embryos were excised and placed in Neurobasal medium (Fisher Sci‐ entific, Waltham, MA, USA). The cortex was dissected and gently dissociated with a fire‐polished glass Pasteur pipette. Cells were seeded onto 12 mm glass coverslips pre‐coated with 0.1 mg/mL poly‐D‐lysine (Sigma‐Aldrich, St. Louis, MO, USA) at a den‐ sity of 80,000 cells/cm2. Neurobasal medium supplemented with Glutamax (Fisher Sci‐ entific, Waltham, MA, USA) and B27 (Fisher Scientific, United States) was used to grow cells in serum‐free conditions. Cultures were maintained at 37 °C in a humified atmos‐ phere, containing 5% CO2 until 15 days in vitro (DIV). Embryos from three different mothers were used for neuronal cultures. In every culture series, experiments were per‐ formed in cultures coming from at least three different litters. 4.6. Synaptosomal Fractionation and Western Blotting 4.6. Synaptosomal Fractionation and Western Blotting Cerebral cortexes from 16‐week‐old mice were homogenized in Krebs‐Ringer buffer (125 mM NaCl, 1.2 mM KCl, 22 mM NaHCO3, 1 mM NaH2PO4, 1.2 mM MgSO4, 1.2 mM CaCl2, 10 mM Glucose, 0.32 M Sucrose; pH 7.4). Initial lysate was first centrifuged at 1000× g for 10 min. Homogenate was centrifuged for 20 min at 16,000× g to obtain the cytosolic fraction and the crude synaptosomal fraction, which was resuspended in Krebs‐Ringer buffer. Protein concentration was measured using the Pierce Coomassie Plus Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). For VPS13A detection in every lysate fraction, 15 μg of protein were subjected to 3‐8% SDS‐PAGE and transferred to Nitrocellulose membrane through the iBlot 2 Gel Transfer Device (Thermo Fisher Scientific, Waltham, MA, USA). Immunoblots were probed with anti‐VPS13A antibody (1:1500, Cat: HPA021662, Sigma‐Aldrich, St. Louis, MI, United States) and horseradish peroxidase‐conjugated (HRP) anti‐β‐actin antibody (1:100,000, Sigma‐Aldrich, St. Louis, MI, USA) or anti‐Tubulin antibody (1:100,000; Cat: T9026, Sigma‐Aldrich, St. Louis, MI, USA) as loading controls. Immunoblots were also probed with anti‐SV2A (1:1000, Cat: sc‐376234, Santa Cruz Biotechnology, Dallas, TX, USA) and anti‐PSD95 (1:1000, Cat: MA1‐045, Thermo Fisher Scientific, Waltham, MA, USA) antibodies as controls of pre‐synaptic and post‐synaptic protein enrichment, re‐ spectively. Membranes were incubated with anti‐rabbit HRP conjugated IgG (H + L) (1:2000, Promega, Madison, WI, USA) or with anti‐mouse HRP conjugated IgG (1:2000, Promega, Madison, WI, USA). Immunoreactive bands were visualized using the Western blotting Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX, USA). Images were acquired using ChemidocTM (Bio‐Rad, Hercules, CA, USA) and quantified by a comput‐ er‐assisted densitometer (ImageLab™, Bio‐Rad, Hercules, CA, USA). Pictures of the whole membranes of all Western blots included in Figures 6 and 7 are provided as Sup‐ plementary Material (Supplementary Figures S4 and S5). 4.5. Quantitative Real‐Time PCR The aqueous phase containing total RNA was isolated from the different mouse brain regions using QIAzol (Qiagen, Hilden, Germany), in accordance with the protocol of the manufacturer. Then, total RNA from the aqueous phase was precipitated with isopropanol, washed with ethanol, and redissolved in RNase‐free water. One μg of RNA was reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RT reaction was performed at 25 °C for 5 min, followed by 42 °C for 60 min. The reaction was terminated by heating at 70 °C for 5 min. cDNA was diluted to 5 ng/μL and 1 μL was used to perform qRT‐PCR. PrimeTime qPCR assays were used as recommended by the provider (assay code Mm.PT.56a.8500899, se‐ quence NM_173028(1) for VPS13A, and assay code Mm.PT.39a.1 sequence NM_008084 for GAPDH; IDT technologies, USA). qRT‐PCR was carried out with a SensiFAST™ SYBR® Hi‐ROX Kit (Meridian Bioscience, Newton, OH, USA), in 20 μL final volume us‐ ing a StepOnePlus thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The amplification program was: 2 min at 95 °C for polymerase activation, followed by 40 cy‐ Int. J. Mol. Sci. 2021, 22, 13018 18 of 23 18 of 23 cles of 5 s at 95 °C for denaturation, 10 s at 60 °C for annealing, and a final 20 s at 72 °C for extension. The expression level was determined using a standard curve and normalized to housekeeper GAPDH gene mRNA levels. In our conditions, GAPDH expression in the mouse brain assessed by qRT‐PCR remain unvaried over time in the different regions analyzed (Supplementary Figure S3). Reactions were performed in triplicate to reduce variability. The ΔΔCt method was used to analyze the data. 4.8. Immunocytochemistry and Sub‐Cellular Localization Analysis Cortical primary cultures grown on glass coverslips were fixed at 15 DIV with 4% PFA for 15 min and washed with 0.1M PBS. After rinsing, coverslips were permeabilized with 0.1 M PBS containing 1% BSA and 0.1% Saponin for 10 min at RT and incubated with blocking solution of 0.1M PBS containing 15% NDS for 30 min at RT. Then, co‐ verslips were incubated at 4 °C overnight with 5% NDS in 0.1 M PBS, containing an‐ Int. J. Mol. Sci. 2021, 22, 13018 19 of 23 19 of 23 ti‐VPS13A antibody (1:200, Cat: PA5‐54483, Invitrogen, Waltham, MA, USA) in combi‐ nation with either AF568 Phalloidin (1:500, Cat: A12380, Fisher Scientific, Waltham, MA, USA) as a marker of cellular processes and synaptic buttons, anti‐TOMM20 antibody (1:250, Cat: ab56783, Abcam, Cambridge, UK) as a marker of mitochondria, or an‐ ti‐calnexin antibody (1:250, Cat: sc‐6465, Santa Cruz Biotechnology, Dallas, TX, USA) as a marker of ER. After washing, coverslips were incubated with 5% NDS in 0.1 M PBS con‐ taining AF488 donkey anti‐rabbit IgG (1:200, Thermo Fisher Scientific, Waltham, MA, USA), Cy3‐conjugated donkey anti‐mouse IgG (1:200, Jackson ImmunoResearch, St. Thomas’ Place, UK), or Cy3‐conjugated donkey anti‐goat IgG (1:200, Jackson Immu‐ noResearch, St. Thomas’ Place, UK), as secondary antibodies for 1 h at RT. Finally, co‐ verslips were washed, incubated with Hoechst (1:10,000), mounted on microscope slides with Prolong, and kept in the dark. g p For VPS13A‐ER marker and VPS13A‐mitochondria marker co‐localization analysis, digital images were taken by a confocal laser scanning microscope (ZEISS LSM880, Zeiss, Oberkochen, Germany) with 2.0 digital zoom and stacks of 0.63 μm. Protein co‐localization was quantified through Mander co‐localization coefficients and co‐localization scatter plots obtained from the ImageJ plugin “Coloc 2” using the Costes threshold algorithm. For each condition, three neurons were analyzed from two inde‐ pendent experiments. 4.9. Amphetamine, Ketamine and Pilocarpine Treatment To pharmacologically manipulate dopaminergic and glutamatergic brain circuits, adult male C57BL6J mice were intraperitoneally treated daily for 8 days with either D‐amphetamine sulphate (3 mg/kg; TOCRIS, Bristol, UK) or ketamine (30 mg/kg; SIG‐ MA, St. Louis, MO, United States), respectively. Fifteen minutes after the last injection, mice were sacrificed and the striatum, frontal cortex, and hippocampus were rapidly dissected out and frozen at −80 °C until use. To activate the cerebral cholinergic system, another group of mice were treated with pilocarpine (45 mg/kg, i.p.). To reduce the pe‐ ripheral convulsant consequences of pilocarpine, the procedure was a first injection of lithium (LiCl, 423 mg/kg, i.p.) 20–23 h prior to the administration of methyl‐scopolamine (1 mg/kg, i.p.), which was injected 30 min before the final pilocarpine administration. Status epilepticus was stopped after approximately 120 min with valium (10 mg/kg, i.p.) [48]. Ten days after pilocarpine treatment, mice were sacrificed and the hippocampus was rapidly dissected out and frozen at −80 °C until use. For all treatments, control ani‐ mals were injected with saline solution. j Each brain area was homogenized in 50 mM Tris–HCl (pH 7.5) containing 10% glycerol, 1% Triton X‐100, 150 mM NaCl, 100 mM NaF, 5 μM ZnCl2, 10 mM EDTA, pro‐ tease inhibitors (phenylmethylsulphonyl fluoride (2 mM), aprotinin (1 μg/mL), leupeptin (1 μg/mL), and sodium orthovanadate (1 mM)). After homogenization, samples were centrifuged at 16,000× g for 15 min at 4 °C, the supernatants were collected, and protein concentration was measured using the Pierce Coomassie Plus Protein Assay (Bradford, Thermo Fisher Scientific, Waltham, MA, USA). To quantify the VPS13A concentration in the supernatants, fifteen micrograms of protein were subjected to Western blot analysis, as explained above. Tubulin was used as a loading control. 4.10. Analysis of VPS13A Protein Stability Conditionally immortalized striatal cells (STHdHQ7/Q7) [49] were grown at 33 °C in a DMEM‐high glucose medium supplemented with 10% fetal bovine serum, 1% non‐essential amino acids, 2 mM L‐glutamine, and 400 μg/mL geneticin, as previously described [50]. Three independent cultures of STHdHQ7/Q7 cells were grown in six‐well plates at a density of 250,000 cells/well for twenty‐four hours. Then, cells were treated with 50 μg/mL cycloheximide (CHX) for 6, 24, or 48 h at 33 °C to inhibit protein synthesis [51]. Concentrations of VPS13A and tubulin were determined in cell lysates by Western blot. Tubulin was used as a loading control. To take into account the tubulin half‐life, first Int. J. Mol. Sci. 2021, 22, 13018 20 of 23 20 of 23 the OD intensity of tubulin bands was corrected relative to a reference tubulin band in the membrane with an intermediate intensity. Next, VPS13A lanes were normalized vs. the corrected OD of the tubulin band in its line. the OD intensity of tubulin bands was corrected relative to a reference tubulin band in the membrane with an intermediate intensity. Next, VPS13A lanes were normalized vs. the corrected OD of the tubulin band in its line. Informed Consent Statement: Not applicable Informed Consent Statement: Not applicable Data Availability Statement: All data presented this study are available from the corresponding authors, upon responsible request. Acknowledgments: We are very grateful to Carmen Andrade (Dept. Biomedical Sciences, UB) for her technical support on immunohistochemistry procedures and to Ana Maria Lopez (María de Maeztu Unit of Excellence, Institute of Neurosciences, University of Barcelona, MDM‐2017‐0729, Ministry of Science, Innovation and Universities) for excellent mouse colony management. We are also grateful to the staff of the Confocal Microscopy Service of the Scientific and Technological Centers of the University of Barcelona (CCiTUB). Conflicts of Interest: The authors declare no conflict of interest. 4.11. Statistical Analysis Quantitative data are presented as mean ± standard error of the mean (SEM). For all parameters, homogeneity of variance was checked using the Levene test. Changes in time course of VPS13A expression were assessed by the one‐way ANOVA test, followed by the Bonferroni post hoc test. When normality was not reached, data were compared with the non‐parametric Kruskal–Wallis test, followed by the Dunn test. Changes in protein concentration in either cell or brain lysates were analyzed by the unpaired Student’s t‐test. Values of p < 0.05 were considered significant. The VPS13A protein stability was calculated by fitting the curve to a one‐phase decay type exponential equation. Analyses were performed with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Supplementary Materials: The following are available www.mdpi.com/article/10.3390/ijms222313018/s1. Supplementary Materials: The following are available online at www.mdpi.com/article/10.3390/ijms222313018/s1. Author Contributions: E.G.‐G., M.M., J.A., and M.J.R. contributed to the conception and design of the study and designed the experiments. E.G.‐G., N.C.‐C., A.C.‐M., M.C.‐C., and A.G. performed all the experiments. D.D.T. performed the scRNAseq data analysis. M.M. and M.J.R. performed the statistical analysis. J.A., M.M., and M.J.R. obtained financial support. E.G.‐G. and M.J.R. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by grants from the Ministerio de Ciencia y Innovación (Spain), under project no. PID2020‐119386RB‐I00 (J.A. and M.J.R.) and RTI2018‐094678‐A‐I00 (A.G.); Insti‐ tuto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades and European Regional Development Fund (ERDF) [CIBERNED, to J.A.], Spain; and ChAc Foundation (J.A., M.M. and M.J.R.) Spain. This research is part of NEUROPA. The NEUROPA Project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under Grant Agree‐ ment No. 863214 (MM). A.G. is a Ramón y Cajal fellow (RYC‐2016‐19466). D.D.T. is supported by the Ramón y Cajal program (RYC‐2017‐23486 / RTI2018‐095580‐A‐I00). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. 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Fernández‐García, S.; Sancho‐Balsells, A.; Longueville, S.; Hervé, D.; Gruart, A.; Delgado‐García, J.M.; Alberch, J.; Giralt, A. Astrocytic BDNF and TrkB regulate severity and neuronal activity in mouse models of temporal lobe epilepsy. Cell Death Dis. 2020 116 2020, 11, 1–17, doi:10.1038/s41419‐020‐2615‐9. 49. Trettel, F.; Rigamonti, D.; Hilditch‐Maguire, P.; Wheeler, V.; Sharp, A.; Persichetti, F.; Cattaneo, E.; MacDonald, M. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 2000, 9, 2799–2809, doi:10.1093/HMG/9.19.2799. Int. J. Mol. Sci. 2021, 22, 13018 23 of 23 23 of 23
https://openalex.org/W2115576167	https://thescipub.com/pdf/ajassp.2012.1538.1541.pdf	English	null	Design of Pass Band Filter in Hybrid Architecture Planar/Non-Radiative Dielectric Waveguide Integration Technology	American journal of applied sciences	2,012	cc-by	1,690	components, this design was described in (Cassivi et al., 2000; 2002). components, this design was described in (Cassivi et al., 2000; 2002). A non radiative dielectric waveguide is currently interesting filter types due to their high Q at low cost technology for fabrication and compatibility with other technology. Hence their electromagnetic models are still under research and therefore not easy accessible. This study aims to provide circuit designers with accurate, easy determinable for a pass band filter. American Journal of Applied Sciences 9 (10): 1538-1541, 2012 ISSN 1546-9239 © 2012 Science Publication American Journal of Applied Sciences 9 (10): 1538-1541, 2012 ISSN 1546-9239 © 2012 Science Publication Design of Pass Band Filter in Hybrid Architecture Planar/Non-Radiative Dielectric Waveguide Integration Technology Harizi Hanen and Gharssallah Ali Unit of Research Circuits and Electronics Systems High Frequency, Faculty of Science, University El ManarTunis, Tunisia Abstract: Problem statement: The expansion of RF, microwave and millimeter devices has revolutionized today’s ommunication and sensor systems. Low-cost, high-performance and mass producible millimeter wave technologies are vital for commercial broadband systems. Challenging issues are commonly faced in the design of low-loss integrated circuits for example high-Q band pass filter, which the planar technique is fundamentally limited in performance. Approach: In this study, we present a design of a nonradiative dielectric waveguide band pass filter based on hybrid architecture of micro strip line and non-radiative dielectric waveguide. Results: The simulation with High Frequency Structure Simulator (HFSS) three dimensional analyses is presented. Conclusion: The non radiative dielectric resolves most of the drawbacks of dielectric waveguide in connection with the radiation loss. Key words: Non-Radiative Dielectric (NRD), Non Radiative Perforated Dielectric (NRPD), Substrate Integrated Non Radiative Dielectric (SINRD), Engraved Non-Radiative Dielectric waveguide (ENRD), High Frequency Structure Simulator (HFSS) Key words: Non-Radiative Dielectric (NRD), Non Radiative Perforated Dielectric (NRPD), Substrate Integrated Non Radiative Dielectric (SINRD), Engraved Non-Radiative Dielectric waveguide (ENRD), High Frequency Structure Simulator (HFSS) MATERIALS AND METHODES Its basic component, the NRD waveguide, consists of a rectangular-section dielectric rod (height a, width b, permittivity εr1) sandwiched between two conducting plates that are at a distance apart less than half the free- space wavelength 20 (Fig. 1). Corresponding Author: Harizi Hanen, Unit of Research Circu University El ManarTunis, Tunisia Proposed by Yoneyama and Nishida (1981) Non- Radiative Dielectric (NRD) guide circuits is nowadays a well-known technology for various millimeter wave applications (Yoneyama, 2009; Yoneyama and Nishida, 1984). Subsequently, Bacha has proposed the model of a hybrid integration of NRD-waveguide and micro strip line (Bacha and Wu 1998a; 1998b). Indeed, basic features and applications of the NRD-guide had been investigated by different research. However, several techniques were proposed, Grigoropoulos and Young present a Non Radiative Perforated Dielectric (NRPD) in (Grigoropoulos and Young, 2003); Cassivi and Wu (2004) introduce the Substrate Integrated Non Radiative Dielectric (SINRD). Other scheme of design called Engraved Non-Radiative Dielectric waveguide (ENRD), proposed to reduce the problem of alignment and mechanical tolerances in fabrication of NRD Corresponding Author: Harizi Hanen, Unit of Research Circuits and Electronics Systems High Frequency, Faculty of Science, University El ManarTunis, Tunisia Proposed by Yoneyama and Nishida (1981) Non- Radiative Dielectric (NRD) guide circuits is nowadays a well-known technology for various millimeter wave applications (Yoneyama, 2009; Yoneyama and Nishida, 1984). Subsequently, Bacha has proposed the model of a hybrid integration of NRD-waveguide and micro strip line (Bacha and Wu 1998a; 1998b). Indeed, basic features and applications of the NRD-guide had been investigated by different research. However, several techniques were proposed, Grigoropoulos and Young present a Non Radiative Perforated Dielectric (NRPD) in (Grigoropoulos and Young, 2003); Cassivi and Wu (2004) introduce the Substrate Integrated Non Radiative Dielectric (SINRD). Other scheme of design called Engraved Non-Radiative Dielectric waveguide (ENRD), proposed to reduce the problem of alignment and mechanical tolerances in fabrication of NRD p g g The required distance between the two metallic plates of NRD wave guide is computed using the following relation: 0 2 r a λ < ε where, λ0 the free-space wavelength εr the relative dielectric constant. The hybrid architecture planar/NRD waveguide integration geometry consist of a micro strip line deposited on the top of ground plane which is one of the two parallel metallic plates of the NRD wave guide. The coupling between the two dissimilar structures is achieved though apertures that are made in the common ground plane. MATERIALS AND METHODES The aperture orientation defines essentially the operating mode in the NRD wave guide. The required distance between the two metallic plates of NRD wave guide is computed using the following relation: 0 2 r a λ < ε where, λ0 the free-space wavelength εr the relative dielectric constant. d El t i S t Hi h F F lt f S i The hybrid architecture planar/NRD waveguide integration geometry consist of a micro strip line deposited on the top of ground plane which is one of the two parallel metallic plates of the NRD wave guide. The coupling between the two dissimilar structures is achieved though apertures that are made in the common ground plane. The aperture orientation defines essentially the operating mode in the NRD wave guide. The hybrid architecture planar/NRD waveguide integration geometry consist of a micro strip line deposited on the top of ground plane which is one of the two parallel metallic plates of the NRD wave guide. 1538 Am. J. Applied Sci., 9 (10): 1538-1541, 2012 Fig. 1: General structure of NRD waveguide and the distribution of the electric and magnetic fields → Electrical Field → Magnetic Field Am. J. Applied Sci., 9 (10): 1538-1541, 2012 Fig. 1: General structure of NRD waveguide and the distribution of the electric and magnetic fields → Electrical Field → Magnetic Field Fig. 2: Structure design of cylindrical NRD waveguide Fig. 2: Structure design of cylindrical NRD waveguide The proposed structure of a design of pass band filter in hybrid architecture planar/NRD is presented in Fig 2. The filter is composed of a series of cylindrical dielectric resonators with a height and different diameter di, coupled by air gaps of length gi. The NRD wave guide has a width b= 4.267 mm and a height a = 4.572 mm and it’s made of Polyflon Nor CALD dielectric substrate εr = 2.55. An absorbing boundary condition is applied in reducing our computional window for this unbounded structure so an electrical rectangular box is simulated. The micro strip line is made of Rogers RT/Duroid 5880 dielectric substrate εr = 2.2 and thickness h = 0.254 mm and the width of wmcr = 0.711 mm. The slot size have a length ls = 5.842 mm and a width ws = 0.508 mm and the position of the slot is given by the parameters lmcr = 1.458 mm and lnrd = 2.616 mm. RESULTS Yoneyama, T. and S. Nishida, 1984. Nonradiative dielectric waveguide. 11: 61-98. This designed filter is simulated using the High Frequency Structure Simulator (HFSS) based on finite element method. Simulated filter response is shown in Fig. 3. Bacha, A. and K. Wu, 1998a. Toward an optimum design of NRD-guide and microstrip-line transition for hybrid-integration technology. IEEE Trans. Microwave Theory Technol. 46: 1796-1800. DOI: 10.1109/22.734585 MATERIALS AND METHODES The Fig. 2 shows the configuration of the gap-coupled cylindrical NRD wave guide filter with geometrical dimensions d1 = 5.705 mm, d2 = 5.928 mm, d3 = 5.957 mm and g1 = 1.211 mm, g2 = 2.651 mm, g3 = 3.199 mm. 1539 Am. J. Applied Sci., 9 (10): 1538-1541, 2012 (a) (b) Fig. 3: Simulation result of the pass band filter with the cylindrical NRD wave guide (a) Scatterin parameters (b) The ripple level in the pass band RESULTS This designed filter is simulated using the High 5.14%. The ripple level in the pass band is higher than the assumed-0.12 dB value and falls down to -0.15 dB. Equally, the return losses in the pass band are below- 15dB level, except for one point where they reach the value of -13.6 dB. (a) The-0.12 dB pass band is equal 3.63% (f1 =27.5 GHz and f2 = 28.5 GHz). The quality factor Q of a filter is a measure of how sharply the performance characteristics transition between pass band and out-of-band behavior. 0f Q f = ∆ Therefore, the quality factor Q of the band pass filter in hybrid architecture planar/NRD waveguide integration technology at frequency as high as 28 GHz is equal to 19.5. (a) (a) (b) REFERNCES Yoneyama, T. and S. Nishida, 1981. Nonradiative dielectric waveguide for millimeter-wave integrated circuits. IEEE Trans. Microwave Theory Technol., 29: 1190-1981. DOI: 10.1109/TMTT.1981.1130529 (b) Fig. 3: Simulation result of the pass band filter with the cylindrical NRD wave guide (a) Scattering parameters (b) The ripple level in the pass band Yoneyama, T., 2009. Recent advances in millimeter- wave NRD-guide circuits. IEICE Trans. Elec., E92: 1106-1110. CONCLUSION The NRD-guide band pass filter in hybrid architecture planar/NRD waveguide integration technology design has been described. Numerical simulation of an entire filter structure have shown that the design has met the specification of an pass band filter in a high frequency and the presented method can be used as a fast, simple and accurate way to design NRD-wave guide filters given its ability of integration with other elements of the circuit. DISCUSION Bacha, A. and K. Wu, 1998b. LSE-mode balun for hybrid integration of NRD-guide and microstrip line. IEEE Microwave Guided wave letters, 5: 199- 201. DOI: 10.1109/75.668729 1540 This band pass filter has been designed for the center frequency f0 = 28GHz and the bandwidth of 1540 Am. J. Applied Sci., 9 (10): 1538-1541, 2012 Cassivi, Y., D. Deslandes and K. Wu, 2000. Engraved NRD-guide for millimeter-wave integrated circuits. IEEE MTT-S Int. Microwave Symp. Dig., 2: 605- 608. DOI: 10.1109/MWSYM.2000.863257 Grigoropoulos, N. and P.R. Young, 2003. Low cost non radiative perforated dielectric waveguide. Proeceedings of the 33rd European Conference on Microwave, Oct. 7-9, IEEE Xpolre Press, pp: 439- 442. DOI: 10.1109/EUMC.2003.1262317 Cassivi, Y., D. Deslandes and K. Wu, 2002. Design considerations of engraved NRD Guide for millimeter-wave integrated circuits. IEEE Trans. Microwave Theory Technol., 50: 165-171. DOI: 10.1109/22.981261 Cassivi, Y. and K. Wu, 2004. Substrate integrated nonradiative dielectric waveguide. IEEE Microw. Wireless Compon. Lett., 14: 89-91. DOI: 10.1109/LMWC.2004.824808 1541
https://openalex.org/W4391980646	https://link.springer.com/content/pdf/10.1007/s11357-024-01088-1.pdf	English	null	Correction to: Ultra‑processed food consumption and nutritional frailty in older age	GeroScience	2,024	cc-by	599	GeroScience (2024) 46:3507 https://doi.org/10.1007/s11357-024-01088-1 GeroScience (2024) 46:3507 https://doi.org/10.1007/s11357-024-01088-1 CORRECTION Correction to: GeroScience (2023) 45:2229–2243 https://doi.org/10.1007/s11357-023-00753-1 The original version of this article unfortunately con- tained an error in the affiliation section.fi Open Access This article is licensed under a Creative Com- mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Crea- tive Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. fi The affiliation 1 should be as follows: Unit of Data Sciences and Technology Innovation for Population Health, National Institute of Gastroenter- ology IRCCS "Saverio de Bellis", Research Hospital, Castellana Grotte, Bari, Italy The corrected affiliation 1 is shown below. Correction to: Ultra‑processed food consumption and nutritional frailty in older age Roberta Zupo · Rossella Donghia · Fabio Castellana · Ilaria Bortone · Sara De Nucci · Annamaria Sila · Rossella Tatoli · Luisa Lampignano · Giancarlo Sborgia · Francesco Panza · Madia Lozupone · Giuseppe Colacicco · Maria Lisa Clodoveo · Rodolfo Sardone Roberta Zupo · Rossella Donghia · Fabio Castellana · Ilaria Bortone · Sara De Nucci · Annamaria Sila · Rossella Tatoli · Luisa Lampignano · Giancarlo Sborgia · Francesco Panza · Madia Lozupone · Giuseppe Colacicco · Maria Lisa Clodoveo · Rodolfo Sardone Published online: 20 February 2024 © The Author(s) 2024 Published online: 20 February 2024 © The Author(s) 2024 Published online: 20 February 2024 © The Author(s) 2024 Correction to: GeroScience (2023) 45:2229–2243 https://doi.org/10.1007/s11357-023-00753-1 The original article can be found online at https://doi.org/ 10.1007/s11357-023-00753-1. The original article can be found online at https://doi.org/ 10.1007/s11357-023-00753-1. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. R. Zupo () · R. Donghia · F. Castellana · S. De Nucci · A. Sila · R. Tatoli · L. Lampignano · R. Sardone Unit of Data Sciences and Technology Innovation for Population Health, National Institute of Gastroenterology IRCCS “Saverio de Bellis”, Research Hospital, Castellana Grotte, Bari, Italy e-mail: roberta.zupo@irccsdebellis.it R. Zupo () · R. Donghia · F. Castellana · S. De Nucci · A. Sila · R. Tatoli · L. Lampignano · R. Sardone Unit of Data Sciences and Technology Innovation for Population Health, National Institute of Gastroenterology IRCCS “Saverio de Bellis”, Research Hospital, Castellana Grotte, Bari, Italy e-mail: roberta.zupo@irccsdebellis.it G. Colacicco Department of Translational Biomedicine and Neuroscience (DiBrain), University of Bari Aldo Moro, Bari, Italy M. L. Clodoveo Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Bari, Italy R. Sardone Local Healthcare Authority of Taranto, Taranto, Italy G. Colacicco Department of Translational Biomedicine and Neuroscience (DiBrain), University of Bari Aldo Moro, Bari, Italy G. Colacicco Department of Translational Biomedicine and Neuroscience (DiBrain), University of Bari Aldo Moro, Bari, Italy I. Bortone Institute of Clinical Physiology, National Research Council (IFC-CNR), Pisa, Italy M. L. Clodoveo Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Bari, Italy G. Sborgia · F. Panza · M. Lozupone Department of Translational Biomedicine and Neuroscience, University of Bari Aldo Moro, Bari, Italy R. Sardone Local Healthcare Authority of Taranto, Taranto, Italy ol.: (01
https://openalex.org/W2037741701	https://europepmc.org/articles/pmc3990998?pdf=render	English	null	Impact of androgen deprivation therapy on the thymus and the production of naïve T-cells	Journal for immunotherapy of cancer	2,013	cc-by	866	POSTER PRESENTATION Impact of androgen deprivation therapy on the thymus and the production of naïve T-cells Ravi A Madan1,2, Hakim Frances3, Caroline Jochems1, Christopher R Heery1, Geraldine C O’Sullivan2, Harpreet Singh2, Ira Surolia2, Kwong Y Tsang1, Ronald Gress3, Jeffery Schlom1,2, James L Gulley1,2 Madan et al. Journal for ImmunoTherapy of Cancer 2013, 1(Suppl 1):P83 http://www.immunotherapyofcancer.org/content/1/S1/P83 Madan et al. Journal for ImmunoTherapy of Cancer 2013, 1(Suppl 1):P83 http://www.immunotherapyofcancer.org/content/1/S1/P83 Madan et al. Journal for ImmunoTherapy of Cancer 2013, 1(Suppl 1):P83 http://www.immunotherapyofcancer.org/content/1/S1/P83 Madan et al. Journal for ImmunoTherapy of Cancer 2013, 1(Suppl 1):P83 http://www.immunotherapyofcancer.org/content/1/S1/P83 Open Access Purpose A clinical trial was conducted employing androgen depri- vation therapy (ADT) +/- vaccine in patients (pts) with ris- ing prostate specific antigen (PSA) after definitive therapy but no disease visible on bone or computed tomography (CT) scan. The primary endpoint was to determine if a whole tumor cell vaccine could prolong PSA recovery, but a key secondary endpoint was to evaluate the impact of ADT on the thymus and production of naïve T-cells. Pre- vious studies suggest that ADT regenerates the thymus (in mice) and increases naïve T-cell production in humans [1]. Impact of androgen deprivation therapy on the thymus and the production of naïve T-cells Ravi A Madan1,2, Hakim Frances3, Caroline Jochems1, Christopher R Heery1, Geraldine C O’Sullivan2, Harpreet Singh2, Ira Surolia2, Kwong Y Tsang1, Ronald Gress3, Jeffery Schlom1,2, James L Gulley1,2 From Society for Immunotherapy of Cancer 28th Annual Meeting National Harbor, MD, USA. 8-10 November 2013 361 ng/dl, respectively. Median time to T recovery after a single 3-month shot of the GnRH agonist was 7 months (mos), range: 3-15 mos. Six mos after ADT, only 2 pts had more than minimal changes in thymus size (thymic index >2) on CT, however significant changes in naïve T-cell populations were detected in 20 evaluable pts. Naïve CD4 cells increased from a med- ian of 16.4% of CD4+ T-cells to 20.5%(p=0.0014) and TRECs increased from 93/100,000 cells to 147/100,000 cells (p=0.0025). Methods These data suggest that ADT could have a significant impact on the production of naïve T-cells despite mini- mal impact on the actual size of the thymus at 6 months. The ultimate clinical impact of a change in immune parameters may not be immediately clear given the short duration of follow-up in this trial. These data further support the hypothesis that immune stimulating therapies could be combined with ADT to enhance immune responses. All pts were treated with a 3 month dose of goserelin (ADT) as part of an intermittent therapy approach, consistent with the standard of care for this population. Once a PSA decline was confirmed at 12 weeks, pts were randomized to treat- ment with vaccine or placebo administered via 2 intrader- mal injections each at 4 sites in the torso. The vaccine consisted of 3 irradiated prostate cancer cell lines. Pts received injections at weeks 1, 3, 5 and then monthly to complete 12 months of therapy. Pts had follow-up evalua- tions of testosterone (T) levels, thymic measurements on CT scan and assessments of naïve T-cells. Thymic evalua- tions were based on a previously established radiographic measure, the thymic index [2]. Changes in naïve CD 4 T-cells, defined as CD45RA+CD31+, and T-cell receptor excision circles (TRECs) were also evaluated. Wilcoxon matched pairs signed rank analyses was used in this analysis. Authors’ details 1 Authors details 1Laboratory of Tumor Immunology & Biology, NCI, Bethesda, MD, USA. 2Medical Oncology Branch, NCI, Bethesda, MD, USA. 3Experimental Transplantation & Immunology, NCI, Bethesda, MD, USA. 1Laboratory of Tumor Immunology & Biology, NCI, Bethesda, MD, USA. 2Medical Oncology Branch, NCI, Bethesda, MD, USA. 3Experimental Transplantation & Immunology, NCI, Bethesda, MD, USA. Published: 7 November 2013 Published: 7 November 2013 References 1. Sutherland JS, et al: J Immunol 2005. 2. McCune JM, et al: J Clin Invest 1998. © 2013 Madan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1Laboratory of Tumor Immunology & Biology, NCI, Bethesda, MD, USA Full list of author information is available at the end of the article Results doi:10.1186/2051-1426-1-S1-P83 Cite this article as: Madan et al.: Impact of androgen deprivation therapy on the thymus and the production of naïve T-cells. Journal for ImmunoTherapy of Cancer 2013 1(Suppl 1):P83. doi:10.1186/2051-1426-1-S1-P83 Cite this article as: Madan et al.: Impact of androgen deprivation therapy on the thymus and the production of naïve T-cells. Journal for ImmunoTherapy of Cancer 2013 1(Suppl 1):P83. 33 pts were evaluable after response to ADT. The median age, PSA and T were 62 years, 2.2 ng/ml and 1Laboratory of Tumor Immunology & Biology, NCI, Bethesda, MD, USA Full list of author information is available at the end of the article 1Laboratory of Tumor Immunology & Biology, NCI, Bethesda, MD, USA Full list of author information is available at the end of the article © 2013 Madan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://openalex.org/W3002167846	https://zenodo.org/records/3632154/files/BDJ_article_47110.pdf	English	null	New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia	Biodiversity data jurnal	2,020	cc-by	3,337	© Bárrios S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. rresponding author: Sara Bárrios (s.barrios@kew.org) Corresponding author: Sara Bárrios (s.barrios@kew.org) Academic editor: Luis Cayuela Received: 07 Oct 2019 \| Accepted: 14 Nov 2019 \| Published: 23 Jan 2020 Citation: Bárrios S, Sustache JA, Goyder D, Hamilton MA (2020) New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia. Biodiversity Data Journal 8: e47110. https://doi.org/10.3897/BDJ.8.e47110 Z B k l id b k b 6AF663C1 0974 43EE A555 7770C464C1D7 ZooBank: urn:lsid:zoobank.org:pub:6AF663C1-0974-43EE-A555-7770C464C1D7 Biodiversity Data Journal 8: e47110 doi: 10.3897/BDJ.8.e47110 Species Conservation Profiles New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia Sara Bárrios , José A Sustache , David Goyder , Martin A Hamilton ‡ Royal Botanic Gardens, Kew, London, United Kingdom § Department of Natural and Environmental Resources of Puerto Rico, San Juan, Puerto Rico Corresponding author: Sara Bárrios (s.barrios@kew.org) Academic editor: Luis Cayuela Received: 07 Oct 2019 \| Accepted: 14 Nov 2019 \| Published: 23 Jan 2020 Citation: Bárrios S, Sustache JA, Goyder D, Hamilton MA (2020) New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia. Biodiversity Data Journal 8: e47110. https://doi.org/10.3897/BDJ.8.e47110 ZooBank: urn:lsid:zoobank.org:pub:6AF663C1-0974-43EE-A555-7770C464C1D7 ‡ § ‡ ‡ Biodiversity Data Journal 8: e47110 doi: 10.3897/BDJ.8.e47110 Species Conservation Profiles Biodiversity Data Journal 8: e47110 doi: 10.3897/BDJ.8.e47110 Species Conservation Profiles Species Conservation Profiles New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia Sara Bárrios , José A Sustache , David Goyder , Martin A Hamilton ‡ Royal Botanic Gardens, Kew, London, United Kingdom ‡ § ‡ ‡ Corresponding author: Sara Bárrios (s.barrios@kew.org) Academic editor: Luis Cayuela Received: 07 Oct 2019 \| Accepted: 14 Nov 2019 \| Published: 23 Jan 2020 Citation: Bárrios S, Sustache JA, Goyder D, Hamilton MA (2020) New island record and conservation status of Puerto Rican Bank endemic plant species, Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov., formally transferred from Marsdenia. Biodiversity Data Journal 8: e47110. https://doi.org/10.3897/BDJ.8.e47110 ZooBank: urn:lsid:zoobank org:pub:6AF663C1 0974 43EE A555 7770C464C1D7 Background Thought to be endemic to the Commonwealth of Puerto Rico, Ruehssia woodburyana (Apocynaceae) was recently discovered at a single location on Norman Island in the British Virgin Islands. Despite an increase in the extent of occurrence and area of occupancy, this species meta-population is very limited with a total of 37 individuals known in the wild. The largest subpopulation, on Mona Island, has only 26 individuals. The species suitable habitat is experiencing a continuing decline due to urban development, grazing by feral ungulates and human-induced forest fires. Conservation action is urgently needed and should be directed towards establishing genetically representative ex situ collections, such as seed for long term storage and live material for propagation. This species is evaluated as Critically Endangered (CR), based on Criteria C2a(i)+D, according to the IUCN Red List Bárrios S et al 2 Categories and Criteria (version 3.1) and guidelines (Subcommittee IUCN Standards and Petitions 2016). Introduction In this paper, we present a species conservation profile for an endemic species to the British Virgin Islands and to the Commonwealth of Puerto Rico. Keywords Apocynaceae, Caribbean Flora, Conservation, Endemism, New combination, Nomenclature, Red List New information Extensive and regular surveys to the region enable the discovery of new plant records for different countries and islands. In this paper, we record a new island record for Ruehssia woodburyana on Norman Island, in the British Virgin Islands and discuss the species conservation status. Marsdenia woodburyana is transferred to the genus Ruehssia to reflect the resurrection of that genus for species of Marsdenia native to the New World. Species information Marsdenia woodburyana Acev.-Rodr., 1999 – Acevedo-Rodriguez 1999: 167. Taxonomy Kingdom Phylum Class Order Family Plantae Tracheophyta Magnoliopsida Gentianales Apocynaceae Taxonomy Taxonomic notes Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov. Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov. Ruehssia woodburyana (Acev.-Rodr.) Goyder, comb. nov. BASIONYM: Marsdenia woodburyana Acev.-Rodr., 1999 – Acevedo-Rodriguez 1999: 167. BASIONYM: Marsdenia woodburyana Acev.-Rodr., 1999 – Acevedo-Rodriguez 1999: 167. TYPE: Puerto Rico, Mun. Guánica, Bosque Estatal de Guánica, Caña Gorda, 26 May 1998, Ramírez & Rosado 27, holotype: US [US00604132]; isotypes: MAPR; MO [MO-169808]; NY [NY00328790]; UPRRP; US (alcohol collection)). TYPE: Puerto Rico, Mun. Guánica, Bosque Estatal de Guánica, Caña Gorda, 26 May 1998, Ramírez & Rosado 27, holotype: US [US00604132]; isotypes: MAPR; MO [MO-169808]; NY [NY00328790]; UPRRP; US (alcohol collection)). TYPE: Puerto Rico, Mun. Guánica, Bosque Estatal de Guánica, Caña Gorda, 26 May 1998, Ramírez & Rosado 27, holotype: US [US00604132]; isotypes: MAPR; MO [MO-169808]; NY [NY00328790]; UPRRP; US (alcohol collection)). Region for assessment: - Global Taxonomic notes All the native New World species of the broadly delimited pan-tropical genus Marsdenia R.Br. (Apocynaceae: Asclepiadoideae) occur in a single clade, according to a recent study using two plastid and two nuclear gene regions (Espírito Santo et al. 2019). The genus New island record and conservation status of Puerto Rican Bank endemic ... 3 3 Ruehssia H.Karst. was resurrected for these species but, to date, only those taxa occurring in Brazil have been transferred, although Cuban taxa will follow shortly (Liede-Schumann pers. comm. 2019, manuscript under review) and it is planned to transfer species from other parts of the Americas in subsequent papers. Ruehssia H.Karst. was resurrected for these species but, to date, only those taxa occurring in Brazil have been transferred, although Cuban taxa will follow shortly (Liede-Schumann pers. comm. 2019, manuscript under review) and it is planned to transfer species from other parts of the Americas in subsequent papers. In order to expedite the range extension of M. woodburyana Acev.-Rodr. to the British Virgin Islands and to permit timely publication of its conservation status, we here propose the formal transfer of this species from Marsdenia to Ruehssia. Geographic range Observed occurrences. Bárrios S et al 4 4 Max Elevation/Depth (m): 150 Max Elevation/Depth (m): 150 Extent of occurrence EOO (km2): Trend: Causes ceased?: Causes understood?: Causes reversible?: 5649 Increase Unknown Unknown Unknown EOO (km2): Trend: Causes ceased?: Causes understood?: Causes reversible?: 5649 Increase Unknown Unknown Unknown Range description Ruehssia woodburyana is a rare plant species restricted to the Commonwealth of Puerto Rico and the British Virgin Islands (BVI). This species was originally described occurring exclusively at Caña Gorda within the Guánica State Forest in Guánica municipality on the island of Puerto Rico (Acevedo-Rodriguez 1999, Acevedo-Rodríguez 2005). Herbarium collections and observations dating from 2007 to 2017 (Suppl. material 1) reveal that this species also grows in other southern coastal municipalities of Puerto Rico: Peñuelas, Lajas and Cabo Rojo (Segarra et al. 2014). There are also herbarium collections from the islands of Mona and Culebra in the Commonwealth of Puerto Rico. In 2017, a team of botanists from the Royal Botanic Gardens Kew, the National Parks Trust of the Virgin Islands, US Fish and Wildlife Service, the Department of Natural and Environmental Resources of Puerto Rico and the University of Puerto Rico (Mayagüez Campus) came across fertile material of a similar vine on Norman Island, an island on the south-western edge of the British Virgin Islands (BVI) archipelago (Hamilton and Barrios 2017). This vine was vouchered (M.A. Hamilton, #1738, K000860152, K!) and later confirmed as R. woodburyana (O. Monsegur pers. comm. 2017). This species extent of occurrence (EOO) was estimated to be 5,649 km and the area of occupancy to be 32 km based on a 2×2 km cell size (Bachman et al. 2011). 2 2 Population Information (Narrative) Originally described from Guánica State Forest as extremely rare, Acevedo-Rodriguez (1999) mentioned a single mature plant and two juveniles. More recent collections from southern municipalities on the island of Puerto Rico, Culebra and Mona islands in the Commonwealth of Puerto Rico and Norman Island in the BVI suggest a total of 37 individuals. The largest sub-population occurs on Mona Island with 26 known individuals. All other subpopulations have between one and five individuals. The area and quality of suitable habitat of this species is in continuing decline due to grazing by feral ungulates and human disturbance, including development and human-induced fires. Locations Number of locations: 7-8 Subpopulations Abundance largest subpopulation: Number of subpopulations: Trend: 26 4 Unknown Abundance largest subpopulation: 26 Number of subpopulations: Trend: 4 Unknown Number of subpopulations: 4 Area of occupancy AOO (km2): Trend: Causes ceased?: Causes understood?: Causes reversible?: 32 Increase Unknown Unknown Unknown New island record and conservation status of Puerto Rican Bank endemic ... 5 5 Justification for number of locations Justification for number of locations The number of locations was calculated to be seven to eight, considering threats posed by human disturbance and human-induced fires at the different sites where the species has been recorded. Trend: Unknown Trend: Unknown Population Number of individuals: 37 Number of individuals: Trend: Causes ceased?: Causes understood?: Causes reversible?: Extreme fluctuations?: 37 Unknown Unknown Unknown Unknown Unknown Number of individuals: Trend: Causes ceased?: Causes understood?: Causes reversible?: Extreme fluctuations?: 37 Unknown Unknown Unknown Unknown Unknown Population Information (Narrative) Habitat System: Terrestrial Bárrios S et al 6 Habitat specialist: Unknown Ecology Generation length (yr): Dependency of single sp?: 0 No Ecology and traits (narrative) The generation length of this vine is unknown. More field observations are required. Trend in extent, area or quality?: Decline (observed) Habitat importance: Habitats: Major Importance - 1.5. Forest - Subtropical/Tropical Dry - 1.5. Forest - Subtropical/Tropical Dry Habitat (narrative) A woody vine which can grow to eight metres long in tropical dry forest (Figs 1, 2, 3, Acevedo-Rodríguez 2005). This species is known to flower only once per year for a short period (Segarra et al. 2014). Figure 1. Flowers of specimen of Ruehssia woodburyana observed and collected on Norman Island, British Virgin Islands. Flowers of specimen of Ruehssia woodburyana observed and collected on Norman Island, British Virgin Islands. Figure 2. Habit (vine) of Ruehssia woodburyana observed in Guánica State Forest, Puerto Rico. Habit (vine) of Ruehssia woodburyana observed in Guánica State Forest, Puerto Rico. New island record and conservation status of Puerto Rican Bank endemic ... 7 New island record and conservation status of Puerto Rican Bank endemic ... 7 Figure 3. Fruit of Ruehssia woodburyana observed in Guánica State Forest, Puerto Rico. Figure 3. Fruit of Ruehssia woodburyana observed in Guánica State Forest, Puerto Ric Fruit of Ruehssia woodburyana observed in Guánica State Forest, Puerto Rico. it of Ruehssia woodburyana observed in Guánica State Forest, Puerto Rico. Threats Threats: - 1.3. Residential & commercial development - Tourism & recreation areas - 4.1. Transportation & service corridors - Roads & railroads - 4.2. Transportation & service corridors - Utility & service lines - 7.1. Natural system modifications - Fire & fire suppression - 11.2. Climate change & severe weather - Droughts Bárrios S et al 8 8 Justification for threats This species is subjected to a variety of threats. Most locations are threatened by human disturbance which is causing habitat degradation and fragmentation, particularly through urban development and fire. In Puerto Rico, human-induced fires are frequent in Guánica State Forest along road PR 333 near Caña Gorda, the type locality. These seriously affect the quality of this species suitable habitat and may preclude the species natural recruitment. The habitat on Norman Island in the BVI was degraded by feral animals in the past, but these have now been removed, promoting the recovery of native vegetation. Despite the presence of feral goats and pigs on Mona Island, these animals are not thought to be impacting the species as feral mammal populations are managed through sports hunting. However, it is noted that no recruitment has been observed at this location in the recent years (J. Sustache pers. comm. 2018). At Laguna Cartagena and Cabo Rojo National Wildlife Refuges, there is no direct evidence of impact due to human-induced fires or feral animals, despite the presence of these threats. Within the municipality of Peñuelas, this species suitable habitat is threatened by the expansion of industrial landfills, service roads and utility lines (O. Monsegur pers. comm. 2018). Climate change might already be impacting this species through more severe droughts and stronger tropical storms. Conservation Conservation action type: Needed Conservation action type: Needed Conservation actions: - 1.2. Land/water protection - Resource & habitat protection - 3.4. Species management - Ex-situ conservation - 4.3. Education & awareness - Awareness & communications Justification for research needed Justification for research needed Justification for research needed Conservation action and research should be directed to develop a better understanding of this species' ecology and population trends and develop ex situ conservation collections. Further surveys are needed to look for potential undetected individuals and subpopulations within the species range, particularly in the US and British Virgin Islands. The areas and habitats, where this species occurs, should be closely managed and monitored. Acknowledgements We are grateful to the National Parks Trust of the Virgin Islands for their continued support and assistance in the field and for providing necessary letters to other government departments to facilitate field visits in the British Virgin Islands. Thanks to Mr Omar Monsegur of the US Fish and Wildlife Service for identifying the new record, supplying images of the species and useful discussion on the species conservation and comments on this manuscript. Justification for conservation actions This species is found within protected areas across its natural range. In the Commonwealth of Puerto Rico, the species is recorded as occurring within the Guánica State Forest, Laguna Cartagena National Wildlife Refuge, Cabo Rojo National Wildlife Refuge and Mona Island Nature Reserve. It is also thought to occur within the Culebra National Wildlife Reserve, but further surveys are needed. Norman Island in the BVI is not a protected area, as it is privately owned. This species is listed as a Critical Element by the Department of Natural and Environmental Resources (DNER 2007). Monsegur (Monsegur 2009) suggests that this species should also be evaluated to be listed under the U.S. Endangered Species Act of 1973. There are no known ex situ collections for this species despite attempts in recent years to collect seed from southern municipalities on the island of Puerto Rico (J. Sustache pers. comm. 2018). New island record and conservation status of Puerto Rican Bank endemic ... 9 9 Other Research needed: - 1.2. Research - Population size, distribution & trends - 1.3. Research - Life history & ecology - 3.4. Monitoring - Habitat trends New island record and conservation status of Puerto Rican Bank endemic ... • Acevedo-Rodriguez P (1999) West Indian novelties I: A new species of Marsdenia (Asclepiadaceae) from Puerto Rico and a new name for Jamaican species of Calyptranthes (Myrtaceae). Brittonia 51 (2): 166‑169. https://doi.org/10.2307/2666625 • Acevedo-Rodríguez P (2005) Vines and climbing plants of Puerto Rico and the Virgin Islands. Contributions from the United States National Herbarium 51: 1‑483. • Bachman S, Moat J, Hill A, de la Torre J, Scott B (2011) Supporting Red List threat assessments with GeoCAT: geospatial conservation assessment tool. ZooKeys 150: 117‑126. https://doi.org/10.3897/zookeys.150.2109 References Kew Bulletin 74: 30 https://doi.org/10.1007/s12225-019-9807-4 • Espírito Santo FdSdE, Rapini A, Ribeiro PL, Liede-Schumann S, Goyder DJ, Fontella- Pereira J (2019) Phylogeny of the tribe Marsdenieae (Apocynaceae), reinstatement of Ruehssia and the taxonomic treatment of the genus in Brazil. Kew Bulletin 74: 30 https://doi.org/10.1007/s12225-019-9807-4 • Hamilton MA, Barrios S (2017) Puerto Rican Bank (British Virgin Islands & Puerto Rico) June-July 2017 fieldwork report. In: Hamilton MA (Ed.) Overseas Fieldwork Committee registration number 559-14. Royal Botanic Gardens, Richmond, Surrey, 156 pp. https:// doi.org/10.13140/RG.2.2.10974.95047 • Hamilton MA, Barrios S (2017) Puerto Rican Bank (British Virgin Islands & Puerto Rico) June-July 2017 fieldwork report. In: Hamilton MA (Ed.) Overseas Fieldwork Committee registration number 559-14. Royal Botanic Gardens, Richmond, Surrey, 156 pp. https:// doi.org/10.13140/RG.2.2.10974.95047 • Monsegur O (2009) Vascular flora of the Guánica dry forest, Puerto Rico. Master of Science Thesis. University of Puerto Rico, Mayagüez Campus • Segarra A, Morales-Pérez A, Franqui R, Ratcliffe B (2014) First report of a South American cetoniine beetle, Gymnetis strigosa (Olivier, 1789) (Coleoptera: Scarabaeidae: Cetoniinae), in Puerto Rico. The Coleopterists Bulletin 68 (2): 217‑218. https://doi.org/10.1649/0010-065x-68.2.217 • Monsegur O (2009) Vascular flora of the Guánica dry forest, Puerto Rico. Master of Science Thesis. University of Puerto Rico, Mayagüez Campus • Segarra A, Morales-Pérez A, Franqui R, Ratcliffe B (2014) First report of a South American cetoniine beetle, Gymnetis strigosa (Olivier, 1789) (Coleoptera: S C ) C 68 (2) 21 218 Segarra A, Morales-Pérez A, Franqui R, Ratcliffe B (2014) First report of a South American cetoniine beetle, Gymnetis strigosa (Olivier, 1789) (Coleoptera: Scarabaeidae: Cetoniinae), in Puerto Rico. The Coleopterists Bulletin 68 (2): 217‑218. https://doi.org/10.1649/0010-065x-68.2.217 • Subcommittee IUCN Standards and Petitions (2016) Guidelines for using the IUCN Red List Categories and Criteria. Version 12. International Union for Conservation of Nature, Gland, Switzerland. • Subcommittee IUCN Standards and Petitions (2016) Guidelines for using the IUCN Red List Categories and Criteria. Version 12. International Union for Conservation of Nature, Gland, Switzerland. References • Acevedo-Rodriguez P (1999) West Indian novelties I: A new species of Marsdenia (Asclepiadaceae) from Puerto Rico and a new name for Jamaican species of Calyptranthes (Myrtaceae). Brittonia 51 (2): 166‑169. https://doi.org/10.2307/2666625 • Acevedo-Rodríguez P (2005) Vines and climbing plants of Puerto Rico and the Virgin Islands. Contributions from the United States National Herbarium 51: 1‑483. • Bachman S, Moat J, Hill A, de la Torre J, Scott B (2011) Supporting Red List threat assessments with GeoCAT: geospatial conservation assessment tool. ZooKeys 150: 117‑126. https://doi.org/10.3897/zookeys.150.2109 10 Bárrios S et al • DNER (2007) Elementos críticos de la división de patrimonio natural - plantas. Unpublished Report Submitted to Gov Puerto Rico 12 • Espírito Santo FdSdE, Rapini A, Ribeiro PL, Liede-Schumann S, Goyder DJ, Fontella- Pereira J (2019) Phylogeny of the tribe Marsdenieae (Apocynaceae), reinstatement of Ruehssia and the taxonomic treatment of the genus in Brazil. Kew Bulletin 74: 30 https://doi.org/10.1007/s12225-019-9807-4 • Hamilton MA, Barrios S (2017) Puerto Rican Bank (British Virgin Islands & Puerto Rico) June-July 2017 fieldwork report. In: Hamilton MA (Ed.) Overseas Fieldwork Committee registration number 559-14. Royal Botanic Gardens, Richmond, Surrey, 156 pp. https:// doi.org/10.13140/RG.2.2.10974.95047 • Monsegur O (2009) Vascular flora of the Guánica dry forest, Puerto Rico. Master of Science Thesis. University of Puerto Rico, Mayagüez Campus • Segarra A, Morales-Pérez A, Franqui R, Ratcliffe B (2014) First report of a South American cetoniine beetle, Gymnetis strigosa (Olivier, 1789) (Coleoptera: Scarabaeidae: Cetoniinae), in Puerto Rico. The Coleopterists Bulletin 68 (2): 217‑218. https://doi.org/10.1649/0010-065x-68.2.217 • Subcommittee IUCN Standards and Petitions (2016) Guidelines for using the IUCN Red List Categories and Criteria. Version 12. International Union for Conservation of Nature, Gland, Switzerland. Supplementary material Suppl. material 1: Google Earth map showing Ruehssia woodburyana known records Authors: Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J. Data type: occurences Download file (42.42 kb) • DNER (2007) Elementos críticos de la división de patrimonio natural - plantas • DNER (2007) Elementos críticos de la división de patrimonio natural - plantas. • DNER (2007) Elementos críticos de la división de patrim Unpublished Report Submitted to Gov Puerto Rico 12 Unpublished Report Submitted to Gov Puerto Rico 12 • Espírito Santo FdSdE, Rapini A, Ribeiro PL, Liede-Schumann S, Goyder DJ, Fontella- Pereira J (2019) Phylogeny of the tribe Marsdenieae (Apocynaceae), reinstatement of Ruehssia and the taxonomic treatment of the genus in Brazil. Supplementary material Suppl. material 1: Google Earth map showing Ruehssia woodburyana known records Authors: Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J. Data type: occurences Download file (42.42 kb) Suppl. material 1: Google Earth map showing Ruehssia woodburyana known records Authors: Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J. Data type: occurences Download file (42.42 kb) uppl. material 1: Google Earth map showing Ruehssia woodburyana known recor Authors: Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J. Data type: occurences Download file (42.42 kb) Authors: Barrios, S.; Hamilton, H.; Monsegur, O.; Sustache, J. Data type: occurences Download file (42.42 kb)
https://openalex.org/W2783558852	https://europepmc.org/articles/pmc5771220?pdf=render	English	null	Correction to: Health effects of milder winters: a review of evidence from the United Kingdom	Environmental health	2,018	cc-by	220	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 1. Hajat S. Health effects of milder winters: a review of evidence from the United Kingdom. Environ Health. 2017;16(S1):109. https://doi.org/10.1186/ s12940-017-0323-4. Correspondence: shakoor.hajat@lshtm.ac.uk Department of Social & Environmental Health Research, London School of Hygiene & Tropical Medicine, 15-17 Tavistock Place, London WC1H 9SH, UK Correction to: Health effects of milder winters: a review of evidence from the United Kingdom Shakoor Hajat Correction After publication of the article [1], it has been brought to our attention that there is an error in the abstract. The line that reads “a 1 °C fall during winter months led to reductions of 4.5%, 3.9% and 11.2%” should say “a 1 ° C fall during winter months led to increases of 4.5%, 3.9% and 11.2%”. Received: 9 January 2018 Accepted: 10 January 2018 Hajat Environmental Health (2018) 17:7 DOI 10.1186/s12940-018-0353-6 Hajat Environmental Health (2018) 17:7 DOI 10.1186/s12940-018-0353-6
https://openalex.org/W3153366633	https://www.researchsquare.com/article/rs-205196/latest.pdf	English	null	Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells	Molecular biology reports	2,021	cc-by	3,978	Ethanol and cocaine increases microtubule stability and decreases [11C]MPC-6827 uptake in SH-SY5Y cells and decreases [11C]MPC-6827 uptake in S cells Naresh Damuka Wake Forest University School of Medicine Miranda E Orr Wake Forest University School of Medicine Paul W Czoty Wake Forest University School of Medicine Jeffrey L Weiner Wake Forest University School of Medicine Thomas J Martin Wake Forest University School of Medicine Michael A Nader Wake Forest University School of Medicine Avinash H Bansode Wake Forest University School of Medicine Buddhika S Liyana Pathirannahel Wake Forest University School of Medicine Akiva Mintz Columbia University Medical Center: Columbia University Irving Medical Center Shannon L Macauley Wake Forest University School of Medicine Suzanne Craft Wake Forest University School of Medicine Kiran Kumar Solingapuram Sai (  ksolinga@wakehealth.edu ) Wake Forest University School of Medicine Short Report Keywords: Microtubule, cytoskeleton, substance use disorder, in vitro binding, biomark Posted Date: February 17th, 2021 Jeffrey L Weiner Wake Forest University School of Medicine Thomas J Martin Wake Forest University School of Medicine Thomas J Martin Wake Forest University School of Medicine Michael A Nader Wake Forest University School of Medicine Avinash H Bansode Wake Forest University School of Medicine Buddhika S Liyana Pathirannahel Wake Forest University School of Medicine Short Report Page 1/11 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Molecular Biology Reports on April 20th, 2021. See the published version at https://doi.org/10.1007/s11033-021-06336-7. Page 2/11 Abstract Microtubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain- penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 selectively bound to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin monomers). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs. Background Cytoskeletal defects, including alterations in microtubule stability, axonal transport and actin dynamics, have been characterized in several psychiatric and neurodegenerative disorders, including alcohol and substance use disorders (AUD/SUDs) and Alzheimer’s disease, suggesting they are a common feature contributing to neurodegeneration. An organized neuronal cytoskeleton is required for nervous system development, maintenance, and regenerative processes after injury. Its three components intermediate filaments, actin filaments, and microtubules (MTs), all play a vital role in neurological processes. MTs are critical to cellular structure; as neuronal backbones they facilitate cell division, axonal transport, and neurotransmission. MTs are hetero-dimer units formed from α- and β-tubulin monomers.[1] Essential biophysical functions, including cellular signaling and axoplasmic transport, depend on the structural integrity of MTs i.e., polymerization with bound and free tubulin units and MT integrity is heavily dysregulated in AUD and SUDs.[2-5] Addictive behaviors lead to many adaptations in postsynaptic spine structure that result in profound alterations in synaptic transmission.[6] At the molecular level, synaptic activity triggers diverse signaling pathways, which, in turn regulate and reorganize cytoskeleton- associated proteins. For example, repeated cocaine administration has been shown to change stathmin, a regulatory protein crucial to MT dynamics,[7,6] causing morphologic changes.[8] Neuronal structural changes may contribute to the progression of AUD and SUDs.[9] Chronic ethanol (EtOH) exposure significantly stabilizes neuronal and acetylated MTs in hepatic PC12 cells,[10] increases dendrite lengths and neurite outgrowth and causes aberrant sprouting of hippocampal neurites.[11] Loss of α and β free tubulin units in the caudate nucleus, cortex, and cerebellum was noted in post-mortem Page 3/11 Page 3/11 brain samples from individuals diagnosed with AUD. Repeated exposure to drugs of abuse like alcohol and cocaine induces structural plasticity[12,13] in many brain circuits and changes in the density and morphology of dendritic spines.[5,14,15] These alterations have significant consequences including cognitive deficits and neurodegeneration.[16,17] Prolonged SUD is also associated with brain injury characterized by impaired synaptogenesis, cellular migration, and neurogenesis—all of which require proper MT functioning.[18] [19] MT agents (MTAs), believed to work primarily by altering MT network integrity, are widely being investigated as drug candidates to treat cancer, brain disorders, and cardiovascular diseases. Thus, MT integrity is important to many neurochemical pathways commonly associated with SUD. However, studies of cytoskeleton-dependent structural plasticity resulting from alcohol and cocaine use have focused predominantly on actin and filament dynamics; molecular level MT impairments remains largely unexplored. Background Positron Emission Tomography (PET) imaging is a sensitiv modality to examine and quantify in vivo MT-based changes in the neurochemical cascades of SUD. MPC-6827 is a small molecule MTA that causes mitotic arrest and cell death. It exerts antitumor (glioblastoma) properties by binding to β-tubulin sites. We reported the automated radiochemical synthesis of [11C]MPC-6827 as the first brain-penetrating, MT-tracking PET ligand and imaged it in vivo in normal rodents and non-human primates.[20] [21] To establish the potential of [11C]MPC-6827 as a PET imaging ligand for various neurological disorders, we need to investigate its mechanism of action. Here, we report our preliminary in vitro evaluations of [11C]MPC-6827 in SH-SY5Y neuroblastoma cells[22-24] with (a) two different abused drugs (alcohol and cocaine), and (b) various MT stabilizing and destabilizing agents. Methods To investigate the effects of cocaine and alcohol on tubulin dynamics, we performed a MT-based assay (Cytoskeleton, Inc., Denver, CO, USA)[25-27] in SH-SY5Y neuroblastoma cells treated with 100 mM EtOH[10] and 1 mM cocaine[8] (n=6/group) for 3 days. This commercially available kit separates large complexes of polymerized MTs attached to nuclei and Golgi bodies into bound and non-polymerized free tubulins. After differential centrifugation, the high-speed pellet supernatant and low-speed pellet samples were isolated and used for western blot analysis. An enhanced Chemiluminescence kit was used to visualize the tubulin bands,[12,11] (Fig. 1a and 2a). Bound/stabilized MT content was significantly higher and unbound/free α/β MTs lower in EtOH-treated cells than control cells treated with PBS. Cells treated with cocaine showed no significant difference in bound MTs and slightly fewer free α/β MTs than untreated cells possibly due to the accrued rate of MT polymerization with substances. Therefore, both EtOH and cocaine compromise MT integrity i.e., increase in bound and decrease in free tubulin units. Having demonstrated these drug-induced changes in MT integrity in SH-SY5Y cells, we next aimed to determine whether [11C]MPC-6827 could also detect similar MT alterations using the same cells. We performed cell binding assays in vitro in SH-SY5Y cells with [11C]MPC-6827 following our previously published protocols.[28-30] The cells were treated with 100 mM EtOH or 1 mM cocaine[8] (n = 6/group) Page 4/11 Page 4/11 for 3 days. We then measured radiotracer binding by adding [11C]MPC-6827 (1-2 µCi/well) and incubating the cells for 5, 30, 60, and 90 min at room temperature (n = 6/time point). To demonstrate tracer specificity, a subgroup of cells (n = 3) was pre-treated with non-radioactive MPC-6827 (1.0 µM), adding radiotracer 60 min later and incubating for 30 min. To demonstrate tracer sensitivity to length of drug exposure, cells were treated with 100 mM EtOH or 1 mM cocaine for 1 h, 1 day, or 3 days and incubated with [11C]MPC-6827 for 30 min at room temperature. All the cells were then washed with PBS and lysed with 1N NaOH. Finally, the lysate from each well was g-counted (PerkinElmer, Waltham, MA, USA) and counts-per-minute (cpm) values were normalized to the amount of radioactivity added to each well. Cpm values were then matched with the protein concentration per well, and the data expressed as %ID/mg of protein present in each well. Methods EtOH- (Fig 1b) and cocaine-treated (Fig 2b) cells demonstrated an ~30(±2) and ~24(±6) percent decrease respectively in radioactive uptake versus non-treated controls over the 30-90 min incubation times. Additionally, uptake in EtOH-treated and cocaine-treated cells increased ~13(±3) and ~12(±2) percent from 5 to 30 min of incubation times respectively and decreased ~53(±2) and ~19(±3) percent by 90 min in EtOH- and cocaine-treated cells; thus demonstrating favorable pharmacokinetics. For the self-blocking assays (Fig 1b), uptake was ~78(±1) percent lower after addition of nonradioactive MPC-6827, demonstrating high specificity. Radioactive uptake was decreased ~21(±1) and 28(±1) percent from 1 h to 3 days EtOH and cocaine exposures (Fig 3) respectively. Therefore, [11C]MPC-6827 uptake decreased selectively with increased exposure to EtOH or cocaine. Moreover, since no significant decrease in radioactivity was observed after 3 days of drug exposure we used the same 3 days exposure in all our assays. MPC-6827 primarily targets the β tubulin site at pharmacological doses.[31-33] The lowered radioactive uptake in EtOH- and cocaine-treated SH-SY5Y cells indicates that [11C]MPC-6827 uptake correlate well with observed bound/free tubulin changes and may be tracking free β tubulin units, as both substance treatments decreased free tubulin content in the same cells. MTAs are categorized as either stabilizing agents (paclitaxel, laulimalide, and EpoD),[34-36] which favor polymerization of tubulin units and inhibit cell proliferation, or destabilizing agents (vinblastine and mertasine),[37-40] which increase free/unbound tubulins and promote apoptotic cell death. To distinguish their effect on MT integrity in SH-SY5Y cells, we performed the same tubulin-based western blot assays on paclitaxel- and vinblastine-treated cells.[41] The paclitaxel-treated cells had more bound/stabilized MTs, and vinblastine-treated cells had more unbound/free MTs than the untreated cells (Fig 4a). To confirm the free tubulin-based binding mechanism of [11C]MPC-6827, SH-SY5Y cells were pretreated with different MTAs at 1.0 µM concentration, 3.0 h prior to addiction of [11C]MPC-6827. Paclitaxel, laulimalide and EpoD decreased radioactive uptake by ~58(±3), ~40(±4), and ~66(±7) percent respectively, while vinblastine, and mertasine increased it by ~77(±6), and 64(±5) percent respectively (Fig 4b), confirming that [11C]MPC-6827 primarily targets primarily free tubulin units. Results Page 5/11 Page 5/11 Results of the preliminary [11C]MPC-6827 in vitro assays with EtOH and cocaine treatments at different incubation times in SH-SY5Y cells indicate that radioactive uptake decreases with increased drug exposure. Tests with various MTAs demonstrate that [11C]MPC-6827 preferentially binds to free/unbound tubulin units with high selectivity. The radioactive uptake results are well-corroborated with observed changes in bound and free tubulin expressions in SH-SY5Y cells with EtOH and cocaine treatments. Taken together, these studies confirm that [11C]MPC-6827 has great potential as an MT imaging agent for defining MT-based mechanisms that underlie the development of alcohol and cocaine addiction. We are currently characterizing its complete in vivo and ex vivo imaging properties in both rodent and nonhuman primate models of AUD/SUDs. Sources: Cocaine hydrochloride and other reagents were purchased from Sigma Aldrich; MPC 6827 hydrochloride was purchased from Tocris a biotech brand; SH-SY5Y cells was purchased from ATCC (American Type Culture Collection) and the precursor for [11C]MPC-6827 was purchased from ABX Inc supplies. (American Type Culture Collection) and the precursor for [11C]MPC-6827 was purchased from ABX Inc supplies. References 1. 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[11C]MPC-6827 uptake in vitro at different incubation times in SH-SY5Y cells with EtOH (100 mM/3 days) and without EtOH treatment (n=6/group); p<0.05 and ns: non-significant Figure 3 [11C]MPC-6827 uptake in vitro with EtOH (100 mM) and cocaine (1 mM) for 1h, 1 day and 3 day exposures in SH-SY5Y cells (n=6); p<0.05 and ns: non-significant Figure 4 Representative bound and free tubulin a. western blots and b. [11C]MPC-6827 uptake in vitro with different MT agents (1 µM/60 min) in SH-SY5Y cells (n=6); p<0.05 Figure 4 Representative bound and free tubulin a. western blots and b. [11C]MPC-6827 uptake in vitro with different MT agents (1 µM/60 min) in SH-SY5Y cells (n=6); p<0.05 Representative bound and free tubulin a. western blots and b. [11C]MPC-6827 uptake in vitro with different MT agents (1 µM/60 min) in SH-SY5Y cells (n=6); p<0.05 Page 11/11 Figure 2 Representative bound and free MT a. western blots and b. [11C]MPC-6827 uptake in vitro at different incubation times in SH-SY5Y cells with cocaine (1 mM/3 days) and without cocaine treatment (n=6); p<0.05 and ns: non-significant Page 10/11 Figure 3 [11C]MPC-6827 uptake in vitro with EtOH (100 mM) and cocaine (1 mM) for 1h, 1 day and 3 day exposures in SH-SY5Y cells (n=6); *p<0.05 and ns: non-significant Figure 3
https://openalex.org/W4297789289	https://proceedings.unisba.ac.id/index.php/BCSPR/article/download/4451/2022	Indonesian	null	Strategi Customer Engagement pada Bisnis UMKM	Bandung Conference Series. Public Relations	2,022	cc-by	4,223	Bandung Conference Series: Public Relations Bandung Conference Series: Public Relations https://doi.org/10.29313/bcspr.v2i2.4451 Avirley Nursyafa, Riza Hernawati Prodi Hubungan Masyarakat, Fakultas Ilmu Komunikasi, Universitas Islam Bandung, Indonesia. Kata Kunci: Customer Engagement, Strategi Komunikasi, UMKM Corresponding Author Email: riza@unisba.ac.id A. Dengan perkembangan zaman saat ini, PT. Arafah Jaya Metalindo membuka sayapnya untuk membuka bisnis baru yaitu dengan mendirikan In Out House. In Out House yang masih dalam pengawasan PT. Arafah Jaya Metalindo Entertaiment yang merupakan anak perusahaan PT. Arafah Jaya Metalindo. PT. Arafah Jaya Metalindo Entertaiment terdiri dari In Out Carwash, In Out Coffee dan Cairo Indo Futsal. Perusahaan ini berjalan di bidang olahraga, Food and Beverages dan kebutuhan kendaraan. Untuk memberikan kesan nyaman, menambah kepuasaan konsumen serta peningkatan keuntungan PT. Arafah Jaya Metalindo Entertaiment. Maka dari itu, perusahaan ini membuka sayapnya ke bidang food and beverages. Pada tanggal 1 September 2019, terbentuklah In Out Coffee dengan harapan konsumen dari In Out Carwash dapat mencuci kendaraannya dan menunggu sambil menikmati minuman dan makanan ringan dari In Out Coffee. ff Perilaku keterlibatan konsumen merupakan manifestasi dari perilaku terhadap fokus perusahaan (selain pembelian) yang bersumber dari momentum motivasi (Van Doorn J. L., 2010). Perilaku keterlibatan konsumen adalah konsep “tindakan nyata” yang mencerminkan kontribusi sumber daya sukarela konsumen (pengetahuan, pengalaman, energi, waktu, dll) dengan pihak lain seperti perusahan (Kumar, 2010 ). Penelitian ini berfokus pada aspek positif dari perilaku keterlibatan. Maka, salah satu hal yang harus difokuskan oleh perusahaan adalah memahami perilaku keterlibatan berkontribusi pada aktivitas pemasaran dan mengecualikan perilaku konsumen seperti konsumsi dan pembuangan produk. Kemudian, “di luar pembelian” mencerminkan sifat keterlibatan non-transaksional, tidak seperti perilaku transaksional seperti membeli produk (Van Doorn J. L., 2010), ”Dari pendorong motivasi” menunjukkan bahwa perilaku keterlibatan berasal dari sikap keterlibatan sebagai anteseden. (Lemon, 2016). Perilaku keterlibatan konsumen adalah konsep yang jelas dalam pemasaran, tetapi menunjukkan kesamaan dengan konsep terkait. Perilaku ini perlu sejalan dengan orientasi konsumen yang relasional atau dapat dikatakan bahwa mempunyai nilai tertentu untuk menjadikannya strategi Customer Engagement. Sifat Customer Engagement sebagai variabel yang menonjol dalam hubungan layanan berasal dari sifat konsep yang interaktif, empiris dan kreatif. Secara khusus, aspek interaktif dan empiris dari konsep tersebut membedakan Customer Engagement dari konsep hubungan lainnya dalam jangkauan yang lebih luas dari hubungan layanan. Ini membutuhkan jaringan untuk memiliki pendahuluan Customer Engagement (yaitu, harus terjadi sebagai pendahulu dari Customer Engagement) yang telah ditemukan termasuk dalam “partisipasi” dan “keterlibatan”. Di sisi lain, konsep terkait lainnya, termasuk “aliran” dan “hubungan”, telah ditemukan tidak perlu dan berpotensi melibatkan pelanggan dalam konteks tertentu. Selain itu, Customer Engagement sebagai hasil dari hubungan dapat mencakup “komitmen”, “kepercayaan”, “ikatan merek sendiri”, “konsumen”, “keterikatan merek emosional”, dan “kesetiaan”. (Brodie, 2011). virleynusyafaa@gmail.com, riza@unisba.ac.id *virleynusyafaa@gmail.com, riza@unisba.ac.id Abstract. Customer Engagement behaviors have emerged as an influential concept in marketing and refer to the driving force behind the motivation to realize consumer behavior towards companies. In a dynamic and interactive business environment, the role of Customer Engagement creates experiences and value that enhance business people. The Customer Engagement strategy is indirectly supported by consumers because Customer Engagement plays an important role in improving the relationship between the company and its consumers. The purpose of this study is to determine the Customer Engagement strategy used by MSME businesses. The object of the research was In Out Coffee, Cakung. The research method used in this research is a case study with a qualitative approach. Collecting data using structured interviews, participatory observation, and documentation. There were five informants in this study. Presentation of research using triangulation of data collection using transcription, data reduction, and analysis of concluding with the logic of deductive understanding. It was found that interaction and creativity helped In Out Coffee's Customer Engagement strategy. It can be stated that the communication strategy through Customer Engagement has been successful. Keywords: Customer Engagement, Communication Strategy, MSME Abstrak. Perilaku Customer Engagement telah muncul sebagai konsep yang berpengaruh dalam pemasaran dan mengacu pada kekuatan pendorong di balik motivasi untuk mewujudkan perilaku konsumen terhadap perusahaan. Dalam lingkungan bisnis yang dinamis dan interaktif, peran Customer Engagement menciptakan pengalaman dan nilai yang meningkatkan pelaku bisnis. Strategi Customer Engagement secara tidak langsung didukung oleh konsumen karena Customer Engagement berperan penting dalam meningkatkan hubungan antara perusahaan dengan konsumennya. Tujuan penelitian ini untuk mengetahui strategi Customer Engagement yang digunakan oleh pelaku usaha UMKM. Objek penelitian dilakukan pada In Out Coffee, Cakung. Metode penelitian yang digunakan dalam penelitian ini adalah studi kasus dengan pendekatan kualitatif. Pengumpulan data menggunakan wawancara terstruktur, observasi partisipatif, dan dokumentasi. Informan dalam penelitian ini berjumlah lima informan. Penyajian penelitian menggunakan triangulasi pengumpulan data menggunakan transkripsi, reduksi data, dan analisis kesimpulan dengan logika pemahaman deduktif. Ditemukan bahwa interaktif dan kreativitas membantu strategi Customer Engagement yang dilakukan oleh In Out Coffee. Dapat dinyatakan bahwa strategi komunikasi melalui Customer Engagement telah berhasil. Kata Kunci: Customer Engagement, Strategi Komunikasi, UMKM Corresponding Author Email: riza@unisba.ac.id 1 1 Strategi Customer Engagement pada Bisnis UMKM\| 657 Pandemi Covid-19 telah memberikan dampak negatif terhadap perekonomian dunia A. Dapat ditarik kesimpulan bahwa Customer Engagement harus memiliki unsur – unsur yang telah dijelaskan sebelumnya untuk mendapatkan Customer Engagement yang sesuai. Sesuai dengan loyalitas pelanggan dalam lingkungan yang interaktif untuk membuat penelitian empiris dengan ini perspektif akan memberikan landasan konseptual untuk mengembangkan konsep Customer Engagement. Hal ini mencerminkan pengalaman interaktif dan kreativitas pelanggan dengan pemangku kepentingan lainnya dalam hubungan layanan tertentu. Menurut Liputan 6 (2019) (diakses pada tanggal 5 Maret 2022), kondisi bisnis terus membaik pada kuartal IV-2018. Hal ini disebabkan adanya peningkatan komponen laba usaha pada nilai indikator 106,25, penggunaan kapasitas pada nilai indikator 105,49, dan rata-rata jumlah jam kerja pada nilai indikator 102,40. Perbaikan situasi dari Q4 hingga 2018 terjadi di 13 kategori bidang usaha. Kondisi usaha yang baik dan optimisme usaha tertinggi terjadi pada kategori usaha penyelenggaraan pemerintahan, pertanahan, dan jaminan sosial wajib, dengan skor ITB 122,58. Hal ini menunjukkan bahwa kondisi bisnis mengalami trend positif dengan bantuan media sosial yang terus berkembang seperti Instagram, Twitter, Facebook, dan lain – lain Pandemi Covid-19 telah memberikan dampak negatif terhadap perekonomian dunia Public Relations 658 Avirley Nursyafa, et al. secara global. Pandemi Covid-19 yang terjadi di Indonesia telah menyebabkan adanya kebijakan – kebijakan baru yang telah mengubah keadaan aktivitas dan kebiasaan masyarakat diluar ruangan menjadi di rumah sesuai dengan anjuran pemerintah untuk melakukan social distancing (Khasanah, 2020). Dengan adanya kebijakan karantina, lockdown, dan pengurangan aktivitas diluar ruangan membuat banyak perusahaan menunda kegiatan operasional bahkan sampai tutup akibat dari pandemi ini. Banyak perusahaan yang terpaksa untuk memPHK karyawannya karena kondisi ini, disamping itu juga adanya penurunan akan permintaan barang dan jasa yang menyebabkan supply bahan baku tersendat berakibat pada penurunan produksi. y p p p Berdasarkan pada perkembangan teknologi yang menyesuaikan dengan kondisi pandemi Covid-19 ini, hampir seluruh perusahaan menggunakan media sosial sebagai tempat untuk menjelaskan produk atau jasanya, sebagai media promosi, sebagai wadah perkenalan kepada khalayak dan tujuan lainnya. Tetapi, ada pula perusahaan yang tidak menggunakan media sosial atau mereka menggunakannya hanya saja tidak fokus pada hal tersebut. Seperti dalam kasus ini adalah Café In Out Coffee yang mempunyai media sosial seperti Instagram hanya sebagai media perkenalan dengan dunia bahwa adanya perusahaan ini. Serta, In Out Coffee pula tidak fokus mendalami Instagram seperti perusahaan lainnya yang konsisten dalam menjalankan media sosial. In Out Coffee merupakan salah satu bisnis UMKM yang terdapat di Cakung, Jakarta Timur yang bergerak di bidang kuliner. A. UMKM In Out Coffee sempat tutup selama 1 tahun saat Covid-19 datang diawal tahun 2020. Lalu, pada Juni 2021, In Out Coffee mencoba untuk buka kembali dan berusaha meraih respon positif dari masyarakat. Berdasarkan dengan fenomena yang telah dijabarkan sebelumnya, maka pertanyaa penelitian yang sesuai dengan fenomena tersebut antara lain, adalah : Berdasarkan dengan fenomena yang telah dijabarkan sebelumnya, maka pertanyaan enelitian yang sesuai dengan fenomena tersebut antara lain, adalah : 1. Bagaimana pengalaman konsumen yang interaktif pada bisnis In Out Coffee ? 2. Bagaimana pengalaman konsumen yang kreativitas pada bisnis In Out Coffee ? 3. Bagaimana strategi Customer Engagement pada In Out Coffee ? 4. Mengapa In Out Coffee menggunakan strategi Customer Engagement ? Dengan pertanyaan penelitian tersebut, diharapkan penelitian ini dapat menemukan fenomena sentral yang sedang dipelajari untuk menemukan apa strategi Customer Engagement pada bisnis UMKM perusahaan In Out Coffee Cakung. Dengan pertanyaan penelitian tersebut, diharapkan penelitian ini dapat menemukan fenomena sentral yang sedang dipelajari untuk menemukan apa strategi Customer Engagement pada bisnis UMKM perusahaan In Out Coffee Cakung. Agar penelitian ini dapat berfokus, tidak meluas, dan terarah sesaui dengan judulnya, maka penulis memberikan batasan pada penelitian yang berjudul “Strategi Customer Engagement Pada Bisnis UMKM (Studi Kasus Pada In Out Coffee, Cakung)” sebagai berikut : Agar penelitian ini dapat berfokus, tidak meluas, dan terarah sesaui dengan judulnya, maka penulis memberikan batasan pada penelitian yang berjudul “Strategi Customer Engagement Pada Bisnis UMKM (Studi Kasus Pada In Out Coffee, Cakung)” sebagai berikut : g g ( ff g) g 1. Peran yang diukur adalah hanya pada bisnis UMKM Perusahaan In Out Coffee Cakung 2. Wawancara daring dilakukan dengan pemilik usaha In Out Coffee, Public Relations In Out Coffee, Karyawan In Out Coffee, dan Konsumen baru yang datang ke In Out Coffee. y y g g 3. Penelitian menggunakan penelitian Kualitatif Studi Kasus untuk meluruskan wawancara daring dan secara langsung dengan informan yang telah di tetaptkan di In Out Coffe Cakung. g 4. Penelitian ini hanya membahas Public Relations Café In Out Coffee dengan konsumen baru yang datang ke In Out Coffee. 4. Penelitian ini hanya membahas Public Relations Café In Out Coffee dengan konsumen baru yang datang ke In Out Coffee. C. Customer Engagement merupakan hubungan emosional melalui interaksi antara produk atau brand dengan konsumen. Interaksi – interaksi ini akan memunculkan ikatan antara konsumen dengan perusahaan yang akan mengembangkan strategi Public Relations kedepannya. Stategi Customer Engagement merupakan bentuk perencanaan interaksi antara konsumen dengan perusahaan atau dalam kasus ini adalah Public Relations. Penggunaan Customer Engagement merupakan hal yang sudah tepat untuk In Out Coffee yang memang memfokuskan pemasarannya secara langsung, tidak seperti café lain yang mengandalkan penggunaan media sosial sebagai media pemasarannya. Customer Engagement yang digunakan oleh Public Relations In Out Coffee merupakan pendekatan secara langsung kepada konsumen untuk mendapatkan umpan balik langsung dari konsumen dan melihat sendiri kepuasan yang telah diterima oleh konsumen atau kekurangan yang diterima oleh konsumen dan menjadikannya sebagai strategi baru untuk masa yang akan datang. Terkait pengalaman konsumen terhadap interaktivitas yang diberikan In Out Coffee berdasarkan hasil observasi dan wawancara yang telah dilaksanakan, peneliti merasa bahwa konsumen dengan baik menerima seluruh interaktivitas yang telah dilakukan oleh In Out Coffee. Dilihat dari penerimaan konsumen terhadap interaksi, cara berbicara, cara menanggapi, mengulang pesanan, menikmati suasana yang ada, serta merekomendasikan In Out Coffee kepada orang lain. Selain itu, konsumen juga menerima program kerja dan ikut serta dalam aktivitas yang dilakukan oleh Public Relations yang diantaranya acara nonton bola bersama dengan konsep menggunakan jersey bola, mengadakan giveaway atau bagi – bagi hadiah dengan minimal pembelian, masuk kedalam ranah K-Pop Fanbase, hingga ke acara Live music yang setiap minggu diadakan. Dalam pengamatan peneliti, ini merupakan hasil yang baik untuk sebuah café juga merupakan strategi Customer Engagement yang cukup jarang ditemukan untuk kasus secara langsung. Dapat diartikan bahwa In Out Coffee melakukan ini agar menaikkan keuntungan melalui soft selling atau penjualan secara halus dengan melakukan program kerja yang sebelumnya telah disebutkan. Pengalaman interaktif dapat dinilai dari rangsangan interaksi seperti produk dan pelayanan yang telah disediakan (Cova, 2002), pesan pengguna atau interaksi konten (Levy, 1999), interaksi yang dimediasi manusia atau komputer (Judee K. Burgoon, 1999), dan interaksi antarpribadi (Brodie, 2011). Penilaian berdasarkan interaksi produk dan pelayanan, konsumen In Out Coffee sudah memenuhi standar dilihat dari pendapat konsumen mengenai pengalamannya. Penilaian berdasarkan interaksi manusia atau komputer, dapat dilihat melalui interaksi media sosial yang hanya saja di penelitian ini tidak dibahas. Dan melalui interaksi konsumen dengan karyawan dan Public Relations In Out Coffee yang diterima dengan baik oleh konsumen In Out Coffee. B. Metodologi Penelitian Unit analisis dalam penelitian ini adalah pemilik In Out Coffee, Public Relations In Out Coffee, serta 3 konsumen random yang ada di In Out Coffee. Metode yang digunakan adalah metode penelitian kualitatif. Lokasi penelitian di Jl. Dr. KRT Radjiman Widyodiningrat No. 7 – 8, RT.13/RW.6, Rw. Ternate, Kec. Cakung, Kota Jakarta Tim Teknik pengumpulan data dalam penelitian ini yaitu : – 8, RT.13/RW.6, Rw. Ternate, Kec. Cakung, Kota Jakarta Timur. Teknik pengumpulan data dalam penelitian ini yaitu : g Teknik pengumpulan data dalam penelitian ini yaitu : nik pengumpulan data dalam penelitian ini yaitu 1. Wawancara. Wawancara dilakukan secara langsung dengan informan yang terkait, dengan tujuan mendapatkan informasi dan data secara mendalam mengenai strategi Customer Engagement In Out Coffee. g g ff 2. Observasi. Merupakan pengamatan selama penelitian berlangsung terkait interaksi, gestur tubuh informan, penilaian suasana, hingga ke kondisi konsumen saat sedang di wawancara. Vol. 2 No. 2 (2022), Hal: 1-4 ISSN: 2828-2167 ISSN: 2828-2167 Strategi Customer Engagement pada Bisnis UMKM\| 659 3. Studi literatur. Adalah pengumpulan data dengan mempelajari berbagai buku, makalah, artikel internet, media cetak dan lain – lain sebagai pendukung penelitian. 3. Studi literatur. Adalah pengumpulan data dengan mempelajari berbagai buku, makalah, artikel internet, media cetak dan lain – lain sebagai pendukung penelitian. g g 4. Studi dokumentasi. Dilakukan dengan melihat dokumen – dokumen sebelumnya yang dapat menyerupai tulisan, berita, potret, surat kabar, artikel dan foto yang sekiranya dapat mendukung penelitian. 4. Studi dokumentasi. Dilakukan dengan melihat dokumen – dokumen sebelumnya yang dapat menyerupai tulisan, berita, potret, surat kabar, artikel dan foto yang sekiranya dapat mendukung penelitian. Analisis data menggunakan reduksi data untuk memilah data yang telah didapatkan selama dilapangan. Penyajian data yaitu menyaring data – data yang telah dipilih, kemudian disusun secara sistematis agar mudah untuk dianalisa. Dan penarikan kesimpulan adalah melakukan kesimpulan dari semua data yang telah diperoleh sebagai hasil dari penelitian. Penelitian ini menggunakan teknik triangulasi untuk mengetahui realitas data yang diperoleh. Uji validitas data dalam penelitian ini menggunakan triangulasi teknik perolehan data. Triangulasi metode akuisisi data dilakukan untuk mengkonfirmasi kebenaran data dengan melihat data dengan cara yang berbeda untuk sumber data yang sama. Validasi triangulasi teknik akuisisi data ini untuk memprediksi kebenaran tentang Strategi Customer Engagement pada Bisnis UMKM Studi Kasus Pada In Out Coffee Cakung dengan memakai teknik seperti wawancara, observasi, dan lain sebagainya. Relations In Out Coffee. ff Berdasarkan hasil temuan penelitian, ditemukan bahwa konsumen In Out Coffee menerima interaksi dengan Public Relations yang memungkinkan bahwa Public Relations memberikan pembahasan yang menarik atau menuangkan kemampuan berbicaranya kepada konsumen. Artinya, Public Relations telah menerapkan penggabungan strategi Customer Engagement dengan kreativitas terhadap pembicaraan yang menarik. Selain cara Public Relations menarik perhatian konsumen melalui pembicaraannya, Public Relations In Out Coffee juga menggunakan program kerja untuk memberikan hiburan kepada konsumennya sebagai bentuk terima kasih dan penarik perhatian konsumen baru. Terlebih dengan aktivitas yang dilakukan oleh In Out Coffee berdasarkan program kerja yang telah ditetapkan, menjadikan In Out Coffee lebih banyak mendapatkan keuntungan dibandingkan tanpa strategi Customer Engagement. Pernyataan ini dilihat dari wawancara dengan NT selaku Public Relations In Out Coffee yang menjelaskan bahwa terdapat kenaikan dengan bantuan pengalaman kreativitas konsumen. g p g Kondisi yang telah disebutkan sebelumnya merupakan hasil dari penerapan strategi Customer Engagement dengan penggabungan kreativitas. Hasilnya memberikan kenyamanan, kepuasan, serta loyalitas konsumen terhadap In Out Coffee. Kenyamanan yang dimaksudkan disini adalah cita rasa makanan dan minuman yang enak untuk lidah konsumen, tempat yang cocok untuk bersantai dan ‘nongkrong’ dengan kerabat, harga makanan dan minuman yang pas untuk kantong anak SMP hingga pekerja, serta In Out Coffee merupakan tempat yang “instagramable” atau bahasa lainnya tempat yang estetik untuk berfoto menurut konsumen. Hal inilah yang memberikan nilai tambahan untuk In Out Coffee selain dari aktivitas yang telah disebutkan sebelumnya. Disebutkan dalam Brodie (2011) teori sebelumnya memperlukan nilai kepuasan pengalaman konsumen dan nilai instrumental yang dalam kasus In Out Coffee adalah pengalaman kreativitas konsumen. Dengan teori dari Brodie (2011), membantu peneliti dalam menemukan pengalaman konsumen yang kreativitas dan menekankan pengalaman konsumen terhadap strategi Customer Engagement yang telah dibuat oleh Public Relations In Out Coffee. Merujuk pada hasil wawancara dengan 5 subjek, ditemukan bahwa Public Relations In Out Coffee menyatakan benar adanya In Out Coffee menggunakan strategi Customer Engagement untuk mendapatkan konsumen. Didukungnya pernyataan dari pemilik In Out Coffee yang menyetujui strategi Customer Engagement tersebut. C. Sedangkan, penilaian berdasarkan interaksi antarpribadi dilihat dari cara menanggapi konsumen terhadap interaksi atau percakapan yang telah dibuat oleh Public Public Relations Public Relations 660 Avirley Nursyafa, et al. Vol. 2 No. 2 (2022), Hal: 1-4 Relations In Out Coffee. Dilihat dari hasilnya, In Out Coffee sukses dalam menggunakan strategi Customer Engagement untuk mendapatkan konsumen dengan cara pendekatan secara halus, penjualan secara halus atau soft selling, pengenalan produk, komunikasi persuasif aktivitas aktivitas yang sedang dilaksanakan, pengenalan media sosial, pembagian hadiah dengan minimal pesanan, hingga event atau acara yang disebutkan oleh Public Relations sebelumnya yaitu Live music, nonton bola bareng, ikut serta dalam acara K-Pop Fanbase, Giveaway Instagram, Photoshoot Challenge, buka bersama K-Pop Fanbase, menggunakan sistem reservasi, dan acara lain yang mungkin tidak sempat disebutkan. Setelah diteliti lebih dalam, strategi Customer Engagement In Out Coffee tidak akan berjalan tanpa adanya kreativitas, interaktivitas, dan strategi komunikasi persuasif dari Public Relations In Out Coffee. Pemikiran dari Public Relations In Out Coffee yaitu NT memberikan dampak besar terhadap pengembangan strategi In Out Coffee. NT menyarankan untuk menjadikan karyawan In Out Coffee lebih memperhatikan kebutuhan konsumennya, menjadi lebih ramah, lebih sopan, dan mau untuk berinteraksi lebih banyak dengan konsumen agar konsumen merasakan kepuasan akan layanan yang didapat dan mendapatkan kesan yang baik setelah pulang. Sejalan dengan strategi komunikasi yang dilakukan oleh Public Relations In Out Coffee, Customer Engagement juga dilakukan untuk memantapkan strategi tersebut. Customer Engagement menurut Brodie (2011) merupakan sebuah proses mengembangkan, melindungi dan memelihara konsumen agar tetap berhubungan dengan perusahaan. Apa yang dilakukan oleh In Out Coffee sudah sesuai dengan teori dari Brodie (2011) untuk memastikan konsumen tetap terikat dengan perusahaan. Secara tidak langsung-pun, konsumen In Out Coffee telah ISSN: 2828-2167 Strategi Customer Engagement pada Bisnis UMKM\| 661 memasarkan In Out Coffee dengan mem-posting story di instagram mereka, serta merekomendasikan In Out Coffee kepada kerabatnya. Maka, teori dari Brodie (2011) yang menyatakan konsumen tidak hanya pembeli perusahaan, tetapi lebih. Dengan kata lain, sebagi media pemasaran bagi perusahaan sangatlah valid untuk konsumen In Out Coffee. Selama penelitian berlangsung, peneliti menemukan Public Relations menggabungkan strategi komunikasi dengan Customer Engagement yang menciptakan interaktivitas dan kreativitas untuk konsumen. Maka, konsumen serta perusahaan menjalin simbiosis mutualisme (hubungan yang saling menguntungkan). Hal ini didasari oleh teori Brodie (2011), yang menyatakan Customer Engagement didasarkan pada pengalaman interaktif dengan perusahaan dan adanya kreativitas konsumen. Yang mana dalam penelitian ini, In Out Coffee telah mencoba untuk memberikan interaksi kepada konsumen dan disambut baik oleh konsumen. Menjadikan konsumen dengan In Out Coffee saling terikat satu sama lain. Relations In Out Coffee. g ff g Berdasarkan teori Brodie (2011) yang menjelaskan bahwa, strategi komunikasi yang telah diterapkan oleh perusahaan dengan gabungan teori Customer Engagement sebagai pendukung akan menciptakan interaktif (Interactive) dan kreativitas (Co – Creative) kepada konsumen. Dengan teori konsep ini, maka perusahaan dapat terikat dengan konsumennya, serta In Out Coffee dapat mengetahui kebutuhan konsumennya. Selain itu, In Out Coffee juga dapat memikirkan kembali strategi apa yang selanjutkan akan dipakai. Maka, In Out Coffee dapat mengembangkan usaha mereka. g g Pemilihan strategi ini merupakan pilihan yang tepat untuk In Out Coffee yang ingin lebih dekat dengan konsumen dan ingin memperluas pasarnya dalam jangkauan konsumen. In Out Coffee tidak hanya sekadar menjual produk dan konsumen menikmati, tetapi lebih dari itu. In Out Coffee ingin konsumen menjadi akrab dengan mereka, dan juga In Out Coffee ingin konsumen menjadikan In Out Coffee sebagai “rumah kedua” mereka. Dengan menggunakan strategi komunikasi untuk memberikan pengalaman interaktivitas dan kreativitas konsumen, In Out Coffee melanjutkan rencananya yaitu dengan menggabungkan strategi komunikasi dengan strategi Customer Engagement yang hasilnya memberikan loyalitas konsumen kepada In Out Coffee, penambahan konsumen baru melalui rekomendasi konsumen lama, kepuasan konsumen yang memberikan kesan untuk kembali, kenyamanan konsumen, hubungan antara In Out Coffee dan konsumen yang mungkin akan berguna di masa yang akan datang, hingga strategi baru yang memungkinkan penggabungan antara pemikiran konsumen dengan pemikiran Public Relations In Out Coffee. Dengan banyaknya keuntungan yang diperoleh In Out Coffee dan konsumen, strategi ini merupakan startegi yang tepat untuk dipertahankan oleh In Out Coffee. Startegi ini menggunakan pendekatan secara langsung kepada konsumen untuk melihat reaksi dari konsumen sebagai bahan pelajaran Public Relations In Out Coffee. g p j ff Berdasarkan konsep teori Brodie (2011), ditemukan bahwa konsumen mendapatkan pengalaman interaktif yang baik, kreativitas yang diterima oleh konsumen, strategi Customer Engagement yang terlihat oleh konsumen, serta alasan penggunaan strategi Customer Engagement yang dilakukan oleh In Out Coffee. Karena hal ini bukanlah dimensi dari sebuah teori, maka dapat disebutkan bahwa teori dari Brodie ini merupakan teori yang valid sesuai dengan kondisi yang ditemukan dilapangan selama penelitian berlangsung. Public Relations Public Relations 662 Avirley Nursyafa, et al. Gambar 1. Alur Komprehennsif Keseluruhan Penelitian 662 Gambar 1. Alur Komprehennsif Keseluruhan Penelitian D. Kesimpulan D. Kesimpulan Berdasarkan pembahasan dalam penelitian ini, peneliti menyimpulkan beberapa hasil penelitian sebagai berikut: Berdasarkan pembahasan dalam penelitian ini, peneliti menyimpulkan beberapa hasil penelitian sebagai berikut: Berdasarkan pembahasan dalam penelitian ini, peneliti menyimpulkan beberapa hasil penelitia sebagai berikut: 1. Pengalaman interaktivitas yang dilakukan oleh Public Relations In Out Coffee mendapatkan reaksi positif dari konsumen. Reaksi yang dimaksud berupa penanggapan percakapan, interaksi yang terjalin, menikmati suasana, mengulang pesanan hingga merekomendasikan In Out Coffee kepada kerabatnya. Pengalaman ini menjadi acuan Public Relations untuk fokus pada hubungan konsumen. Dengan ini, memungkinkan konsumen akan memberikan loyalitasnya kepada perusahaan. y y p p 2. Kreativitas yang dimasukkan dalam strategi Customer Engagement merupakan hal yang cukup jarang untuk ditemui. Pengalaman konsumen akan kreativitas In Out Coffee memberikan kesan baik pada perusahaan memberikan efek positif pada kelanjutan hubungan antara konsumen dengan In Out Coffee. Hal ini dapat dilihat dari keikutsertaan konsumen terhadap aktivitas yang dilaksanakan oleh In Out Coffee, selain itu Public Relations In Out Coffee juga memberikan komunikasi persuasif yang menarik perhatian konsumen untuk tetap mendengarkan dan memberikan umpan balik. Dengan ini, maka pengalaman kreativitas konsumen terhadap In Out Coffee telah memberikan dampak baik untuk hubungan antara konsumen dan In Out Coffee. g ff 3. Strategi Customer Engagement merupakan sebuah rencana pendekatan kepada konsumen untuk menghasilkan hubungan antara konsumen dengan perusahaan. Pada penelitian ini, In Out Coffee menggunakan strategi Customer Engagement dengan pendekatan secara langsung melalui interaktivitas (interaksi, percakapan, perkenalan, gestur tubuh, dan lain sebagainya) dan melalui kreativitas (desain interior atau eksterior, aktivitas yang melibatkan konsumen, giveaway, dan lain sebagainya) yang membuahkan hasil simbiosis mutualisme atau hubungan yang saling menguntungkan. Vol. 2 No. 2 (2022), Hal: 1-4 ISSN: 2828-2167 ISSN: 2828-2167 Strategi Customer Engagement pada Bisnis UMKM\| 663 4. Alasan Public Relations In Out Coffee menggunakan strategi ini, karena strategi ini merupakan hal yang mudah untuk dilakukan, low budget atau tidak mengeluarkan dana yang lebih, menjadikan konsumen lebih dekat dengan karyawan, menambah relasi, serta memberikan dampak positif kepada perusahaan di masa yang akan datang. 4. Alasan Public Relations In Out Coffee menggunakan strategi ini, karena strategi ini merupakan hal yang mudah untuk dilakukan, low budget atau tidak mengeluarkan dana yang lebih, menjadikan konsumen lebih dekat dengan karyawan, menambah relasi, serta memberikan dampak positif kepada perusahaan di masa yang akan datang. Acknowledge Terima kasih kepada Allah SWT, Kedua Orang Tua, Keluarga Besar H. Moesa, Beranda.Lab, Bedug Subuh, serta pihak – pihak Universitas Islam Bandung yang telah membantu peneliti selama pembuatan jurnal berlangsung. Terima kasih kepada Allah SWT, Kedua Orang Tua, Keluarga Besar H. Moesa, Beranda.Lab, Bedug Subuh, serta pihak – pihak Universitas Islam Bandung yang telah membantu peneliti selama pembuatan jurnal berlangsung. Daftar Pustaka [1] Brodie, R. J. (2011). Customer Engagement : Conceptual Domain, Fundamental Propositions, and Implications for research. J. Serv. Res, 14(3), 252 - 271. [2] Cova, A. C. (2002). Retour Sur Le Concept d'Experience : Pour une Vue Plus Modeste et Plus Complete du Marketing. Actes des 7e Journees de Recherche en Marketing de Bourgogne, 156 - 172. [3] Judee K. Burgoon, J. A. (1999). Testing the Interactivity Model : Communication Process, Partner Assessments, and the Quality of Collaborative Work. Journal of Management Informations Systems, 16(3), 33 - 56. [4] Khasanah, D. R. (2020). Pendidikan Dalam Masa Pandemi Covid-19. Jurnal Sinestesia, 10(01), 41 - 48. [5] Kumar, V. A. (2010 ). Undervalued or Overvalued Customers : Capturing Total Customer Engagement Value. Journal of Service Research, 13(3), 297 - 310. [6] Lemon, K. N. (2016). Understanding Customer Experience Throughout the Custom Journey. Journal of Marketing, 80(6), 69 - 96. [7] Levy, B. L. (1999). Interactivity, Online Journalism, and English Language We Newspapers in Asia. Journalism and Mass Communication Quarterly, 76(1), 138 - 151 [7] Levy, B. L. (1999). Interactivity, Online Journalism, and English Language Web Newspapers in Asia. Journalism and Mass Communication Quarterly, 76(1), 138 - 151. [8] Van Doorn, J. L. (2010). Customer Engagement Behavior : Theoretical Foundations and h i i l f h 13(3) 253 266 Newspapers in Asia. Journalism and Mass Communication Quarterly, 76(1), 138 - 151. [8] Van Doorn, J. L. (2010). Customer Engagement Behavior : Theoretical Foundations and Research Directions Journal of Service Research 13(3) 253 266 [8] Van Doorn, J. L. (2010). Customer Engagement Behavior : Theoretical Foundations an Research Directions. Journal of Service Research, 13(3), 253-266. [9] Van Doorn, J. L. (2010). Customer Engagement Behavior : Theoretical Foundations an Research Directions. 13(3), 253 - 266. [10] Priyono, Agung, Ahmadi, Dadi. (2021). Strategi Komunikasi Marketing Public Relation Barli Coffee. Jurnal Riset Public Relations, 1(1), 90 - 95 [11] [11] www.liputan6.com. (2019, Febuary 06). Keyakinan Pelaku Usaha Turun, Kondisi Bisnis Malah Meningkat pada Kuartal IV 2018. (D. A. Putra, Editor) Retrieved March 5, 2022, from Liputan 6: https://www.liputan6.com/bisnis/read/3888994/keyakinan-pelaku- usaha-turun-kondisi-bisnis-malah-meningkat-pada-kuartal-iv-2018 Public Relations Public Relations
https://openalex.org/W1744979136	https://zenodo.org/records/1073577/files/9860.pdf	English	null	Investigation of the Effect of Cavitator Angle and Dimensions for a Supercavitating Vehicle	Journal of Aerospace Sciences and Technologies	2,023	cc-by	6,766	World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 NOMENCLATURE Angle of attack (deg) c Angle of attack of the cavitator (deg) Control vector Cavitation Number Density (Kg/m3) CD Coefficient of drag D Maximum diameter of the cavity (m) dc Diameter of the cavitator (m) F Force (N) Fr Froude number Fx Force acting in X- direction in body frame (N) Fy Force acting in Y- direction in body frame (N) Fz Force acting in Z- direction in body frame (N) H Angular momentum (Nms) Ixx Moment of Inertia about XX axis in body frame (Kg m2) Iyy Moment of Inertia about YY axis in body frame (Kg m2) Izz Moment of Inertia about ZZ axis in body frame (Kg m2) Ixz Product of Inertia about XZ axis (Kg m2) L Maximum length of the cavity (m) m Mass (Kg) M Moment (Nm) Mx Moment acting about X- axis in body frame (Nm) My Moment acting about Y- axis in body frame (Nm) Mz Moment acting about Z- axis in body frame (Nm) P Free stream pressure (N/m2) Pv Vapor pressure (N/m2) p Angular rate with respect to Xaxis in body frame (rad/s) q Angular rate with respect to Y axis in body frame (rad/s) International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2 NOMENCLATURE Angle of attack (deg) c Angle of attack of the cavitator (deg) Control vector Cavitation Number Density (Kg/m3) CD Coefficient of drag D Maximum diameter of the cavity (m) dc Diameter of the cavitator (m) F Force (N) Fr Froude number Fx Force acting in X- direction in body frame (N) Fy Force acting in Y- direction in body frame (N) Fz Force acting in Z- direction in body frame (N) H Angular momentum (Nms) Ixx Moment of Inertia about XX axis in body frame (Kg m2) Iyy Moment of Inertia about YY axis in body frame (Kg m2) Izz Moment of Inertia about ZZ axis in body frame (Kg m2) Ixz Product of Inertia about XZ axis (Kg m2) L Maximum length of the cavity (m) m Mass (Kg) M Moment (Nm) Mx Moment acting about X- axis in body frame (Nm) My Moment acting about Y- axis in body frame (Nm) Mz Moment acting about Z- axis in body frame (Nm) P Free stream pressure (N/m2) Pv Vapor pressure (N/m2) p Angular rate with respect to Xaxis in body frame (rad/s) q Angular rate with respect to Y axis in body frame (rad/s) International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, International Scholarly and Scientific Research & Innovation 6(8) 2012 M.Tech student, Department of Aerospace Engineering, Indian Institute of Technology Kanpur, Kanpur, India (sriram.aeropsn@gmail.com) Professor, Department of Aerospace Engineering, Indian Institute of Technology Kanpur, Kanpur, India (akg@iitk.ac.in) International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Keywords—High speed underwater vehicle, Non-Linear Dynamic Inverse (NDI),six-dof modeling, Supercavitation, Torpedo. Investigation of the Effect of Cavitator Angle and Dimensions for a Supercavitating Vehicle Sri Raman A., A.K.Ghosh Angular rate with respect to Z axis in body frame (rad/s) Non dimensional radius of the cavity Radius of the cavity (m) Thrust (N) Volume (m3) Free stream velocity (m/s) X component of velocity in body frame (m/s) Y component of velocity in body frame (m/s) Z component of velocity in body frame (m/s) Component of weight acting in X- direction (N) Component of weight acting in Y- direction (N) Component of weight acting in Z- direction (N) Location of center of gravity from leading edge (m) Location of center of buoyancy from leading edge (m) Non-dimensional axial location Axial location (m) State vector Angular rate with respect to Z axis in body frame (rad/s) Non dimensional radius of the cavity Radius of the cavity (m) Thrust (N) Volume (m3) Free stream velocity (m/s) X component of velocity in body frame (m/s) Y component of velocity in body frame (m/s) Z component of velocity in body frame (m/s) Component of weight acting in X- direction (N) Component of weight acting in Y- direction (N) Component of weight acting in Z- direction (N) Location of center of gravity from leading edge (m) Location of center of buoyancy from leading edge (m) Non-dimensional axial location Axial location (m) State vector Abstract—At very high speeds, bubbles form in the underwater vehicles because of sharp trailing edges or of places where the local pressure is lower than the vapor pressure. These bubbles are called cavities and the size of the cavities grows as the velocity increases. A properly designed cavitator can induce the formation of a single big cavity all over the vehicle. Such a vehicle travelling in the vaporous cavity is called a supercavitating vehicle and the present research work mainly focuses on the dynamic modeling of such vehicles. Cavitation of the fins is also accounted and the effect of the same on trajectory is well explained. The entire dynamics has been developed using the state space approach and emphasis is given on the effect of size and angle of attack of the cavitator. Control law has been established for the motion of the vehicle using Non-linear Dynamic Inverse (NDI) with cavitator as the control surface. I. INTRODUCTION A TORPEDOis a self-propelled underwater missile weapon with an explosive warhead, launched above or below the water surface. Most of the torpedoes are propelled under the water by a propeller with an exception of very few torpedoes which are propelled by rocket engines. Since the density of the water is much higher when compared to that of air, the drag experienced by the torpedo is also very high when compared to conventional missile. This demands very high thrust for higher velocities under the water. As we increase the velocity of the torpedo under the water by a suitable propulsion unit, the local pressure on the surface of the torpedo falls below the vapor pressure and this leads to the formation of bubbles which are called the cavities. If we still increase the velocity of the torpedo at the cost of propulsion unit, the size of the cavity begins to grow and at one condition there will be only one bubble which encloses the whole the torpedo. This condition is called supercavitation and is generally achieved by a cavitator. When a supercavity is formed over the torpedo, even though the torpedo cruises in water the torpedo is surrounded by a vapor bubble. Because of this, the actual wetted area of the torpedo reduces and this results in nearly total elimination of skin friction and the overall drag to an order. Due to the reduction in drag, the thrust required to cruise in the supercavitating regime is also reduced. Using this advantage, underwater vehicles can reach very high velocities as cruising in air when the drag reduces in virtue of supercavitation. Supercavitation can still be divided into natural supercavitation and ventilated supercavitation. A M.Tech student, Department of Aerospace Engineering, Indian Institute of Technology Kanpur, Kanpur, India (sriram.aeropsn@gmail.com) M.Tech student, Department of Aerospace Engineering, Indian Institute of Technology Kanpur, Kanpur, India (sriram.aeropsn@gmail.com) Professor, Department of Aerospace Engineering, Indian Institute of Technology Kanpur, Kanpur, India (akg@iitk.ac.in) scholar.waset.org/1307-6892/9860 1454 International Scholarly and Scientific Research & Innovation 6(8) 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Thevector equations can be rewritten in scalar form and then consist of three force equations and three moment equations which are coupled in nature. II.MODELING THE MOTION OF THE SYSTEM The analysis of physical behavior of any system can be done by mathematical modeling. The vehicle is treated as a rigid body and is having six degrees of freedom in space. The six degrees of freedom in space will result in six equations of motion, in which the inertial forces associated with one degree of freedom, are balanced by the corresponding Aerodynamic/Hydrodynamic and gravity forces. cal Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 The above is the governing equations of motion for any rigid body and the properties mass, inertia; force and moments are measured in the body reference frame. Apart from the above, the three kinematic equations and the three velocity relations between the inertial and body fixed frame is also required to completely solve the initial value problem which governs the motion of any six degree freedom system. Unlike conventional vehicles, here the force and moment model will be different due to the presence of cavity which will have a considerable effect on the dynamics of the motion. I. INTRODUCTION Natural supercavitation is achieved while increasing the velocity of the vehicle whereas the ventilated supercavitation is due to the increase in cavity pressure. The cavity pressure in the ventilated cavitation is generally increased by blowing gas behind a sharp trailing edge and the amount of gas which has to be injected is a critical parameter. In this paper we will be dealing only with the cavitation which is formed due to velocity of the vehicle alone. During the motion of a supercavitating vehicle, its motion dynamics is different than that of normal torpedoes because of the huge reduction in wetted area. Although there are many research papers which deal with the supercavitation, most of them deal with the shape and stability of the formed cavity with and without ventilation in static cases. LEE Qi-taoet al [1] experimentally studied the pitching motion of a supercavitating vehicle in a high speed water tunnel with an emphasis of planing and investigation of loads during the pitching motion. Bálint Vanek [2] in his doctoral thesis explains about control oriented modeling and stability augmentation for supercavitating vehicles. Daijin Li et al [3] have theoretically given a control law for motion of the supercavitating vehicles in vertical plane. In this paper we will be discussing the theoretical modeling of naturally supercavitating vehicles at high velocities with particular emphasis on the effect of cavitator dimensions, cavitator angle and the effect of cavitation of fins on the dynamics of the vehicle. ! "" # "" $ / 0 1 0 +' (* / 0 1 0 )( (* / 0 1 0 +' (* 2 0 3 4+ 0 ) 2 0 )+ 0 +5 )5 2 0 3 4)* 0 + , "" - "" . ! "" # "" $ / 0 1 0 +' ( / 0 1 0 )( (* / 0 1 0 +' (* 2 0 3 4+ 0 ) 2 0 )+ 0 +5 )5 2 0 3 4)* 0 *+ , "" - "" . III. FORCE AND MOMENT MODELING Assuming the motion is in the vertical plane, the component of forces due to the formation of cavity acting on the cavitator disk inclined to the stream with an angle can be approximately calculated as [8] G C 6 0 ?@ 6?JJ 0 B?A6 C DEK => 6 ?BL76 M N O P C+Q R M ;<9 5C56 ?BL76 M N O ) / /V _`a5bD / /V acdbD _`abD / /V _`a5bD / /V acdbD _`abD Where Fx0 is the drag experienced by the cavitator at zero angle of attack. By applying the theorem of momentum, Where Fx0 is the drag experienced by the cavitator at zero angle of attack. By applying the theorem of momentum, C+Q R M ;<9 5C56 ?BL76 M N O ) C+Q R M ;<9 5C56 ?BL76 M N O ) ef g D h?UJ 6 0 5 i j 7 g @/ ;<5k D 5 ef g D h?UJ 6 0 5 i j 7 Waid [6] also found the expressions for cavity dimensions based on experiments for a ventilated supercavity, which are given by International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Where, anical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 g @/ ;<5k D 5 G B?@ 6S?SSM DFC ?TLU 6V?WXM 0 B G B?@ 6S?SSM DFC ?TLU 6V?WXM 0 B Savchenko et al [5] have given the relations for supercavity profile based on water tunnel experiments which is the universally accepted supercavity profile. The results were also validated by ZHANG Xue-weiet al [7]. In this paper we will be using this result for calculating the supercavity profile. In the supercavitating regime Fx and Fz are the additional terms which have to be added. The values of Fx and Fz along with other forces due to the fins of the vehicle which extends out of the cavity constitute the entire force model of the vehicle. Depending upon the fin configuration and the local flow angle, cavities will form over the surface of the fin and the characteristics of the cavity will vary based on the angle of attack experienced by the fin. III. FORCE AND MOMENT MODELING The cavitating flows are characterized by the Cavitation number () and to a small extent they also depend on the Froude Number (Fr). But the dependence on Froude number is basically for the ventilated supercavitation at low velocities. At high velocities natural supercavity forms and the effect of Froude number is negligible and can be ignored. The reference frame for our analysis is depicted in Fig. 1. From the Newton’s second law, Fig. 1 Reference frame system International Science Index, Aeros 6 789 8: ;<9 5 The supercavitation regime corresponds to very small magnitudes of < 0.1. For small depths supercavity forms when the velocity of the vehicle is greater than 50 m/s. Trajectory simulation of such a high speed supercavitating vehicle requires a precise force and moment models and these force and moment models depend on the dimensions of the cavity also. Based on the Riabouchinsky model, Garabedian [4] gave expressions for the drag and cavity dimensions of a supercavitating vehicle. => ?@7AB 0 6 C DE=> 6 FG D H=> 6 EI B 6 => ?@7AB 0 6 C DE=> 6 FG D H=> 6 EI B 6 Fig. 1 Reference frame system 1455 nternational Scholarly and Scientific Research & Innovation 6(8) 2012 scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 scholar.waset.org/1307-6892/9860 1455 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Reichardt [5] gave a semi-empirical relation to predict the drag and dimensions of the supercavity, which are valid for < 0.1. III. FORCE AND MOMENT MODELING Computational Fluid Dynamic simulations [9], [10] have shown that partial cavity exists till the angle of attack is 2 degrees and beyond that supercavity evolves. B 0 LYS Z [ F \ 7? 5 L?JT] 0 ?@UAY 7? ?7LJ6Y 7?5F ^ 7? 5 L?JT] 0 ?@UAY 7? ?7LJ6Y 7?5F ^ 7? International Science Index, Aerospace and Mechanical Engine Where, Y 7 D F 7 D Fig. 3 Variation of fin force.Source : Kirshner [10] Garabedian’s result is taken for the calculation of drag for zero angle of attack. But, as the vehicle cruises, the angle of attack keeps on changing until steady state is achieved. When the angle of attack changes the cavity axis also shifts and the entire cavity profile changes as shown in Fig. 2. Because of this the resultant force splits up to give the drag force in the X- direction and a small lift force in the Z- direction. So the effect of angle of attack of the cavitator has to be calculated. Fig. 2 Deformation of cavity axis due to inclined cavitator Z X c V hf(x) x Fig. 3 Variation of fin force.Source : Kirshner [10] Fig. 3 Variation of fin force.Source : Kirshner [10] Fig. 3 Variation of fin force.Source : Kirshner [10] The information from the literature [9], [10] gives us the force coefficients for a specific wedge type fin with some sweep angle. But since the flow is supercavitating the difference in force coefficients between a wedge shaped swept back fin and a flat plate fin will be small and thus the same data has been taken into account for the fin cavitation. Fig. 2 Deformation of cavity axis due to inclined cavitator scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 1456 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Similarly, the moment generated by Fz must also be taken into account in the moment modeling. Rests of the moments are same as that of a non-supercavitating vehicle. regime are given in table II. For the complete supercavitating regime simulation the procedure as explained in section III is followed along with the use of data from table II. Fig. III. FORCE AND MOMENT MODELING 4 Variation of fin force coefficients.Source : Kirshner [10] gineering Vol:6, No:8, 2012 waset.org/Publication/9860 TABLE I STANDARD PROPERTIES OF THE PROJECTILE Property Value Unit m 0.2 Kg Ixx 1×10-5 Kg m2 Iyy 2.45×10-5 Kg m2 Izz 2.45×10-5 Kg m2 Ixy 0 Kg m2 Ixz 0 Kg m2 Vol 2.5132×10-5 m3 T 100 N Xcg (from L.E) 7×10-2 m Xcb (from L.E) 7.25×10-2 m TABLE I STANDARD PROPERTIES OF THE PROJECTILE F u th h w International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 TABLE II LONGITUDINAL PARAMETERS REQUIRED FOR THE SIMULATION TABLE II LONGITUDINAL PARAMETERS REQUIRED FOR THE SIMULATION Property Value (non cavitating) Property Value (non cavitating) Cd0 0.7351 CLq 27.49 Cd 0.02 Cm0 0 Cdq 0 Cm -13.44 CL0 0 Cmq -60.13 CL 6.283 All data are based on fin reference area International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Public It is known from section III that the longitudinal characteristics for the supercavitating regime depends on the cavitation number and the angle of attack. Hence it varies with respect to time and the whole cavity dynamics has been programmed accordingly. Fig. 4 Variation of fin force coefficients.Source : Kirshner [10] IV. CONFIGURATION OF THE MODEL VEHICLE The model which is taken into account here as shown in Fig. 5 is a small projectile which is capable of clearing underwater mines up to a depth of 40m. Generally the wall thickness of such a mine will be around 5 mm and calculations have shown that projectiles with a velocity of around 40m/s will have enough kinetic energy to pierce through the mine. V.OPEN LOOP SIMULATION The outlines of the aforementioned discussions are depicted in Fig.6. The coupled differential equations are solved by the classical Runge-Kutta method in MATLAB®. International Science Index, Aerospace a Fig. 6 Schematic of the procedure Output Initial Values of Parameters Model Definition Control Input 6 DOF Equations of Motion Force & Moment Calculation Cavity & its Dynamics Numerical Solver (Classical RK4) 6 DOF Equations of Motion will have enough kinetic energy to pierce through the mine. Fig. 5 Configuration of the model vehicle The mass and inertial properties of the projectile are given in table I. The hydrodynamic parameters which are required for the longitudinal simulation in the non-supercavitating Fig. 6 Schematic of the procedure Output Initial Values of Parameters Model Definition Control Input 6 DOF Equations of Motion Force & Moment Calculation Cavity & its Dynamics Numerical Solver (Classical RK4) Fig. 5 Configuration of the model vehicle Fig. 5 Configuration of the model vehicle International Science Index Initial Values of Parameters Numerical Solver (Classical RK4) Output Model Definition Cavity & its Dynamics Force & Moment Calculation Control Input Fig. 5 Configuration of the model vehicle The mass and inertial properties of the projectile are given in table I. The hydrodynamic parameters which are required for the longitudinal simulation in the non-supercavitating Fig. 6 Schematic of the procedure International Scholarly and Scientific Research & Innovation 6(8) 2012 scholar.waset.org/1307-6892/9860 1457 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Simulations have shown that for the present configuration which is taken into account the minimum diameter of the cavitator comes to be 9 mm for zero cavitator angle of attack. Below which the cavity interacts with the body of the projectile and that leads to distortion of the cavity and the projectile becomes unstable. Since this projectile is launched from a depth of 5 m, for cavitator angles more than 0.5 degrees, the pitching moment generated consequences the projectile to come out of the water level. So it is evident that with a proper control law we can control the vehicle with cavitator as the only control surface. In Fig. 10, the super cruise velocity sustains for more time for dc=1 degree than other cavitator angles. So, the cavitator angle also assists in cruising in the supercavitating regime. International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 erospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Fig. 7 Comparison of longitudinal characteristics of the vehicle with and without cavitation of fin Cavitator angle = 0 deg, Cavitator Dia = 9mm On examining the results obtained in Fig. 7, the sudden change in velocity is due to the loss of supercavitation. At this point the projectile will experience a huge impact like drag force of magnitude 50N which is considerably a large force for a projectile of this size. This impact force reduces the velocity of the vehicle and because of this the vehicle comes to the non-supercavitating equilibrium condition. The force experienced by the vehicle due to the loss of supercavitation is an important phenomenon if the vehicle is still allowed to cruise in the non-supercavitating regime. X (m) Launch velocity = 40m/s, Launch Depth = 5m, Cavitator angle = 0 deg, Cavitator Dia = 9mm Fig. 8 Variation in trajectory due to fin cavitation Fig. 8 Variation in trajectory due to fin cavitation 0 20 40 60 80 100 120 140 160 180 -80 -60 -40 -20 0 20 40 X (m) Z (m) -2 deg 0 deg 1 deg TRAJECTORY VARIATION WITH RESPECT TO CAVITATOR ANGLE Launch velocity = 40m/s, Launch depth = 5m, Cavitator dia = 9 mm Fig. 9 Trajectory variation with cavitator angle 0 20 40 60 80 100 120 140 160 180 -80 -60 -40 -20 0 20 40 X (m) Z (m) -2 deg 0 deg 1 deg TRAJECTORY VARIATION WITH RESPECT TO CAVITATOR ANGLE Launch velocity = 40m/s, Launch depth = 5m, Cavitator dia = 9 mm TRAJECTORY VARIATION WITH RESPECT TO CAVITATOR ANGLE Drastic changes occur for the angle of attack of the vehicle for the fin cavitating model at the point of loss of supercavitation. A peak in pitch rate is also noticed at this point. This point is like a critical point in which if the missile doesn’t have enough thrust to overcome the sudden drag force experienced, it will lead to total instability of the vehicle Although we concentrate on the design of cavitator, for any supercavitating vehicle stability is achieved by the fins. For complete dynamic stability and control over the projectile, the fins must extend over the cavity. V.OPEN LOOP SIMULATION Also for angles more than 5 degrees the cavity interacts with the body leading to instability of the missile. Using the procedure discussed in this paper, one can design a proper guidance system with cavitator as a control surface rather than conventional control surfaces. Fig. 7 Comparison of longitudinal characteristics of the vehicle with and without cavitation of fin 0 0.5 1 1.5 2 2.5 3 0 50 100 u (m/s) 0 0.5 1 1.5 2 2.5 3 -0.05 0 0.05 w (m/s) 0 0.5 1 1.5 2 2.5 3 -0.4 -0.2 0 q (rad/s) 0 0.5 1 1.5 2 2.5 3 -40 -20 0 θθθθ (deg) 0 0.5 1 1.5 2 2.5 3 -0.05 0 0.05 t (s) αααα (deg) Non-Cavitating fin Cavitating fin LONGITUDINAL CHARACTERISTICS WITH AND WITHOUT CAVITATION OF FIN Launch velocity = 40m/s, Launch Depth = 5m, Cavitator angle = 0 deg, Cavitator Dia = 9mm LONGITUDINAL CHARACTERISTICS WITH AND WITHOUT CAVITATION OF FIN Fig. 8 Variation in trajectory due to fin cavitation 0 20 40 60 80 100 120 140 160 180 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 X (m) Z (m) Non-Cavitating fin Cavitating fin COMPARISON OF TRAJECTORIES WITH AND WITHOUT CAVITATION OF FIN Launch velocity = 40m/s, Launch Depth = 5m, Cavitator angle = 0 deg, Cavitator Dia = 9mm COMPARISON OF TRAJECTORIES WITH AND WITHOUT CAVITATION OF FIN 0 20 40 60 80 100 120 140 160 180 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 X (m) Z (m) Non-Cavitating fin Cavitating fin COMPARISON OF TRAJECTORIES WITH AND WITHOUT CAVITATION OF FIN Launch velocity = 40m/s, Launch Depth = 5m, Cavitator angle = 0 deg, Cavitator Dia = 9mm International Scholarly and Scientific Research & Innovation 6(8) 2012 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Fig. 12Velocity variation with cavitator diameter Launch velocity = 40m/s, Launch depth = 5m, Cavitator angle = 0 de Fig. 10 Effect of cavitator angle on velocity Launch velocity = 40m/s, Launch depth = 5m, Cavitator dia = 9 mm VI. CONTROL LAW USING NDI TECHNIQUE International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Even though it extends over the cavity, because of high velocities, cavities form over the surface of fins. The trajectory of the projectile is simulated both by including and neglecting the effect of cavitation on fins. The variation of trajectory due to the effect of cavitation of fins is shown in Fig.8. As mentioned earlier, the emphasis of the present work is to find the effect of cavitator angle of attack and cavitator dimensions on the system dynamics. Fig. 9 best explains the variation of trajectory for various cavitator deflection angles. Fig. 9 Trajectory variation with cavitator angle scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 1458 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Fig. 10 Effect of cavitator angle on velocity Fig. 12Velocity variation with cavitator diameter 0 0.5 1 1.5 2 2.5 3 40 45 50 55 60 65 t (s) Vel (m/s) -2 deg 0 deg 1 deg VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR ANGLE Launch velocity = 40m/s, Launch depth = 5m, Cavitator dia = 9 mm 0 0.5 1 1.5 2 2.5 3 30 35 40 45 50 55 60 65 t (s) Vel (m/s) dc = 9 mm dc = 12 mm dc = 15 mm VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR DIAMETER Launch velocity = 40m/s, Launch depth = 5m, Cavitator angle = 0 deg World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Fig. 12Velocity variation with cavitator diameter 0 0.5 1 1.5 2 2.5 3 30 35 40 45 50 55 60 65 t (s) Vel (m/s) dc = 9 mm dc = 12 mm dc = 15 mm VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR DIAMETER Launch velocity = 40m/s, Launch depth = 5m, Cavitator angle = 0 deg VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR ANGLE Fig. VI. CONTROL LAW USING NDI TECHNIQUE The next major importance is on the size of the cavitator. It has been observed from Fig.11 that the advantage of cavitator exists only if the size of the cavitator is the minimum size for which it is capable of inducing a full cavity around the whole vehicle. Smaller the size of the cavitator the lesser the drag, but cavitators of size less than 9 mm are unable induce single cavity around the whole vehicle. As we increase the size of the cavitator, the drag also increases thereby reducing the velocity of the vehicle. This causes the cavity to collapse sooner taking the vehicle to the normal non-supercavitating phase. As depicted in Fig. 12 when the cavitator is 12 mm and 15 mm, even though when the projectile is fired at 40 m/s, it is unable to sustain in the supercavitating regime and immediately falls to the normal phase. Non-Linear Dynamic Inverse (NDI) is a control technique which accounts for all the non-linearities present in the system and thus it is one of the best methods to design controllers for highly non-linear systems. The concept of NDI is to generate control signals based on the error signal generated from the desired state and current state received from the feedback. This is achieved by inverting the governing equations for which the states needs to be controlled. In this method, the number of control inputs has to be equal to the number of states which are to be controlled. The states which remain uncontrolled will simply behave like open loop dynamics. If there are many states which depend on one particular input, then only one of any of the states can be controlled. International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/ The governing equations which are described in section I can be written in the state form as Fig. International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 12Velocity variation with cavitator diameter 0 0.5 1 1.5 2 2.5 3 30 35 40 45 50 55 60 65 t (s) Vel (m/s) dc = 9 mm dc = 12 mm dc = 15 mm VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR DIAMETER Launch velocity = 40m/s, Launch depth = 5m, Cavitator angle = 0 deg VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR DIAMETER 65 VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR ANGLE VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR DIAMETER Fig. 10 Effect of cavitator angle on velocity 0 0.5 1 1.5 2 2.5 3 40 45 50 55 60 65 t (s) Vel (m/s) -2 deg 0 deg 1 deg VARIATION OF TIME HISTORY OF VELOCITY WITH RESPECT TO CAVITATOR ANGLE Launch velocity = 40m/s, Launch depth = 5m, Cavitator dia = 9 mm International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 e Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 VI. CONTROL LAW USING NDI TECHNIQUE 11Trajectory variation with cavitator diameter 0 20 40 60 80 100 120 140 160 180 -45 -40 -35 -30 -25 -20 -15 -10 -5 t (s) Vel (m/s) dc = 9 mm dc = 12 mm dc = 15 mm TRAJECTORY VARIATION WITH RESPECT TO CAVITATOR DIAMETER Increasing cavitator diameter Launch velocity = 40m/s, Launch depth = 5m, Cavitator angle = 0 deg International Science Index, Aerospace and Mecha TRAJECTORY VARIATION WITH RESPECT TO CAVITATOR DIAMETER l& /lF m This can be written as l& nl 0 Qlm n 0 Q Where the l& o pq is the state vector includes all the state variables m o pr isthe control vector. The functionnl is an s Bvector which represents all the non-linear dynamics of the system and Ql is an s matrix representing the control distribution function for the state vector t . Here n and m are the number of state variables which can be controlled and number of number of control variables respectively. It has to be noted that Ql has to be invertible for this NDI to be implemented. This makes clear that Ql has to be square which further implies that the number of states which can be controlled is equal to number of control variables. Fig. 11Trajectory variation with cavitator diameter At first, the state equation is inverted as follows, m QluSvl& nlw scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 1459 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 Fig. 14 Pitch angle control using cavitator 0 2 4 6 8 10 12 14 16 18 20 -15 -10 -5 h (m) 0 2 4 6 8 10 12 14 16 18 20 -4 -2 0 2 θθθθ (deg) 0 2 4 6 8 10 12 14 16 18 20 0 0.5 1 δδδδc (deg) t (s) dc = 9mm dc = 12mm PITCH ANGLE CONTROL USING CAVITATOR Launch velocity = 40 m/s, Launch Depth = 5 m PITCH ANGLE CONTROL USING CAVITATOR PITCH ANGLE CONTROL USING CAVITATOR Although it looks simple in inverting, if the dimensions ofQl matrix increase, then singularities might arise during inversion and it has to be taken care while modeling the system. VII. SUMMARY AND FUTURE WORK The mathematical model for the analysis of longitudinal characteristics of a supercavitating vehicle is developed and is used for the analysis of the effect of cavitator angle of attack and dimensions on the system dynamics. The variation of states due to the cavitation of fin has been figured out and the trajectory variation is well explained. Acquired Or Simulated Data Fig. 13 Schematic of the control system For every supercavitating vehicles there exists a cavitator of minimum diameter which is capable of inducing a single cavity over the vehicle. Only at this condition the performance of the supercavitating vehicle will be maximum. The prime advantage of the supercavitating vehicles are to maintain the cavity around them which will help in cruising with less thrust when compared to conventional torpedoes. So the objective of the control system designed is to keep the flow supercavitating, i.e., the cavitation number () hasto be maintained below 0.1. This can be achieved by either controlling the velocity or by maintaining the depth so that the hydrostatic pressure (P) is less. Since the hydrostatic pressure increases tremendously with depth, it is undesirable to control the velocity to keep the cavitation number below the limits. So here the depth is maintained by controlling the pitch angle. It has been found that cavitator can play a pivotal role in the control of supercavitating vehicles, since its effect on the trajectory due to the variation of its angle and dimensions are considerable. Control law established using the NDI technique has shown that a naturally supercavitating vehicle can be controlled with cavitator as the only control surface. The idea of this paper can be extended to formulate a design algorithm to optimally design the size of the cavitator for a particular vehicle configuration to achieve maximum performance. maintain the pitch angle of 0 degrees is around 0.4 degrees, which is reasonably a good value and the rate at which the controller has to deflect is also very less since the cavitator deflection curve is smooth. Also, from the obtained results one can observe that the 9mm cavitator has given good performance when compared to the 12mm cavitator. This again validates the statement “the advantage of the cavitator exists only if its size is minimum enough to induce a single cavity around the vehicle”, and is true for the control also. ACKNOWLEDGMENT The authors would like to thank the Naval Research Board, India for their grateful support and assistance to carry out the research. International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 International Science Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 Fig. 13 Schematic of the control system l}D~ mxyz Desired Output Acquired Or Simulated Data NDI Actual System & its dynamics Control Input ( ) Launch velocity = 40 m/s, Launch Depth = 5 m Fig. 14 Pitch angle control using cavitator International Scholarly and Scientific Research & Innovation 6(8) 2012 VI. CONTROL LAW USING NDI TECHNIQUE After inversion the control required for the state to be controlled is found by replacing the inherent(or actual) dynamics with the desired dynamics. h (m) mxyz QluSvl& xyz nlw l& xyz {\|lxyz l}D~ Where Where Herelxyz l}D~ is the error signal and {\| is the proportional controller gain.Here only the proportional controller is used, but the other controllers, differential or integral or their combination may also be used and their effect will be reflected in the system performance. A simplified block diagram of control algorithm is shown in fig. 13. cience Index, Aerospace and Mechanical Engineering Vol:6, No:8, 2012 waset.org/Publication/9860 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 [5] Vladimir N.Semenenko - Artificial Supercavitation – Physics and Calculations – DTIC, ADP012080. [6] Travis Jon Schauer., An Experimental study of ventilated supercavitating vehicle, M.S. Thesis, University of Minnesota, 2003. [7] ZHANG Xue-wei, WEI Ying-jie, ZHANG Jia-zhong, WANG Cong, YU Kai-ping, “Experimental research on the shape characteristics of natural and ventilated supercavitation,” Journal of Hydrodynamics, Ser.B, 2007,19(5): pp. 564-571. [8] Birkhoff, G.,Zarantonello, E.H.,Jets, Wakes and Cavities, Academic Press Inc. Publishers,New York, 1957. [9] A. J. Kurdila, R. Lind, J. Dzielski, A. Jammulamadaka, and A. Goel, “Dynamics and control of supercavitating vehicles," Office of Naval Research - Supercavitating High speed bodies, Tech. rep. 2003. Research - Supercavitating High speed bodies, Tech. rep. 2003 p g g p p [10] Ivan N. Kirschner, David C. Kring, Ann W. Stokes, Neal E. Fine and [10] Ivan N. Kirschner, David C. Kring, Ann W. Stokes, Neal E. Fine and James S. Uhlman, Jr, “Control strategies for supercavitating vehicles,” Journal of Vibration and Control, 2002, Vol 8, No.2, pp. 219-242. g James S. Uhlman, Jr, “Control strategies for supercavitating vehicles,” Journal of Vibration and Control, 2002, Vol 8, No.2, pp. 219-242. [11] Lane S.H., Stengel R.F., Flight Control Design using Nonlinear Inverse Dynamics, Automatica, Vol.24,1988, pp. 471-483 [12] Knapp, R.T., Daily J.W., Hammitt, F.G., Cavitation, McGraw-Hill, New York, 1970. [13] Franc.J.P.,Michel.J.M., Fundamentals of Cavitation, Kluwer Academic Publishers, Dordrecht, 2004. [14] Nelson, Flight Stability and Automatic Control, Tata McGraw Hill, New Delhi, 2007. [15] Tewari, A., Modern Control Design with Matlab and Simulink, John Wiley and Sons Ltd, Kundli, 2003. [16] Logvinovich.G.V., Hydrodynamics of free boundary flows, IPST Press, Jerusalem, 1972. [17] Vladimir N.Semenenko., Fundamentals of calculation and designing the underwater supercavitating vehicles, Special Lecture course, Institute of hydromechanics, National Academy of sciences of Ukraine. 2 waset.o [18] Davies.T.V., Steady two dimensional cavity flow past an aerofoil using linearized theory. Quarterly Journal of mechanics and applied mathematics,VolXXIII,Part 1, 1970. o:8, 2012 [19] David R. Stinebring, Michael L. Billet, Jules W. Lindau, Robert F. Kunz - Developed Cavitation-Cavity Dynamics– DTIC, ADP012075. l:6, No [20] Santosh Kumar Burnwal, Modeling& Simulation of High Speed Supercavitating Vehicle, M.Tech Thesis, Indian Institute of Technology Kanpur, 2011. [21] “Supercavitating flows”, RTO EN-010/AVT-058, January 2002. [22] Selected lecture notes on “Ship Hydrodynamics” course, Faculty of Naval architecture and Ocean Engineering, Istanbul Technical University. [23] Selected Lecture notes on “Performance of marine vehicles at sea”, Dept. of Ocean engineering and Naval Architecture, IIT Kharagpur. International Scholarly and Scientific Research & Innovation 6(8) 2012 REFERENCES [1] LEE Qi-tao, XUE Lei-ping, HE You-sheng, “Experimental study of ventilated supercavities with a dynamic pitching model,” Journal of Hydrodynamics,2008, 20(4): pp. 456-460. [2] Bálint Vanek, Control methods for high speed supercavitating vehicles, Ph.D Thesis, University of Minnesota, 2008. [3] Daijin Li, Yuwen Zhang, Kai Luo&Jianjun Dang, “Motion Control of Underwater Supercavitating Projectiles,” Modern Applied Science, 2009, Vol 3, No.2, pp. 60-65. [4] Garabedian, P.R., “Calculation of axially symmetric cavities and jets,” Pac. J. Math., 1956, 6,No. 4, pp. 611-684. [4] Garabedian, P.R., “Calculation of axially symmetric cavities and jets,” Pac. J. Math., 1956, 6,No. 4, pp. 611-684. scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 1460 scholar.waset.org/1307-6892/9860 World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol:6, No:8, 2012 scholar.waset.org/1307-6892/9860 International Scholarly and Scientific Research & Innovation 6(8) 2012 1461
https://openalex.org/W2031841569	https://www.scielo.br/j/rcf/a/NsPG7Jz44VLXBh7yH3XmXQf/?lang=pt&format=pdf	Portuguese	null	Evidenciação de instrumentos financeiros derivativos nas demonstrações contábeis: uma análise das empresas brasileiras	Revista Contabilidade & Finanças	2,005	cc-by	8,565	Leandro Luís Darós • José Alonso Borba Leandro Luís Darós • José Alonso Borba 68 EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS* LEANDRO LUÍS DARÓS Bacharel em Administração e Ciências Contábeis pela UFSC – SC Mestrando do Programa de Pós-Graduação em Contabilidade da UFSC – SC E-mail: ldaros@hotmail.com JOSÉ ALONSO BORBA Prof. Dr. do Depto. de Ciências Contábeis e do Programa de Pós-Graduação em Contabilidade da UFSC – SC E-mail: jalonso@cse.ufsc.br * Artigo originalmente apresentado no XXVIII ENANPAD, setembro de 2004, Curitiba-PR. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Recebido em 12 Abr. 2005 • Aceito em 23 Jun. 2005 1 INTRODUÇÃO Board, em 1999, o IAS 392 pelo IASB – International Accounting Standards Board e os estudos, no Bra- sil, realizados especialmente pelos pesquisadores Alexsandro Broedel Lopes, Luiz Nelson Carvalho e Jorge Vieira da Costa Junior. Ainda, assim, no exte- rior, diversas outras pesquisas têm discutido ques- tões relacionadas com a divulgação, transparência e o impacto dos derivativos tanto nas Demonstra- ções Contábeis quanto nos seus usuários, como, por exemplo, os trabalhos de Guay (1999), Blankley, Lamb, Schroeder (2000), Seow e Tam (2002), Ishi- kawa (2003). Board, em 1999, o IAS 392 pelo IASB – International Accounting Standards Board e os estudos, no Bra- sil, realizados especialmente pelos pesquisadores Alexsandro Broedel Lopes, Luiz Nelson Carvalho e Jorge Vieira da Costa Junior. Ainda, assim, no exte- rior, diversas outras pesquisas têm discutido ques- tões relacionadas com a divulgação, transparência e o impacto dos derivativos tanto nas Demonstra- ções Contábeis quanto nos seus usuários, como, por exemplo, os trabalhos de Guay (1999), Blankley, Lamb, Schroeder (2000), Seow e Tam (2002), Ishi- kawa (2003). Demonstrar as alterações patrimoniais e gerar informações é um dos objetivos da Contabilidade. Essa tarefa tem se tornado cada vez mais comple- xa nos últimos anos devido, principalmente, ao de- senvolvimento e volatilidade do sistema ﬁ nanceiro e pela soﬁ sticação e complexidade das transações econômicas. Dentro desse contexto, um tipo espe- cial de operação ﬁ nanceira, os instrumentos ﬁ nan- ceiros derivativos, se desenvolveram com grande rapidez e criaram diﬁ culdades de seu reconheci- mento e mensuração por parte da Contabilidade. Criados com a ﬁ nalidade de antecipar preços futu- ros de seus ativos objeto e proteger investidores e produtores contra riscos, os derivativos são, hoje, papéis largamente comercializados no mercado ﬁ - nanceiro internacional. Contratos futuros, a termo, opções e swaps são exemplos desses contratos derivativos. A complexidade de muitos deles, que por vezes são estruturados sobre soﬁ sticados sis- temas de cálculos, tem despertado a atenção da contabilidade para entender e reconhecer seus efeitos sobre a situação patrimonial das empresas e, concomitantemente, identiﬁ car a maneira correta de evidenciá-los nas Demonstrações Contábeis. Assim, com base nas recomendações e mo- delos de contabilização da Instrução Normativa 235 da CVM, o presente trabalho busca analisar como as operações com instrumentos ﬁ nanceiros deriva- tivos vêm sendo evidenciadas nas Demonstrações Contábeis de empresas brasileiras não ﬁ nanceiras. 1 Statement of Financial Accounting Standars – SFAS 133 – Accounting for derivatives instruments and hedging activies – Junho/1998. 2 IAS – International Accounting Standard – IAS 39 – Financial instruments: recognition and measurement. 3 GAO: General Accounting Ofﬁ ce, Special Report to US Congress on Financial Instruments and Capital Markets, 1998. p. 18. ABSTRACT Dentro da perspectiva de governança corpo- rativa e de acordo com a Instrução Normativa 235 da Comissão de Valores Mobiliários – CVM, este trabalho apresenta uma pesquisa empírica sobre as formas de evidenciação de instrumentos ﬁ nan- ceiros derivativos nas Demonstrações Contábeis divulgadas no Brasil. Foram analisadas as Demons- trações Contábeis das vinte maiores empresas brasileiras não ﬁ nanceiras classiﬁ cadas de acordo com a receita líquida. Como resultado constatou-se que a grande maioria das empresas não atende às determinações da CVM e não evidencia, de forma clara, concisa e objetiva, as informações referentes às suas operações envolvendo instrumentos ﬁ nan- ceiros derivativos. As principais divergências estão na falta de avaliação dos instrumentos a preços de mercado, ausência de divulgação das políticas de investimento em derivativos e falta de controle do risco de mercado, câmbio e crédito. In the context of ﬁ nancial statement disclosure and in accordance with determinations by the Brazil- ian Securities and Exchange Commission, this study presents rules and ways to disclose derivatives in ﬁ nancial statements. In the empirical analysis, we examined the ﬁ nancial statements of the twenty big- gest public companies arranged by revenue. We veriﬁ ed that many companies do not comply with the Brazilian Securities and Exchange Commission’s determinations and do not present a clear, concise and objective disclosure of related ﬁ nancial informa- tion. The main problems were related to the lack of evaluation in terms of fair value, lack of dissemina- tion of investment policies in derivatives and quan- titative and qualitative control of market, credit and exchange risks. Keywords: Financial Statements; Derivatives; Disclosure. Palavras-chave: Demonstrações Financeiras; Derivativos; Evidenciação. . Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 69 R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 1 INTRODUÇÃO A relevância deste trabalho concentra-se em analisar de que forma as empresas brasileiras não ﬁ nanceiras evidenciam operações com instrumen- tos ﬁ nanceiros derivativos em suas Demonstrações Contábeis já que transações desse tipo não fazem parte de seu contexto operacional. Por que adqui- rem contratos dessa natureza, como contabilizam e comunicam perdas e ganhos com esses contratos são questões discutidas no decorrer do presente trabalho. De acordo com Benston e Mian (1997), o tama- nho, crescimento e importância do mercado de de- rivativos indicam um amplo uso desses instrumen- tos ﬁ nanceiros pelas empresas. Contudo, apesar do seu uso intensivo, os contadores têm carência de orientações para a sua evidenciação, reconheci- mento, mensuração e classiﬁ cação. Em decorrência disso, a proﬁ ssão contábil tem enfrentado diﬁ culda- des na formulação de métodos claros e compreen- síveis a respeito do reconhecimento e apresentação desses instrumentos. Ainda assim, um trabalho desta natureza se tor- na necessário pelas razões apresentadas a seguir: • deve-se considerar a elevada importância dos instrumentos ﬁ nanceiros derivativos no contexto do mercado ﬁ nanceiro interna- cional. As transações com esses produtos alcançam alguns trilhões de dólares e eles são peças importantes numa estratégia de gestão de risco realmente eﬁ caz no âmbito internacional3; Um dos aspectos que tem prejudicado o tra- balho de demonstrar o impacto dos derivativos nas empresas é o fato de publicações e instruções de contabilização de operações com instrumentos ﬁ - nanceiros derivativos serem bastante recente em nosso país. • o mercado ﬁ nanceiro internacional vem enfrentando modiﬁ cações signiﬁ cativas nos últimos anos causadas pela quebra de barreiras ao ﬂ uxo de capitais e desre- gulamentações nacionais. Dentro desse contexto, a gestão do risco passou a ser um ponto crítico na estratégia da maioria das instituições ﬁ nanceiras internacionais e os derivativos se tornaram instrumen- tos fundamentais nesse novo modelo de gestão; Apesar da CVM exigir desde 1995 a eviden- ciação em notas explicativas desse tipo de opera- ção, havia uma lacuna no meio contábil sobre como contabilizar e reconhecer operações com derivati- vos. Uma grande contribuição para a solução desse problema foram as emissões, em 1998, do SFAS1 133 pelo FASB – Financial Accounting Standards R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Leandro Luís Darós • José Alonso Borba 70 • os efeitos dos derivativos não são trans- parentes nas Demonstrações Financeiras básicas. 1 INTRODUÇÃO IV demonstração das origens e aplicações de recursos.” Assim, todas as empresas sociedades anô- nimas são obrigadas a publicar as Demons- trações Contábeis, de onde são coletados os dados. No que diz respeito às empresas de capital aberto, justiﬁ ca-se sua utilização por essas empresas possuírem papéis ne- gociados em Bolsa, ou seja, são empresas que devem apresentar informações aos acionistas de seus negócios, mais especi- ﬁ camente, nesse caso, sobre derivativos; Assim, todas as empresas sociedades anô- nimas são obrigadas a publicar as Demons- trações Contábeis, de onde são coletados os dados. No que diz respeito às empresas de capital aberto, justiﬁ ca-se sua utilização por essas empresas possuírem papéis ne- gociados em Bolsa, ou seja, são empresas que devem apresentar informações aos acionistas de seus negócios, mais especi- ﬁ camente, nesse caso, sobre derivativos; 4 LOPES, A. B.; CARVALHO, L. N. G.. Contabilização de operações com derivativos: uma comparação entre o SFAS 133 e o arcabouço emanado pelo COSIF. Caderno de Estudos, São Paulo, FIPECAFI, n. 20, jan/abr., 1999, p. 22. 1 INTRODUÇÃO Com a inexistência de uma abor- dagem completa para a contabilização desses produtos, há uma inconsistência geral no modelo de evidenciação e con- tabilização de cada entidade considerada internacionalmente4; 1. Empresas brasileiras: busca-se analisar as empresas de um determinado espaço geo- gráﬁ co e que respeitem a uma mesma legis- lação. Não faria sentido, por exemplo, analisar empresas de diferentes países com legisla- ções diferentes, o que, sem dúvida, prejudi- caria a uniformidade das informações. Assim, as empresas brasileiras foram escolhidas pelo fato de o trabalho estar sendo realizado no Brasil e pela conveniência de obtenção de informações relativas a essas empresas. • a crescente utilização de instrumentos ﬁ nan- ceiros derivativos no mercado internacional com operações de hedge, especulação e arbitragem associada às crises de insti- tuições respeitadas como, por exemplo, o Banco inglês Barings, trouxeram esses pro- dutos para o centro da atenção de institui- ções ﬁ nanceiras e órgãos reguladores; 2. ç Empresas Sociedade Anônima de Capital Aberto: a fonte de coleta de dados são as demonstrações contábeis das empresas. O artigo 176 da Lei 6.404 de 1976, que trata das Sociedades Anônimas, diz que: “Ao ﬁ m de cada exercício social, a diretoria fará ela- borar, com base na escrituração mercantil da companhia, as seguintes demonstra- ções ﬁ nanceiras, que deverão exprimir com clareza a situação do patrimônio da compa- nhia e as mutações ocorridas no exercício: • a preciﬁ cação de instrumentos ﬁ nanceiros derivativos exige um instrumental quantita- tivo extremamente avançado que parece fugir ao domínio da maioria dos proﬁ ssio- nais do mercado ﬁ nanceiro e dos contado- res em particular. Dessa forma, a diﬁ culda- de no entendimento dos mecanismos de formação de preço dos derivativos leva a equívocos de interpretação por parte da Contabilidade; I balanço patrimonial; I balanço patrimonial; I balanço patrimonial; II demonstração dos lucros ou prejuízos acumulados; III demonstração do resultado do exercício; e • Ishikawa (2003) pontua que a introdução do “valor justo” na contabilidade para avaliar instrumentos ﬁ nanceiros derivativos está profundamente relacionada com a neces- sidade de evidenciar as diversas caracte- rísticas qualitativas e quantitativas desses ativos, fundamentalmente, em decorrência da soﬁ sticação, diversiﬁ cação e expansão desses mercados e a conseqüente prolife- ração de seu uso. 2 METODOLOGIA 3. 3. Empresas não ﬁ nanceiras: optou-se por analisar as empresas não ﬁ nanceiras, pois realizar operações com derivativos não faz parte de seu contexto operacional. Assim, busca-se analisar por que empresas não ﬁ nanceiras utilizam derivativos e como os evidenciam nas Demonstrações Contábeis; Para a consecução dos objetivos foi realiza- do um estudo exploratório descritivo que segun- do Lakatos e Marconi (1992) “é toda pesquisa que busca constatar algo num organismo ou num fenô- meno”. Como esse trabalho analisa as formas de evidenciação de derivativos nas Demonstrações Contábeis esse tipo de estudo é o mais adequado para se atingirem os objetivos da pesquisa. ç 4. Por ﬁ m, entenda-se como empresa brasi- leira de capital aberto aquelas registradas como Sociedades Anônimas de Capital Aberto na Comissão de Valores Mobiliários e na Bolsa de Valores de São Paulo e com ações negociadas no mercado. ç 4. Por ﬁ m, entenda-se como empresa brasi- leira de capital aberto aquelas registradas como Sociedades Anônimas de Capital Aberto na Comissão de Valores Mobiliários e na Bolsa de Valores de São Paulo e com ações negociadas no mercado. A população deﬁ nida para análise na pesquisa são as empresas brasileiras, Sociedades Anônimas de Capital Aberto, não Financeiras. Justiﬁ ca-se a escolha dessas empresas pelos critérios abaixo elencados: 4 LOPES, A. B.; CARVALHO, L. N. G.. Contabilização de operações com derivativos: uma comparação entre o SFAS 133 e o arcabouço emanado pelo COSIF. Caderno de Estudos, São Paulo, FIPECAFI, n. 20, jan/abr., 1999, p. 22. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 71 em função da receita líquida constante na Demons- tração do Resultado do Exercício do ano de 2002. Para estabelecer-se o tamanho da amostra, consi- derou-se a representatividade da amostra em rela- ção a população em função da receita líquida anual constante na Demonstração de Resultado de Exer- cício de 31 de dezembro de 2002. Deﬁ nida a população, para a seleção da amos- tra de pesquisa foi utilizado o software de informa- ções ﬁ nanceiras e econômicas EconomáticaTM. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 2 METODOLOGIA De acordo com a deﬁ nição da amostra e os recursos do programa, foram estabelecidos os parâmetros apresentados no Quadro 1: Através desse método, foram selecionadas 195 empresas, ordenadas em ordem decrescente PARÂMETRO LIMITADOR RESTRIÇÃO DEFINIÇÃO País Igual a Brasil Empresas registradas em Bolsas brasileiras Bolsa Igual a BRA-Bovespa Empresas cujo os papéis são negociados na Bovespa Tipo de ativo Igual a Ação Tipo de ativo Setor Econômico Diferente de Finanças e Seguros/ Empresas que não pertencem Fundos/Outros a nenhum desses três setores Ativo/Cancelado Igual a Ativo Papéis que são válidos para negociação Receita líquida do último balanço Maior que R$ 0,00 Receita auferida no último balanço Fonte: Elaborado pelos autores com base em informações do Economática. Quadro 1 – Critérios de Seleção da Amostra Quadro 1 – Critérios de Seleção da Amostra Assim, trabalhou-se com uma representativida- de de 67,2758 % o que representa um número de 20 empresas do total de 195 empresas da população. Isso signiﬁ ca que 20 empresas tomadas como amos- tra representam 67,2758% da receita liquida total da população. A despeito da amostra ser não probabilís- tica, optou-se por trabalhar-se com essas empresas pois são as vinte maiores empresas brasileiras não ﬁ nanceiras com a receita líquida bastante concentra- da. Contudo, enfatiza-se, os resultados não podem (estatisticamente) ser generalizados para toda popu- lação. As empresas selecionadas como amostragem estão relacionadas no Quadro 2 a seguir: ORDEM EMPRESA SETOR 8 COMPANHIA Siderurgia e Metalurgia SIDERÚRGICA NACIONAL 9 VARIG Transporte Serviços 10 USIMINAS Siderurgiae Metalurgia 11 LIGHT Energia Elétrica 12 IPIRANGA PETRÓLEO Petróleo e Gás 13 TELEMAR Telecomunicações 14 ELETROBRÁS Energia Elétrica 15 COSIPA Siderurgia e Metalurgia 16 COMPANHIA Siderurgia e Metalurgia SIDERÚRGICA TUBARÃO 17 PÃO DE AÇÚCAR Comércio 18 GLOBEX Comércio 19 BRASKEM Química 20 CESP Energia Elétrica Fonte: Elaborado pelos autores com base em informações do Economática Quadro 2 – Empresas Analisadas ORDEM EMPRESA SETOR 1 PETROBRAS Petróleo e Gás 2 TELESP Telecomunicações 3 COMPANHIA VALE Mineração DO RIO DOCE 4 EMBRAER Veículos e peças 5 BRASIL TELECOM Telecomunicações 6 ELETROPAULO Energia Elétrica 7 CEMIG Energia Elétrica Continua Fonte: Elaborado pelos autores com base em informações do Economática Leandro Luís Darós • José Alonso Borba 72 trumentos ﬁ nanceiros é a Instrução Normativa 235 da CVM publicada em 23 de março de 19955. Selecionada a amostra, os dados das empre- sas foram coletados nas Demonstrações Contábeis encerradas em 31 de dezembro de 2002. 5 As instituições ﬁ nanceiras e assemelhadas devem respeitar as normas determinadas pelo COSIF – plano contábil das instituições ﬁ nanceiras do sistema ﬁ nanceiro nacio- nal do Banco Central do Brasil. Como o objeto de estudo deste trabalho são as empresas não ﬁ nanceiras, serão analisadas as determinações da CVM que é quem regula as Sociedades Anônimas não Financeiras. 2 METODOLOGIA São caracterizados como passivos ﬁ nanceiros as obrigações contratuais de: a) pagamento de determinada importância em moeda ou em instrumentos ﬁ nanceiros e Os dados foram analisados individualmente de empresa para empresa e, em seguida, comparados entre si. Foram feitas análises quantitativas e quali- tativas dos dados. Na análise quantitativa buscou-se veriﬁ car quantas empresas enquadravam-se dentro dos termos da legislação. Já na análise qualitativa foram comparadas as demonstrações das empre- sas, suas diferenças e similaridades e também o conteúdo das informações apresentadas. b) troca de resultados ﬁ nanceiros ou instru- mentos ﬁ nanceiros. A norma considera como deve ser avaliado o valor de mercado do instrumento derivativo: I o valor que se pode obter com a negocia- ção do instrumento ﬁ nanceiro em um mer- cado ativo, em que comprador e vendedor possuam conhecimento do assunto e inde- pendência entre si, sem que corresponda a uma transação compulsória ou decorrente de um processo de liquidação, ou 2 METODOLOGIA Por De- monstrações Contábeis entendam-se os seguintes documentos: Balanço Patrimonial, Demonstração do Resultado do Exercício, Demonstração dos Lu- cros ou Prejuízos Acumulados ou Demonstração de Mutações do Patrimônio Líquido, Demonstração das Origens e Aplicação dos Recursos, acompa- nhadas de Notas Explicativas e o Parecer dos Audi- tores Independentes. Segundo essa norma, as companhias abertas que possuam instrumentos ﬁ nanceiros, reconhecidos ou não como ativo ou passivo em seu balanço patri- monial, devem evidenciar, em nota explicativa, anexa às suas Demonstrações Financeiras e às informações trimestrais — ITR, o valor de mercado desses instru- mentos ﬁ nanceiros. Devem demonstrar, ainda, em nota explicativa, os critérios e as premissas adotados para determinação desse valor de mercado, bem como as políticas de atuação e controle das operações nos mercados derivativos e os riscos envolvidos. Salienta-se que na pesquisa utilizou-se ape- nas dados primários coletados diretamente das demonstrações contábeis das empresas. Essas demonstrações foram obtidas através do sistema de informações da CVM disponibilizado na Internet através das Demonstrações Financeiras Padroniza- das do ano de 2002. A norma deﬁ ne como instrumento ﬁ nanceiro “todo contrato que dá origem a um ativo ﬁ nanceiro em uma entidade e a um passivo ﬁ nanceiro ou títu- lo representativo do patrimônio em outra entidade”. Pela instrução normativa são considerados como ativos ﬁ nanceiros: Com base nas informações sobre a legislação e nos modelos de evidenciação de derivativos cons- tantes em instruções da CVM foram estabelecidos itens que deveriam constar nas demonstrações con- tábeis das empresas. O primeiro item trata da pre- sença ou não da nota explicativa sobre instrumen- tos ﬁ nanceiros derivativos. A seguir, analisou-se a política de divulgação dos objetivos, ﬁ nalidades e intenções da empresa com o uso de derivativos, as premissas utilizadas para determinação dos valores justos e o uso de quadro comparativos mostrando as diferenças entre o valor contábil e o de merca- do dos derivativos. Logo após, foram estabelecidos itens de análise do controle de risco que a empresa efetua sobre três aspectos: risco de crédito, risco de taxa de câmbio e risco de produto. a) disponibilidades; b) direitos contratuais recebíveis em moeda ou em instrumentos ﬁ nanceiros de outra entidade; c) direitos contratuais de troca de resultados ﬁ nanceiros (swaps) ou instrumentos ﬁ nan- ceiros; d) títulos representativos de participação no patrimônio de outra entidade. 4 ANÁLISE DE COMO AS EMPRESAS BRASILEIRAS EVIDENCIAM DERIVATIVOS EM SUAS DEMONSTRAÇÕES CONTÁBEIS a) o valor que se pode obter com a nego- ciação de outro instrumento ﬁ nanceiro de natureza, prazo e risco similares, em um mercado ativo, conforme referido no inciso I deste artigo ou a) o valor que se pode obter com a nego- ciação de outro instrumento ﬁ nanceiro de natureza, prazo e risco similares, em um mercado ativo, conforme referido no inciso I deste artigo ou a) o valor que se pode obter com a nego- ciação de outro instrumento ﬁ nanceiro de natureza, prazo e risco similares, em um mercado ativo, conforme referido no inciso I deste artigo ou g b) o valor presente líquido dos ﬂ uxos de caixa futuros a serem obtidos, ajustado com base na taxa de juros vigente no mercado, na data do balanço, para instrumentos ﬁ nan- ceiros de natureza, prazo e risco similares. Foi demonstrada no capítulo anterior, a norma que dispõe sobre a evidenciação de instrumentos ﬁ nanceiros derivativos nas Demonstrações Contá- beis. Segue-se, agora, a análise empírica de como essas informações vêm sendo demonstradas ao mercado através de uma amostra das vinte maio- res empresas brasileiras com atividade ﬁ m não ﬁ - nanceira. A companhia aberta deverá evidenciar, sempre que relevante: De acordo com a Instrução Normativa 235/95 da CVM, são analisados aspectos de divulgação dos objetivos, ﬁ nalidades e intenções da empresa com o uso de derivativos, premissas utilizadas para determinação dos valores justos dos derivativos e controle de risco que a empresa efetua sobre três aspectos: risco de crédito, risco de taxa de câmbio e risco de produto. a) o valor de mercado de todos os instrumen- tos ﬁ nanceiros, reconhecidos ou não como ativo/passivo em seu balanço patrimonial; b) os critérios e premissas adotados para de- terminação desse valor e ç c) as políticas de atuação e de controle das operações nos mercados derivativos e dos riscos envolvidos. Dado o grau de internacionalização, porte das empresas e universalização do uso de derivativos, foi adotado o pressuposto de que todas as empre- sas analisadas utilizam derivativos. O artigo 4o da Instrução CVM no 235/95 dispen- sa a divulgação dos valores de mercado de duplica- tas a receber e a pagar. A CVM entende, também, ser desnecessária a divulgação dos valores de mercado das demais contas a receber e a pagar com prazo compatível com as operações normais da compa- nhia. 3 NORMAS CONTÁBEIS QUE TRATAM DE EVIDENCIAÇÃO DE DERIVATIVOS No Brasil, a principal norma que dispõe sobre a evidenciação nas Demonstrações Contábeis de Companhias Abertas do valor de mercado dos ins- p q ç , II na ausência de um mercado ativo para um determinado instrumento ﬁ nanceiro: . Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 73 4.1 Presença da Nota Explicativa sobre Instrumentos Financeiros Derivativos Inicialmente, buscou-se identiﬁ car nas De- monstrações Contábeis das empresas a presença da nota explicativa que trata de instrumentos ﬁ nan- ceiros derivativos, conforme determina a instrução CVM 235/95. A dispensa acima, contudo, não se aplica a contas a receber de entidades governamentais ou outras decorrentes de contratos de longo prazo, cuja possibilidade de recebimento no prazo de até três meses não esteja efetivamente assegurada. Nesse caso, deverá ser informado, em nota explicativa, o valor de mercado ou, na ausência dessa informação, deverá ser indicada uma estimativa de desconto em função do custo de seu ﬁ nanciamento, de acordo com o prazo previsto para o seu recebimento. Pela veriﬁ cação das demonstrações, cons- tatou-se que das vinte empresas analisadas todas possuem notas explicativas sobre instrumentos ﬁ - nanceiros. Assim, as empresas atendem à legislação bra- sileira que determina a presença dessa nota nas De- monstrações. A análise desse tópico é mais quan- titativa do que qualitativa já que a qualidade das informações, constantes nas notas explicativas, foram analisadas nos itens seguintes. A CVM estabeleceu para as instituições ﬁ nan- ceiras e não ﬁ nanceiras modelos de nota explicativa. O modelo proposto não esgota todas as necessida- des de divulgação ou, ainda, de sua própria identiﬁ - cação e, portanto, deverá ser adaptado e analisado, criteriosamente, pela administração da companhia e seus auditores independentes. 4 ANÁLISE DE COMO AS EMPRESAS BRASILEIRAS EVIDENCIAM DERIVATIVOS EM SUAS DEMONSTRAÇÕES CONTÁBEIS Essa dispensa está baseada no fato de as con- tas a receber e a pagar, bem como os demais itens monetários, serem ajustados a valor presente confor- me requerido pela Instrução CVM no 191/92. 4.4 Quadros Demonstrativos Os quadros demonstrativos servem como comparação dos valores contábeis registrados pelas empresas com as avaliações dos valores de mercado. Essa comparação presta-se a veriﬁ car as perdas ou ganhos da empresa com os derivati- vos. Nesse item, 9 (45%) das empresas apresentam quadros comparativos. Aqui, surge uma questão interessante: se apenas 5 (25%) das empresas ava- liam seus investimentos em derivativos a preços de mercado como 9 (45%) delas apresentam quadros comparativos? É uma boa pergunta e deixa dúvida se os valores constantes nos quadros comparativos são de fato ﬁ dedignos. Já a Embraer expôs a seguinte justiﬁ cativa para o uso de derivativos em suas operações: Os instrumentos derivativos contratados pela Empresa têm o propósito de proteger as opera- ções da empresa contra riscos de variação cam- bial e de ﬂ utuação na taxa de juros e não são utilizados para ﬁ ns especulativos. Assim, apesar de 8 (40%) das empresas não fazerem qualquer referência, em suas notas expli- cativas, sobre suas intenções com instrumentos derivativos, as que as apresentam o fazem de for- ma clara. Saliente-se que essa informação é im- portante para o investidor veriﬁ car se a empresa especula no mercado ﬁ nanceiro utilizando deriva- tivos ou se utiliza esses instrumentos como meio de proteção contra riscos decorrentes de suas ati- vidades. Saliente-se que apenas 5 (25%) das empresas possuem informações sobre premissas de avalia- ção e a grande maioria delas com informações de baixa qualidade e padronizadas como a apresenta- da acima. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 4.2 Divulgação dos Objetivos, Fins e Intenções com o Uso de Derivativos A seguir, analisou-se nas Demonstrações Con- tábeis a presença dos objetivos, ﬁ ns e intenções da empresa com o uso de instrumentos ﬁ nanceiros de- rivativos. Com esse item buscou-se identiﬁ car quais os motivos para as empresas utilizarem esse tipo de instrumento no decorrer de suas atividades e sua comunicação ao mercado. As informações contidas nesses modelos de nota explicativa podem ser reunidas em uma úni- ca nota às Demonstrações Contábeis/informações trimestrais ou divididas em duas ou mais notas ex- plicativas. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Leandro Luís Darós • José Alonso Borba 74 Como resultado veriﬁ cou-se que 12 (60%) das empresas analisadas divulgam essa informa- ção ao mercado. A Cosipa, uma das empresas que evidenciou seus objetivos com derivativos nas De- monstrações Contábeis, apresentou o seguinte pa- rágrafo na nota explicativa referente a instrumentos ﬁ nanceiros: avaliações. Entretanto, considerável julgamen- to foi requerido na interpretação dos dados de mercado para se produzir a mais adequada es- timativa do valor justo. Como conseqüência, as estimativas apresentadas a seguir não indicam, necessariamente, os montantes que poderão ser realizados no mercado de troca corrente. O uso de diferentes hipóteses e/ou metodologias de mercado pode ter um efeito material nos valores estimados de realização. A companhia possui operações envolvendo instrumentos ﬁ nanceiros exclusivamente em co- nexão com suas atividades e com o objetivo de reduzir a exposição aos riscos de mercado, de moeda e taxas de juros, de seus ativos opera- cionais. A nota evidencia uma informação de pouca re- levância e não diz como realmente a avaliação foi feita. A empresa fala em “aplicação de metodolo- gias apropriadas de avaliações” porém, que meto- dologias são essas? Com base em que elas foram realizadas? Ainda assim, a nota diz que “as estima- tivas apresentadas a seguir não indicam, necessa- riamente, os montantes que poderão ser realizados no mercado de troca corrente”. Ora, a instrução da CVM é clara quando exige que os instrumentos de- rivativos sejam evidenciados a valores de mercado. Se a própria nota apresenta ressalva aﬁ rmando que os valores não são os de realização, ﬁ ca evidente a falta de informação. Já a Embraer expôs a seguinte justiﬁ cativa para o uso de derivativos em suas operações: 4.3 Premissas de Avaliação Na avaliação das opções a valor de mercado utilizou- se o modelo “Black & Scholes”. Fonte: Demonstrações Contábeis do ano de 2002 da Telesp. Fonte: Demonstrações Contábeis do ano de 2002 da Telesp. gerou um ganho de cerca de R$ 177, líquido de impostos. A tabela 1, divulgada pela Telesp, apesar de sucinta, é bastante explicativa e apresenta a posi- ção da empresa em relação a taxas cambiais, inclu- sive com o modelo de avaliação utilizado (Black & Scholes) e a exposição líquida. Dessa forma, apesar de poucas empresas apre- sentarem quadros comparativos, as 9 (45%) que o ﬁ zeram evidenciam informações objetivas e com pa- rágrafos anexos explicando os quadros e os modelos utilizados para a avaliação. Uma informação impor- tante que deveria constar nos quadros comparativos é o ganho ou perda com derivativos e as diferenças entre os valores de aquisição e o valor atual conforme os critérios adotados para mensuração. Destaque-se que esse item está diretamente relacionado à avalia- ção de derivativos a valores justos ou de mercado. Outra empresa que também divulgou quadros comparativos com explicações foi a Companhia Si- derúrgica Tubarão – CST, que está reproduzido na Tabela 2 abaixo. O valor de mercado dos ﬁ nanciamentos foi determinado com base no valor presente do ﬂ uxo de caixa futuro, ajustado pela taxa de juros vigen- te no mercado para instrumentos ﬁ nanceiros de natureza, prazo e risco similares. 4.3 Premissas de Avaliação As premissas de avaliação mostram como a empresa mensura o valor dos derivativos divulga- dos nas Demonstrações Contábeis. De acordo com a Instrução da CVM 235/95, as empresas devem ajustá-los a valor de mercado e divulgar as premis- sas utilizadas nesse processo de avaliação. Das empresas analisadas, 5 (25%) divulgaram essa informação, no entanto, de maneira vaga ou incompleta deixando dúvidas se a avaliação foi de fato realizada. As notas sobre esse tema, curiosa- mente, apresentam um padrão e são semelhantes à apresentada abaixo por uma das empresas: A Telesp, por exemplo, apresenta o seguinte quadro comparativo de suas operações envolvendo taxas cambiais. Os valores justos dos ativos e passivos ﬁ - nanceiros da Companhia foram determinados através de informações disponíveis no mercado e de aplicação de metodologias apropriadas de A exposição líquida (excesso) pelo valor con- tábil e de mercado da Sociedade ao risco de taxa de câmbio em 31 de dezembro de 2002 e 2001, é demonstrada na tabela a seguir: EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 75 Tabela 1 – Exposição Líquida 2002 2001 CONSOLIDADO Posição cambial Valor de Mercado Posição cambial Valor de Mercado Empréstimos e ﬁ nanciamentos 4.169.662 3.318.458 4.004.032 4.090.102 Fornecedores 74.857 74.857 – – Posição ativa em “swap” cambial 4.244.132 3.790.553 2.974.226 3.077.006 Posição ativa em opções cambiais – 1.027.937 (i) Exposição líquida 387 1.869 (i) Em 31 de dezembro de 2001 a Sociedade tinha US$ 443.300 de valor nominal em diversas estruturas de opções cambiais, tais como “call”, “call spread” e “operação gaivota”. Naquela data, o valor de mercado das operações de opções apresenta um ativo de R$ 23.600 caso a empresa resolvesse desfazer- se das mesmas enquanto que o valor contábil ativo considerado para tais operações é de R$ 32.600. Na avaliação das opções a valor de mercado utilizou- se o modelo “Black & Scholes”. Fonte: Demonstrações Contábeis do ano de 2002 da Telesp. Tabela 1 – Exposição Líquida (i) Em 31 de dezembro de 2001 a Sociedade tinha US$ 443.300 de valor nominal em diversas estruturas de opções cambiais, tais como “call”, “call spread” e “operação gaivota”. Naquela data, o valor de mercado das operações de opções apresenta um ativo de R$ 23.600 caso a empresa resolvesse desfazer- se das mesmas enquanto que o valor contábil ativo considerado para tais operações é de R$ 32.600. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 4.5 Controle de Risco de Crédito Tabela 3 – Demonstrações de Derivativos OPERAÇÕES DE SWAP 2002 2001 Posição ativa Variação cambial + Taxa de juros 200.340 96.657 Posição passiva 101% do CDI 151.066 102.620 Fonte: Demonstrações Contábeis do ano de 2002 da Petróleos Ipiranga. Tabela 3 – Demonstrações de Derivativos Fonte: Demonstrações Contábeis do ano de 2002 da Petróleos Ipiranga. A nota da CESP sobre gerenciamento de risco de crédito de clientes revela que não foi feito ne- nhum tipo de controle mais avançado pela empresa e tampouco avaliação de clientes. As informações da empresa contribuem pouco para a tomada de decisão do investidor, pois não apresentam a posição líquida da empresa e os re- sultados obtidos com derivativos até o momento. A Globex Utilidades S.A. proprietária de mar- ca Ponto Frio que, pelo mercado de varejo em que atua, deveria ter um grande controle de risco de seus clientes, apesar da pulverização, não apresen- ta nenhum tipo de informação relevante ao mercado sobre esse ponto. Das empresas analisadas 15 (75%), fazem re- ferência a riscos de alterações no câmbio em suas atividades, mas a qualidade das informações deixa a desejar. Apesar de a Demonstração do Resultado do Exercício revelar que muitas empresas têm gran- de parte de suas receitas em vendas para o exterior, poucas empresas divulgam ao mercado informa- ções sobre essas receitas em moeda estrangeira. Pela análise das vinte empresas constatou- se que nenhuma delas utiliza critérios conforme os descritos na instrução 235 da CVM ou se utiliza não os divulga ao mercado. A CVM sugere, inclusive, a classiﬁ cação de clientes com bases em avaliações de ratings de empresas especializadas em análise de risco. Na análise das demonstrações não foi ve- riﬁ cada a presença de nenhum tipo de avaliação de risco de inadimplência de clientes e tampouco di- vulgado o grau de concentração das vendas. Por outro lado, a Telemar, de forma bastante sensata e com propriedade, apresenta a seguinte nota relativa ao tema: Cerca de 44% (2001 – 35%) da dívida conso- lidada, excluindo pessoas ligadas, é expressa em moeda estrangeira (moeda dos Estados Unidos e cesta de moedas do BNDES). Como resulta- do, a Companhia está exposta a riscos cambiais que podem afetar negativamente seus negócios, situação patrimonial e ﬁ nanceira e resultado das operações, bem como sua capacidade de honrar as obrigações do serviço da dívida. 4.5 Controle de Risco de Crédito Para redu- zir a exposição da Companhia a riscos cambiais, a administração celebra contratos de “Swap”s, opções e “forwards” (contratos a termo). As ope- rações de ”swap” cambial transferem o risco de variação de moedas estrangeiras para a variação do CDI. As operações de opções cambiais são de compra e de venda de moeda dos Estados Unidos, nas quais a Companhia paga e recebe prêmios no ato da contratação. Tais prêmios são registrados em “Despesas antecipadas” e “Demais obrigações” e apropriados ao resul- 4.5 Controle de Risco de Crédito Em novembro de 2002, a Companhia efetuou operação com instrumento derivativo na modali- dade “Swap” de CDB para dólar norte-america- no. Em 31 de dezembro, a operação correspon- dia a R$ 18.087, equivalentes a US$ 5.119 mil e O risco de crédito é a possibilidade de a em- presa vir a incorrer em perdas por conta de proble- mas ﬁ nanceiros com seus clientes, ocasionando R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Tabela 2 – Valores de Mercado de Ativo e Passivo CONSOLIDADO 2002 2001 Ativo Contábil Mercado Contábil Mercado Disponibilidades e aplicações ﬁ nanceiras 369.040 369.040 153.441 153.441 Passivo Contábil Mercado Contábil Mercado Adiantamentos sobre contrato de câmbio 816.138 804.907 591.391 593.241 Financ. de Matérias-primas e sobressalentes 13.595 13.620 19.433 19.433 Securitização de recebíveis de exportação 735.029 689.876 676.100 663.307 Outros ﬁ nanciamentos 1.476.787 1.265.494 955.590 896.791 Fonte: Demonstrações Contábeis do ano de 2002 da Companhia Siderúrgica Tubarão. Tabela 2 – Valores de Mercado de Ativo e Passivo Leandro Luís Darós • José Alonso Borba 76 a inadimplência de seus compromissos. Poucas empresas fazem a análise de risco de seus clien- tes (apenas 8 – 40% – das empresas) e, em geral, apresentam justiﬁ cativas para não fazê-lo, como é o exemplo da CESP. montante de R$ 77.187 (2001 – R$ 146), decor- rente das diferenças das variações nos indexa- dores contratados sobre os indexadores referen- ciais estão registrados em despesas ﬁ nanceiras. As operações com instrumentos ﬁ nanceiros deri- vativos podem assim ser demonstradas: O risco surge da possibilidade de a Compa- nhia vir a incorrer em perdas resultantes da diﬁ - culdade de recebimento de valores faturados a seus clientes. Este risco é avaliado pela Compa- nhia como baixo, tendo em vista o concentrado número de seus clientes, garantias contratuais e serem concessionárias para prestação de ser- viços públicos de distribuição de energia e não haver histórico de perdas signiﬁ cativas na reali- zação de seus recebíveis. Tabela 3 – Demonstrações de Derivativos OPERAÇÕES DE SWAP 2002 2001 Posição ativa Variação cambial + Taxa de juros 200.340 96.657 Posição passiva 101% do CDI 151.066 102.620 Fonte: Demonstrações Contábeis do ano de 2002 da Petróleos Ipiranga. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 4.6 Controle de Risco de Taxa de Câmbio adicional de energia, por preços e condições negociados com a contraparte. As sobras ou faltas de energia devem ser vendidas ou com- pradas no mercado de energia no curto prazo e, portanto, estão sujeitas à volatilidade dos pre- ços desse mercado que durante 2002 variaram de R$ 4,85 a R$ 297,05. 4.6 Controle de Risco de Taxa de Câmbio O risco de taxa de câmbio é a possibilidade de a companhia vir a incorrer em perdas por conta de ﬂ utuações nas taxas de câmbio, que reduzam valo- res nominais faturado ou aumentem valores capta- dos no mercado. A Petróleos Ipiranga apresentou a seguinte nota sobre seu risco cambial: A companhia celebra contratos de “swap” para troca do indexador de seus empréstimos e ﬁ nanciamentos em moeda estrangeira pela varia- ção dos Certiﬁ cados de Depósitos Interbancários – CDI, com instituições ﬁ nanceiras sediadas no País, em montantes suﬁ cientes para compensar esses eventuais impactos. Em 31 de dezembro de 2002, os ganhos das operações de “swap” no R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 77 de 2003 a novembro de 2005, correspondendo a aproximadamente 26% “over” (45% “over” na controladora) de cobertura para o risco cambial, considerando que os contratos de opções estão limitados às taxas contratadas. A taxa média de câmbio de proteção à variação cambial do dó- lar dos Estados Unidos dos contratos de opções é entre R$ 2,70 e R$ 2,90 para US$ 1,00, com um prêmio médio de 2,85%. A posição resumida dessas operações se apresenta como segue: tado de acordo com o regime de competência, pelo prazo contratual. As operações de “forwar- ds” deﬁ nem uma taxa de câmbio pré-ﬁ xada a ser adquirida quando do vencimento contratual. O valor nominal total de derivativos em moeda estrangeira (“swaps” e opções) e aplicações em moeda dos Estados Unidos em 31 de dezembro de 2002 e de 2001 eram, respectivamente, US$ 557.891 mil e US$ 517.000 mil (no Consolidado e na Controladora), com vencimentos de janeiro Tabela 4 – Operações com Derivativos CONTA Valor dos contratos Ganho (perda) Ganho (perda) com de derivativos com derivativos derivativos contabilizados 2002 2001 2002 2001 2002 2001 Aplicação em moeda estrangeira 71.412 24.632 24.632 Swap de moeda estrangeira 910.461 84.641 (21.278) 84.641 (21.278) Opções 989.324 816.781 22.395 (56.181) (29.705) (56.181) Termo (“forward”) 382.866 37.608 37.608 Fonte: Demonstrações Contábeis do ano de 2002 da Telemar. Tabela 4 – Operações com Derivativos Como se observa acima, a nota da Telemar é clara e evidencia a real posição da empresa em rela- ção aos riscos. É lamentável que poucas empresas tenham notas semelhantes à da Telemar. R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 4.7 Controle de Risco de Preço dos Produtos Tabela 5 – Instrumentos Derivativos • não divulgação das políticas de atuação sobre os riscos a que estão sujeitos os instrumentos ﬁ nanceiros e derivativos, os tipos de riscos envolvidos (riscos de mercado, taxa de câmbio e de crédito) e o controle das operações no mercado de derivativos; • não divulgação das políticas de atuação sobre os riscos a que estão sujeitos os instrumentos ﬁ nanceiros e derivativos, os tipos de riscos envolvidos (riscos de mercado, taxa de câmbio e de crédito) e o controle das operações no mercado de derivativos; • não divulgação das políticas de atuação sobre os riscos a que estão sujeitos os instrumentos ﬁ nanceiros e derivativos, os tipos de riscos envolvidos (riscos de mercado, taxa de câmbio e de crédito) e o controle das operações no mercado de derivativos; Como se observa pelos exemplos acima, na proteção contra riscos de mercado a nota da Vale do Rio Doce é bem mais completa e realizada com acurácia do que a da Light. Assim, a analise qualita- tiva comparativa é importante para se determinar a aderência às normas exigidas pelos órgãos regula- dores e pelo mercado. • imprecisões na divulgação da nota, faltan- do não só a comparação entre o valor con- tábil e o valor de mercado, como também informações sobre os critérios de avaliação adotados para determinação desse valor de mercado; 4.7 Controle de Risco de Preço dos Produtos Já a companhia Vale do Rio Doce apresenta a seguinte nota relativa ao risco de preço: O risco de preço de produtos é a possibilidade de a companhia vir a incorrer em perdas por conta de ﬂ utuações de preços de seus produtos, nos mer- cados interno e externo. Do total de empresas da amostra, apenas 6 (30%) ﬁ zeram referência a esse tópico em suas notas explicativas. Os preços do minério de ferro, principal pro- duto da Companhia, são ﬁ xados através de ne- gociações anuais entre produtores e consumido- res, apresentando notável estabilidade ao longo do tempo. A Companhia não contrata operações para proteção contra variações no preço do mi- nério de ferro. A Companhia utiliza instrumen- tos de “hedge” para gerenciar sua exposição às mudanças de mercado do ouro e alumínio. As operações com derivativos permitem ﬁ xar lucro médio mínimo para a produção futura. A Com- panhia gerencia ativamente as posições con- tratadas, sendo os resultados destas atividades acompanhados mensalmente, a ﬁ m de permitir que seja feitos ajustes nas metas e estratégias em resposta às condições de mercado. A tabela abaixo informa o portfólio de derivativos de ouro da Companhia em 2002 e 2001. A Light apresentou a seguinte nota a respeito de risco de mercado: As quantidades de energia elétrica com- pradas pela Companhia são baseadas em es- timativas do consumo de energia nos períodos subseqüentes. Parte da energia comprada esta contratada através dos denominados contratos iniciais, assinados em período anterior ao da pri- vatização da Companhia, que ﬁ xa determinados volumes de compra até o ano de 2002, sendo reduzidos em 25% a partir de 2003 até sua ex- tinção. A Companhia por sua opção pode efetu- ar compras através dos denominados contratos bilaterais, para atender eventual necessidade R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Leandro Luís Darós • José Alonso Borba 78 Tabela 5 – Instrumentos Derivativos TIPO 2002 2001 Quant. (oz) Faixa de Ganho/perda Último Quant. (oz) Faixa de Ganho/perda preço não realizado vecto. preço não realizado US$/oz em R$ milhões US$/oz em R$ milhões Puts compradas 428.000 270-355 11 Dez/07 422.000 270-340 25 Calls vendidas 595.000 316-407 (63) Dez/07 718.000 308-366 (8) Instrumentos Híbridos 20.000 – (1) Nov/06 25.000 – – Total (53) 17 Fonte: Demonstrações Contábeis do ano de 2002 da Companhia Vale do Rio Doce. 5 CONSIDERAÇOES FINAIS A análise das Demonstrações Contábeis das vinte maiores empresas brasileiras, no que se refere à evidenciação de instrumentos ﬁ nanceiros deriva- tivos, revela que a grande maioria das companhias abertas, ainda, não informam, de acordo com a Instrução Normativa 235 da CVM, corretamente e de forma clara, o mercado de suas operações com derivativos. • informação da inexistência de instrumen- tos ﬁ nanceiros, enquanto o balanço patri- monial revelava o contrário; • informação de que o valor contábil é o mesmo (ou próximo ao) valor de merca- do, provavelmente por não terem efetuado qualquer cálculo nesse sentido. Desde que a matéria foi disciplinada pela CVM em 1995, ainda há diferenças entre as práticas de di- vulgação de instrumentos ﬁ nanceiros pelas compa- nhias abertas brasileiras e as determinações da Instru- ção Normativa 235/95. Essas diferenças podem ser, assim, resumidas, conforme entendimento da própria CVM e constatações no decorrer deste trabalho: Pela análise quantitativa das empresas, che- gou-se à seguinte conclusão em termos de eviden- ciação das vinte empresas analisadas, demonstra- das na Tabela 6: R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Tabela 6 – Evidenciação de Derivativos – em % Controle de Risco Itens – Empresas Nota Objetivos, Premissas Crédito Taxa de Preço dos Explicativa Finalidades de Avaliação Quadros Câmbio Produtos e Intenções Apresentam 100 60 25 45 40 75 30 Não apresentam 0 40 75 55 60 25 70 Fonte: Elaborado pelos autores com base no resultado da pesquisa. Tabela 6 – Evidenciação de Derivativos – em % Tabela 6 – Evidenciação de Derivativos – em % EVIDENCIAÇÃO DE INSTRUMENTOS FINANCEIROS DERIVATIVOS NAS DEMONSTRAÇÕES CONTÁBEIS: UMA ANÁLISE DAS EMPRESAS BRASILEIRAS 79 nistração da companhia para gestão desses riscos, e em que contexto estão inseridos os instrumentos ﬁ nanceiros. Tais informações não foram prestadas pela maioria das empresas analisadas. Apesar de os instrumentos ﬁ nanceiros deriva- tivos prestarem-se, principalmente, a estratégias de gestão de risco, muitas vezes eles são utilizados para especulação e operações ﬁ nanceiras de cur- to prazo. Assim, invariavelmente, os instrumentos ﬁ nanceiros sujeitam a empresa a outros fatores de risco para os quais ela não estava originariamente exposta. Essas informações, obrigatoriamente, de- veriam estar presentes nas notas explicativas ela- boradas pelas companhias abertas. Outro ponto que deve ser tratado diz respei- to a procedimentos contábeis. REFERÊNCIAS BIBLIOGRÁFICAS BENSTON, G.; MIAN, S.. Financial reporting of derivatives: an analysis of the issues, ev aluations proposals, and a suggested solution. The Journal of Financial Engineering, 4, 1997, 217-246. ISHIKAWA, Junji. A social science of contemporary value-based accounting: economic foundations of accounting for ﬁ nancial in- struments. Critical Perspectives on Accounting. 2003. ISHIKAWA, Junji. A social science of contemporary value-based accounting: economic foundations of accounting for ﬁ nancial in- struments. Critical Perspectives on Accounting. 2003. LAKATOS, Eva Maria; MARCONI, Marina de Andrade. Metodologia do trabalho cientíﬁ co. 4. ed. São Paulo: Atlas, 1992, p. 82. BRASIL. Lei 6404 de 1976. Dispõe sobre as Sociedades por Ações. LOPES, Alexsandro Broedel. A informação contábil e o mercado de capitais. São Paulo: Thomson, 2002. BLANKLEY, Alan; REINHOLD, Lamb; SCHOROEDER, Richard. Compliance with SEC disclosure requirements about market risk. Journal of Derivatives, v. 7, issue 3, Spring 2000. ; CARVALHO, Luiz Nelson G.. Contabilização de operações com derivativos: uma comparação entre o SFAS 133 e o arcabouço emanado pelo COSIF. Caderno de estudos, São Paulo, FIPECAFI, n. 20, jan/abr., 1999. COMISSÃO DE VALORES MOBILIÁRIOS. Instrução Normativa 235 de 23 de março de 1995. Dispõe sobre a divulgação, em nota explicativa, do valor de mercado dos instrumentos ﬁ nanceiros, reconhecidos ou não nas demonstrações ﬁ nanceiras das com- panhias abertas e dá outras providências. COMISSÃO DE VALORES MOBILIÁRIOS. Instrução Normativa 235 de 23 de março de 1995. Dispõe sobre a divulgação, em nota explicativa, do valor de mercado dos instrumentos ﬁ nanceiros, reconhecidos ou não nas demonstrações ﬁ nanceiras das com- panhias abertas e dá outras providências. SEOW, Gim S.; TAM, Kinsun. The usefulness of derivative-related Accounting Disclosures. Review of Quantitative Finance and Ac- counting, 19, 2002, p. 273-291. GAO: General Accounting Ofﬁ ce, Special Report to US Congress on Financial Instruments and Capital Markets, 1998. p. 18. Statement of Financial Accounting Standars – SFAS 133 – Accounting for derivatives instruments and Hedging activies – Junho/1998. GUAY, Wayne. R. The impact of derivatives on ﬁ rm risk: an em- pirical examination of new dervivative users. Journal of Account- ing and Economics, 26, p. 319-351. 1999. 5 CONSIDERAÇOES FINAIS Devem ser divulga- das informações no que respeita à política contábil dispensada ao instrumento ﬁ nanceiro (critério de mensuração, classiﬁ cação contábil recebida, regis- tro dos efeitos patrimoniais, entre outros), de modo que os usuários da informação tenham todos os elementos necessários às suas análises. A nota explicativa para instrumentos ﬁ nancei- ros deve conter, de modo integrado, uma descrição qualitativa dos fatores de risco de mercado que afe- tam os negócios de uma companhia aberta (risco de preços de suas mercadorias, risco de taxa de juros, risco de taxa de câmbio, risco de crédito, risco in- ﬂ acionário, risco de liquidez etc.) e, como a informa- ção mais relevante, a estratégia adotada pela admi- Ainda assim, a despeito da enorme quantidade de desvios e incorreções encontrados nas Demons- trações Contábeis analisadas, todas de grandes e tradicionais empresas brasileiras, saliente-se, por ﬁ m, que em nenhuma delas o parecer dos auditores independentes fez qualquer referência ao fato. SILVA NETO, Lauro de Araújo. Opções do tradicional ao exótico. 2. ed., São Paulo: Atlas, 1996. BIBLIOGRAFIA COMPLEMENTAR ASSAF NETO, Alexandre. Mercado ﬁ nanceiro. 2. ed. São Paulo: Atlas, 1999. ASSAF NETO, Alexandre. Mercado ﬁ nanceiro. 2. ed. São Paulo: Atlas, 1999. 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Manual de contabilidade. 6. ed. São Paulo: Atlas, 2003. ROSS, Stephen A. et al. Administração ﬁ nanceira. São Paulo: Atlas, 1995. LOPES, Alexsandro Broedel. LIMA, Iran Siqueira. Perspectivas para a pesquisa em contabilidade: o impacto dos derivativos. Revista Contabilidade & Finanças FIPECAFI – FEA – USP, São Paulo, FIPECAFI, v. 15, n. 26, p. 25-41, maio/agosto 2001. HWANG, Angela L. J.. Comparative analysis of accounting treat- ments for derivatives. Journal of Accounting Education, 20, 2002, p. 205-233. SOARES, Jairo da Rocha. A Nova Feição à Contabilidade de Instrumentos Financeiros: Aplicações do IASC e US GAAP. ENANPAD. 2000. .Contabilidade e controle de operações com derivativos. São Paulo: Pioneira, 1999. .Contabilidade e controle de operações com derivativos. São Paulo: Pioneira, 1999. SANVICENTE & MELLAGI FILHO. Mercado de capitais e estra- tégias de investimento. São Paulo: Atlas, 1996. MASHEANE, Motseoa. Derivatives: accounting and economic is- sues. Journal of Accounting Education, v. 16, ns. 3/4, p. 591-598, 1998. SILVA NETO, Lauro de Araújo. Opções do tradicional ao exótico. 2. ed., São Paulo: Atlas, 1996. Nota: Endereço dos autores: R. Cont. Fin. – USP, São Paulo, n. 39, p. 68 – 80, Set./Dez. 2005 Nota: Endere Universidade Federal de Santa Catarina Centro Sócio-Econômico Campus Universitário – Trindade Florianópolis – SC 88040-900
https://openalex.org/W2774242418	http://real.mtak.hu/105140/1/Halgren_et_al_PNAS_2019.pdf	English	null	The Generation and Propagation of the Human Alpha Rhythm	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	16,650	The generation and propagation of the human alpha rhythm Milan Halgrena,1,2, István Ulbertb,c, Hélène Bastujid,e, Dániel Fabóf, Lorand Er}ossc,g, Marc Reyh,i,j, Orrin Devinskyk, Werner K. Doylek, Rachel Mak-McCullyl, Eric Halgrenm, Lucia Wittnerb, Patrick Chauvelh,i,j, Gary Heitn, Emad Eskandara,3, Arnold Mandello, and Sydney S. Casha aDepartment of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; bInstitute of Cognitive Neuroscience and Psychology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest 1051, Hungary; cFaculty of Information Technology and Bionics, Péter Pázmány Catholic University, Budapest 1088, Hungary; dLyon Neuroscience Research Center, Université Claude Bernard, 69100 Villeurbanne, France; eUnité d’Hypnologie, Service de Neurologie Fonctionnelle et d’Épileptologie, Hôpital Neurologique, Hospices Civils de Lyon, 69003 Lyon, France; fEpilepsy Centrum, National Institute of Clinical Neurosciences, 1145 Budapest, Hungary; gDepartment of Functional Neurosurgery, National Institute of Clinical Neurosciences, 1145 Budapest, Hungary; hDivision is Institut de Neurosciences des Systèmes, Aix-Marseille Université, 13007 Marseille, France; iINSERM, Institut de Neurosciences des Systèmes, 13005 Marseille, France; jAssistance Publique–Hôpitaux de Marseille, Timone Hospital, 13005 Marseille, France; kComprehensive Epilepsy Center, New York University School of Medicine, New York, NY 10016; lDepartment of Psychology, University of California, Berkeley, CA 94720; mDepartment of Neurosciences and Radiology, University of California San Diego, La Jolla, CA 93093; nDepartment of Neurosurgery, Permanente Medical Group, Redwood City, CA 94063; and oDepartment of Psychiatry, University of California San Diego, La Jolla, CA 92093 Edited by Gyorgy Buzsáki, New York University Neuroscience Institute, New York, NY, and approved October 11, 2019 (received for review July 30, 2019) patients (SI Appendix, Fig. S1 and Table S1) (ECoG patients [Pts.] E1 to E5; E1, E3, and E4 participated in an eye-closure task, whereas E2 and E5 did not and were recorded during quiet wakefulness). Strikingly, alpha oscillations propagated as trav- eling waves from anterosuperior cortex toward posteroinferior areas (Figs. 1 and 2 and SI Appendix, Figs. S2–S4) (19). To quantify this propagation, we used a 2-pass 3rd-order 0-phase-shift Butter- worth filter between 7 and 13 Hz to extract alpha-band activity. The Hilbert transform was then applied to find the analytic signal and, from this, the instantaneous amplitude and phase of ongoing al- pha activity; only time points with the highest 20% of alpha-band amplitude (averaged across array channels at each time point) were analyzed further. The alpha rhythm is the longest-studied brain oscillation and has been theorized to play a key role in cognition. Still, its physiology is poorly understood. Published under the PNAS license. Data deposition: The data underlying our figures have been made publicly available on figshare, DOI: 10.6084/m9.figshare.9927125.v2. Custom scripts are freely available at https://github.com/mhalgren/AlphaGen. Macaque data are publicly available at http:// neurotycho.org/data/20120813ktanesthesiaandsleepchibitoruyanagawa. Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 1To whom correspondence may be addressed. Email: mhalgren@mit.edu. 2Present address: Department of Brain & Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139. 2Present address: Department of Brain & Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139. Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on J 3Present address: Department of Neurological Surgery, Albert Einstein College of Medi- cine, Bronx, NY 10461. 3Present address: Department of Neurological Surgery, Albert Einstein College of Medi- cine, Bronx, NY 10461. The generation and propagation of the human alpha rhythm In this study, we used microelectrodes and macroelectrodes in surgical epilepsy patients to measure the intracortical and thalamic generators of the alpha rhythm during quiet wakefulness. We first found that alpha in both visual and somatosensory cortex propagates from higher-order to lower-order areas. In posterior cortex, alpha propagates from higher-order anterosuperior areas toward the occipital pole, whereas alpha in somatosensory cortex propagates from associative regions toward primary cortex. Several analyses suggest that this cortical alpha leads pulvinar alpha, complicating prevailing theories of a thalamic pacemaker. Finally, alpha is dominated by currents and firing in supragranular cortical layers. Together, these results suggest that the alpha rhythm likely reflects short-range supragranular feed- back, which propagates from higher- to lower-order cortex and cortex to thalamus. These physiological insights suggest how alpha could mediate feedback throughout the thalamocortical system. y To visualize the spatial progression of alpha oscillations across the array, we found the circular difference between the mean phase across all contacts and each individual contact at each point in time (Fig. 1B). This yielded a distribution of differences of each contact’s phase from the grid’s mean phase across all alpha \| oscillations \| intracranial EEG \| laminar \| thalamocortical alpha \| oscillations \| intracranial EEG \| laminar \| thalamocortical alpha \| oscillations \| intracranial EEG \| laminar \| thalamocortical ha \| oscillations \| intracranial EEG \| laminar \| thalamocortical Significance The alpha rhythm dominates the electroencephalogram during quiet wakefulness, but the brain structures which generate it are not known. Using rare intracranial recordings in epilepsy patients, we find that alpha rhythms propagate toward the back of the brain and that alpha waves in cortex (particularly superficial layers) lead alpha oscillations in the thalamus. These findings shed light on how the human alpha rhythm coordi- nates activity throughout the brain. Author contributions: I.U., E.H., and S.S.C. designed research; I.U., H.B., D.F., L.E., M.R., O.D., W.K.D., R.M.-M., E.H., L.W., P.C., G.H., E.E., and S.S.C. performed research; M.H. and A.M. analyzed data; and M.H. and S.S.C. wrote the paper. The authors declare no competing interest Significance A l t A lpha oscillations (7 to 13 Hz) (1) are the most salient elec- troencephalogram (EEG) event during wakefulness and may be fundamental for top-down cognitive processes (2, 3), such as attention (4), perception (5, 6), functional inhibition (7, 8), and working memory (9). However, the underlying neural structure(s) and circuits which generate alpha are intensely controversial. Studies have pointed to the thalamus as the pri- mary alpha pacemaker, with the classic posterior alpha rhythm driven by the pulvinar and/or lateral geniculate nucleus (LGN) (4, 10–12). Within the cortex, it’s widely assumed that alpha originates from infragranular layers driven by layer V pyramidal cells (13–17). Despite the prevalence of these hypotheses, the studies used to support them are not definitive; previous elec- trophysiological literature has either used a distant reference susceptible to volume conduction (4, 13, 15), was performed in vitro (14), or relied on extracranial recordings (18) (Discussion). Crucially, none of these hypotheses have been directly tested via invasive recordings in humans. We therefore analyzed focal mi- croelectrode and macroelectrode recordings from human neo- cortex and thalamus in surgical epilepsy patients to characterize alpha’s generation during quiet wakefulness. The alpha rhythm dominates the electroencephalogram during quiet wakefulness, but the brain structures which generate it are not known. Using rare intracranial recordings in epilepsy patients, we find that alpha rhythms propagate toward the back of the brain and that alpha waves in cortex (particularly superficial layers) lead alpha oscillations in the thalamus. These findings shed light on how the human alpha rhythm coordi- nates activity throughout the brain. Author contributions: I.U., E.H., and S.S.C. designed research; I.U., H.B., D.F., L.E., M.R., O.D., W.K.D., R.M.-M., E.H., L.W., P.C., G.H., E.E., and S.S.C. performed research; M.H. and A.M. analyzed data; and M.H. and S.S.C. wrote the paper. The authors declare no competing interest. The authors declare no competing interest. This article is a PNAS Direct Submission. Published under the PNAS license. Author contributions: I.U., E.H., and S.S.C. designed research; I.U., H.B., D.F., L.E., M.R., O.D., W.K.D., R.M.-M., E.H., L.W., P.C., G.H., E.E., and S.S.C. performed research; M.H. and A.M. analyzed data; and M.H. and S.S.C. wrote the paper. Results We then found the circular mean of this difference (across time points): If a contact is leading an oscillation, it will have a positive circular distance with respect to the grid’s spatial mean phase; if a contact is lagging, it will have a negative phase difference with the grid’s average phase. Fig. 2B was then generated by finding the mean circular distance between each contact’s phase and the grid’s mean phase at each time point, or the av- erage advance/delay of a given contact. This method allows one to measure traveling waves oblique to the grid’s implantation and sidestep the selection of a potentially biasing reference contact. However, this analysis only yields a single, average gradient in each patient and makes it hard to assess statistical significance within subjects. We therefore performed a second test to confirm that alpha oscillations had a consistent propagation direction across time in individual patients. This was done by finding the direction of the average spatial phase gradient across the grid of electrodes at each time point, reflecting the average direction of alpha propagation at a single time point, and then determining if the distribution of gradient directions throughout time was nonuniform (20) (P ≤10−17 in each patient, Rayleigh test) (Fig. 2C, Materials and Methods, and SI Appendix, Fig. S4). Estimated median speeds of these waves (derived from the phase gradient) were just under 1 m/s (median speed across patients: 0.9134 ± 0.1563 m/s). (SI Appendix, Fig. S4 and SI Methods). Open-source time points. We then found the circular mean of this difference (across time points): If a contact is leading an oscillation, it will have a positive circular distance with respect to the grid’s spatial mean phase; if a contact is lagging, it will have a negative phase difference with the grid’s average phase. Fig. 2B was then generated by finding the mean circular distance between each contact’s phase and the grid’s mean phase at each time point, or the av- erage advance/delay of a given contact. This method allows one to measure traveling waves oblique to the grid’s implantation and sidestep the selection of a potentially biasing reference contact. However, this analysis only yields a single, average gradient in each patient and makes it hard to assess statistical significance within subjects. We therefore performed a second test to confirm that alpha oscillations had a consistent propagation direction across time in individual patients. PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23773 Results We analyzed electrocorticographic (ECoG) recordings of spon- taneous alpha oscillations (4.54 ± 0.87 min, mean ± SD) in the occipital, posterior temporal, and posterior parietal cortices of 5 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1913092116/-/DCSupplemental. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1913092116/-/DCSupplemental. First published November 4, 2019. First published November 4, 2019. 23772–23782 \| PNAS \| November 19, 2019 \| vol. 116 \| no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1913092116 100 ms time ECoG channel le n n a h c G o C E Raw Alpha Phase Alpha phase relative to grid mean phase 24 ms Subtract mean spatial phase (across channels) at each time point from each channel Circular mean across time Average alpha phase relative to grid mean phase - - 3 3 - 3 3 0 ms 96 ms 300 uV A B Fig. 1. Analysis stream for ECoG traveling waves. (A) Alpha propagates as a traveling wave in the raw broadband data. (B) Analysis stream for visualizing traveling waves in ECoG. We start with raw alpha phase of the grid over time. Then, for each time point, we find the circular distance (distance on the unit circle) between each contact and the grid’s mean phase (across all contacts) at that time point. Lastly, we find the circular mean of this difference to get each contact’s average phase advance or delay. A B NEUROSCIENCE Subtract mean spatial phase (across channels) at each time point from each channel Average alpha phase l ti t id Alpha phase relative to grid mean phase Fig. 1. Analysis stream for ECoG traveling waves. (A) Alpha propagates as a traveling wave in the raw broadband data. (B) Analysis stream for visualizing traveling waves in ECoG. We start with raw alpha phase of the grid over time. Then, for each time point, we find the circular distance (distance on the unit circle) between each contact and the grid’s mean phase (across all contacts) at that time point. Lastly, we find the circular mean of this difference to get each contact’s average phase advance or delay. ECoG recordings in a healthy macaque during eye closure demonstrated a highly similar propagation direction and speed (21, 22) (Fig. 2B). time points. Halgren et al. Results This was done by finding the direction of the average spatial phase gradient across the grid of electrodes at each time point, reflecting the average direction of alpha propagation at a single time point, and then determining if the distribution of gradient directions throughout time was nonuniform (20) (P ≤10−17 in each patient, Rayleigh test) (Fig. 2C, Materials and Methods, and SI Appendix, Fig. S4). Estimated median speeds of these waves (derived from the phase gradient) were just under 1 m/s (median speed across patients: 0.9134 ± 0.1563 m/s). (SI Appendix, Fig. S4 and SI Methods). Open-source To determine if the thalamus coordinated these traveling al- pha waves, we utilized stereo EEG (SEEG) to make bipolar local-field-potential gradient (LFPg) macroelectrode depth re- cordings (n = 9 patients, 36 ± 7.5 min, mean ± SD) during quiet wakefulness. Recordings were made simultaneously from the cortex and the pulvinar, a thalamic nucleus which projects broadly to posterior cortical regions (23) and postulated to drive cortical alpha (4, 24) (Fig. 3A) (SEEG Pts. S1 to S9). The use of a bipolar derivation (i.e., referencing each contact to its neigh- bor) ensured that activity was locally generated and not volume conducted from a distal structure. Cortical coverage was pre- dominantly posterior (108 of 124 cortical contacts posterior to the central sulcus), similar to our ECoG patients (Fig. 3B). We first verified that alpha traveling waves could be measured in these cortical depth recordings by applying the same method used to quantify alpha propagation in our ECoG data (i.e., measuring how much each individual channel’s alpha phase led or lagged the mean phase across all channels). Analysis was re- stricted to occipital and posterior temporal/parietal channels which were in unambiguously lateral cortex; this ensured that the LFPg’s polarity was consistent across sites (always surface Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23773 Halgren et al. Pt. E1 ECoG channel le n n a h c G o C E 24 ms Time Pt. E4 Pt. E2 Pt. E3 0 1 2 2 3 1 Speed (m/s) superior inferior posterior anterior Snapshots of Raw Alpha Phase 2 0 2 2 4 C B A Pt. E5 Phase Delay Phase Advance Wave Starts Wave Ends - - 3 3 Macaque D Propagation Direction Propagation Speed - 5 5 Fig. 2. Results Alpha propagates from anterosuperior to posteroinferior cortex. (A) Alpha-phase snapshots from Pt. L1 demonstrate propagation from the grid’s top- right (anterosuperior) to bottom-left corner (posteroinferior). (B) Average circular distance of each contact’s alpha phase from the spatial mean phase during eye closure. In all patients, alpha propagates toward posteroinferior areas. Overlaid arrow is the direction of the grid’s average phase gradient. Color runs from ±π 3 in Pts. E1, E2, and E5 and macaque; and from ±π 5 in Pts. E3 and E4. (C) Average probability distribution of traveling wave directions across time such that the bottom left contact is the most posteroinferior (±SEM across patients). (D) Average probability distribution of traveling wave speeds (±SEM across patients). Pt. E1 ECoG channel le n n a h c G o C E 24 ms Time Pt. E2 Pt. E3 Snapshots of Raw Alpha Phase B A Phase Delay Phase Advance Wave Starts Wave Ends - - 3 3 - 5 5 Fig. 2. Alpha propagates from anterosuperior to posteroinferior cortex. (A) Al right (anterosuperior) to bottom-left corner (posteroinferior). (B) Average circula closure In all patients alpha propagates toward posteroinferior areas Overlaid a Pt. E4 C Pt. E5 Macaque D A B Macaque C Macaque C D D Propagation Speed Propagation Direction superior inferior posterior ant 2 0 2 2 4 Propagation Direction 0 1 2 2 3 1 Speed (m/s) ior superior 2 2 1 Fig. 2. Alpha propagates from anterosuperior to posteroinferior cortex. (A) Alpha-phase snapshots from Pt. L1 demonstrate propagation from the grid’s top- right (anterosuperior) to bottom-left corner (posteroinferior). (B) Average circular distance of each contact’s alpha phase from the spatial mean phase during eye closure. In all patients, alpha propagates toward posteroinferior areas. Overlaid arrow is the direction of the grid’s average phase gradient. Color runs from ±π 3 in Pts. E1, E2, and E5 and macaque; and from ±π 5 in Pts. E3 and E4. (C) Average probability distribution of traveling wave directions across time such that the bottom left contact is the most posteroinferior (±SEM across patients). (D) Average probability distribution of traveling wave speeds (±SEM across patients). despite driving one another (26). Thalamocortical coherence spectra often exhibited robust alpha peaks (Fig. 4E), indicating that alpha rhythms in the posterior cortex and pulvinar are functionally coupled (peak alpha coherence in thalamocortical channel pairs with significant alpha coherence: 0.3346 ± 0.012, mean ± SEM). Results As a first means of determining whether neo- cortical alpha led thalamic alpha (or vice versa), we detected alpha bursts in both thalamic and cortical channels. Briefly, a putative burst was detected when alpha-band power (7 to 13 Hz, hilbert.m) exceeded 3 times the average of theta-band (4 to 6 Hz) and beta-band (15 to 25 Hz) power. Burst starts and stops were defined as when alpha-band power was at least 2 times the theta/ beta average, and all bursts less than 400 ms were then rejected. Lastly, alpha bursts which were less than 50 ms apart were merged. For each thalamocortical channel pair, we determined if cortical bursts started significantly before thalamic bursts (or vice versa). By the binomial test, 28 of 362 thalamocortical channel pairs (P < 0.05, Bonferroni corrected across all 362 channel pairs) had a significant lead in alpha onset. In all 28 of these channel pairs, cortical alpha bursts led thalamic alpha bursts (Fig. 4C and SI Appendix, Fig. S8). Importantly, a previous study applying a similar burst-detection algorithm to analyze sleep spindles in the same subjects and channels during sleep found the opposite di- rectionality—i.e., that thalamic spindles led cortical spindles (25). As a second measure of cortical alpha leading thalamic alpha power, we measured the cross-covariance of alpha amplitude (as derived from the amplitude envelope of the analytic signal) in thalamocortical channel pairs with statistically significant alpha positive, given that we subtracted medial from lateral contacts for each bipolar pair). Just as with our ECoG recordings, we only analyzed time points with the top 20% of cortical alpha power (averaged across channels at each time point). The alpha phase of anterosuperior contacts led ones closer to the occipital pole, replicating our ECoG recordings (Fig. 4A and SI Appendix, Fig. S6). This demonstrates that these traveling waves are not reference- dependent and suggests that the alpha rhythms recorded in our depth patients are analogous to the ones recorded in our ECoG patients. As we then wished to examine the pulvinar’s role in cortical alpha, all further analyses were biased toward thalamic activity by only analyzing the 2-s epochs with the 20% most thalamic alpha-band power (averaged across all thalamic chan- nels). First, we characterized the prevalence of alpha rhythms in both cortex and pulvinar. Results This was done by detecting which channels had peaks between 7 and 13 Hz in their power spectra (peaks were detected via the peakfinder algorithm; Materials and Methods). Surprisingly, power spectra from cortical contacts had alpha-band peaks more frequently (63.4%; 78 of 123 of cortical channels) than ones in the pulvinar (34.6%; 9 of 26 of thalamic channels) (Fig. 4B, Materials and Methods, and SI Appendix, Fig. S7) Thalamic and cortical power spectra also sometimes had different peak frequencies (Fig. 4B); while this could be con- strued as evidence for separate thalamic and cortical alpha generators, this is not necessarily the case. Empirically, spindles (believed to be thalamocortically driven) have higher frequencies in the thalamus than the cortex (25). Analytically, weakly cou- pled oscillators can also exhibit different peak frequencies, positive, given that we subtracted medial from lateral contacts for each bipolar pair). Just as with our ECoG recordings, we only analyzed time points with the top 20% of cortical alpha power (averaged across channels at each time point). The alpha phase of anterosuperior contacts led ones closer to the occipital pole, replicating our ECoG recordings (Fig. 4A and SI Appendix, Fig. S6). This demonstrates that these traveling waves are not reference- dependent and suggests that the alpha rhythms recorded in our depth patients are analogous to the ones recorded in our ECoG patients. As we then wished to examine the pulvinar’s role in cortical alpha, all further analyses were biased toward thalamic activity by only analyzing the 2-s epochs with the 20% most thalamic alpha-band power (averaged across all thalamic chan- nels). First, we characterized the prevalence of alpha rhythms in both cortex and pulvinar. This was done by detecting which channels had peaks between 7 and 13 Hz in their power spectra (peaks were detected via the peakfinder algorithm; Materials and Methods). Surprisingly, power spectra from cortical contacts had alpha-band peaks more frequently (63.4%; 78 of 123 of cortical channels) than ones in the pulvinar (34.6%; 9 of 26 of thalamic channels) (Fig. 4B, Materials and Methods, and SI Appendix, Fig. S7) Thalamic and cortical power spectra also sometimes had different peak frequencies (Fig. 4B); while this could be con- strued as evidence for separate thalamic and cortical alpha generators, this is not necessarily the case. Empirically, spindles (believed to be thalamocortically driven) have higher frequencies in the thalamus than the cortex (25). 23774 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 Results By averaging the mean cortical and thalamic HGP with respect to the phase of thalamic alpha LFPg phases across channel pairs, it is apparent that cortical HGP leads thalamic HGP (Fig. 4F). In individual thalamocortical channel pairs, this could be quantified by measuring the cross-covariance between the thalamic and cortical HGP profiles and seeing if the peak was positive/negative or examining which had minimal HGP at an earlier thalamic alpha phase. Cortex led thalamus in all 5 channel pairs, as derived by both measures, more than expected by chance (P = 0.0313, 1-tailed binomial test of cortex leading thalamus against thalamus leading cortex). This lag (difference between HGP minima, as seen in Fig. 4F) was on average ∼40°, or ∼11 ms, assuming an alpha period of 100 ms. This time delay is physiologically plausible and similar to how much the thalamus leads cortex during sleep spindles (25). NEUROSCIENCE B g p p ( ) As a final directional measure, we measured the Granger causality (GC) spectrum (which quantifies the amount of in- formation one time series contains about another across fre- quencies) of the LFPg between all pairs of cortical and thalamic contacts (34) (Fig. 4G). Corticothalamic causality in the alpha band was found to be significantly greater than thalamocortical causation for almost every thalamocortical channel pair (across all patients), with a significant difference between thalamocortical and corticothalamic causation (P ≤0.01 for each channel pair, Wilcoxon signed rank test, Bonferroni corrected within patients; 143 of 163 [87.73%] pairs with greater corticothalamic than thalamocortical causality; P < 1.83 × 10−24 across all significantly different channel pairs, binomial test). To ensure that this wasn’t due to our cortical channels having greater alpha power, we re- peated our Granger analysis only using thalamocortical channel pairs in which the thalamic lead had greater normalized alpha power. This actually increased the percentage of channel pairs with significantly greater corticothalamic than thalamocortical alpha causality (74 of 82 [90.24%], significantly more than chance as determined by the binomial test, P < 7.2 × 10−15). Fig. 3. Robust alpha rhythms can be recorded in human pulvinar and cor- tex. (A) Representative 6-s LFPg traces of simultaneous thalamic and cortical alpha activity. Prominent, largely continuous alpha rhythms can be recorded in various locations within the pulvinar as well as posterior cortex. (B) Cor- tical implant locations in all SEEG patients displayed on Pt. S3′s brain. Results Analytically, weakly cou- pled oscillators can also exhibit different peak frequencies, Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 Halgren et al. lateral occipital cortex medial pulvinar 1 s 300 -200 uV 30 -30 uV Pt S8 Pt S2 medial occipital cortex lateral pulvinar 17 -17 uV 200 -380 uV A B Fig. 3. Robust alpha rhythms can be recorded in human pulvinar and cor- tex. (A) Representative 6-s LFPg traces of simultaneous thalamic and cortical alpha activity. Prominent, largely continuous alpha rhythms can be recorded in various locations within the pulvinar as well as posterior cortex. (B) Cor- tical implant locations in all SEEG patients displayed on Pt. S3′s brain. Each color signifies a different patient. quantified by its MI. SR and MI were not significantly correlated, trending toward anticorrelation (i.e., smoother waveforms had more PAC), the opposite of what would be expected if our PAC was spurious (r = −0.2124, P = 0.0602) (31, 32). Notably, thalamic alpha was rarely coherent with its own HGP (coherence: 0 of 14 intrathalamic contact pairs, MI: 3 of 14, P < 0.05, Bonferroni corrected within patients); instead, thalamic alpha rhythms were predominantly synchronous with cortical HGP (coherence: 9 of 14, MI: 10 of 14; mean peak alpha coherence between thalamic LFPg and cortical HGP channel pairs with significant coherence of 0.3535 ± 0.026) (Fig. 4 D and E), supporting cortical generation. B HGP i i f f l fi i (33) A Because HGP is an imperfect proxy for neuronal firing (33), the failure to find consistent local thalamic PAC could reflect a limitation of our recordings rather than a cortical origin for al- pha [but it should be noted that thalamic HGP is modulated by thalamic sleep spindles in the same recordings (25)]. To resolve this ambiguity, we further analyzed the minority of thalamic channels (5 of 42 intrathalamic contact pairs) in which alpha LFPg was phasic with HGP in at least 1 thalamic and cortical channel. These channel pairs gave us the opportunity to examine average HGP in both the thalamus and cortex at different tha- lamic alpha phases. Unlike the relative LFPg phase, which is uninterpretable in the thalamus due to its nonlaminar structure, differences in mean HGP with respect to the alpha LFPg phase can be interpreted as lags of putative population spiking activity (25). PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23775 Halgren et al. Results Each color signifies a different patient. coherence (189 of 362) (27). This analysis was largely equivocal, but weakly favored cortical leading thalamic alpha (SI Appendix, Fig. S9 and SI Methods). g ) To further determine whether cortical or thalamic activity was driving these rhythms, we extracted high gamma power (HGP), a rough proxy for neural firing, in both structures (n = 5; 70 to 120 Hz in Pts. S1 to S3 due to a low sampling rate, 70–190 Hz in Pts. S8 and S9; Pts. S4 to S7 were excluded due to low sampling rates; note that using the same 70- to 120-Hz HGP band in Pts. S8 and S9 didn’t substantially change the results; Materials and Methods). If a given structure is generating alpha oscillations (and if local HGP reflects neural firing), its HGP should be synchronous with its alpha-band LFPg and exhibit phase-amplitude coupling (PAC) (28). PAC was assessed by using 2 methods: Tort’s Modulation Index (MI) (29) and the coherence between the time-domain LFPg and HGP (30) (Fig. 4E and SI Appendix, Fig. S10 and SI Methods). To ensure that this PAC wasn’t spuriously driven by sharp waveforms (31), we measured the sharpness ratio (SR; SI Appendix, SI Methods) of each channel’s alpha and then measured the correlation of this with the strength of each channel’s PAC Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 To determine which cortical layers generate the alpha rhythm, we utilized laminar microelectrodes (35) in the occipital, tem- poral, and parietal cortex to record current-source density (CSD; n = 3), HGP, and multiunit activity (MUA) (n = 2) across gray- matter layers during quiet wakefulness (11.32 ± 0.48 min, mean ± SD) (35) (Fig. 5) (laminar Pts. L1 to L3). The CSD is the second spatial derivative of the monopolar field potential, which yields a volume-conduction-free measure of local transmembrane Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on J PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23775 Halgren et al. x e d n I C G -250 5 2 1 0 5 2 5 2 1 - 0 Time Relative to Thal. Results Alpha Peak (ms) B 2 5 10 15 20 5 7 10 13 15 20 5 7 10 1315 20 5 7 10 1315 20 5 7 10 13 15 20 12 0 4 8 20 0 10 15 5 12 0 4 8 8 0 4 6 2 Pt. S3 Pt. S4 Pt. S5 Pt. S8 E C Cortex -> Thalamus Thalamus -> Cortex Coherence of Ctx LFPg Thal HGP Ctx HGP F Frequency (Hz) r e w o P . m r o N ). u . a ( . p m A Coherence of Thal LFPg Thal HGP Ctx HGP 0 0.2 0.4 0.6 0.8 1.0 2 5 10 15 20 Frequency (Hz) Granger Causality between... Ctx LFPg Thal HGP Ctx HGP Thal LFPg Timelocked to Thal. Alpha Peaks Ctx LFPg with... Thal LFPg with... G 7 2 20 15 10 5 13 .085 .12 Frequency (Hz) .05 D A - 8 8 Cortical HGP precedes Thalamic HGP - 2 2 - 0 Thalamic Alpha Phase (radians) 4 0 4- Normalized HGP (z-score) 0 0 7 0 5 3 0 5 3 - 0 -700 0 5 10 Lag/Lead Time (ms) 0 0.2 0.4 0.6 0.8 1.0 # of chanel pairs Thalamus -> Cortex Cortex -> Thalamus Fig. 4. Cortical alpha leads thalamic (pulvinar) alpha. (A) Average alpha-phase lag/leads in bipolar contacts (n = 5). Note that anterosuperior channels lead inferoposterior ones, in accord with our ECoG recordings. (B) Power spectra of the thalamic (color) and cortical (gray) channel with the greatest alpha power. (C) The difference in start times between all cortical and thalamic alpha bursts (start time in thalamus to start time in cortex) in the 28 channel pairs with a significant thalamic or cortical lead (P < 0.05, Bonferroni corrected, binomial test). Alpha bursts start (on average) in cortex for all 28 channel pairs. (D) Cortical and thalamic LFPg and HGP from representative channels locked to peaks in thalamic alpha LFPg—cortical, but not thalamic, HGP is phasic with thalamic LFPg. (E) Coherence spectra of thalamic and cortical LFPg with thalamic and cortical HGP and LFPg from the same channel pair in D; the coherence of thalamic LFPg with cortical HGP (but not thalamic HGP) suggests that the cortex may drive thalamic alpha activity. Results (F) Normalized thalamic and cortical HGP at different thalamic alpha phases averaged across channels; note that cortical HGP slightly leads thalamic HGP. (G) GC spectra averaged over all thalamocortical contact pairs; corticothalamic causality shows a strong alpha peak. Amp., amplitude; a.u., arbitrary units; ctx, cortical; norm., normalized; thal, thalamic. x e d n I C G -250 5 2 1 0 5 2 5 2 1 - 0 Time Relative to Thal. Alpha Peak (ms) B 2 5 10 15 20 5 7 10 13 15 20 5 7 10 1315 20 5 7 10 1315 20 5 7 10 13 15 20 12 0 4 8 20 0 10 15 5 12 0 4 8 8 0 4 6 2 Pt. S3 Pt. S4 Pt. S5 Pt. S8 E C Cortex -> Thalamus Thalamus -> Cortex Coherence of Ctx LFPg Thal HGP Ctx HGP F Frequency (Hz) r e w o P . m r o N ). u . a ( . p m A Coherence of Thal LFPg Thal HGP Ctx HGP 0 0.2 0.4 0.6 0.8 1.0 2 5 10 15 20 Frequency (Hz) Granger Causality between... Ctx LFPg Thal HGP Ctx HGP Thal LFPg Timelocked to Thal. Alpha Peaks Ctx LFPg with... Thal LFPg with... G 7 2 20 15 10 5 13 .085 .12 Frequency (Hz) .05 D A - 8 8 Cortical HGP precedes Thalamic HGP - 2 2 - 0 Thalamic Alpha Phase (radians) 4 0 4- Normalized HGP (z-score) 0 0 7 0 5 3 0 5 3 - 0 -700 0 5 10 Lag/Lead Time (ms) 0 0.2 0.4 0.6 0.8 1.0 # of chanel pairs Thalamus -> Cortex Cortex -> Thalamus A Fig. 4. Cortical alpha leads thalamic (pulvinar) alpha. (A) Average alpha-phase lag/leads in bipolar contacts (n = 5). Note that anterosuperior channels lead inferoposterior ones, in accord with our ECoG recordings. (B) Power spectra of the thalamic (color) and cortical (gray) channel with the greatest alpha power. (C) The difference in start times between all cortical and thalamic alpha bursts (start time in thalamus to start time in cortex) in the 28 channel pairs with a significant thalamic or cortical lead (P < 0.05, Bonferroni corrected, binomial test). Alpha bursts start (on average) in cortex for all 28 channel pairs. 23776 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 Results (D) Cortical and thalamic LFPg and HGP from representative channels locked to peaks in thalamic alpha LFPg—cortical, but not thalamic, HGP is phasic with thalamic LFPg. (E) Coherence spectra of thalamic and cortical LFPg with thalamic and cortical HGP and LFPg from the same channel pair in D; the coherence of thalamic LFPg with cortical HGP (but not thalamic HGP) suggests that the cortex may drive thalamic alpha activity. (F) Normalized thalamic and cortical HGP at different thalamic alpha phases averaged across channels; note that cortical HGP slightly leads thalamic HGP. (G) GC spectra averaged over all thalamocortical contact pairs; corticothalamic causality shows a strong alpha peak. Amp., amplitude; a.u., arbitrary units; ctx, cortical; norm., normalized; thal, thalamic. currents surrounding the laminar probe (35, 36). MUA [filtered online at 200 to 5,000 Hz, then filtered offline at 300 to 3,000 Hz and rectified (35)] and HGP (filtered offline at 70 to 190 Hz) are also spatially focal and reflect neural firing (37). By quantifying both transmembrane currents [which generally reflect postsynaptic events (38)] and firing within each cortical layer, we can determine which laminae generate the alpha currents and firing measured extracortically with ECoG, magnetoencephalography, and EEG (36, 39–41). Similar to our previous analyses, we only utilized artifact-free 2-s epochs with the 20% most alpha-band power. Despite being recorded from various regions of cortex, alpha-band currents in all patients were strongest within superficial cortical 0.5 mm I II III IV V VI source ECoG 500 ms sink Laminar I II III IV V VI no histology Pt. L3 I II III IV V VI I II III IV V VI 2 B 3 A 1 Pt. L2 Pt. L1 supramarginal gyrus posterior superior temporal gyrus lip of lateral occipital sulcus Fig. 5. Laminar recordings of cortical alpha. (A) Nissl stains of the explanted tissue surrounding the laminar probe in Pts. L1 and L3, in addition to repre- sentative laminar CSD traces from each layer in each subject. Note that despite being made in distinct cortical locations, alpha oscillations were always strongest in layer I/II. Furthermore, in L3, the trace of a simultaneously recorded overlying ECoG contact is near identical to the underlying laminar layer I. (B) Locations of each laminar probe in all patients. Adapted with permission from ref. 86. Results more HGP less HGP 0 15 I II III V VI IV V I I V V III II I I II III IV V VI I II III IV V VI 0 250 -250 -125 125 V I I V V III II I I II III V VI IV 2550 1950 1350 750 150 0 Normalized Power Time Relative to Alpha Sink (ms) Pt. L1 Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Fig. 6. Alpha CSD and HGP are maximal in supragranular cortex. (A) Average CSD and HGP waveforms of a single channel on the same time axes as B (±SEM across alpha sinks). (B and C) CSD (B) and HGP (C) averaged on current sinks in channels 3 and 6 in Pts. L2 and L3, respectively; white and black dashed lines indicate layer IV boundaries and the time of the alpha sink, respectively. (D) Z score of the MI be- tween alpha phase and HGP across all channels (Ch.). (E) Average alpha power throughout the cortical depth (±SEM across epochs). (F) Power spectra of the channel with greatest alpha power in each subject (±SEM across epochs). C Time Relative to Alpha Sink (ms) C 0 250 -250 -125 125 HGP Locked to Alpha Sinks more HGP less HGP I II III IV V VI I II III IV V VI 0 250 -250 -125 125 Time Relative to Alpha Sink (ms) 250 125 0 250 125 NEUROSCIENCE D Tort's Modulatio . h C d e t alu d o M - P G H Phase Providing Ch. V I I V V III II I I II III V VI IV D Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H Phase Providing Ch. Phase Providing Ch. 0 15 I II III V VI IV V I I V V III II I V I I V V III II I I II III V VI IV D . n Index (z-score) Phase Providing Ch. 0 15 I II III V VI IV V I I V V III II I g p p Lastly, we tested if the traveling alpha waves recorded with ECoG corresponded to the supragranular alpha oscillations recorded with laminar probes. To demonstrate this, we made combined ECoG-laminar recordings in Pt. Results Consequently, this sink–source configura- tion comports with previous studies (39, 43) reporting that firing is maximal during the surface-negative trough of the alpha rhythm and maximal during its surface-positive peak. that alpha-band firing is located in layers I through III. Interestingly, while HGP was modulated by alpha throughout layers I through III, significant MUA modulation was restricted to layer III (Fig. 7). It’s not clear whether this reflects differential sensitivity to noise or suggests that MUA and HGP have divergent neural generators. In accord with the latter hypothesis, recent laminar recordings found that HGP was driven by dendritic processes as well as spiking, consistent with our HGP modulation being superficial to MUA (the latter reflecting firing at the soma) (42). Despite this difference, the MUA and HGP profiles averaged across all laminar chan- nels and triggered on alpha troughs were highly similar (Pt. L2: r = 0.6756, P = 0; Pt. L3: r = 0.4493, P = 0), and both measures A B imply that the firing of supragranular (and not infragranular) pyramidal cells are phasic with the human alpha rhythm. Fur- thermore, this supragranular firing was maximal during very superficial sinks and minimal during superficial sources, con- sistent with active synaptic and/or voltage-gated currents in layers I/II. The sink-over-source current dipole associated with increased firing would be recorded with ECoG/(M)EEG as surface-negative, as the negative end of the dipole (a sink of current flowing away from the extracellular space) is closer to the surface electrode. Consequently, this sink–source configura- tion comports with previous studies (39, 43) reporting that firing is maximal during the surface-negative trough of the alpha rhythm and maximal during its surface-positive peak. ( h t p e D la citr o C μ ) m Time Relative to Alpha Sink (ms) Frequency (Hz) C D E F 15 10 5 0 3150 8 4 0 12 0 250 -250 -125 125 CSD Power Spectra 12 8 4 16 24 20 Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H Phase Providing Ch. Alpha Power vs. Depth HGP Locked to Alpha Sinks Phase Providing Ch. Results 0 250 -250 -125 125 0 250 -250 -125 125 Time Relative to Alpha Sink (ms) T t' M d l ti I d ( ) MUA Locked to Alpha Sinks more MUA less MUA A B Time Relative to Alpha Sink (ms) Patient 2 Patient 3 CSD CSD A Fig. 6. Alpha CSD and HGP are maximal in supragranular cortex. (A) Average CSD and HGP waveforms of a single channel on the same time axes as B (±SEM across alpha sinks). (B and C) CSD (B) and HGP (C) averaged on current sinks in channels 3 and 6 in Pts. L2 and L3, respectively; white and black dashed lines indicate layer IV boundaries and the time of the alpha sink, respectively. (D) Z score of the MI be- tween alpha phase and HGP across all channels (Ch.). (E) Average alpha power throughout the cortical depth (±SEM across epochs). (F) Power spectra of the channel with greatest alpha power in each subject (±SEM across epochs). II III I II III V VI IV I VI Time Relative to Alpha Sink (ms) Tort's Modul . h C d e t alu d o M - P G H IV V Phase Providing Ch. B B I II III VI I II III V VI IV ion Index (z-score) 0 15 . h C d e t alu d o M - P G H V IV Phase Providing Ch. Z-score Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 layers (Pt. L1: P < 2.27 × 10−25; Pt. L2: P < 1.9 × 10−22; Pt. L3: P < 1.66 × 10−5; largest P values of Wilcoxon sign rank comparing mean alpha power in supragranular versus granular and infra- granular channels across epochs, Bonferroni corrected) (Figs. 5 and 6 B and E and SI Appendix, Fig. S11). II III IV V Phase Providing Ch. II Phase Providing Ch. Fig. 7. MUA is modulated in layer III. (A) MUA averaged on current sinks on the channels with the greatest alpha power (channels 3 and 6, respectively), which is most clearly modulated within lower layer III. (B) Tort’s MI between alpha CSD phase and MUA amplitude over all laminar contact pairs—note that firing is correlated with alpha phase in both superficial and deep cortex. Ch., channel. Averaging HGP on alpha current sinks as well as measuring Tort’s MI between alpha CSD and HGP (Fig. Results (F) Power spectra of the channel with greatest alpha power in each subject (±SEM across epochs). II III I II III VI 0 250 -250 -125 125 0 250 -250 -125 125 I II III V VI IV I II III V VI IV I VI Time Relative to Alpha Sink (ms) Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H IV V Phase Providing Ch. MUA Locked to Alpha Sinks more MUA less MUA 0 15 . h C d e t alu d o M - P G H V IV Phase Providing Ch. Z-score A B Time Relative to Alpha Sink (ms) Patient 2 Patient 3 CSD CSD Fig. 7. MUA is modulated in layer III. (A) MUA averaged on current sinks on the channels with the greatest alpha power (channels 3 and 6, respectively), which is most clearly modulated within lower layer III. (B) Tort’s MI between alpha CSD phase and MUA amplitude over all laminar contact pairs—note that firing is correlated with alpha phase in both superficial and deep cortex. Ch., channel. II III I II III VI 0 250 -250 -125 125 0 250 -250 -125 125 I II III V VI IV I II III V VI IV I VI Time Relative to Alpha Sink (ms) Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H IV V Phase Providing Ch. MUA Locked to Alpha Sinks more MUA less MUA 0 15 . h C d e t alu d o M - P G H V IV Phase Providing Ch. Z-score A B Time Relative to Alpha Sink (ms) Patient 2 Patient 3 CSD CSD Fig. 7. MUA is modulated in layer III. (A) MUA averaged on current sinks on the channels with the greatest alpha power (channels 3 and 6, respectively), which is most clearly modulated within lower layer III. (B) Tort’s MI between alpha CSD phase and MUA amplitude over all laminar contact pairs—note that firing is correlated with alpha phase in both superficial and deep cortex. Ch., channel. Results (B and C) CSD (B) and HGP (C) averaged on current sinks in channels 3 and 6 in Pts. L2 and L3, respectively; white and black dashed lines indicate layer IV boundaries and the time of the alpha sink, respectively. (D) Z score of the MI be- tween alpha phase and HGP across all channels (Ch.). (E) Average alpha power throughout the cortical depth (±SEM across epochs). (F) Power spectra of the channel with greatest alpha power in each subject (±SEM across epochs). II III 0 -250 -125 I II III V VI IV I Time Relative to Alpha To . h C d e t alu d o M - P G H IV Phase Providing A B Patient 2 Fig. 7. MUA is modula the channels with the g Cortical Layer A B I II III IV V VI 0 250 -250 -125 125 HGP CSD CSD Locked to Alpha Sinks sink source I II III IV V VI 0 250 -250 -125 125 Time Relative to Alpha Sink (ms) Pt. L2 Pt. L3 that alpha-band firing is located in layers I through III. Interestingly, while HGP was modulated by alpha throughout layers I through III, significant MUA modulation was restricted to layer III (Fig. 7). It’s not clear whether this reflects differential sensitivity to noise or suggests that MUA and HGP have divergent neural generators. In accord with the latter hypothesis, recent laminar recordings found that HGP was driven by dendritic processes as well as spiking, consistent with our HGP modulation being superficial to MUA (the latter reflecting firing at the soma) (42). Despite this difference, the MUA and HGP profiles averaged across all laminar chan- nels and triggered on alpha troughs were highly similar (Pt. L2: r = 0.6756, P = 0; Pt. L3: r = 0.4493, P = 0), and both measures imply that the firing of supragranular (and not infragranular) pyramidal cells are phasic with the human alpha rhythm. Fur- thermore, this supragranular firing was maximal during very superficial sinks and minimal during superficial sources, con- sistent with active synaptic and/or voltage-gated currents in layers I/II. The sink-over-source current dipole associated with increased firing would be recorded with ECoG/(M)EEG as surface-negative, as the negative end of the dipole (a sink of current flowing away from the extracellular space) is closer to the surface electrode. Results rce k ar B 3 supramarginal gyrus posterior superior temporal gyrus B A posterior superior temporal gyrus 2 1 posterior superior temporal gyrus lip of lateral occipital sulcus I II III IV V VI no histology I II III IV V VI Pt. L2 Pt. L1 Fig. 5. Laminar recordings of cortical alpha. (A) Nissl stains of the explanted tissue surrounding the laminar probe in Pts. L1 and L3, in addition to repre- sentative laminar CSD traces from each layer in each subject. Note that despite being made in distinct cortical locations, alpha oscillations were always strongest in layer I/II. Furthermore, in L3, the trace of a simultaneously recorded overlying ECoG contact is near identical to the underlying laminar layer I. (B) Locations of each laminar probe in all patients. Adapted with permission from ref. 86. 23776 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 Halgren et al. ( h t p e D la citr o C μ ) m Cortical Layer Time Relative to Alpha Sink (ms) Frequency (Hz) A B C D E F 15 10 5 I II III IV V VI 0 3150 8 4 0 12 0 250 -250 -125 125 0 250 -250 -125 125 CSD Power Spectra 12 8 4 16 24 20 HGP CSD Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H Phase Providing Ch. Alpha Power vs. Depth CSD Locked to Alpha Sinks HGP Locked to Alpha Sinks sink source Phase Providing Ch. more HGP less HGP 0 15 I II III V VI IV V I I V V III II I I II III IV V VI 0 250 -250 -125 125 I II III IV V VI I II III IV V VI 0 250 -250 -125 125 V I I V V III II I I II III V VI IV 2550 1950 1350 750 150 0 Normalized Power Time Relative to Alpha Sink (ms) Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Fig. 6. Alpha CSD and HGP are maximal in supragranular cortex. (A) Average CSD layers (Pt. L1: P < 2.27 × 10−25; Pt. L2: P < 1.9 × 10−22; Pt. Results L3: P < 1.66 × 10−5; largest P values of Wilcoxon sign rank comparing mean alpha power in supragranular versus granular and infra- l h l h B f i d) (Fi 5 that alpha-band firin while HGP was mod significant MUA mo not clear whether t suggests that MUA accord with the latte that HGP was driv consistent with our H latter reflecting firi the MUA and HG nels and triggered o r = 0.6756, P = 0; P imply that the firin pyramidal cells are thermore, this sup superficial sinks an sistent with active layers I/II. The sink increased firing w surface-negative, a current flowing aw the surface electro tion comports with p maximal during the and maximal during Lastly, we tested ECoG corresponde recorded with lami combined ECoG-la alpha rhythm at bo (44) (Fig. 8). Robu rhythm given the el ECoG from perir implanted laminar recordings exhibited averaging the lami strong supragranula gated as a traveling similar median spe cortex. If the suprag ( h t p e D la citr o C μ ) m Cortical Layer Time Relative to Alpha Sink (ms) Frequency (Hz) A B C D E F 15 10 5 I II III IV V VI 0 3150 8 4 0 12 0 250 -250 -125 125 0 250 -250 -125 125 CSD Power Spectra 12 8 4 16 24 20 HGP CSD Tort's Modulation Index (z-score) . h C d e t alu d o M - P G H Phase Providing Ch. Alpha Power vs. Depth CSD Locked to Alpha Sinks HGP Locked to Alpha Sinks sink source Phase Providing Ch. more HGP less HGP 0 15 I II III V VI IV V I I V V III II I I II III IV V VI 0 250 -250 -125 125 I II III IV V VI I II III IV V VI 0 250 -250 -125 125 V I I V V III II I I II III V VI IV 2550 1950 1350 750 150 0 Normalized Power Time Relative to Alpha Sink (ms) Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Fig. 6. Alpha CSD and HGP are maximal in supragranular cortex. (A) Average CSD and HGP waveforms of a single channel on the same time axes as B (±SEM across alpha sinks). Halgren et al. Results L3 to record the same alpha rhythm at both local (laminar) and global (ECoG) scales (44) (Fig. 8). Robust alpha oscillations [likely the classical mu rhythm given the electrodes’ locations (45)] were measured with ECoG from perirolandic cortex and with a simultaneously implanted laminar probe from the supramarginal gyrus; both recordings exhibited nearly identical alpha peaks (Fig. 8E), and averaging the laminar CSD on ECoG alpha troughs revealed strong supragranular sinks and sources (Fig. 8F). Alpha propa- gated as a traveling wave throughout the grid (Fig. 8 A–D) with a similar median speed (0.62 m/s) to traveling waves in posterior cortex. If the supragranular alpha measured with laminar probes ( h t p e D la citr o C μ ) m E 1 10 5 0 3150 Alpha Power vs. Depth 2550 1950 1350 750 150 Pt. L1 Pt. L2 Pt. L3 E F Frequency (Hz) F 15 8 4 0 12 CSD Power Spectra 12 8 4 16 24 20 h 0 Normalized Power Pt. L1 Pt. L2 Pt. L3 Alpha Power vs. Depth layers (Pt. L1: P < 2.27 × 10−25; Pt. L2: P < 1.9 × 10−22; Pt. L3: P < 1.66 × 10−5; largest P values of Wilcoxon sign rank comparing mean alpha power in supragranular versus granular and infra- granular channels across epochs, Bonferroni corrected) (Figs. 5 and 6 B and E and SI Appendix, Fig. S11). Averaging HGP on alpha current sinks as well as measuring Tort’s MI between alpha CSD and HGP (Fig. 6 B–D) suggested h t p e D la citr o C Frequency (Hz) 3150 4 0 12 8 4 16 24 20 2550 1950 1350 0 Normalized P Pt. L1 Pt. L2 Pt. L3 Pt. L1 Pt. L2 Pt. L3 Fig. 6. Alpha CSD and HGP are maximal in supragranular cortex. (A) Average CSD and HGP waveforms of a single channel on the same time axes as B (±SEM across alpha sinks). (B and C) CSD (B) and HGP (C) averaged on current sinks in channels 3 and 6 in Pts. L2 and L3, respectively; white and black dashed lines indicate layer IV boundaries and the time of the alpha sink, respectively. (D) Z score of the MI be- tween alpha phase and HGP across all channels (Ch.). (E) Average alpha power throughout the cortical depth (±SEM across epochs). Results 100 ms SG G IG minar (layer I CSD) sink source -250 -125 0 5 2 1 0 5 2 CSD Triggered on ECoG Alpha Troughs Wave Starts Wave Ends Posterior ECoG Anterior ECoG B F ECoG I II III IV V VI Time Relative to ECoG Alpha Trough (ms) 8 12 16 20 Laminar (layer I CSD) ECoG power spectrum Frequency (Hz) power spectrum r e y a L la citr o C Wave Starts Posterior ECoG Wave Ends Anterior ECoG C A B C D E F sink source -250 -125 0 5 2 1 0 5 2 E CSD Triggered on ECoG Alpha Troughs F ECoG I II III IV V VI Time Relative to ECoG Alpha Trough (ms) 4 8 12 16 20 Laminar (layer I CSD) ECoG power spectrum ( ) Frequency (Hz) power spectrum r e y a L la citr o C Fig. 8. Simultaneous ECoG–laminar recordings reveal traveling alpha waves which propagate through supragranular cortex. (A) Average circular distance of each ECoG (circles) and layer I laminar (diamond) contact’s alpha phase from the spatial mean phase throughout the ECoG grid. Note that the laminar’s alpha phase is intermediate to neighboring ECoG contacts, suggesting that ECoG and the laminar probe recording the same traveling wave at different scales. (B) Representative drawing of a traveling alpha wave (as measured with ECoG) propagating through superficial layers (as measured by a laminar probe). (C) Example traces from ECoG contacts posterior (red) and anterior (blue) to the laminar probe. Alpha phase in the laminar is intermediary to the ECoG contacts. (D) Distribution of traveling wave directions; mu waves propagate from posterior (higher-order) toward anterior (lower-order) cortex. (E) Power spectra from simultaneous laminar and ECoG recordings; they share a near-identical alpha peak. (F) Laminar CSD averaged on troughs in the nearest ECoG contact. Note that alpha activity is superficial. underlies the traveling alpha waves recorded with ECoG, we should be able to observe traveling alpha waves propagating through the supragranular layers of the laminar probe with a phase between its neighboring ECoG contacts (Fig. 8C). To establish this, we measured the average phase advance/delay of the CSD in the laminar’s most superficial channel with respect to the grid’s mean phase and found a phase lag intermediate to that of neighboring ECoG contacts (Fig. Results 8A; this was also demon- strated by measuring the phase of the coherence between lami- nar and ECoG in SI Appendix, Fig. S13). posterior toward the occipital pole), the reversed propagation direction is consistent with alpha propagating from higher- to lower-order cortex in both systems, as associative (higher-order) somatosensory areas are posterior to primary somatosensory cortex (46–48). Future studies should record somatosensory al- pha from more patients to confirm that this effect is robust. 23778 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 Results 6 B–D) suggested PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23777 Lam E superior inferior posterior anterior 2 0 2 A D 2 4 6 2 0 4 Power (a.u.) Propagation Direction - 4 4 100 ms SG G IG Laminar (layer I CSD) sink source -250 -125 0 5 2 1 0 5 2 E superior inferior posterior anterior 2 0 2 CSD Triggered on ECoG Alpha Troughs Wave Starts Wave Ends Posterior ECoG Anterior ECoG A B D F 2 4 6 2 0 ECoG I II III IV V VI Time Relative to ECoG Alpha Trough (ms) 4 8 12 16 20 Laminar (layer I CSD) ECoG power spectrum Power (a.u.) Frequency (Hz) power spectrum r e y a L la citr o C Wave Starts Posterior ECoG Wave Ends Anterior ECoG C Propagation Direction - 4 4 Fig. 8. Simultaneous ECoG–laminar recordings reveal traveling alpha waves which propagate through supragranular cortex. (A) Average circular distance of each ECoG (circles) and layer I laminar (diamond) contact’s alpha phase from the spatial mean phase throughout the ECoG grid. Note that the laminar’s alpha phase is intermediate to neighboring ECoG contacts, suggesting that ECoG and the laminar probe recording the same traveling wave at different scales. (B) Representative drawing of a traveling alpha wave (as measured with ECoG) propagating through superficial layers (as measured by a laminar probe). (C) Example traces from ECoG contacts posterior (red) and anterior (blue) to the laminar probe. Alpha phase in the laminar is intermediary to the ECoG contacts. (D) Distribution of traveling wave directions; mu waves propagate from posterior (higher-order) toward anterior (lower-order) cortex. (E) Power spectra from simultaneous laminar and ECoG recordings; they share a near-identical alpha peak. (F) Laminar CSD averaged on troughs in the nearest ECoG contact. Note that alpha activity is superficial. Discussion How does this compare to the cortical generation of other rhythms? Slow waves, delta and theta in humans, are also strongest in superficial layers (65–67); this may indicate that, in humans, supragranular cortex plays a privileged role in sustaining low-frequency oscil- latory activity. Laminar recordings made in homologous areas and states across species will be necessary to determine if this is unique to humans, extends to primates, or is true across species. An important limitation of our study is that only 1 of our laminar NEUROSCIENCE ( ) Simultaneous recordings from human cortex and pulvinar provided several measures suggesting that cortical alpha leads thalamic alpha during quiet wakefulness. Thalamic alpha was less common than cortical alpha, and discrete alpha bursts in cortex led alpha bursts in the thalamus. Furthermore, thalamic alpha LFPg was synchronous with cortical HGP (putative firing) more often than thalamic HGP; when thalamic alpha was syn- chronous with both cortical and thalamic HGP, HGP in the cortex led HGP in the thalamus. However, because HGP is an imperfect proxy for spiking (33, 42), this finding must be con- firmed with single unit recordings in animal models. Finally, thalamic alpha was Granger caused by cortical alpha. A potential weakness of these bivariate causal analyses is the possibility of a third structure (not recorded from) driving alpha oscillations in both pulvinar and posterior cortex, such as the LGN (10). While this is a possibility, the LGN is an unlikely cortical pacemaker, as its major projections are limited to striate and circumstriate cortex (23), and we found that alpha oscilla- tions propagate toward (and not from) the occipital pole. Be- cause the LGN and pulvinar are the 2 thalamic nuclei with the most robust projections to posterior cortex capable of driving visual alpha, these findings agree with strong cortical influences on thalamic (pulvinar) alpha during quiet wakefulness (though this conclusion only follows for posterior/visual alpha). While this appears to contradict animal studies in which the pulvinar drove cortical alpha (4), it’s consonant with findings that the cortex can still generate alpha in vitro (14) and actually shows increased alpha-band power when the pulvinar is inactivated (24), as well as alpha coherence within the cortex exceeding thalamocortical alpha coherence (55). These findings can be reconciled with other studies supporting thalamic alpha gener- ators (4, 56) in a number of ways. Discussion Alpha also propa- gated from associative (posterior) to primary (anterior) areas within somatosensory cortex, leading to a reversed physical propagation direction but the same hierarchical one. Interestingly, previous scalp studies of human traveling alpha waves have also found varying propagation directions (2, 49), in contrast to the consistent an- terior–posterior directionality we observed. This may be due to scalp recordings of alpha reflecting a volume-conducted mixture of traveling alpha waves with different directions traversing dis- tinct cortical hierarchies, consonant with our demonstration of posterior-to-anterior alpha propagation in somatosensory cortex. Alternatively, propagation direction might change with task or behavioral state (49, 50); our macaque analysis provided some evidence for this, as eye opening induced a clear (though much smaller) peak of directional propagation from posterior to an- terior areas (SI Appendix, Fig. S4). Recent studies which used ECoG to study alpha traveling waves also appeared to find that alpha within posterior cortex generally propagates toward the occipital pole, although they do not explicitly draw this conclusion (43, 51). That their propagation directions were less consistent could be due to differences in state, as much of our data were collected during eye closure (which greatly increases alpha power), whereas theirs was collected during tasks. Alpha traveling waves might serve as a mechanism to internally scan the attentional field, tag distinct visual features with different phases (52), or facilitate plasticity between upstream and downstream areas (53, 54). matrix projections target superficial cortical layers (59) (in which we found alpha power to be strongest). Nuclei besides pulvinar or LGN, and particularly those with diffuse projections such as the medial dorsal or ventromedial nuclei, could also be involved in generating visual cortical alpha (60). Though direct measure- ments and causal manipulation of spiking activity in animal models are needed to prove this, the simplest explanation for our findings is a leading role for the cortex driving alpha in the pulvinar during quiet wakefulness. Functionally, corticothalamic alpha might inhibit the thalamus to gate feed-forward processing and suppress irrelevant neural assemblies akin to its putative role within the cortex (7), as low-frequency corticothalamic activity can inhibit thalamic firing (61). Laminar microelectrode recordings demonstrate that alpha oscillations reflect layer I/II currents [postsynaptic (38)] and layer I through III firing (presynaptic), demonstrating that supragranular layers are the source of alpha LFPs and HGP recorded via ECoG and (M)EEG. Discussion However, inferring the neural circuit mechanisms which generate the alpha rhythm from our laminar recordings is more complex. As supragranular pyramidal cells (which is where we observed alpha-phasic firing) are known to make feedback projections to layers I/II (where we recorded driving currents), our recordings support layer II/III pyramidal cells as the primary alpha generators within the cortex during quiet wakefulness. Our ECoG recordings also support this, as the short-range feedback projections subserved by supragranular pyramidal cells (62) are a likely intracortical mechanism for me- diating the continuously propagating top-down traveling waves measured by using ECoG. These projections would enable os- cillations which propagate continuously from high- to low-order cortex (i.e., not in the saltatory manner that might be expected if mediated by long-range feedback) at <1 m/s [the conduction velocity of intracortical fibers (63)] (Fig. 9). While most models posit that layer V (infragranular) pyramidal cells drive alpha within the cortex (13, 14), these are based mostly on monopolar LFP recordings (39, 64), which (unlike CSD) are prone to vol- ume conduction from deep sources. Some studies in macaque cortex appear to circumvent this by reporting significant alpha- band CSD-MUA coupling in deep layers of V1, V2, and V4 (41). However, it should be noted that these studies did not report CSD alpha power across the cortical depth [unlike another study which found that supragranular cortex had the most alpha power in each macaque primary sensory area (39)], and prior to cal- culating their directional measures, they aligned their data to peaks/troughs in the channel with the most monopolar LFP al- pha power. As this channel was likely infragranular [volume conduction leads to monopolar alpha power being spuriously maximal in deeper cortex (39)], this may have biased their results toward granular/infragranular generators. Importantly, CSD al- pha power being greatest in superficial layers is consistent with alpha-reflecting currents on the apical dendrites of supragranular or infragranular pyramidal cells; our paper resolves this ambiguity by measuring the coherence between alpha currents and MUA throughout the cortical depth and finding significant modulation of only supragranular firing by alpha currents [though a study in macaques did report modulation of granular MUA by superficial alpha CSD (39)]. Further work employing causal manipulation of infragranular cortex in animal models, as well as controlling for behavioral state and eye closure, will be needed to determine the role of deeper layers in alpha generation. Discussion Our results suggest that alpha contributes to feedback processing within and across brain regions and structures (Fig. 9). The an- atomical propagation of posterior alpha traveling waves from anterosuperior to posteroinferior cortex implies a functional pro- gression from higher-order to lower-order visual areas, matching Interestingly, the direction of propagation was posterior to anterior (Fig. 8D). While this may appear to conflict with our recordings of posterior alpha (which propagated anterior to Higher order cortex Pulvinar Top-down influences (attention, predictive coding, plasticity, etc.) Lower order cortex SG G IG V3 V2 V1 MT Higher Order Lower Order Travelling Wave A B MT V3 V2 V1 * * * * * * * * * * * * short-range feedback Fig. 9. Tentative model for how alpha’s physiology could mediate feedback. (A) Alpha propagates as a traveling wave from higher-order (middle temporal, visual area 3) toward lower-order visual areas 1/2 cortical areas. (B) Alpha is strongest within supragranular cortex and may carry top-down information via short-range feedback connections to constrain lower-level processing; for instance, alpha may play a role in resolving ambiguous visual imagery, such as the picture of a woman and a horse’s snout shown above. Cortical alpha in layer VI might influence alpha activity within the pulvinar. Reproduced with per- mission from ref. 87, which is licensed under CC BY 4.0. Higher order cortex Pulvinar Top-down influences (attention, predictive coding, plasticity, etc.) Lower order cortex SG G IG V3 V2 V1 MT Higher Order Lower Order Travelling Wave A B MT V3 V2 V1 * * * * * * * * * * * * short-range feedback A B Top-down influences (attention, predictive coding, plasticity, etc.) SG Fig. 9. Tentative model for how alpha’s physiology could mediate feedback. (A) Alpha propagates as a traveling wave from higher-order (middle temporal, visual area 3) toward lower-order visual areas 1/2 cortical areas. (B) Alpha is strongest within supragranular cortex and may carry top-down information via short-range feedback connections to constrain lower-level processing; for instance, alpha may play a role in resolving ambiguous visual imagery, such as the picture of a woman and a horse’s snout shown above. Cortical alpha in layer VI might influence alpha activity within the pulvinar. Reproduced with per- mission from ref. 87, which is licensed under CC BY 4.0. 23778 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 alpha’s putative role as a feedback rhythm (13). Halgren et al. PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23779 Discussion There may be separable tha- lamic and cortical alpha pacemakers which become differentially active and coupled under different behavioral conditions (4, 57)—for instance, this effect may be dependent on eye closure, which was not controlled for in these recordings. Alternatively, the oscillatory circuit required to generate alpha may require both thalamic and cortical cells, or the pulvinar could enable an intracortical alpha pacemaker with tonic (nonrhythmic) excitation/ inhibition without being a direct pacemaker (58), perhaps co- ordinating cortical alpha phase in a task-relevant fashion (4). Diffuse thalamocortical matrix projections might mediate this, as Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on J PNAS \| November 19, 2019 \| vol. 116 \| no. 47 \| 23779 cross-spectral density between x and y and Sxx is the autospectral density of x (74) (SI Appendix, SI Methods). recordings was made from occipital cortex (and therefore reflected the classical posterior alpha rhythm). Despite this, all 3 laminar recordings showed a very similar profile of alpha cur- rents and HGP/MUA (Figs. 5 and 7 and SI Appendix, Fig. S11). Though there are different sensory alphas in the cortex, our work complements other findings which find broadly similar alpha physiology across areas (39, 68, 69). However, careful work will need to be done to tease apart how alpha varies between regions. In all, our microelectrode recordings strongly suggest that cor- tical alpha reflects short-range intracortical feedback mediated by supragranular pyramidal cells within superficial layers. To derive alpha and high-gamma amplitude as well as alpha phase, we used the Hilbert transform. First, data were filtered by using a 2-pass 4th- order IIR Butterworth filter. Then, the analytic signal z(t) was found by ap- plying the Hilbert transform to the filtered signal of each channel. The phase series φ(t) was found by taking the angle of the analytic signal, and the amplitude A(t) of every channel was found by taking the real component of the analytic signal. As a second measure of the alpha rhythm’s effects on neural HGP and MUA, we used Tort’s MI (29) (Figs. 6D and 7B and SI Appendix, Fig. S10B) with a nonparametric trial shuffling procedure to assess significance (SI Appendix, SI Methods). This supports an integrative function for alpha, due to the termination of widespread associative connections in superficial layers (62) and the modulatory role of layer I/II apical dendrites (66, 70). Materials and Methods Traveling wave analyses were performed separately for the strip and grid ECoG arrays in the macaque, as well as the 2 strips in Pt. E1, as the large cortical distances between the electrodes would make phase differences difficult to interpret. Patients. Implantations were performed on patients with pharmacologically resistant epilepsy undergoing surgery to locate and resect seizure foci. Laminar and ECoG recordings were made from hospitals in the United States and Hungary, and thalamocortical depth recordings were performed in France (SI Appendix, Table S1). Seventeen patients (10 female, ages 15 to 50) were informed of potential risks and told that they had no obligation to participate in the study, as well as being informed that their decision to participate wouldn’t affect their clinical care. Experiments were made with fully informed consent as specified by the Declaration of Helsinki and were approved by local institutional review boards (IRBs). These boards included the Partners Health Care IRB, the New York University Medical Center IRB, the Stanford University IRB, the RIKEN Ethics Committee, and the Hungarian Medical Scientific Council. All decisions concerning macroelectrode place- ment were made solely on a clinical basis, whereas laminar microelectrodes were inserted into cortex likely to be resected. We then wished to measure the directionality of these traveling waves. To do this, we employed the phase-gradient ∇, found by using MATLAB’s gradient function (but with subtractions replaced with circular distances). To prove that there was a consistent directionality of propagation across time, at each time point, we found the mean direction of the gradient throughout the grid. Using Rayleigh’s test for nonuniformity demonstrated that each patient had a significant propagation direction (SI Appendix, Fig. S4). To generate Fig. 2D, we binned the traveling-wave directions across time into 100 bins normalized within patients (i.e., divided the count of each bin by the total number of time points) and then averaged across patients (with the error bar being the SEM across patients). To find instantaneous speeds across time, the grid’s instantaneous fre- quency first needed to be estimated. This was done by taking the first de- rivative of the instantaneous alpha phase over time (SI Appendix, SI Methods). Instantaneous speeds were then calculated as follows: Each channel’s instantaneous frequency was divided by the magnitude of its phase gradient ð ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ∇2 x + ∇2 y q Þ, yielding the instantaneous speed at each channel and time point. Discussion A supragranular origin for the alpha rhythm is also in accord with its putative role in neural inhibition (68), as layers I/II contain a dense interneuronal network which strongly inhibits the apical dendrites of excitatory cells throughout the cortical column (71). This short-range inhibition would allow higher-order cortex to modulate the gain of lower-order areas throughout visual cor- tex, providing a laminar circuit for top-down processes such as attention. Further studies which combine cognitive tasks with in- vasive recordings are needed to understand the implications of our findings for alpha’s behavioral role, as the physiology we de- scribe is consistent with a breadth of potential functions for alpha. In all, we find that alpha acts within the nervous system by propagating from cortex to thalamus and higher-order to lower- order cortex, likely via short-range supragranular feedback pro- jections. These intracortical and corticothalamic dynamics could allow alpha to sculpt activity throughout the neural hierarchy. Traveling Waves. We utilized 4.54 ± 0.87 min (mean ± SD) of ECoG recordings made for clinical purposes. These arrays had 2-mm contact diameters and 1-cm intercontact spacing and were referenced to 1 to 4 inactive electrodes placed outside of the dura facing the skull. In Pts. E1, E3, and E4, patients were instructed to open and close their eyes with an audial cue at 15-s in- tervals using Presentation software (Neurobehavioral Systems). We only utilized activity during eye closure in these patients (except for SI Appendix, Fig. S4). In Pts. E2 and E5 (who didn’t participate in the eye-closure task), we analyzed spontaneous activity during quiet wakefulness. We also analyzed 16.5 min of open-source ECoG recordings made from a macaque monkey during an eye-closure task. Eyes were closed via a sleep mask for 10 min, and the sleep mask was then removed for 10 min of data. The macaque was included in group statistics with other patients due to its high similarity with human activity (SI Appendix, Fig. S4). Further details concerning the ma- caque recording can be found at Neurotycho.org (21). Time-domain data in Pts. E1 and E5 were spatially interpolated in missing channels [using inpaint_nans (75)] prior to further analysis. To localize contacts to the pial surface, we aligned a preoperative MRI with a structural MRI or computed tomography (CT). These contact locations were then displayed on the reconstructed cortical surface, created by using Freesurfer (76), of each individual patient (Figs. Discussion 1 and 2) (77, 78). Materials and Methods Then, at each time point, the median speed and frequency across all channels was found, and time points with a speed or frequency in the top or bottom 0.5th percentile were rejected to eliminate outliers. The distribution of these median speeds across time was then plotted as a normalized histogram (bin width of 0.01 m/s) for each patient and presented in SI Appendix, Fig. S4. These normalized histograms were averaged and plotted in Fig. 2C. Patients were numbered according to their modality (E# for ECoG, S# for SEEG/macroelectrode depth, and L# for laminars). Numbering for patients was started anew for each measurement modality, and no patients had more than 1 kind of electrode (ECoG, SEEG, or laminar) analyzed with the exception of L3 (no corresponding ECoG number). All recordings other than those during our eye-closure task were made of spontaneous activity during quiet wakefulness, in which the patient was not engaged in a cognitive task. 23780 \| www.pnas.org/cgi/doi/10.1073/pnas.1913092116 General Analysis Procedures. Recordings were analyzed by using custom MATLAB scripts with the CircStat (72) and Fieldtrip (73) Toolboxes. The data were then subsampled into 2-s artifact-free epochs, and, consistent with ECoG and SEEG, the 20% of epochs with the most alpha-band CSD am- plitude across all channels were utilized for further analysis. In Pt. L1, all artifact- free epochs (not just those with the most alpha amplitude) were used due to a relatively short recording session. We observed no significant modulation of HGP or MUA in Pt. L1, probably due to technical issues with the recording such as gliosis or faulty electrodes. Cortical and thalamic power spectral peaks were found by using the peakfinder (83) algorithm with a selectivity (the minimum difference a local maxima must have from the nearest local minima to be considered a peak) of the power spectrum’s range divided by 5. A channel was considered to have an alpha peak if it had at least 1 peak between 7 and 13 Hz. To phase-align ongoing alpha activity (Fig. 4D), we picked the thalamic contact with the greatest alpha-band power and averaged the rest of our data (cortical and thalamic wide-band LFP and HGP) to alpha-band peaks in this channel. Alpha-band peaks were found by bandpass filtering from 7 to 13 Hz (2-pass 3rd-order Butterworth), taking the angle of the analytic signal (hilbert.m) to find the phase, and then finding peaks in this series. LFP was high-pass filtered at 2 Hz (2-pass 3rd-order Butterworth) prior to averaging on alpha peaks. NEUROSCIENCE CSD was measured by taking the first spatial derivative of the LFPg (in effect, the second spatial derivative of the monopolar field potential) and then applying a 5-point Hamming filter (35, 36). The Vaknin approximation (adding pseudochannels of zeros to the LFPg above and below the array) was used to estimate the CSD on the second-most deep and superficial channels of the laminar probe (84). HGP was derived by filtering from 70 to 120 Hz in Pts. S1 to S3 (due to a Nyquist frequency of 128 Hz) and 70 to 190 Hz in Pts. S8 and S9. The sampling rate in Pts. S4 to S7 (128 Hz) was too low to measure HGP. Although the former frequency band was somewhat lower than the usual definition of HGP (70 to 190 Hz), we observed similar results in patients with both usual and reduced HGP bands (SI Appendix, Fig. S10). General Analysis Procedures. Recordings were analyzed by using custom MATLAB scripts with the CircStat (72) and Fieldtrip (73) Toolboxes. Further use of contact, channel, or site all refer to these bipolar channel pairs. Recordings were made at 256 Hz in Pts. S1 to S3, 128 Hz in Pts. S4 to S7, and 1,024 Hz in Pts. S8 and S9. For further details, see ref. 81. Laminar Recordings. Laminar microelectrodes were implanted perpendicular to the cortex in noneloquent tissue that was ultimately resected (35) (SI Appendix, SI Methods). Each laminar probe spanned the cortical depth with a length of 3.5 mm and diameter of 0.35 mm. Contacts had 40-μm diameters, spaced at 175 μm in Pt. L1 and 150 μm in Pts. L2 and L3. Recordings were made during 11.32 ± 0.48 min (mean ± SD) of quiet wakefulness. We analyzed spontaneous activity (36 ± 7.5 min, mean ± SD) during wakefulness prior to the onset of sleep, the time of which was determined behaviorally as well as electrographically by a qualified sleep stager using standard methods (82). The last 3 min of wakefulness before the onset of sleep was rejected to further avoid the analysis of sleep or excess drowsiness. The LFPg, or the first derivative of the field potential (i.e., each contact referenced to its neighbor) and MUA were recorded simultaneously at 2,000 and 20,000 Hz and filtered online from 0.2 to 500 Hz and 200 to 5,000 Hz, respectively. Data from faulty channels (2 in each patient) were linearly in- terpolated from the channels directly above and below them. Prior to further analyses, we split the data into nonoverlapping 2-s epochs. We bandpass filtered (2-pass 3rd-order Butterworth) thalamic activity in the alpha band, then found the absolute value of its analytic signal (hilbert.m) to find single-trial thalamic alpha amplitude. Then, the 20% of epochs with the most total alpha-band amplitude (summing across thalamic bipolar pairs and samples) were used for further processing. Line noise was eliminated from both the LFPg and MUA by band-stop filtering (4-Hz bandwidth) at 60 Hz in Pts. L1 and L2 and 50 Hz in Pt. L3 (4th-order Butterworth). The LFPg was then high-pass filtered at 0.5 Hz in Pts. L2 and L3 and 3.5 Hz in Pt. L1 due to a low-frequency vascular artifact (2-pass 2nd-order Butterworth), HGP from 70 to 190 Hz, and MUA from 300 to 3,000 Hz (4th-order Butterworth). MUA was then rectified and resampled at 2,000 Hz. General Analysis Procedures. Recordings were analyzed by using custom MATLAB scripts with the CircStat (72) and Fieldtrip (73) Toolboxes. Furthermore, a previous study employing the same recordings demonstrated that 70- to 120-Hz HGP re- liably decreased during K-complexes identical to 70- to 190-Hz power (81). Lastly, reanalyzing Pts. S8 and S9 using the 70- to 120-Hz band yielded highly similar results. Seven of 14 (coherence) or 10 of 14 (MI) thalamic channels exhibited significant PAC between thalamic alpha LFPg and the HGP of at least 1 cortical channel (compare to 9 of 14 coherence and 10 of 14 MI with 70- to 190-Hz band). Furthermore, 0 of 14 (coherence) or 4 of 14 (MI) thalamic channels had significant PAC between thalamic alpha LFPg and thalamic HGP (0 of 14 [coherence] and 3 of 14 [MI] with the 70- to 190-Hz band). We localized laminar contacts to cortical layers by performing histology on explanted tissue in Pts. L1 and L3 and identifying a putative layer IV sink in Pt. L2 (SI Appendix, Fig. S12). Data Availability. The data underlying our figures are available on figshare (85). Macaque data are publicly available at http://neurotycho.org/data/ 20120813ktanesthesiaandsleepchibitoruyanagawa. Code Availability. Custom scripts are publically available on GitHub (https:// github.com/mhalgren/AlphaGen). ACKNOWLEDGMENTS. We thank Erica Johnson, Nathan Meng, Richárd Fiáth, Qianli Xu, and Adam Niese for insightful comments, hypotheses, and technical support; and Project Tycho for making their macaque dataset publicly available. This study was supported by US Office of Naval Research Grant N00014-13-1-0672; NIH Grants R01-MH-099645, R01-EB-009282, R01- NS-062092, and K24-NS-088568; the Massachusetts General Hospital Execu- tive Council on Research; Hungarian National Brain Research Program Grants KTIA_13_NAP-A-IV/1- 4,6 and 2017-1.2.1-NKP-2017-00002; European Union Grant FP7 600925 NeuroSeeker; and Hungarian Government Grants OTKA PD101754 and OTKA K119443. The SR was measured as described in Cole et al. (31) (SI Appendix, SI Methods). GC analyses were performed by using the Multivariate GC Toolbox (34). Frequency-resolved GC values were assessed between each corticothalamic channel pair in the nonoverlapping 2-s epochs with the greatest 20% of 10. M. L. Lorincz, K. A. Kékesi, G. Juhász, V. Crunelli, S. W. Hughes, Temporal framing of thalamic relay-mode firing by phasic inhibition during the alpha rhythm. Neuron 63, 683–696 (2009). 2. J. Ito, A. R. Nikolaev, C. van Leeuwen, Spatial and temporal structure of phase syn- chronization of spontaneous alpha EEG activity. Biol. Cybern. 92, 54–60 (2005). 11. S. W. Hughes et al., Thalamic gap junctions control local neuronal synchrony and influence macroscopic oscillation amplitude during EEG alpha rhythms. Front. Psychol. General Analysis Procedures. Recordings were analyzed by using custom MATLAB scripts with the CircStat (72) and Fieldtrip (73) Toolboxes. General Analysis Procedures. Recordings were analyzed by using custom MATLAB scripts with the CircStat (72) and Fieldtrip (73) Toolboxes. Prior to further analysis, the raw data were visually inspected for artifacts due to machine noise, patient movement, or epileptiform activity. Epochs containing these artifacts (as judged by an expert neurologist) were removed prior to further analysis. To ensure that this effect was specific to the alpha band, we reapplied our main analysis to 2-Hz filtered bands with 1-Hz spacing from 1 to 35 Hz and found the resultant vector length of propagation direction across all time points. A clear alpha peak was observed (SI Appendix, Fig. S3). Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on January 6, 2020 Unless otherwise specified, all analyses of alpha-band effects refer to the 7- to 13-Hz band. Error bars correspond to the SEM. Downloaded at MTA TTKKUTATOKOZPONTI KONYVTAR on J Power and cross-spectral densities were found via the multitaper method. This was performed by applying a Hanning taper and then taking the Fourier transform of the zero-meaned data. Corticothalamic Interactions. SEEG (79) was performed on 9 patients to characterize epileptogenic activity and inform possible resections. SEEG macroelectrode depth probes had 10–15 contacts; each contact was 2 mm long and 0.8 mm in diameter with 1.5-mm intercontact spacing. The probes themselves were ∼5 cm long, with the exact length varying between electrodes. In Pts. S1 to S7, contact locations were found by stereotactic teleradiographs Coherence (Fig. 4E and SI Appendix, Figs. S10A and S13) was calculated by using the ft_connectivity function, which defines the coherence between S mean subtracted time series x and y as Cohðx, yÞ = Sxy SxxSyy , where Sxy is the Halgren et al. thalamic alpha amplitude (derived from the analytic signal computed with hilbert.m) (SI Appendix, SI Methods). thalamic alpha amplitude (derived from the analytic signal computed with hilbert.m) (SI Appendix, SI Methods). from within the stereotactic frame. These coordinates were then superimposed on a T1 MRI of the subject. An atlas (80) was then overlaid to determine the anatomical positions of thalamic and cortical contacts (Fig. 3A). 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https://openalex.org/W2213996336	https://epub.ub.uni-muenchen.de/26401/1/Remodeling_of_nuclear_landscapes_during_human_myelopoietic_cell_differentiation.pdf	English	null	Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments	Epigenetics & chromatin	2,015	cc-by	15,225	© 2015 Hübner et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract This model has been experimentally substantiated by super-resolution fluorescence microscopy in mamma- lian somatic cell lines [2–5] and in bovine preimplanta- tion embryos [6] and can be summarized as follows: Chromosome territories (CTs) are built up from chroma- tin domain clusters (CDCs), which can perform locally Hübner et al. Epigenetics & Chromatin (2015) 8:47 DOI 10.1186/s13072-015-0038-0 Hübner et al. Epigenetics & Chromatin (2015) 8:47 DOI 10.1186/s13072-015-0038-0 Abstract Background: Previous studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization. Results: Common human hematopoietic progenitors, myeloid precursor cells, differentiated monocytes and granu- locytes analyzed by super-resolution fluorescence microscopy and electron microscopy revealed profound differ- ences with respect to global chromatin arrangements, the nuclear space occupied by the interchromatin compart- ment and the distribution of nuclear pores. In contrast, we noted a consistent organization in all cell types with regard to two co-aligned networks, an active (ANC) and an inactive (INC) nuclear compartment delineated by functionally relevant hallmarks. The ANC is enriched in active RNA polymerase II, splicing speckles and histone signatures for transcriptionally competent chromatin (H3K4me3), whereas the INC carries marks for repressed chromatin (H3K9me3). Conclusions: Our findings substantiate the conservation of the recently published ANC-INC network model of mammalian nuclear organization during human myelopoiesis irrespective of profound changes of the global nuclear architecture observed during this differentiation process. According to this model, two spatially co-aligned and func- tionally interacting active and inactive nuclear compartments (ANC and INC) pervade the nuclear space. Keywords: Myelopoiesis, Somatic cell differentiation, Nuclear architecture, Active nuclear compartment, Interchromatin compartment, Perichromatin region, Super-resolution microscopy, Electron microscopy, Chromatin domain, Chromatin density classification for a functionally defined nuclear organization based on two co-aligned three-dimensional networks: an active and an inactive nuclear compartment (ANC and INC). This model has been experimentally substantiated by super-resolution fluorescence microscopy in mamma- lian somatic cell lines [2–5] and in bovine preimplanta- tion embryos [6] and can be summarized as follows: Chromosome territories (CTs) are built up from chroma- tin domain clusters (CDCs), which can perform locally for a functionally defined nuclear organization based on two co-aligned three-dimensional networks: an active and an inactive nuclear compartment (ANC and INC). Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co‑aligned active and inactive nuclear compartments Barbara Hübner1,3, Mariana Lomiento2, Fabiana Mammoli2, Doris Illner1,4, Yolanda Markaki1, Sergio Ferrari2, Marion Cremer1* and Thomas Cremer1* Correspondence: marion.cremer@lrz.uni‑muenchen.de; Thomas.Cremer@lrz.uni‑muenchen.de 1 Department Biology II, Biocenter, Ludwig Maximilians University (LMU), Grosshadernerstr. 2, 82152 Martinsried, Germany Full list of author information is available at the end of the article Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM Figure 1 exemplifies typical nuclear phenotypes of DAPI stained progenitor cells (upper panel), monoblast and myeloblast precursor cells (mid panel), and mono- cytes and granulocytes (bottom panel), represented by xy mid-sections of nuclei acquired with 3D-SIM. Inset magnifications of representative nuclear areas in pro- genitor and precursor cells reveal a network of chro- matin domain clusters (CDCs). CDCs are dispersed throughout the nucleus and pervaded by finely branched IC channels with occasional enlargements into wider IC lacunae. Changes of global nuclear landscapes are most apparent during the transition from precursors toward mature monocytes and granulocytes. Horseshoe-shaped nuclei of monocytes are characterized by aggregations of CDCs into compacted chromatin islets, surrounded by wide interchromatin channels and lacunae. Chromatin in multilobulated nuclei of granulocytes appears mostly restricted to a rather uniformly arranged, densely com- pacted layer at the nuclear periphery. The interior of each nuclear lobe is filled by an ample contiguous IC lacuna with a few decondensed chromatin loops expanding from the compact chromatin layer toward the interior. In this study, we aimed to test the ANC-INC network model during various stages of human myeloid cell dif- ferentiation as a naturally occurring somatic cell differen- tiation system. Myelopoiesis starts from self-renewable pluripotent stem cells, the common progenitor cells. By asymmetric cell divisions resulting daughter cells form lineage restricted precursors with increasing commit- ment, finally leading to terminally differentiated post- mitotic cell types [8, 9]. This maturation process is associated with major changes of the nuclear landscapes [10, 11] and tightly controlled changes of gene expression patterns [12–17]. Proteins located on the cell surface can be used for a refined identification of cells within distinct maturational stages [9, 18]. Myelopoiesis is the part of hematopoiesis involved in the differentiation of multipotent myeloid progeni- tors into erythrocytes, megakaryocytes, monocytes and granulocytes. For our study, we chose five human cell types from the myeloid differentiation pathway, namely (1) CD34+ cells from umbilical cord blood representing common myeloid and lymphoid progenitors, (2) myelo- blasts and monoblasts representing lineage committed precursor cells, as well as (3) monocytes and granulo- cytes representing differentiated cells (see Fig. 1, upper left panel for an allocation of these cell types within the myeloid differentiation pathway). y At all differentiation stages, IC channels penetrate the heterochromatin layer beneath the nuclear enve- lope (Fig. 2a, arrows). Background Accumulated evidence from different studies argues for an inseparable intertwining between nuclear structure and function [1]. We have recently proposed a model Correspondence: marion.cremer@lrz.uni‑muenchen.de; Thomas.Cremer@lrz.uni‑muenchen.de 1 Department Biology II, Biocenter, Ludwig Maximilians University (LMU), Grosshadernerstr. 2, 82152 Martinsried, Germany Full list of author information is available at the end of the article Page 2 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 constrained movements and interact dynamically with each other in ways, whose functional implications are still not well understood. The compacted core of CDCs is enriched in repressive histone marks and lined by a peripheral layer of decondensed chromatin, called the perichromatin region (PR). The PR is enriched in epige- netic marks for transcriptionally competent chromatin and represents the chromatin compartment, where tran- scription, splicing, chromatin replication and DNA repair occur. The PR is co-aligned with a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores, permeates the nuclear space between CDCs and serves a role in nuclear import and export functions. More extended sites of this channel system, called IC lacunae, harbor splicing speckles and other nuclear bodies. The PR and the IC together form the ANC, whereas the INC is constituted by the compact interior of CDCs. Constrained motions of CDCs and chromatin loops expanding into the interior of the IC can lead to transient or consistent contacts between random or non-random sites of different CDCs (for a detailed review see [7]). fluorescence microscopy in each spatial dimension resulting in an approximately eightfold increased volu- metric resolution (for review see [21]). TEM provides a resolution, which is superior to any current approach of super-resolution fluorescence microscopy [22]. How- ever, the capability of 3D-SIM for the simultaneous, high- resolution targeting of differently fluorescence-labeled macromolecules involved in functionally relevant struc- tures, such as RNA polymerase II, nuclear bodies, or epi- genetic histone marks, makes this approach an ideal tool for quantitative, high-resolution studies of the nuclear topography of such targets and their spatial nuclear rela- tionships [2, 3, 5, 6]. Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM Their exit points appear as little holes on the nuclear surface (Fig. 2b) that were previ- ously shown to be directly connected to nuclear pores [5, 7, 23, 24]. We used these holes, mirroring nuclear pores, to study their topography in 3D reconstructions of 3D-SIM image stacks. Their number is distinctly reduced in monocytes and even more in granulocytes compared to progenitor and precursor cells (Fig. 2b, for quantifi- cation see Additional file 1). These images as well as the section galleries shown in Additional file 2 also demon- strate the variations in the global nuclear morphology in the different cell types: nuclei of progenitors exhibit an f Nuclei were imaged by transmission electron micros- copy (TEM) and 3D structured illumination microscopy (3D-SIM), a super-resolution fluorescence microscopic approach [19, 20]. 3D-SIM allows optical sectioning with a twofold resolution improvement over conventional Page 3 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Fig. 1 3D-SIM recorded chromatin landscapes of nuclei representing various myelopoietic differentiation stages. Upper left panel allocation of the analyzed cell types (framed) within the myeloid differentiation pathway. Remaining panels representative xy mid-sections of DAPI stained nuclei recorded with 3D-SIM, exemplifying the transforming global nuclear landscapes during myeloid cell differentiation. A network of chromatin domain clusters (CDCs) permeated by finely branched IC channels is seen in progenitor and precursor cells. Monocytes are characterized by compacted chromatin islets formed by tight aggregations of CDCs embedded within wide IC channels. Granulocytes show a rather uniformly arranged com- pacted chromatin layer at the nuclear periphery around a central IC lacuna. Arrows in inset magnification point to few decondensed chromatin sites expanding from the compact chromatin layer. Scale bars 2 µm, insets 0.5 µm Fig. 1 3D-SIM recorded chromatin landscapes of nuclei representing various myelopoietic differentiation stages. Upper left panel allocation of the analyzed cell types (framed) within the myeloid differentiation pathway. Remaining panels representative xy mid-sections of DAPI stained nuclei recorded with 3D-SIM, exemplifying the transforming global nuclear landscapes during myeloid cell differentiation. A network of chromatin domain clusters (CDCs) permeated by finely branched IC channels is seen in progenitor and precursor cells. Monocytes are characterized by compacted chromatin islets formed by tight aggregations of CDCs embedded within wide IC channels. Granulocytes show a rather uniformly arranged com- pacted chromatin layer at the nuclear periphery around a central IC lacuna. Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM Arrows in inset magnification point to few decondensed chromatin sites expanding from the compact chromatin layer. Scale bars 2 µm, insets 0.5 µm overall roundish shape with invaginations at the surface. Monoblast and myeloblast nuclei are ellipsoid with typi- cally deep and complex invaginations which can pervade the whole nucleus in myeloblasts. Monocytes are char- acterized by horseshoe-shaped nuclei with an irregular surface; nuclei of granulocytes are divided into several interconnected lobes. sections after osmium ammine B staining, a DNA-spe- cific staining procedure (Fig. 3). In line with SIM, TEM images reveal a change of landscapes with progressive differentiation from a fine granular network of CDCs/ IC channels in progenitor cells to a dense and more lump-like chromatin pattern in granulocytes (Fig. 3a). However, compared with the range of DAPI staining intensities observed in 3D-SIM images, TEM sections present a more contrasted, black and white appearance Nuclear landscapes recorded with 3D-SIM were com- pared with landscapes of the same cell types in TEM Page 4 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Fig. 2 IC channels and nuclear pores. a upper and mid panel: In all studied cell types IC channels penetrate peripheral (hetero)chromatin toward the nuclear envelope (arrows) as shown in vertical (yz) sections of 3D-SIM acquisitions. Bottom panel xy mid-sections from the respective nuclei for comparison. b surface rendering of 3D reconstructions (Amira) of the same nuclei shown in (a) reveal small “chromatin holes” mirroring exit point of IC channels (nuclear pores, [23, 24]) at the nuclear envelope. Scale bars 2 µm, insets 0.5 µm Fig. 2 IC channels and nuclear pores. a upper and mid panel: In all studied cell types IC channels penetrate peripheral (hetero)chromatin toward the nuclear envelope (arrows) as shown in vertical (yz) sections of 3D-SIM acquisitions. Bottom panel xy mid-sections from the respective nuclei for comparison. b surface rendering of 3D reconstructions (Amira) of the same nuclei shown in (a) reveal small “chromatin holes” mirroring exit point of IC channels (nuclear pores [23 24]) at the nuclear envelope Scale bars 2 µm insets 0 5 µm Fig. 2 IC channels and nuclear pores. a upper and mid panel: In all studied cell types IC channels penetrate peripheral (hetero)chromatin toward the nuclear envelope (arrows) as shown in vertical (yz) sections of 3D-SIM acquisitions. Bottom panel xy mid-sections from the respective nuclei for comparison. Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM b surface rendering of 3D reconstructions (Amira) of the same nuclei shown in (a) reveal small “chromatin holes” mirroring exit point of IC channels (nuclear pores, [23, 24]) at the nuclear envelope. Scale bars 2 µm, insets 0.5 µm (See figure on next page.) Fig. 3 Chromatin landscapes of nuclei of various myelopoietic differentiation stages visualized by TEM in osmium ammine B stained physical sections and assessment of the chromatin/IC interface length. a A transition from a fine network of CDCs/IC channels toward a dense and more lump-like pattern was observed with progressive myeloid differentiation. b Thresholded masks for the delineation of osmium ammine B stained chromatin (pink) and the IC (gray) in the respective sections. Scale bars 2 µm, insets 0.5 µm. c The interface length between the thresholded chro- matin and the IC is reduced in differentiated cell types (1 progenitor; 2 monoblast; 3 myeloblast; 4 monocyte; 5 granulocyte). n Number of analyzed nuclei; error bars standard deviation; p < 0.001 for monocytes and granulocytes versus respective precursors and progenitors of osmium ammine B positive and negative regions. Thresholded TEM images (Fig. 3b) of mid-sections of representative nuclei used to quantitatively determine the extension of the interface between chromatin and the IC as a measure for the IC surface show a signifi- cant reduction (p < 0.001) in monocytes and granulo- cytes compared to their precursors and progenitors (Fig. 3c). Page 5 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Page 6 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Fig. 4 Topological chromatin density mapping of differentiating myelopoietic cell nuclei. a Upper panel: DAPI stained mid-sections of representa- tive nuclei acquired by 3D-SIM. Mid panel same sections after chromatin density classification, based on seven DAPI intensity classes, are displayed in false colors, ranging from class 1 (blue) representing pixels close to background intensity, largely reflecting the interchromatin compartment (IC), up to class 7 (white) representing pixels with highest density. Bottom panel inset magnifications in progenitors, monoblasts and myeloblasts (precur- sors) reveal a loosely arranged network of small chromatin domain clusters (CDCs) comprising a compacted core part (classes 5–6/7) and a sur- rounding low-density layer (class 2–4), the perichromatin region. Largely DNA-free class 1 regions meander between CDCs as part of the IC system. Monocytes are characterized by closed up CDCs forming larger islets, surrounded by a decondensed perichromatin layer (classes 2–3). Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM Bottom panel inset magnifications in progenitors, monoblasts and myeloblasts (precur- sors) reveal a loosely arranged network of small chromatin domain clusters (CDCs) comprising a compacted core part (classes 5–6/7) and a sur- rounding low-density layer (class 2–4), the perichromatin region. Largely DNA-free class 1 regions meander between CDCs as part of the IC system. Monocytes are characterized by closed up CDCs forming larger islets, surrounded by a decondensed perichromatin layer (classes 2–3). In the lobed granulocyte nucleus, an extended class 1 region, representing the large IC lacuna in the interior of each lobe is lined by a small rim of decondensed chromatin (classes 2–4) at the interface with the highly compacted peripheral chromatin layer (classes 5–7). b classification scheme. c relative 3D signal distributions of the DAPI intensity classes for each cell type. Note the shift toward higher intensity classes with progressing differentiation but similar values for class 1 despite profoundly different nuclear landscapes. n Number of analyzed nuclei; error bars standard deviation Remodeling of global nuclear landscapes during human myeloid cell differentiation studied with 3D‑SIM and TEM In the lobed granulocyte nucleus, an extended class 1 region, representing the large IC lacuna in the interior of each lobe is lined by a small rim of decondensed chromatin (classes 2–4) at the interface with the highly compacted peripheral chromatin layer (classes 5–7). b classification scheme. c relative 3D signal distributions of the DAPI intensity classes for each cell type. Note the shift toward higher intensity classes with progressing differentiation but similar values for class 1 despite profoundly different nuclear landscapes. n Number of analyzed nuclei; error bars standard deviation f Fig. 4 Topological chromatin density mapping of differentiating myelopoietic cell nuclei. a Upper panel: DAPI stained mid-sections of representa- tive nuclei acquired by 3D-SIM. Mid panel same sections after chromatin density classification, based on seven DAPI intensity classes, are displayed in false colors, ranging from class 1 (blue) representing pixels close to background intensity, largely reflecting the interchromatin compartment (IC), up to class 7 (white) representing pixels with highest density. Bottom panel inset magnifications in progenitors, monoblasts and myeloblasts (precur- sors) reveal a loosely arranged network of small chromatin domain clusters (CDCs) comprising a compacted core part (classes 5–6/7) and a sur- rounding low-density layer (class 2–4), the perichromatin region. Largely DNA-free class 1 regions meander between CDCs as part of the IC system. Monocytes are characterized by closed up CDCs forming larger islets, surrounded by a decondensed perichromatin layer (classes 2–3). In the lobed granulocyte nucleus, an extended class 1 region, representing the large IC lacuna in the interior of each lobe is lined by a small rim of decondensed chromatin (classes 2–4) at the interface with the highly compacted peripheral chromatin layer (classes 5–7). b classification scheme. c relative 3D signal distributions of the DAPI intensity classes for each cell type. Note the shift toward higher intensity classes with progressing differentiation but similar values for class 1 despite profoundly different nuclear landscapes. n Number of analyzed nuclei; error bars standard deviation Fig. 4 Topological chromatin density mapping of differentiating myelopoietic cell nuclei. a Upper panel: DAPI stained mid-sections of representa- tive nuclei acquired by 3D-SIM. Mid panel same sections after chromatin density classification, based on seven DAPI intensity classes, are displayed in false colors, ranging from class 1 (blue) representing pixels close to background intensity, largely reflecting the interchromatin compartment (IC), up to class 7 (white) representing pixels with highest density. Nuclear landscapes of human myelopoietic cells delineated by chromatin density classification Monocytes represent an intermediate state. Quantitative comparisons (Fig. 4c and Additional file 3) demonstrate a significant shift toward higher DAPI intensity classes in nuclei of differentiated cells, while the fraction of class 1 (IC) is similar in all cell types irrespective of their highly divergent global appear- ance of the IC compartment. p g Despite their distinctly different nuclear phenotypes on a global level, nuclei from all cell types show a consist- ent overrepresentation (relative signal enrichment) of the “active” marks H3K4me3, RNA Pol II Ser2P/Ser5P and SC35 in the low-density classes representing the ANC and a corresponding underrepresentation (relative signal depletion) in high density classes considered as the INC (Figs. 5, 6, 7 and Additional files 4, 5). Notably, SC35 is largely restricted to the IC compartment (class 1) (Fig. 6b and Additional file 5B), and RNA Pol II Ser2P/Ser5P sig- nals are both higher enriched in the IC compartment compared to chromatin bound H3K4me3 signals (Fig. 5b and Additional file 4B). The relative signal enrichment/ depletion along chromatin density classes for the “silent” mark H3K9me3 (Fig. 7) shows a broader variability even between nuclei of the same cell type (data not shown). On average, in progenitor and precursor cells H3K9me3 sig- nals show the expected underrepresentation (depletion) in classes 1 and 2, while they are fairly proportionally distributed within all other classes. Monocytes show a consistent overrepresentation (enrichment) of H3K9me3 in classes 6–7, while the averaged distribution profile is similar to the DAPI profile in the other classes. Despite these heterogenous results, the relative signal distribu- tions clearly reveal that the majority of the H3K9me3 signals are located in the higher chromatin classes in all cell types. In granulocytes, we failed to detect positive H3K9me3 immunostaining. Failure of H3K9me3 immu- nostaining in granulocytes was previously described [29, 30] and may be due to a lack or a masking of H3K9me3 epitopes. The access of immunoglobulins required for immunodetection with an estimated size of about 20 nm [31] may in addition be hampered when IC channels fall below a critical throat size of 15–20 nm [32] (an overview on all measured parameters for a comparative topol- ogy in relation to chromatin density maps is provided in Additional file 6). Nuclear landscapes of human myelopoietic cells delineated by chromatin density classification on seven DAPI intensity classes of equal intensity vari- ance. DAPI was previously shown to fulfill the require- ments as an appropriate marker for global chromatin representation despite its reported binding preference to AT-rich DNA [5]. Figure 4a presents examples of nuclear A previously described segmentation algorithm [5, 6] was used to obtain 3D chromatin density maps for 3D-SIM acquisitions of whole nuclei from each cell type based Page 7 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 (See figure on next page.) Fig. 5 Comparative topology of H3K4me3 and RNA Pol II Ser2P, markers for transcriptionally permissive/active chromatin in relation to chroma- tin density maps. a 3D-SIM light optical mid-sections from 3D acquisitions of whole nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained H3K4me3 (green) and RNA Pol II Ser2P (red). All cell types show a preferential localization of H3K4me3 and RNA Pol II Ser2P at decondensed chromatin sites or at the surface of compacted chromatin domain clusters. Scale bars 2 µm; insets 0.5 µm. b graphs highlighted with yellow background: relative signal distribution of H3K4me3 (green) and RNA Pol II Ser2P (red) within respective DAPI defined DNA intensity classes. p < 0.001 for DAPI vs. H3K4me3 and RNA Pol II Ser2P in all cell types. Graphs highlighted with light-blue background quanti- fied levels of relative enrichment (positive values) or depletion (negative values) of H3K4me3 (green) and RNA Pol II Ser2P (red) signals relative to the classified DAPI signals. All cell types show a similar profile with a distinct overrepresentation of both markers in low chromatin density classes and a corresponding underrepresentation in high density classes. Note the stronger enrichment of RNA Pol II Ser2P compared to H3K4me3 in class 1 (IC compartment). n Number of analyzed nuclei; error bars standard deviation Polymerase II, phosphorylated in the carboxy-terminal domain either at Ser2 (RNA Pol II Ser2P) or at Ser5 (RNA Pol II Ser5P). RNA Pol II Ser2P is involved in transcrip- tional elongation, while RNA Pol IISer5P is considered as the transcription initiating form [28].f landscapes before (DAPI, upper panel) and after classifi- cation (lower panel) with seven intensity classes plotted in false colors. Class 1 represents regions close to back- ground intensities, assigned to the largely DNA-free interchromatin compartment (IC). Classes 2–4 represent decondensed chromatin of low staining intensity, classes 5–7 the high intensity classes (Fig. 4b). Nuclear landscapes of human myelopoietic cells delineated by chromatin density classification While this clas- sification is a deliberate reduction of the whole range of DAPI intensities (compared, e.g., to 255 gray levels in 8-bit images), it provides a robust tool of statistical com- parisons between different samples [5, 6]. In progeni- tors and precursor cells, the compact core of chromatin domain clusters (CDCs) is represented by aggregations of pixels with highest density assigned to classes 5–7. These compact cores are lined by a layer of pixels assigned to class 2–4, representing the decondensed perichromatin region. Largely DNA-free class 1 regions expand between CDCs as part of the IC system. In the lobed granulocyte nuclei, an extended class 1 region extending into class 2 represents a large IC lacuna in the interior of each lobe, lined by a small rim of decondensed chromatin, while a broad layer of highly compacted chromatin (classes 5–7) resides at the periphery of lobes. Monocytes represent an intermediate state. Quantitative comparisons (Fig. 4c and Additional file 3) demonstrate a significant shift toward higher DAPI intensity classes in nuclei of differentiated cells, while the fraction of class 1 (IC) is similar in all cell types irrespective of their highly divergent global appear- ance of the IC compartment. landscapes before (DAPI, upper panel) and after classifi- cation (lower panel) with seven intensity classes plotted in false colors. Class 1 represents regions close to back- ground intensities, assigned to the largely DNA-free interchromatin compartment (IC). Classes 2–4 represent decondensed chromatin of low staining intensity, classes 5–7 the high intensity classes (Fig. 4b). While this clas- sification is a deliberate reduction of the whole range of DAPI intensities (compared, e.g., to 255 gray levels in 8-bit images), it provides a robust tool of statistical com- parisons between different samples [5, 6]. In progeni- tors and precursor cells, the compact core of chromatin domain clusters (CDCs) is represented by aggregations of pixels with highest density assigned to classes 5–7. These compact cores are lined by a layer of pixels assigned to class 2–4, representing the decondensed perichromatin region. Largely DNA-free class 1 regions expand between CDCs as part of the IC system. In the lobed granulocyte nuclei, an extended class 1 region extending into class 2 represents a large IC lacuna in the interior of each lobe, lined by a small rim of decondensed chromatin, while a broad layer of highly compacted chromatin (classes 5–7) resides at the periphery of lobes. Linking chromatin density maps with functionally relevant markershi The functional link between nuclear landscapes defined by chromatin density and biologically relevant markers was established by quantitatively mapping the relative spatial distribution of histone H3K4me3, representing transcrip- tional competent chromatin, and of histone H3K9me3, conveying a silent chromatin state [25], to the seven DAPI intensity classes. In addition we mapped immuno-stained SC35, an integral protein of splicing speckles, involved in co-transcriptional splicing [26] and in transcriptional elongation [27]. We also studied the topography of RNA Page 8 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Page 9 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Page 10 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 (See figure on previous page.) Fig. 6 Comparative topology of SC35 and RNA Pol II Ser2P, markers for transcriptional activity in relation to chromatin density maps. a 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno- stained SC35 (green) and RNA Pol II Ser2P (red). SC35, an integral part of splicing speckles, is seen almost exclusively in the IC compartment, while RNA Pol II Ser2P shows a preferential localization at decondensed chromatin sites or at the surface of compacted chromatin domain clusters (compare Fig. 5). Scale bars 2 µm; insets 0.5 µm b graphs highlighted with yellow background: relative signal distribution of SC35 (green) and RNA Pol II Ser2P (red) within respective DAPI defined DNA intensity classes. Graphs highlighted with light-blue background quantified levels of relative enrich- ment (positive values) or depletion (negative values) of SC35 (green) and RNA Pol II Ser2P (red) signals relative to the DAPI signals confirm the massive enrichment of SC35 signals in class 1 reflecting the IC compartment. n Number of analyzed nuclei; error bars standard deviation; p < 0.001 for DAPI vs. SC35 and RNA Pol II Ser2P, and for SC35 vs. RNA Pol II Ser 2P decondensation and compaction in granulocytes upon transient exposure to hypotonic and normotonic medium were observed (Fig. 9a). Osmium ammine B stained TEM sections of progenitors, precursors, monocytes and gran- ulocytes, kept in hypotonic medium (~90 mOsm) for 1 min before fixation, demonstrate that hypotonic con- ditions trigger a rather uniform distribution of decon- densed chromatin throughout the nuclear space in all cell types (Fig. 9b). Linking chromatin density maps with functionally relevant markershi These observations argue against a crowding of space-occupying nuclear bodies within the central IC lacunae of lobes and provide indirect evidence for a higher order chromatin organization, which allows a rapid, transient chromatin de- and re-condensation (see “Discussion”).fi A lowered overall transcriptional activity was previ- ously described for monocytes and resting granulocytes compared to their respective precursor cells [16, 33]. In line with their attenuated transcriptional activity we observed a profound shrinkage of nucleoli in monocytes and granulocytes (Additional file 7) and a decrease of RNA Pol II Ser2P/Ser5P signals defined both by recorded pixels and by custom defined spots (Fig. 8). Recording the number of all signal pixels compensates for differences in size of individually detected spots taking into account that a cell with few large spots can have the same num- ber of positive pixels compared to a cell with many small spots. The counted number of RNA Pol II spots relates to potential “transcription factories”. Current attempts of quantification should be considered with the caveat that the values depend on the threshold setting of recorded images (for a detailed discussion see [6]). Co-staining of granulocytes with DAPI (high affin- ity to AT-rich regions) and 7-AAD (high affinity to GC rich regions) that were fixed after 30 s incubation in hypotonic conditions reveals remarkable differences of the staining patterns. 7-AAD denotes the lobe interior, while DAPI strongly stains the peripheral rim (Fig. 9c). This radial polarization of the two dyes illustrates a pref- erential expansion of GC enriched (gene dense) DNA segments toward the nuclear interior. This GC enriched DNA likely represents the transcriptionally competent chromatin fraction residing as ANC at the interface between the compact, peripheral chromatin layer and the internal lacunae in granulocytic lobes. Transient chromatin decondensation in granulocytic lobes Living cells are adapted to an osmolarity of about 270 mOsm. Previous studies have demonstrated that a transient incubation of cells in medium with higher or lower osmolarities exerts massive effects on chromatin compaction, which are fully reversible upon restoring normotonic conditions [34]. Hypertonic medium results in increased chromatin compaction, while hypotonic conditions lead to chromatin decondensation [34, 35].h The huge IC lacunae of granulocytes observed under normotonic conditions may serve as a storage compart- ment and be tightly filled by macromolecular complexes and nuclear bodies, whose nature was not analyzed in the present investigation. Linking chromatin density maps with functionally relevant markershi In a live cell experiment, we tested effects of chromatin decondensation triggered by hypotonic conditions on the appearance of IC lacunae in granulocytes by exposing cells to a transient change from normotonic to hypotonic medium (~90 mOsm). Within <1 min in hypotonic medium chromatin expands into the large IC lacunae of granulocytic lobes and fills their space, resulting in a wide dissolution of the highly compacted, distinct peripheral chromatin layer typically seen in these lobes under normotonic conditions. This state can be rapidly restored (<1 min) after reincubation in normotonic medium. Repeated cycles of chromatin Radial arrangement of gene‑dense and gene‑poor chromatin is maintained in granulocytic lobes A radial gene density correlated arrangement of chroma- tin segments with the preferential localization of gene- poor chromatin at the nuclear periphery and gene-dense chromatin toward the nuclear interior has previously been described in several studies and was consistently confirmed for a large number of cell types and species (for review see [36]). For a further comparison of the topography of gene-dense and gene-poor chromatin regions in granulocytic lobes, we performed 3D-fluores- cence in situ hybridization (3D-FISH) with differentially labeled sets of pooled BAC clones from human chromo- somes HSA #1 and HSA #12 delineating gene-dense and gene-poor segments of these chromosomes (previously Page 11 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Fig. 7 Comparative topology of H3K9me3, a global marker for transcriptionally repressed (hetero)chromatin and H3K4me3 in relation to chromatin density maps. a 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained H3K4me3 (green) and H3K9me3 (red). H3K4me3 marks decondensed chromatin sites and lines compacted CDCs (compare Fig. 5). H3K9me3 marks highly compacted chromatin clusters but is also seen at decondensed sites (arrows). Scale bars 2 µm; insets 0.5 µm. b Graphs highlighted with yellow background relative signal distribution of H3K4me3 (green) and H3K9me3 (red) within respective DAPI defined DNA intensity classes. p < 0.001 for DAPI vs. H3K4me3 and H3K4me3 vs. H3K9me3. Graphs highlighted with light-blue background quantified levels of relative enrichment (positive values) or depletion (negative values) of H3K4me3 (green) and H3K9me3 (red) signals relative to DAPI signals reveal a relative depletion of H3K9me3 signals in classes 1 and 2 in undifferentiated cells (progenitors and precursors) and a relative enrichment of H3K9me3 signals in classes 6 and 7 in monocytes. In both cases the signals are distributed similar to the DAPI intensity classified distributions for the remaining classes. n Number of analyzed nuclei; error bars standard deviation Fig. 7 Comparative topology of H3K9me3, a global marker for transcriptionally repressed (hetero)chromatin and H3K4me3 in relation to chromatin density maps. a 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained H3K4me3 (green) and H3K9me3 (red). H3K4me3 marks decondensed chromatin sites and lines compacted CDCs (compare Fig. 5). H3K9me3 marks highly compacted chromatin clusters but is also seen at decondensed sites (arrows). Scale bars 2 µm; insets 0.5 µm. (See figure on next page) Fig. 9 Reversible decondensation of chromatin in myelopoietic cells triggered by hypotonic conditions. a Live cell observation of granulocytes with Hoechst33342 stained DNA recorded by spinning disc LSM during repeated circles of normotonic (270 mOsm) and hypotonic (90 mOsm) con- ditions. The compacted chromatin rim surrounding large interior IC lacunae is seen under normotonic conditions (0 min). Within <1 min in hypo- tonic conditions a nuclear phenotype appears with (decondensed) chromatin expanding into the IC lacunae. This effect is reversible upon restoring normotonic conditions (2 min) and can be repeated over several cycles (7 and 8 min). Scale bar 10 µm. b comparison of representative myelopoietic cell nuclei seen under normotonic (upper panel) and hypotonic conditions (lower panel) in osmium ammine B stained TEM sections. Inset magni- fications of hypotonic TEM sections demonstrate a similar, rather even distribution of (decondensed) chromatin throughout the nucleus in all cell types, with loss of larger IC channels and IC lacunae, in particular evident in monocytes and granulocytes. Scale bars 2 µm; insets 0.5 µm. c left panel: Different staining intensities after simultaneous DNA staining of granulocytes fixed after 30 s incubation in hypotonic conditions with DAPI (red) and 7-AAD (green). 7-AAD (high affinity to GC-rich regions) denotes the lobe interior while DAPI (high affinity to AT-rich regions) strongly stains the peripheral rim. This radial divergence of the two dyes illustrates a preferential expansion of GC enriched (gene dense) DNA segments toward the nuclear interior. z-projections of 400 nm axial distance are shown. Right panel 3D distance measurements of DAPI (red) and 7-AAD (green) signals to the nuclear border of granulocyte lobes confirm their significantly distinct radial distribution (p < 0.001, assessed by Mann–Whitney rank sum test). The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. n number of analyzed nuclei; error bars standard error of means Radial arrangement of gene‑dense and gene‑poor chromatin is maintained in granulocytic lobes b Graphs highlighted with yellow background relative signal distribution of H3K4me3 (green) and H3K9me3 (red) within respective DAPI defined DNA intensity classes. p < 0.001 for DAPI vs. H3K4me3 and H3K4me3 vs. H3K9me3. Graphs highlighted with light-blue background quantified levels of relative enrichment (positive values) or depletion (negative values) of H3K4me3 (green) and H3K9me3 (red) signals relative to DAPI signals reveal a relative depletion of H3K9me3 signals in classes 1 and 2 in undifferentiated cells (progenitors and precursors) and a relative enrichment of H3K9me3 signals in classes 6 and 7 in monocytes. In both cases the signals are distributed similar to the DAPI intensity classified distributions for the remaining classes. n Number of analyzed nuclei; error bars standard deviation Page 12 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Fig. 8 Decrease of RNA Pol II Ser2P/Ser5P signals during myelopoie- sis. Quantification of both the number of pixels (upper graph) and of spots (see “Methods” part, lower graph) reveals a distinct decrease of RNA Pol II Ser2P and Ser5P during differentiation, in particular in granulocytes. n number of analyzed nuclei; error bars standard devia- tion lobe. The results demonstrate a gene density correlated radial arrangement with gene-dense chromatin oriented toward the IC lacuna and gene-poor chromatin enriched toward the nuclear border (Fig. 10c).h The maintenance of the conventional gene density cor- related chromatin arrangement in multilobulated nuclei of granulocytes further confirms the general validity of such a radial arrangement. The only reported exception of an inverted architecture so far is rod cell nuclei of noc- turnal mammals, where these cells act as micro-lenses for effective light transmission [39]. Local similarities of nuclear organization despite major global differences of nuclear architectures in human myelopoietic cell types y y 3D structured illumination microscopy (3D SIM), complemented by transmission electron microscopy (TEM), was used for a comparative study of nuclear landscapes during myelopoietic cell differentiation, including CD34+ progenitor cells, myeloid precur- sors (monoblasts and myeloblasts) and mature mono- cytes and granulocytes. Nuclei of all cell types carry a higher order chromatin network, composed of chroma- tin domain clusters (CDCs) with a compact core and a less condensed chromatin periphery, the perichromatin region (PR). The entire chromatin network is permeated by a co-aligned network of IC channels and occasional larger IC lacunae. IC channels are connected to nuclear pore complexes (NPCs). The topography of higher order chromatin arrangements and the nearly DNA-free inter- chromatin compartment (IC), however, differs starkly in different cell types. In CD34+ progenitor and precur- sor cells, this topography resembles the pattern previ- ously observed by 3D SIM in various somatic cell lines [2, 5, 35]. In contrast, monocytes show larger aggregates of closely packed CDC islets, surrounded by wide IC described in [37, 38]). We selected HSA #1 and #12 since they represent chromosomes with an overall inter- mediate gene density (19 and 18 genes/Mb, respec- tively, http://www.ncbi.nlm.nih.gov/genome/guide/ human/), however, with distinct segmental differences of regional gene density within size windows of several Mbs (Fig. 10a, b). Confocal image stacks, recorded from gran- ulocyte nuclei, were used for 3D distance measurements of the respective probe signals obtained with regard to the DAPI delineated border of a granulocytic nuclear Page 13 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Page 14 of 21 Fig. 10 Distinct radial positioning of gene-poor and gene-dense segments of chromosomes 1 and 12 with regard to the border of granulocyte nuclear lobes. a ideograms of chromosomes 1 and 12 with regional gene density (left bar). Localization of individual BAC clones representing either gene-dense (green) or gene-poor segments (red) used in this study is marked by an asterisk. b left panel: respective 2D-FISH control experiments with the expected banded pattern on metaphase chromosomes; right panel: z-projections of 3D-FISH experiments. c 3D measurements for the distance distributions of signals delineating gene-dense (green) and gene-poor (red) segments, respectively, reveal their significantly distinct radial distribution both for chromosome 1 (top) and for chromosome 12 (bottom); (p < 0.005 for both curves, assessed by Mann–Whitney rank sum test). Local similarities of nuclear organization despite major global differences of nuclear architectures in human myelopoietic cell types a ideograms of chromosomes 1 and 12 with regional gene density (left bar). Localization of individual BAC clones representing either gene-dense (green) or gene-poor segments (red) used in this study is marked by an asterisk. b left panel: respective 2D-FISH control experiments with the expected banded pattern on metaphase chromosomes; right panel: z-projections of 3D-FISH experiments. c 3D measurements for the distance distributions of signals delineating gene-dense (green) and gene-poor (red) segments, respectively, reveal their significantly distinct radial distribution both for chromosome 1 (top) and for chromosome 12 (bottom); (p < 0.005 for both curves, assessed by Mann–Whitney rank sum test). The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. Nuclear counterstain (DAPI) is denoted in blue. n number of analyzed nuclei; error bars standard error of means channels, and multilobulated nuclei of granulocytes are characterized by a compact peripheral chromatin layer around a large internal IC lacuna, from which sparse, narrow channels penetrate the compact peripheral chro- matin layer toward comparatively infrequent NPCs. The peripheral layer of granulocytic lobes likely represents a higher order chromatin organization of densely arranged CDCs with a strongly collapsed IC channel system, but other more intertwined chromatin arrangements have to be considered. Despite these major differences between nuclear landscapes of different myelopoietic cell types at a global scale, we noted a consistent organization between all cell types at a more local scale with regard to a functional compartmentalization into an active and an inactive nuclear compartment (ANC and INC). This consistency is most evident for the ANC, exemplified in our study by its relative enrichment with H3K4me3, a histone mark indicating transcriptional competence, RNA Pol II Ser2P/Ser5P representing initiating and elon- gating forms of active RNA Pol II [28] and SC35, a pro- tein involved in transcriptional elongation [27] and RNA splicing [40]. SC35 signals are strongly enriched within the largely DNA-free IC, whereas H3K4me3 is mostly channels, and multilobulated nuclei of granulocytes are characterized by a compact peripheral chromatin layer around a large internal IC lacuna, from which sparse, narrow channels penetrate the compact peripheral chro- matin layer toward comparatively infrequent NPCs. The peripheral layer of granulocytic lobes likely represents a higher order chromatin organization of densely arranged CDCs with a strongly collapsed IC channel system, but other more intertwined chromatin arrangements have to be considered. Local similarities of nuclear organization despite major global differences of nuclear architectures in human myelopoietic cell types The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. Nuclear counterstain (DAPI) is denoted in blue. n number of analyzed nuclei; error bars standard error of means Fig. 10 Distinct radial positioning of gene-poor and gene-dense segments of chromosomes 1 and 12 with regard to the border of granulocyte nuclear lobes. a ideograms of chromosomes 1 and 12 with regional gene density (left bar). Localization of individual BAC clones representing either gene-dense (green) or gene-poor segments (red) used in this study is marked by an asterisk. b left panel: respective 2D-FISH control experiments with the expected banded pattern on metaphase chromosomes; right panel: z-projections of 3D-FISH experiments. c 3D measurements for the distance distributions of signals delineating gene-dense (green) and gene-poor (red) segments, respectively, reveal their significantly distinct radial distribution both for chromosome 1 (top) and for chromosome 12 (bottom); (p < 0.005 for both curves, assessed by Mann–Whitney rank sum test). The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. Nuclear counterstain (DAPI) is denoted in blue. n number of analyzed nuclei; error bars standard error of means Fig. 10 Distinct radial positioning of gene-poor and gene-dense segments of chromosomes 1 and 12 with regard to the border of granulocyte nuclear lobes. a ideograms of chromosomes 1 and 12 with regional gene density (left bar). Localization of individual BAC clones representing either gene-dense (green) or gene-poor segments (red) used in this study is marked by an asterisk. b left panel: respective 2D-FISH control experiments with the expected banded pattern on metaphase chromosomes; right panel: z-projections of 3D-FISH experiments. c 3D measurements for the distance distributions of signals delineating gene-dense (green) and gene-poor (red) segments, respectively, reveal their significantly distinct radial distribution both for chromosome 1 (top) and for chromosome 12 (bottom); (p < 0.005 for both curves, assessed by Mann–Whitney rank sum test). The ordinate denotes the normalized sum of voxel intensities for a respective fluorochrome, the abscissa the relative distance to the nuclear border. Nuclear counterstain (DAPI) is denoted in blue. n number of analyzed nuclei; error bars standard error of means Fig. 10 Distinct radial positioning of gene-poor and gene-dense segments of chromosomes 1 and 12 with regard to the border of granulocyte nuclear lobes. Nuclear organization and function in myeloid cells Nuclear organization and function in myeloid cells Monocytes and granulocytes have an overall reduced transcriptional activity of a large number of genes com- pared to their precursors [16], in our study reflected by the relative paucity of active RNA Pol II signals and tiny nucleoli. Both, monocytes and in particular granulocytes are, however, capable of a rapid transcriptional upregu- lation of numerous specific genes upon environmental stimulation, such as exposure to bacterial lipopolysac- charides or lectin stimulation [16, 17, 44–46]. It may be speculated that an architectural configuration with a diminished surface area of the interface between decon- densed chromatin and the IC, as it is established in nuclear lobes of mature granulocytes and in monocytes, reduces the dwell time of transcription factors in search for their specific targets [32] and may speed up regula- tion of genes. g g g We still lack information on the possible redistribu- tion of functionally relevant DNA between the ANC and INC during cell differentiation from precursors to (ter- minally) differentiated cells. We have raised this impor- tant problem in a recent review [7]: “With respect to the topography of regulatory and coding sequences we consider several scenarios: (a) regulatory sequences of genes may be exposed within the ANC independent of whether genes are active or inactive. (b) Only regulatory sequences, which are in active use in a given cell, may be located within the ANC, whereas regulatory sequences of genes shut off in this cell are retracted into the INC represented by the compact and largely inaccessible inte- rior of CDCs. In this case shifts of regulatory sequences embedded within the INC into the ANC should occur prior to the activation of the respective genes, while repositioning of such sequences from the ANC into the INC should occur, when these genes become inactive. (c) Similar considerations apply to coding sequences”. It should be noted that nascent RNA was shown to be pref- erentially synthesized in a narrow layer of decondensed chromatin, called the perichromatin region located at the periphery of chromatin domain clusters (for review see [42]). Accordingly, a potential repositioning of genes between the ANC and INC may typically take place at scales below 200 nm (see also [7]). Experimental studies Monocytes and granulocytes can migrate into tis- sues to the site of bacterial invasion within minutes by squeezing through the tight endothelial wall of blood vessels. Local similarities of nuclear organization despite major global differences of nuclear architectures in human myelopoietic cell types Despite these major differences between nuclear landscapes of different myelopoietic cell types at a global scale, we noted a consistent organization between all cell types at a more local scale with regard to a functional compartmentalization into an active and an inactive nuclear compartment (ANC and INC). This consistency is most evident for the ANC, exemplified in our study by its relative enrichment with H3K4me3, a histone mark indicating transcriptional competence, RNA Pol II Ser2P/Ser5P representing initiating and elon- gating forms of active RNA Pol II [28] and SC35, a pro- tein involved in transcriptional elongation [27] and RNA splicing [40]. SC35 signals are strongly enriched within the largely DNA-free IC, whereas H3K4me3 is mostly overrepresented within the decondensed chromatin layer which forms the interface between the core of CDCs and the IC. RNA Pol II signals show an intermediate localiza- tion, with enrichments both within the IC and the PR. H3K9me3 was employed as a marker for silent chroma- tin and compared to the “active” marks shows a higher variability between and even within cell types. Nuclei of progenitors and precursors, but not of monocytes, show on average the expected underrepresentation of H3K9me3 in the lowest DAPI intensity classes while this epigenetic mark largely reflects the DAPI distribu- tion profile in the higher classes. Notably, H3K9me3 is involved in fine tuning of expression levels at promoters and enhancers for large-scale repression [25].f Differences in the topography of epigenetic mark- ers and other proteins between individual cells and cell populations, respectively, may reflect a stochastic cell- to-cell variability, functional differences between cells at different stages of differentiation or during the cell cycle, as well as technical issues. The classification of DAPI intensity classes and the assignment of signal pix- els from immuno-stained markers depends on param- eters such as the chosen threshold. Cell-to-cell variability Page 15 of 21 Page 15 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 of staining efficiency has also to be taken into account as a caveat against an overinterpretation of differences detected even between nuclei from cell neighbors and in the interpretation of differences obtained from different experiments. However, we wish to emphasize the high reproducibility of the major result, which has emerged from our current approach of a quantitative assessment of the nuclear topography of chromatin and functional markers. Local similarities of nuclear organization despite major global differences of nuclear architectures in human myelopoietic cell types The whole set of data so far obtained with this approach, including nuclei from mouse, human and bovine somatic cells, mouse embryonic stem cells and bovine preimplantation embryos obtained by in vitro fer- tilization and somatic cell nuclear transfer [2, 3, 5, 6] and our present study, consistently demonstrates an enrich- ment of H3K4me3, RNA Pol II Ser2P/5P and SC35 in DAPI intensity classes attributed to the ANC. An enrich- ment of epigenetic markers assigned to transcriptionally competent chromatin was also observed in a most recent study of nuclei from the mouse cardiomyocyte cell-line HL-1 performed with super-resolved, single-molecule localisation microscopy of DNA binding dyes together with immunodetection of H3K14ac [41]. The fact that a highly non-random enrichment of such epigenetic mark- ers with the PR was detected by super-resolved micros- copy based on different physical principles rules out that our findings may result from artifacts of the complex algorithms involved in 3D-SIM generated images. to solve these issues pose an enormous challenge. Mul- ticolor 3D-FISH experiments combined with super- resolution fluorescence microscopy can be considered, yet such experiments bear the danger that destruction of fine details of chromatin structure prohibit a clear answer at this size scale [3]. Recently, it has become pos- sible to visualize specific DNA sequences in nuclei of liv- ing cells [43]. Yet, despite their enormous promise, these new technologies have not reached the state of routine applications. Nuclear organization and function in myeloid cells Efficiently squeezing through the tight endothe- lial wall of blood vessels requires a high deformability of nuclei. Cytoskeletal filaments, in particular actin [47] and the specific composition of the nuclear envelope have been found important for nuclear shape determination and deformability in human granulocytes and mono- cytes [45, 48]. The particular chromatin organization in these cell types may favor a high malleability of nuclear shapes. The large internal IC lacunae in granulocyte lobes are apparently not packed with larger, space filling and rather insolvable complexes, such as nuclear bodies. This was shown by incubation of living human granulocytes in hypotonic medium resulting in a rapid shift of chro- matin from the peripheral chromatin layer into the IC lacunae. This effect was fully reversible, when the cells were again incubated in normotonic medium. While we lack evidence for the actual organization of compacted chromatin in the periphery of granulocytic lobes, these observations provide indirect evidence for a higher order chromatin organization, which allows a rapid, tran- sient chromatin de- and re-condensation. It has been speculated that the possibility of a rapid expansion and Page 16 of 21 Page 16 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 since their peripheral chromatin layer is pervaded by few and narrow IC channels. This topography provides good opportunities to decide whether mRNAs and RNPs, respectively, can take the entire layer of peripheral chro- matin for their passage toward NPCs or whether this pas- sage occurs preferentially along such narrow IC channels. re-compaction of chromatin domains plays a major role in gene regulation [49]. In line with this assumption, it has been argued that chromatin domains may be built up from smaller globules like Russian matryoshka dolls [50, 51]. It is interesting to note that chromatin domains can be modeled as a polymer analog of a 3D Peano curve [first described by Guiseppe Peano (1858–1932)] [52] which fill higher dimensional space as entirely unknot- ted structures. Such an organization prevents chromatin entanglements during the de- and re-condensation of chromatin domains. Direct evidence for such an organi- zation is lacking and currently very difficult to obtain [53]. Conventional TEM sections suggest that the interior of chromatin domain clusters is not solid as suggested by the liquid drop model, but composed of fibrous chro- matin arrangements, which arguably provide sufficient space for RNP export through such clusters [35]. Nuclear organization and function in myeloid cells For a long time, it has been argued that both 10-nm beads-on- a string chromatin fibers and 30 nm thick higher order chromatin fibers exist in vivo [56]. Despite undisputable evidence for the formation of 30 nm chromatin fibers in vitro [57], studies based on cryo-EM and X-ray scatter- ing have argued that the interior of chromatin domains is so densely packed that nucleosome interactions between 10 nm thick chromatin fibers prevent the formation of 30 nm thick fibers [58–60]. To what extent 30 nm chro- matin fibers may play a role in chromatin compaction in vivo is still an unsettled question. These problems sound a note of caution against naïve biological inter- pretations of images without a careful consideration of potential artifacts. A safeguard against misleading inter- pretations is the application of a set of different methods including novel correlative fluorescence—electron-micro- scopic (CFEM) approaches, as well as live cell studies. [ ] The ANC-INC network model argues that the IC chan- nel system serves as a system for nuclear import–export functions [23] (for review see [7]). This hypothesis is in part based on a study, which reported that interchroma- tin channels in mammalian cell nuclei ensure “a steady and continuous wave of mRNPs traveling toward the nuclear pore complex (NPC)” [54]. It implies the assump- tion that the compact core of chromatin domain clusters is much less accessible for both RNPs produced within the ANC as well as for factors which enter the nucleus through NPCs to form aggregates involved in transcrip- tion, splicing, DNA replication and repair preferentially within the ANC. Multi-scale fluorescence cross-cor- relation spectroscopy analysis of the mobility of inert monomers, trimers and pentamers of GFP, as well as GFP fusions with other proteins in nuclei of living cells [32] indicated that the nuclear interior acts like a sponge like structure. Chromatin domains may act as obstacles for individual factors and aggregations. In this type of nuclear organization, the protein–chromatin interactions at the obstacle surface occur in a particle-size depend- ent manner. Accordingly, the decondensed chromatin surface surrounding lacunas may become more easily accessible for functional macromolecules and macromol- ecule complexes than the much more compacted interior of CDCs. In a most recent study multifocus microscopy (MFM) was carried out to capture 3D single-molecule real-time images in living cells [55]. It was concluded that β-actin mRNAs freely access the entire nucleus. Nuclear organization and function in myeloid cells The limited resolution imposed by conventional light micros- copy employed in this study, however, does not exclude a diffusion along IC channels. Further high-resolution microscopic studies, e.g. by super-resolved MFM, are necessary to decide whether RNPs can take any routes throughout the peripheral chromatin layer for their pas- sage toward nuclear pores or whether this passage occurs along IC channels that end directly at nuclear pores. Human granulocytes may serve as a useful model sys- tem for such studies assessing the potential importance of IC channels for nuclear import and export functions Immunodetection Antibodies used for immunodetection: primary anti- bodies against RNA Pol II Ser2P/Ser5P (rat monoclonal; kindly provided by D. Eick LMU Munich), H3K4me3 (rabbit polyclonal; Abcam, ab8580), H3K9me3 (mouse monoclonal; Active Motif, 39285), SC35 (mouse mono- clonal; Sigma-Aldrich, S4045), nucleoli (human nucleolar positive control; Antibodies Incorporated) and second- ary antibodies against rabbit coupled with DyLight488 (raised in donkey; Jackson Immuno Research), against mouse and rat coupled with Alexa594 (raised in goat, respectively donkey; Molecular Probes) and against human coupled with FITC or Alexa594 (raised in goat; Jackson Immuno Research or Molecular Probes, respec- tively). A detailed protocol for fixation and immunode- tection procedure meeting the requirements for 3D-SIM imaging is provided in Additional file 8. Isolation of hematopoietic cells of defined differentiation stages g Human CD34+ cells (progenitors) were purified from umbilical cord blood (CB) samples by magnetic cell sort- ing. Samples were obtained from the Emilia Romagna Cord Blood Bank (ERCB) at the policlinic S. Orsola-Mal- pighi in Bologna. Myeloid precursors (monoblasts and myeloblasts) were obtained by in vitro differentiation of CB derived CD34+ cells as described in [15]. Separation of monoblasts and myeloblasts was achieved by selection for the surface antigen CD14: monoblasts are CD14+, myeloblasts are CD14−. Monocytes were isolated from peripheral blood using either magnetic cell sorting or Histopaque gradients. Granulocytes were isolated from peripheral blood using Histopaque gradients (for a detailed description see Additional file 8). Chromatin density classification by 3D assessment of DAPI intensity classesi For chromatin density quantification signals of DAPI stained DNA were segmented into seven classes with equal intensity variance using an in-house algorithm described in [5, 6]. A hidden Markov random field model classification was used, combining a finite Gauss- ian mixture model with a spatial model (Potts model), implemented into the statistics software R [63, 64].This approach allows threshold-independent signal inten- sity classification at the voxel level, not only based on the intensity of an individual voxel but also considering the classification of surrounding voxels. In our study the influence of the neighboring voxels was set to 0 as this yielded a better correspondence of the segmented images with the original images. Prior to chromatin density clas- sification 3D nuclear masks were generated using the same segmentation algorithm, but without implementing a mask, followed by setting an appropriate threshold in ImageJ and if necessary further processing using dilate/ erode functions and manual corrections. Care was taken that invaginations of the nuclear surface were main- tained in the mask. All signals outside the nucleus were deleted from the mask. For the allocation of immuno- detected marker signals (H3K4me3, RNA Pol II Ser 2P/ Ser5P, SC35, H3K9me3) in relation to chromatin den- sity the marker channels were thresholded and the indi- vidual voxels correlated with the corresponding voxel in the DAPI channel and assigned to the respective DNA Conclusionsh This study demonstrates consistent hallmarks of nuclear landscapes present in all cell types of the human mye- lopoietic lineage irrespective of major differences of global chromatin arrangements. These hallmarks include the presence of co-aligned networks of an active (ANC) and an inactive (INC) nuclear compartment. The ANC starts with interchromatin compartment (IC) channels connected to nuclear pores and pervades the nuclear interior together with its co-aligned INC. ANC and INC differ with regard to functionally relevant hallmarks, including a highly significant enrichment of RNA poly- merase II and histone signatures for transcriptionally competent chromatin in the ANC, whereas the INC is enriched in marks for repressed chromatin. These results are in line with the functional organization of nuclei previously observed in a variety of mammalian spe- cies, including bovine blastomeres, mouse embryonic stem cells, and various somatic cells, reviewed in [7]. We conclude that the ANC-INC network model of nuclear organization reveals evolutionary conserved principles of the functional nuclear organization. To which extent these principles may be valid for eukaryote species in general remains to be seen. Page 17 of 21 Page 17 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 respective OTFs using immersion oil of different refrac- tive indices (RI). Best results were typically obtained with OTFs measured on red, green (both 110 nm diameter) and blue (170 nm diameter) fluorescent FluoSpheres (Invitrogen) using RI 1.512, and sample acquisition with RI 1.514 for depth adjustment in the region of optimal reconstruction a few µm into the sample. Images from the different color channels were corrected for chromatic aberration in SoftWoRx with alignment parameters obtained from calibration measurements with 0.2 µm diameter TetraSpeck beads (Invitrogen). If necessary (e.g. for large cells in axial direction) the alignment parame- ters were manually adjusted to compensate for the larger distance from the coverslip. To normalize all image stacks for subsequent image processing and data analysis, the original 32-bit images were shifted to positive values and transformed to 16-bit. Then the stack specific mode gray value (representing the peak of the background noise) was subtracted, unless the images were used for chro- matin density classification described below. All further image processing was done in ImageJ (http://rsb.info.nih. gov/ij/). 3D models of image stacks were generated in Amira (FEI Visualization Sciences Group). Sample preparation for TEM and osmium ammine B staining Fixation of cells under normotonic conditions was per- formed with 4 % paraformaldehyde/1× PBS for 10 min, fixation under hypotonic conditions was performed by incubating the cells in 0.3× PBS for 1 min prior to fixa- tion with 4 % paraformaldehyde/0.3× PBS for 10 min, followed in both cases by washing with 1× PBS. Fur- ther preparation of samples and cutting of ultrathin sec- tions (100 nm) was essentially performed as described in [35]. Staining of DNA in 2.8 mM osmium ammine B was essentially done according to [65]. For a detailed descrip- tion of TEM sample preparation and osmium ammine B staining see Additional file 8. Ultrathin sections were imaged on a FEI Morgani 268 operated at 80 kV. 8-bit or 16-bit gray scale 2D images were acquired at several mag- nifications with an average gray value of 50–60 %, taking care not to cut off signals on both sides of the spectrum (high and low values). The alignment of different mag- nifications of each nucleus to each other was performed in Adobe Photoshop using the rotate, resize, warp and distort functions. Further image processing was done in ImageJ. 3D‑FISH and 3D distance measurement of fluorescence signals to the nuclear border Pooled BAC probes, assigned to either gene-dense or gene-poor segments of human chromosomes 1 and 12 (19 clones for gene-dense segments of chr. 1 and of chr. 12, 9 and 12 clones, respectively, for gene-poor segments; compare Fig. 10), were differently labeled with haptens and used for the delineation of the respective sequences in human granulocytes. Probe setup, fixation in 4 % PFA/0.5 × PBS, pretreatment of cells, hybridization, DAPI staining and detection by Cy3- or Cy5-conjugated antibodies were performed as previously described [38, 66]. Nuclei were scanned using a laser scanning confocal microscope (Leica SP5) equipped with a 63×/1.4 plan- apochromat oil objective. Stacks of 8-bit gray scale 2D images collected sequentially for all fluorochromes were obtained with a pixel size of 50 nm and an axial distance of 200 nm between optical sections. Images were pro- cessed with ImageJ. Chromatic aberration was corrected with alignment parameters obtained from the measure- ment of multi-colored fluorescent beads and an in-house Live cell observation of granulocytes Live cell observations of granulocytes during changes between normotonic (live cell medium) and hypotonic conditions (0.3× PBS) were performed on an Axio Observer D1 (Zeiss) with an UltraView VoX spinning disk unit (PerkinElmer) and an 63×/1.4 plan-apochro- mat oil objective. Cells seeded into glass bottom dishes (MatTek) as described above for immunodetection were stained with 0.3 µg/ml Hoechst 33342 in live cell medium (DMEM without phenol red (Invitrogen), 50 mM Hepes, 10 % FCS) for 30–45 min. After replacing the medium with fresh live cell medium, stacks of 8-bit gray scale images were acquired with an axial distance of 300 nm between optical sections using the 405 nm laser line for excitation and FITC filter settings for emission detec- tion. After completion of image acquisition the medium was exchanged with 0.3× PBS and image stacks were acquired again, resulting in an incubation time of ~1 min in 0.3× PBS. Subsequently the 0.3× PBS was changed back to live cell medium and image stacks were captured again. Another image stack was acquired after an addi- tional 3 min incubation (i.e. total incubation in medium: 5 min) before repeating the cycle. Quantification of RNA Polymerase II positive pixels or spots The number of RNA Polymerase II positive pixels was summed up from the number of pixels in the seven density classes obtained in the density classification described above. The number of spots within the seg- mented nuclear mask was determined in Volocity (Perki- nElmer) using the “find spots” function: an appropriate offset for spot intensity was chosen in “extended focus” view and the minimum distance between spots was set to 0 µm. The total number of summed up pixels per cell and the number of “spots” were tested for statistical signifi- cance between cell types using a Wilcoxon rank sum test with continuity correction. 3D‑SIM Super-resolution structured illumination imaging was performed on a DeltaVision OMX V3 system (Applied Precision Imaging/GE Healthcare) equipped with a 100×/1.4 UPlan S Apo oil immersion objective (Olym- pus), Cascade II:512 EMCCD cameras (Photometrics) and 405, 488 and 593 nm lasers [61]. Raw data image stacks were acquired with 15 raw images per plane (5 phases, 3 angles) and an axial distance of 125 nm and then computationally reconstructed with a Wiener fil- ter setting of 0.002 and channel specific optical transfer functions (OTFs) using SoftWoRx (Applied Precision) [24, 62]. The reconstruction process generates 32-bit data sets with the pixel number doubled in the lateral axes, resulting in the pixel size being halved from 79 to 39.5 nm to meet the Nyquist sampling criterion. The level of spherical aberration was minimized and matched to the Page 18 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 intensity class. Statistical significance was tested by a Wilcoxon rank sum test with continuity correction. Over-/underrepresentations (relative depletion/enrich- ment) of the marker signals were calculated by setting the difference between the relative amount of signals in the marker channel and the corresponding DAPI channel in relation to the relative amount of signals in the DAPI channel. subtracting the perimeter of the nucleus (determined from the mask in Volocity, (PerkinElmer) from the perimeter of the chromatin threshold, set on the cor- responding original image in Volocity. The results were normalized for the area of the nuclei. The Wilcoxon rank sum test with continuity correction was used for testing for statistical significance. 2D: 2 dimensional; 3D: 3 dimensional; 3D-FISH: 3D fluorescence in situ hybridi- zation; 3D-SIM: 3D structured illumination microscopy; 4D: 4 dimensional; 7-AAD: 7-aminoactinomycin D; ANC: active nuclear compartment; BAC: bacte- rial artificial chromosomes; BSA: bovine serum albumin; CB: cord blood; CDCs: chromatin domain clusters; CFEM: correlative fluorescence—electron-micro- scopic; CTs: chromosome territories; DAPI: 4′,6-diamidino-2-phenylindole; eADS: enhanced absolute 3D distances to surfaces; EM: electron microscopy; Ethics statement Human CD34+ cells were purified upon donor’s informed written consent from umbilical cord blood (CB) samples, collected after normal deliveries, accord- ing to the institutional guidelines for discarded material (Clearance of Ethical Committee for Human experimen- tation of Modena: Secretary office Saverio Santachiara, santachiara.saverio@policlinico.mo.it, approval date: 18.01.2005; approval file number # 793/CE). Additional file 5. Comparative topology of SC35 and RNA Pol II Ser5P, markers for transcriptional activity in relation to chromatin density maps. (A) 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained SC35 (green) and RNA Pol II Ser5P (red). SC35, an integral part of splicing speckles is seen almost exclusively in the IC compartment while RNA Pol II Ser5P shows a preferential localization at decondensed chromatin sites or at the surface of compacted chromatin domain clusters (compare additional file 4). Scale bars: 2 µm; insets 0.5 µm (B) graphs highlighted with yellow background: relative signal distribution of SC35 (green) and RNA Pol II Ser5P (red) within respective DAPI defined DNA intensity classes. p < 0.001 for DAPI vs. SC35 and RNA Pol II Ser5P, and for SC35 vs. RNA Pol II Ser 5P, except for SC35 vs. RNA Pol II Ser5P in granulocytes (p = 0.004). Graphs highlighted with light-blue background: quantified levels of relative enrichment (positive values) or depletion (negative values) of SC35 (green) and RNA Pol II Ser5P (red) signals relative to the DAPI signals confirm massive enrichment of SC35 signals in class 1 reflecting the IC compartment. n = number of analysed nuclei; error bars = standard deviation. Additional file 2. S Additional file 2. Section galleries of nuclei shown in Fig. 2. Galleries of light optical serial sections (axial distance = 125 nm between each optical section) of whole 3D-SIM 3D acquisitions of the DAPI stained nuclei shown in Fig. 2. For the progenitor cell every fifth image (axial distance = 625 nm), for the monoblast every second image (axial distance = 250 nm), for myeloblasts, monocytes and granulocytes every third image (axial distance = 375 nm) is included. Nuclei of progeni- tors exhibit an overall roundish shape with invaginations at the surface. Monoblast nuclei are of ellipsoid shape with typically deep and complex invaginations. Nuclei of myeloblasts are similar to monoblast nuclei; how- ever, typically they are slightly thicker, and invaginations often pervade the whole nucleus. Monocytes are characterized by horseshoe-shaped nuclei with an irregular surface. Nuclei of granulocytes are divided into several interconnected lobes. Additional file 7. Nucleolar phenotypes during myelopoiesis. Light optical mid-sections of whole 3D-SIM acquisitions show DAPI stained DNA (gray) and nucleoli (green) delineated by a human-anti-nucleolus antibody in representative cell nuclei. Scale bars: 2 µm, insets 0.5 µm. While all cell types contain similar numbers of nucleoli (monoblasts 1-3, myeloblasts 2-3, monocytes 2-4 and granulocytes 1-2; data not shown) they are distinctly shrinked in size in monocytes and in granulocytes compared to their precursors. Additional file 8. Methods. Quantification of the chromatin/IC interface length in TEM sections Nuclear masks of osmium ammine B stained nuclei were generated as described above for the masks used for the chromatin density classification of 3D-SIM images. The IC/chromatin interface length was calculated by Page 19 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 plugin for ImageJ. For the measurement of the shortest absolute 3D distances of all BAC signals to the surface of the segmented nuclear border, an in-house software (“enhanced absolute 3D distances to surfaces,” eADS) was used, previously described in detail in [38]. For a detailed description see Additional file 8. Additional file 4. Comparative topology of H3K4me3 and RNA Pol II Ser5P, markers for transcriptionally permissive/active chromatin in relation to chromatin density maps. (A) 3D-SIM light optical mid-sections from whole 3D acquisitions of nuclei and representative inset magnifications delineating DAPI stained DNA (gray), immuno-stained H3K4me3 (green) and RNA Pol II Ser5P (red). All cell types show a preferential localization of H3K4me3 and RNA Pol II Ser5P at decondensed chromatin sites or at the surface of compacted chromatin domain clusters. Scale bars: 2 µm; insets 0.5 µm. (B) graphs highlighted with yellow background: relative signal distribution of H3K4me3 (green) and RNA Pol II Ser5P (red) within respec- tive DAPI defined DNA intensity classes. p < 0.005, except for H3K4me3 vs. RNA Pol II Ser5P in progenitors (p = 0.059). Graphs highlighted with light- blue background: quantified levels of relative enrichment (positive values) or depletion (negative values) of H3K4me3 (green) and RNA Pol II Ser5P (red) signals relative to the intensity classified DAPI signals. All cell types show a similar profile with a distinct overrepresentation of both markers in low chromatin density classes and a corresponding underrepresenta- tion in high density classes. Note the stronger enrichment of RNA Pol II Ser5P compared to H3K4me3 in class 1 (IC compartment). n = number of analysed nuclei; error bars = standard deviation. Simultaneous DAPI and 7AAD staining Granulocytes on coverslips were incubated in 0.5× PBS for 30 s and then fixed with 4 % paraformaldehyde/0.5× PBS for 10 min. The cells were stained with DAPI and 7AAD and images were acquired, processed and evalu- ated as described above for 3D-FISH. Additional files Additional file 1. Exit points of IC channels at the nuclear surface in mye- lopoietic cell nuclei. Exit points of IC channels connected to nuclear pores were previously shown to appear as little holes in the nuclear envelope [5,7,23,24]. 3D reconstructions using Amira software (compare Fig. 2B) of whole 3D-SIM 3D acquisitions of DAPI stained nuclei from progenitors, precursors, monocytes and granulocytes were used for a quantitation of such exit points. Upper graph: Number per nucleus; lower graph: average nuclear surface area harboring one exit point. Number of exit points is significantly decreased in monocytes and granulocytes compared to their respective precursors (p < 0.001). Displaying the results as nuclear surface area harboring one channel exit demonstrates the profound difference between granulocytes and the other four cell types (p ≤ 0.001). n = num- ber of analyzed nuclei; error bars = standard deviation. Additional file 6. Overview of all measured parameters for a compara- tive topology in relation to chromatin density maps. Graphs highlighted with yellow background: relative marker signal distribution within respec- tive DAPI defined DNA intensity classes. Graphs highlighted with light-blue background: quantified levels of relative enrichment (positive values) or depletion (negative values) marker signals relative to the DAPI signals. This summary demonstrates the similarity of the distributions in all cell types (progenitor = light blue, monoblast = yellow, myeloblast = red, mono- cyte = green, granulocyte = dark blue). n = number of analysed nuclei; error bars = standard deviation. Acknowledgements f 16. Newburger PE, Subrahmanyam YV, Weissman SM. Global analysis of neutrophil gene expression. Curr Opin Hematol. 2000;7:16–20. We are indebted to D. Eick (LMU Munich) for the RNA Polymerase II Ser2P/ Ser5P antibodies and to T. Ried (National Cancer Institute, Bethesda; MD) for providing BAC clones from chromosomes 1 and 12. The Center of Advanced Light Microcopy (CALM) of the LMU Biocenter (headed by H. Leonhardt) was essential for the 3D SIM studies. We are indebted to R. Beckmann for provid- ing access to the TEM and to S. Fakan for providing EM sample preparation equipment. This work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft; DFG) to TC (SFB 684, YM and BH), from the Deutscher Akademischer Austauschdienst (DAAD; Vigoni programme) to TC and SF (BH and ML), from AIL MODENA to SF (ML) and from the Centro di studi e richerche Enrico Fermi to ML. 17. Surmiak M, Kaczor M, Sanak M. Proinflammatory genes expression in granulocytes activated by native proteinase-binding fragments of anti- proteinase 3 IgG. J Physiol Pharmacol. 2015;66:609–15. 18. Attar A. Changes in the cell surface markers during norm 18. Attar A. Changes in the cell surface markers during normal hematopoie- sis: a guide to cell isolation. Global J Hematol Blood Transfu. 2014;1:20–8. 19. Heintzmann R, Cremer C. 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Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Develop- ment and Its Functional Implications. PLoS One. 2015;10:e0124619. 24. Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumina- tion microscopy. Science. 2008;320:1332–6. Abbreviations Three- dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenetics Chromatin. 2014;7:8. 6. Popken J, Brero A, Koehler D, Schmid VJ, Strauss A, et al. Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos. Nucleus. 2014;5:555–89. Authors’ contributions 7. Cremer T, Cremer M, Hubner B, Strickfaden H, Smeets D, et al. 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BH carried out most IF experiments and sample processing for TEM, all image recording by confocal microscopy, spinning disk microscopy, 3D-SIM and TEM, performed quantitative data evaluation and made substantial contribu- tions to the conception and design of the study; ML carried out most cell preparations/separations, helped with IF experiments and sample processing for TEM and was involved in conceiving the study; FM helped with the cell preparation/separation; DI performed all FISH experiments and quantita- tive evaluation of FISH data; YM helped with setting up the IF protocol and 3D-SIM imaging and was involved in conceiving the study; SF was involved in conceiving the study; MC drafted and wrote essential parts of the manuscript and made substantial contributions to conception of the study; TC developed and designed the basic concept of the study, essentially contributed to data interpretation and wrote parts of the manuscript. 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Kim YC, Wu Q, Chen J, Xuan Z, Jung YC, et al. The transcriptome of human CD34+ hematopoietic stem-progenitor cells. Proc Natl Acad Sci USA. 2009;106:8278–83. Author details 1 14. Manfredini R, Zini R, Salati S, Siena M, Tenedini E, et al. The kinetic status of hematopoietic stem cell subpopulations underlies a differential expres- sion of genes involved in self-renewal, commitment, and engraftment. Stem Cells. 2005;23:496–506. 1 Department Biology II, Biocenter, Ludwig Maximilians University (LMU), Grosshadernerstr. 2, 82152 Martinsried, Germany. 2 Department of Life Sci- ences, University of Modena (Unimore), Modena, Italy. 3 Present Address: School of Biological Sciences (SBS), Nanyang Technological University (NTU), Singapore, Singapore. 4 Present Address: Bundeswehr Institute of Radiobiol- ogy, Munich, Germany. 15. Montanari M, Gemelli C, Tenedini E, Zanocco Marani T, Vignudelli T, et al. Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14 + human normal myeloid precursors. Cell Death Differ. 2005;12:1588–600. Abbreviations Additional file 3. DAPI intensity classification profiles from individual nuclei. Four representative chromatin density profiles based on seven DAPI intensity classes are shown for each cell type demonstrating the similarity of profiles within a given cell type and the overall shift towards higher intensity classes in differentiated cell types (monocytes and granu- locytes). For comparison the average profiles are repeated from Fig. 4. 2D: 2 dimensional; 3D: 3 dimensional; 3D-FISH: 3D fluorescence in situ hybridi- zation; 3D-SIM: 3D structured illumination microscopy; 4D: 4 dimensional; 7-AAD: 7-aminoactinomycin D; ANC: active nuclear compartment; BAC: bacte- rial artificial chromosomes; BSA: bovine serum albumin; CB: cord blood; CDCs: chromatin domain clusters; CFEM: correlative fluorescence—electron-micro- scopic; CTs: chromosome territories; DAPI: 4′,6-diamidino-2-phenylindole; eADS: enhanced absolute 3D distances to surfaces; EM: electron microscopy; Page 20 of 21 Page 20 of 21 Hübner et al. Epigenetics & Chromatin (2015) 8:47 Hübner et al. Epigenetics & Chromatin (2015) 8:47 4. Rouquette J, Genoud C, Vazquez-Nin GH, Kraus B, Cremer T, et al. Reveal- ing the high-resolution three-dimensional network of chromatin and interchromatin space: a novel electron-microscopic approach to recon- structing nuclear architecture Chromosome Res 2009;17:801–10 4. 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https://openalex.org/W2907584214	http://pjms.org.pk/index.php/pjms/article/download/5/30	English	null	Marginal accuracy of provisional crowns using three material systems and two techniques: A scanning electron microscope study	Pakistan journal of medical sciences	2,019	cc-by	4,308	Original Article Original Article Marginal accuracy of provisional crowns using three material systems and two techniques: A scanning electron microscope study doi: https://doi.org/10.12669/pjms.35.1.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 55 Correspondence: Dr. Talib Amin Naqash, Assistant Professor, College of Dentistry, King Khalid University, Abha 62529, Saudi Arabia. E-mail: go4talib@yahoo.com * Received for Publication: November 3, 2018 * 1st Revision Received: November 14, 2018 * 2nd Revision Received: December 18, 2018 * Final Revision Accepted: * December 21, 2018 INTRODUCTION Provisional (interim/ temporary) restorations are used to safeguard and sedate the pulp of prepared abutments, promote periodontal healing and health, rehabilitate oral function, provide positional stability, evaluate parallelism of abutments, and enhance esthetics.1-3 Interim coverage of a prepared tooth during various stages of treatment is an important step in the fabrication of fixed dental Marginal accuracy of provisional crowns using three material systems and two techniques: A scanning electron microscope study Talib Amin Naqash1, Mohammed Alfarsi2, Muhammad Waqar Hussain3 Objective: The most important desideratum of a provisional crown is an adequate marginal fit that is essential for maintaining optimal periodontal health, reducing the sensitivity of freshly prepared dentin and protection of the pulp. The purpose of this in vitro study was to compare the vertical marginal accuracy of provisional crown materials using three different material systems (chemically activated PMMA powder-liquid system, light activated UDMA single paste system, and chemically activated Bis-GMA two paste auto mix system) and two different techniques (direct and indirect). p y ) q ( ) Methods: Two customized stainless steel dies, simulating prepared and unprepared tooth were used to fabricate 40 provisional crowns. Additional silicone elastomeric impression and a vacuum-formed polypropylene sheet were used as a matrix. Ten crowns, each of the three material systems used in the study (n = 10 × 3) were fabricated using the direct technique and ten crowns from chemically activated PMMA powder-liquid system (n = 10 × 1) using an indirect technique. Scanning electron microscope (SEM) was used to measure vertical marginal discrepancies at x100 magnification. The results were analyzed using descriptive statistics and comparisons between various groups were made using one way analysis of variance (ANOVA) after checking the normality of data using Shapiro Wilk’s Test. Post Hoc Tukey HSD Test was used to determine the statistical difference between the means of independent group pairs. Results: The mean marginal discrepancies of Bis-GMA composite resin, UDMA composite resin, and PMMA acrylic resin using direct technique were 67.15 µm, 71.01 µm, and 84.56 µm respectively. PMMA acrylic resin showed a mean marginal discrepancy of 103.03 µm using the indirect technique. Conclusion: This study has shown that provisional crowns fabricated with Bis-GMA composite resin material (two paste auto mix system) registered the best marginal accuracy. Provisional crowns fabricated with indirect technique recorded less marginal opening than with direct technique. KEYWORDS: Bis-phenol A glycidyl methacrylate (Bis-GMA), Polymethyl methacrylate (PMMA), Urethane dimethylacrylate (UDMA), Scanning electron microscope (SEM), Stainless steel die. doi: https://doi.org/10.12669/pjms.35.1.5 How to cite this: Naqash TA, Alfarsi M, Hussain MW. Marginal accuracy of provisional crowns using three material systems and two techniques: A scanning electron microscope study. Pak J Med Sci. 2019;35(1):55-60. doi: https://doi.org/10.12669/pjms.35.1.5 Naqash TA, Alfarsi M, Hussain MW. Marginal accuracy of provisional crowns using three material systems and two techniques: A scanning electron microscope study. Pak J Med Sci. 2019;35(1):55-60. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION Provisional (interim/ temporary) restorations are used to safeguard and sedate the pulp of prepared abutments, promote periodontal healing and health, rehabilitate oral function, provide positional stability, evaluate parallelism of abutments, and enhance esthetics.1-3 Interim coverage of a prepared tooth during various stages of treatment is an important step in the fabrication of fixed dental 55 Talib Amin Naqash et al. prostheses and is currently recognized to have a fundamental role in the determination of success or failure of permanent restorations. Fig.1: Master dies with a common base simulating unprepared (a) and prepared (b) tooth; stainless steel top simulating an impression tray (c); and stone replica of prepared tooth (d). Marginal accuracy is one of the most important factors that determines the success of a provisional restoration; an acceptable accuracy at the margin is indispensable in maintaining gingival health and protecting the tooth from physical, chemical, bacterial, and thermal injuries.4 Marginal failure might lead to micro-leakage, postoperative sensitivity, pulpal inflammation, recession, and recurrent dental caries.5 The material system and the fabrication technique involved influence the marginal accuracy of the provisional restorations. The materials commonly used for custom fabrication are chemically activated acrylic resins (PMMA), light-activated composite resins (UDMA), and chemically activated composite resins (Bis-GMA). The techniques commonly used for fabrication of interim restorations include direct and indirect techniques. Fig.1: Master dies with a common base simulating unprepared (a) and prepared (b) tooth; stainless steel top simulating an impression tray (c); and stone replica of prepared tooth (d). • To evaluate and compare the marginal accuracy of provisional crowns fabricated using chemically polymerized Bis-GMA composite resin (two paste auto mix system), light polymerized UDMA composite resin (single paste system), and chemically polymerized PMMA acrylic resin (powder-liquid system) by the direct technique. Previous studies conducted to assess the degree of marginal gap formation materials have presented conflicting results. In addition, newly available resin systems are making the selection of an accurate material for provisional crowns arduous. Several studies have found an acceptable marginal accuracy of provisional crowns fabricated with PMMA acrylic resins.6,7 Other studies have revealed better results using Bis-GMA composite resins in terms of appropriate marginal accuracies.8,9 There is also some evidence suggesting light-polymerized materials might have better marginal accuracy.10 Some researchers have demonstrated the pre- eminence of the indirect technique of making provisional restorations extra orally11,12 while others have advocated the intraoral direct technique.13,14 METHODS An in vitro method was used to simulate a clinical technique, in which the provisional crowns were formed directly on the prepared tooth using a matrix or an external surface form. The present study was exempted from institutional review board due to non-involvement of human subjects. The purpose of this in vitro study was to compare the vertical marginal accuracy of commercially available provisional restorative crown materials using three different material systems and two different techniques with the following objectives: Two customized stainless steel master dies were made with a common stainless steel base, into which the dies could be accurately inserted and made interchangeable (Table-I; Fig.1a, 1b). The first die, which simulated an unprepared tooth was used to create a matrix. The second die with smaller axial and vertical dimension simulating the prepared tooth was used to fabricate the provisional crown restoration. An offset angle was placed in the second die (axio-occlusal line angle) for accurate reseating q g j • To evaluate and compare the marginal accuracy of provisional crowns fabricated using chemically polymerized PMMA acrylic resin (powder-liquid system) by the direct and indirect technique. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 56 Table-I: Dimensions of master stainless steel dies. Stainless Steel Dies Height Taper Diameter Shoulder Offset angle Die simulating unprepared tooth 10mm 00 10mm - - Die simulating prepared tooth 8mm 60 - 1mm 300 Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 56 Talib Amin Naqash et al. of the provisional crown. A shoulder finish line was machined in the second die, placed 1mm above the stainless steel base. was mixed in the ratio of 1 gm of powder to 0.45 cc of liquid, for 15 seconds, to produce a creamy mixture. Bis-GMA composite resin was dispensed directly from the cartridge by means of an auto- mixing tip using a dispensing gun. A stainless steel top which simulated an impression tray was machined with an internal dimension 2 mm larger than the external dimension of the die simulating the unprepared tooth (Fig.1c). Metal flanges were machined in the top and orientation notches in the base to ensure consistent repositioning. Vents in the top allowed extrusion of excess material. PMMA acrylic resin was placed in the impression after the material had lost its sheen and was in a dough stage. METHODS Bis-GMA composite resin was dispensed directly into the impression, from the cartridge, by means of an auto-mixing tip using a dispensing gun. The base containing the die and the top containing the impression were then seated; checked for correct positioning with the help of the orientation grooves. Firm finger pressure was applied to the top until the initial setting time mentioned by the manufacturer had elapsed. To mimic the direct technique, PMMA crown was removed once from the master die and reseated again to mimic clinical situation amid to exothermic reaction that might cause pulpal damage. An additional silicone elastomeric impression (Aquasil, soft putty/ regular set, LV-Dentsply, France.) was made in the stainless steel top of the die which simulated the unprepared tooth. The impression was used as a matrix to fabricate PMMA provisional crowns using the direct and indirect technique, and Bis-GMA provisional crowns using the direct technique. A transparent, thermoplastic, vacuum-formed polypropylene matrix was fabricated over the die which simulated the unprepared tooth. The transparent matrix was used to fabricate light polymerized UDMA temporary crowns using the direct technique. The provisional crown was removed from the die and excess was trimmed from the cavosurface margin with a scalpel (No. 11 blade), within 30 seconds, using a ×20 binocular microscope (Barska Co., CA, USA.). The crown was placed in an inverted position and allowed to cure in air at 720 F. This procedure was repeated for all crowns (n = 10 × 2; 10 PMMA and 10 Bis-GMA crowns, direct technique). Elite Double 22 (Zhermack, Italy.) duplication silicone was used to fabricate the stone replica of the mounted die simulating the prepared tooth (Fig.1d), using Type IV Die Stone (Denflo, Prevest, India). Chemically polymerized PMMA provisional crowns were subsequently made on the stone replica by the indirect technique using the impression in the stainless steel top as a matrix. Fabrication of UDMA temporary crowns using the direct technique (Fig.2c): UDMA composite resin-filled transparent matrix was adapted on the master stainless steel die simulating the prepared tooth and photo-polymerized for 10 seconds with LED light cure unit (B.G Light, Bluedent, Bulgaria.). METHODS The materials compared in this study are representative of three chemical types currently available in the market: (1) Revotek LC (GC Corporation, Tokyo, Japan), a UDMA composite resin; (2) Protemp (3M ESPE, Minnesota, USA), a Bis-GMA composite resin; (3) Temporary Cold-V Major (Prodotti Dentari S.p.A., Italy.), a PMMA acrylic resin. Fig.2: Fabrication of PMMA (a), Bis-GMA (b), UDMA (c) provisional crown using direct technique and PMMA (d) provisional crown by indirect technique. y Provisional crowns were made according to the manufacturers’ directions with regard to mixing, manipulation, proportioning, time of removal, and duration of irradiation. Test specimens were made in the following manner: Fabrication of PMMA and Bis-GMA temporary crowns using the direct technique (Fig.2a, 2b): The die simulating the prepared tooth was positioned in the cylindrical space present in the stainless steel base. Manufacturers’ directions for the mixing of each material were followed. PMMA acrylic resin Fig.2: Fabrication of PMMA (a), Bis-GMA (b), UDMA (c) provisional crown using direct technique and PMMA (d) provisional crown by indirect technique. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 57 57 Talib Amin Naqash et al. Table-II: Test of Normality of Data. Table-II: Test of Normality of Data. Data Group Shapiro-Wilk Test Statistic p-value PMMA Direct 0.989 0.995 Bis-GMA Direct 0.916 0.321 UDMA Direct 0.947 0.635 PMMA Indirect 0.954 0.715 Fig.3: Vertical marginal opening of PMMA (a), Bis-GMA (b), and UDMA (c) provisional crowns using direct technique; PMMA provisional crown (d) by indirect technique. level of p<0.05. The results were analyzed using descriptive statistics and making comparisons between various groups using one way analysis of variance (ANOVA) after checking the normality of data using Shapiro Wilk’s Test. Post Hoc Tukey HSD Test was used to determine the statistical difference between the means of independent group pairs. level of p<0.05. The results were analyzed using descriptive statistics and making comparisons between various groups using one way analysis of variance (ANOVA) after checking the normality of data using Shapiro Wilk’s Test. Post Hoc Tukey HSD Test was used to determine the statistical difference between the means of independent group pairs. Fig.3: Vertical marginal opening of PMMA (a), Bis-GMA (b), and UDMA (c) provisional crowns using direct technique; PMMA provisional crown (d) by indirect technique. RESULTS The Shapiro–Wilk test was used to check the normality of the data (Table-II). According to the Shapiro-Wilk test, data of each group showed insignificant deviation from the normal distribution (p>0.05 for each group). Therefore it was concluded that the data followed the normal distribution and hence parametric tests like ANOVA was applicable (Table-III). The crown was then removed from the master die, trimmed and light-cured for 20 seconds per surface. Ten such crowns were made (n = 10 × 1; 10 UDMA crowns, direct technique). Fabrication of PMMA temporary crowns using the indirect technique (Fig.2d): PMMA acrylic resin was placed in the stainless steel top containing the impression after the material had lost its sheen and was in a dough stage. The stone replica of mounted die simulating the prepared tooth was lubricated with petroleum jelly. The top was then seated on the stone replica in a similar fashion as mentioned in the direct technique. The procedure was repeated for all crowns (n = 10 × 1; 10 PMMA crowns, indirect technique). In the PMMA direct technique, the marginal discrepancy was found to be maximum with mean value 103.03±3.47, while for Bis-GMA direct technique the marginal discrepancy was found to be minimum with mean value 67.15±1.81. Highly significant difference was observed in mean marginal discrepancy among the various groups. g y g g Post Hoc Tukey HSD Test was used to determine the statistical difference between the means of independent group pairs. (Table-IV). Testing Procedure: Each provisional crown was seated on the stainless steel master die simulating the prepared tooth. A force of 7.4 pounds was applied in a vertical direction using a seating device. The force was applied for one minute, after which the measurements were made immediately. The marginal discrepancy was determined immediately after fabrication using analytical scanning electron microscope (JSM-6360LA, Jeol Ltd., Japan.) by measuring the space (marginal opening) between the margin of the provisional crown and finish line of the test die at four 900 locations determined by the random positioning of the grid (Fig.3). An accelerating voltage of 20kV under x100 magnification was used for evaluation of marginal accuracy. The highest difference was observed between PMMA Direct and Bis-GMA Direct groups (diff=35.88, p<0.001), which was followed by the difference between PMMA Direct and UDMA Direct groups (diff=32.01, p<0.001). RESULTS All the differences between various group pairs were highly significant except for the pair Bis-GMA Table-III: Comparison of marginal accuracy among various groups. Group Marginal Discrepancy F-value p-value Mean (µm) SD PMMA Direct 103.03 3.47 324.05 <0.001 Bis-GMA Direct 67.15 1.81 UDMA Direct 71.02 2.64 PMMA Indirect 84.56 3.19 Table-III: Comparison of marginal accuracy among various groups. Table-III: Comparison of marginal i The mean marginal opening was calculated for each crown from four measurements. Data was analyzed using IBM SPSS 24.0 at a significance Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 58 Table-IV: Comparison of marginal accuracy between various group pairs. Group PMMA Indirect Bis-GMA Direct UDMA Direct Mean Diff. p-value Mean Diff. p-value Mean Diff. p-value* PMMA Direct 18.47 <0.001 35.88 <0.001 32.01 <0.001 PMMA Indirect 17.41 <0.001 13.55 <0.001 Bis-GMA Direct -3.87 0.022 * p-values are calculated using Post Hoc Tukey HSD Test. Table-IV: Comparison of marginal accuracy between various group pairs. the shrinkage. Bis-GMA has two aromatic rings in its molecule and a low mobility, characteristics that interfere with the degree of conversion. Aliphatic molecular chemistry gives UDMA greater mobility and flexibility than Bis-GMA; thereby, increasing the degree of conversion and subsequent greater polymerization shrinkage.17 2) Polymerization shrinkage depends upon the molecular weight of organic monomer; the lesser the molecular weight, the greater the shrinkage. UDMA has a molecular weight of 470g/mol as compared to Bis-GMA (512g/mol).18i Direct and UDMA Direct where the difference was relatively less but still found to be significant (diff=- 3.87, p=0.022). Direct and UDMA Direct where the difference was relatively less but still found to be significant (diff=- 3.87, p=0.022). Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk DISCUSSION Congruous with nearly all areas of dental management where material science plays a crucial role, there is presently no ideal provisional material suitable for all clinical conditions; however, there are many materials and techniques that have been used successfully for this purpose.1,12 Vertical marginal discrepancy used in this study has been defined by Holmes et al. as the vertical misfit or gap, measured parallel to the path of the draw of the casting, at various points along the margin between the casting and the respective abutment.15 The size of the vertical marginal opening for a provisional crown should be held at about 50–120 microns, similar to that of the definitive fixed prostheses, in order to provide proper maintenance of healthy periodontal and pulpal tissues.9,16 The above findings are comparable to the results of the study conducted by Young et al. where the marginal accuracy of Bis-GMA composite resin provisional crowns fabricated by the direct technique was found significantly superior to that of PMMA acrylic resin.8 y The mean vertical marginal discrepancy of provisional crowns fabricated using chemically polymerized PMMA resin by the direct technique (84.56µm) was higher when compared to the mean vertical marginal discrepancy of provisional restorations fabricated by the indirect technique (103.03µm). The probable reason for this finding could be related to the separation of provisional crowns from the master die before the material was completely set (amid to the exothermic reaction that might cause pulpal inflammation) and later reseating the crown for complete polymerization. This method of separating the resin mix from the master dies before the final set could have caused distortion as there was no supporting substructure.i The materials used in this study showed mean marginal discrepancy values of 67.15–103.03 μm immediately after fabrication. Among the material systems used for fabrication of provisional crowns by the direct technique, chemically polymerized PMMA acrylic resin showed the highest marginal discrepancy (103.03µm). This could be attributed to greater polymerization shrinkage observed with PMMA acrylic resin (6% - 8%) as compared to Bis-GMA and UDMA composite resins (1-2%).2,17 Further, it was observed that the mean vertical marginal discrepancy of provisional crowns fabricated by the direct technique using light polymerized UDMA composite resin (71.01µm) was slightly greater than that found with chemically polymerized Bis-GMA composite resin (67.15 µm). This could be attributed to an increased polymerized shrinkage of UDMA composite resin as compared to Bis-GMA composite resin. DISCUSSION Reasons being: 1) Polymerization shrinkage depends upon the degree of conversion of monomers during polymerization; the greater the degree of polymerization the greater The materials used in this study showed mean marginal discrepancy values of 67.15–103.03 μm immediately after fabrication. Among the material systems used for fabrication of provisional crowns by the direct technique, chemically polymerized PMMA acrylic resin showed the highest marginal discrepancy (103.03µm). This could be attributed to greater polymerization shrinkage observed with PMMA acrylic resin (6% - 8%) as compared to Bis-GMA and UDMA composite resins (1-2%).2,17 p ( ) Further, it was observed that the mean vertical marginal discrepancy of provisional crowns fabricated by the direct technique using light polymerized UDMA composite resin (71.01µm) was slightly greater than that found with chemically polymerized Bis-GMA composite resin (67.15 µm). This could be attributed to an increased polymerized shrinkage of UDMA composite resin as compared to Bis-GMA composite resin. Reasons being: 1) Polymerization shrinkage depends upon the degree of conversion of monomers during polymerization; the greater the degree of polymerization the greater The findings of the current study are in agreement with the study conducted by Crispin et al.11 and Monday et al.12 where they demonstrated that the indirect techniques produced a more acceptable gingival margin for provisional restoration than the direct technique. The purpose of this study was to test accuracy and not to establish adequacy. Although all of the materials and techniques used in this study may be clinically adequate, some are significantly more accurate than others. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 59 59 Talib Amin Naqash et al. 6. Koumjian JH, Holmes JB. Marginal accuracy of provisional restorative materials. J Prosthet Dent. 1990;63:639-642. doi: 10.1016/0022-3913(90)90320-C Limitations of the study. The effect of oral fluids on the polymerization of the provisional materials was not taken into account. In addition, the specimens were not thermo-cycled (experimentally aged) which could result in an increased marginal discrepancy. The results obtained are applicable to single crowns and the data reported may vary from multiple units. / ( ) 7. Ehrenberg D, Weiner GI, Weiner S. Long-term effects of storage and thermal cycling on the marginal adaptation of provisional resin crowns: a pilot study. J Prosthet Dent. 2006;95:230-236. doi: 10.1016/j.prosdent.2005.12.012 j p 8. Young HM, Smith CT, Morton D. Comparative in vitro evaluation of two provisional restorative materials. J Prosthet Dent. 2001;85:129-132. CONCLUSION The vertical marginal discrepancy of provisional crowns ranged from 67.15–103.03 μm immediately after fabrication. Provisional crowns fabricated with Bis-GMA composite resin material using direct technique recorded the least marginal discrepancy followed by UDMA composite resin material. PMMA acrylic resin crowns made using direct technique demonstrated maximum marginal opening. PMMA provisional crowns fabricated with direct technique exhibited more marginal discrepancy than fabricated with indirect technique. p y g 10. Nivedita S, Prithviraj DR. A comparative study to evaluate the marginal accuracy of provisional restorations fabricated by light polymerized resin and autopolymerized resin: A scanning electron microscope study. J Indian Prosthodont Soc. 2006;6:122-127. doi: 10.1016/j.prosdent.2006.02.030 /j p 11. Crispin BJ, Watson JF, Caputo AA. The marginal accuracy of treatment restorations: a comparative analysis. J Prosthet Dent. 1980;44:283-290. doi: 10.1016/0022-3913(81)90389-9 ( ) 12. Monday JJ, Blais D. Marginal adaptation of provisional acrylic resin crowns. J Prosthet Dent. 1985;54(2):194-197. DOI: 10.1016/0022-3913(85)90285-9i ( ) 13. Robinson FB, Hovijitra S. Marginal fit of direct temporary crowns. J Prosthet Dent. 1982;47:390-392. doi: 10.1016/ S0022-3913(82)80087-5i ( ) 14. Miller SD. The anterior fixed provisional restoration: a direct method. J Prosthet Dent. 1983;50:516-519. doi: 10.1016/0022- 3913(83)90573-5 Conflicts of Interest: None. 18. Gajewski VE, Pfeifer CS, Froes-Salgado NR, Boaro LC, Braga RR. Monomers used in resin composites: degree of conversion, mechanical properties and water sorption/ solubility. Braz Dent J. 2012;23:508-514 Source of Funding: Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia (G.R.P-385-39). DISCUSSION doi: 10.1067/mpr.2001.112797i 9. Nejatidanesh F, Lotfi HR, Savabi O. Marginal accuracy of interim restorations fabricated from four interim autopolymerizing resins. J Prosthet Dent. 2006;95:364-367. ACKNOWLEDGEMENT 15. Holmes JR, Bayne SC, Holland GA, Sulik WD. Considerations in measurement of marginal fit. J Prosthet Dent. 1989;62:405-408. doi: 10.1016/0022-3913(89)90170-4i 1. King Khalid University, College of Dentistry, Abha, Saudi Arabia. 1. King Khalid University, College of Dentistry, Abha, Saudi Arabia. ( ) 16. Christensen GJ. Marginal fit of gold inlay castings. J Prosthet Dent. 1966;16:297-305. doi: 10.1016/0022-3913(66)90082-5 2. Dr. Ali Alshehri and Dr. Shkir, Department of Physics, King Khalid University, Abha, Saudi Arabia. 17. Filho JD, Poskus LT, Guimaraes JG, Barcellos AA, Silva EM. Degree of conversion and plasticization of dimethacrylate- based polymeric matrices: influence of light curing mode. J Oral Sci. 2008;50:315-321. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 60 REFERENCES TAN: Research methodology, review of literature and preparation of manuscript. 1. Burns DR, Beck DA, Nelson SK. A review of selected dental literature on contemporary provisional fixed prosthodontic treatment: report of the Committee on Research in Fixed Prosthodontics of Academy of Fixed Prosthodontics. J Prosthet Dent. 2003;90:474-497. doi: 10.1016/S0022- 3913(03)00259-2 1. Burns DR, Beck DA, Nelson SK. A review of selected dental literature on contemporary provisional fixed prosthodontic treatment: report of the Committee on Research in Fixed Prosthodontics of Academy of Fixed Prosthodontics. J Prosthet Dent. 2003;90:474-497. doi: 10.1016/S0022- 3913(03)00259-2 p p p MA: Review of the manuscript and supervision of the research project. p j MWH: Data entry and analysis. p j MWH: Data entry and analysis. p j MWH: Data entry and analysis. j MWH: Data entry and analysis. ( ) 2. Barghi N, Simmons EW Jr. The marginal integrity of the temporary acrylic resin crown. J Prosthet Dent. 1976;36:274- 277. doi: 10.1016/0022-3913(76)90182-7 ( ) 3. Waerhaug J. Temporary restorations: advantages and disadvantages. Dent Clin North Am. 1980;24:305-316. Authors: 1. Dr. Talib Amin Naqash, MDS. Assistant Professor, 2. Dr. Mohammed Alfarsi, PhD. Assistant Professor, 3. Dr. Muhammad Waqar Hussain, MDS. Assistant Professor, 1-3: Department of Prosthetic Dentistry, College of Dentistry, King Khalid University, Abha,Saudi Arabia. 1. Dr. Talib Amin Naqash, MDS. Assistant Professor, 2. Dr. Mohammed Alfarsi, PhD. Assistant Professor, 3. Dr. Muhammad Waqar Hussain, MDS. Assistant Professor, 1-3: Department of Prosthetic Dentistry, College of Dentistry, King Khalid University, Abha,Saudi Arabia. g 4. Chiche G. Improving marginal adaptation of provisional restorations. Quintessence Int. 1990;21:325-329. 4. Chiche G. Improving marginal adaptation of provisional restorations. Quintessence Int. 1990;21:325-329. 5. Browne RM, Tobias RS. Microbial micro leakage and pulpal inflammation: A review. Endod Dent Traumatol. 1986;2:177-183. 5. Browne RM, Tobias RS. Microbial micro leakage and pulpal inflammation: A review. Endod Dent Traumatol. 1986;2:177-183. Pak J Med Sci January - February 2019 Vol. 35 No. 1 www.pjms.org.pk 60
https://openalex.org/W4235404092	https://www.scielo.br/j/fp/a/H4Jkxp4w8nVcjrRv9thqH5j/?lang=pt&format=pdf	Portuguese	null	Errata: Frequência semanal de um programa de intervenção motora para bebês de berçário	Fisioterapia e Pesquisa	2,016	cc-by	88	DOI: 10.1590/1809-2950/14923223022016a DOI: 10.1590/1809-2950/14923223022016a 458Errata A versão do artigo “Frequência semanal de um programa de intervenção motora para bebês de berçário” publicado no volume 23, número 2, 2016, disponibilizada inicialmente continha erros em relação aos nomes das autoras Kelly Andara de Azevedo e Paula Ribeiro Demarco. Onde se lia: Laís Rodrigues Gerzson1, Bruna Maciel Catarino2, Kelly Andara3, Paula Demarco4, Míriam Stock Palma5, Carla Skilhan de Almeida6 Leia-se: Laís Rodrigues Gerzson¹, Bruna Maciel Catarino2, Kelly Andara de Azevedo3, Paula Ribeiro Demarco4, Míriam Stock Palma5, Carla Skilhan de Almeida6 458
https://openalex.org/W4292551984	https://www.duo.uio.no/bitstream/10852/95552/1/2022%2bYBaMn2O5.pdf	English	null	Why is Mn charge ordered and AFM coupled in YBaMn2O5?	Journal of solid state chemistry	2,022	cc-by	5,202	1. Introduction Syntheses and characterizations. Polycrystalline YBaMn2O5þw was sintered from precursors obtained by liquid mixing in melted citric acid monohydrate [11]. Annealed Y2O3 (99.99%, Fluka) was dry-mixed with the citric acid (BDH AnalaR reagent, <0.01% sulfated ash, <0.002% Ca), in amount of 2 mol per mole of cation charge of each starting component, and dissolved upon melting assisted by water added just to cover the bottom of the beaker. Manganese lumps (99.9%, Ventron) were cleaned of oxide in a 1:1 diluted hydrochloric acid, washed, and dried. The ob- tained 8.3786 g of Mn was dissolved in 50 mL water upon gradual ad- ditions of in total 80 mL of 65% HNO3 and then added dropwise into the above melt to a complete release of nitrous gasses. After cooling below 100 C, redistilled water was added on top of the viscous melt, and barium carbonate (Fluka, <0.2% Sr) was dissolved upon warming. The resulting clear melt of brown color was decomposed in a drying oven at 180 C into an organic-based solid xerogel, which spontaneously incin- erated overnight (unexpected, see typical synthesis details in Ref. [12]). The product was milled, pressed into cylindrical pellets of 15 mm in diameter and calcined for 24 h at 860 C in a ﬂowing atmosphere of 10.01(2) mole % H2 in Ar (AGA, certiﬁed analysis) wetted in a saturated solution of KBr at 18 C. The water-formation equilibrium then ﬁxes the oxygen partial pressure in the reaction atmosphere to log(pO2/bar) 18.5. Because small amounts of Y2O3 were detected in the calcination product, a second calcination was performed on the rehomogenized sample under ﬂowing atmosphere of the above-used 10% H2/Ar gas mixed with Ar (AGA, 6 N purity) to an Ar/H2 molar ratio of 78(3) and wetted at 19 C to stabilize log(pO2/bar) 16.6. This yielded a pure mixture of the YBaMn2O5-type and YBaMn2O5.5-type phases of brown color. A R T I C L E I N F O Keywords: Liquid-mixing synthesis Charge order A-site ordered double perovskite Synthesis conditions to obtain single-phase YBaMn2O5 are reported and its crystal structure reﬁned from neutron powder diffraction data collected between 25 and 600 C in the paramagnetic state. The structure parameters are used to reason why the phase maintains ordered ionic charges of Mn instead of mixing them at an elevated temperature, why this Mn2þ and Mn3þ ordering is of the checkerboard type in all three dimensions, and why these ions would couple antiferromagnetic upon cooling. Why is Mn charge ordered and AFM coupled in YBaMn2O5? Pavel Karen Department of Chemistry, University of Oslo, Blindern, N-0315, Oslo, Norway https://doi.org/10.1016/j.jssc.2022.123469 Received 15 July 2022; Accepted 1 August 2022 Available online 11 August 2022 0022-4596/© 2022 The Author. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jssc.2022.123469 Received 15 July 2022; Accepted 1 August 2022 Available online 11 August 2022 0022-4596/© 2022 The Author. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Journal of Solid State Chemistry 315 (2022) 123469 Journal of Solid State Chemistry 315 (2022) 123469 shed by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Available online 11 August 2022 0022-4596/© 2022 The Author. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Table 1 Table 1 Sintering conditions that led to single-phase YBaMn2O5þw samples. Table 1 Sintering conditions that led to single-phase YBaMn2O5þw samples. w * t/C Ar/H2 ** log(pO2/bar) porosity 0.016 1000 1940(37) 10.98(2) 60% 0.010 1100 2246(118) 9.36(6) 37% 0.003 1200 2277(155) 8.09(5) 5.7% 0.006 1200 2551(48) 7.94(2) 5.6% * Standard deviations are less than 1 unit of the last decimal. ** Volume ratio; standard deviations refer to gas-mixing variations over time. ng conditions that led to single-phase YBaMn2O5þw samples. * Standard deviations are less than 1 unit of the last decimal. Volume ratio; standard deviations refer to gas-mixing variations over time. Volume ratio; standard deviations refer to gas-mixing variations over time. 21.8 C (details in Table 1). A phase-pure product was obtained upon free cooling inside the furnace in the ﬂowing atmosphere, the wetting of which was switched off below 1000 C in order to exclude oxygen that would be needed to oxidize this large and highly sintered sample. Powder-diffraction checks were performed with a D5000 diffractometer and CuKα1 radiation. Wet chemical analyses of oxygen content. The oxygen content in YBaMn2O5þw was determined iodometrically. A ﬁnely powdered sample of about 0.15 g and 0.8 g KI were weighed into a 10 mL glass ampoule, 5 mL H2O was added and the ampoule was ﬂushed by Ar. After adding 4 mL of concentrated hydrochloric acid, the ampoule was immediately sealed. The digestion of the samples lasted less than a minute when aided by heating at the bottom of the ampoule. The formed triiodide solutions were titrated under argon atmosphere with a 0.1 M thiosulfate solution of concentration standardized on KIO3 of standard-substance quality (Merck). Close to the equivalence point, soluble starch was added as the indicator. Blank analyses were performed in order to correct for the small amount of iodine produced in the acidic environment employed for the digestion process. Fig. 1. P4/nmm unit cell of the paramagnetic YBaMn2O5 at 298 K. Neutron powder diffraction (NPD). Data were collected at 25, 100, 200, 300, 400, 500 and 600 C in the cryofurnace of the D2B instrument at the Institut Laue–Langevin, Grenoble, France. The two-axis diffractometer was equipped with 128 position-sensitive 3He counters, and the Ge monochromator was in orientation hkl 335. The wavelength λ ¼ 1.59421 Å was standardized on CeO2. Coarsely powdered YBaMn2O5.006 (~10 g) was sealed in a vanadium container under He atmosphere. Table 1 Whereas the four equal basal Mn–O distances are the shortest bonds in the small pyramid of d4 Mn3þ (a dz2-stabilizing Jahn–Teller distortion), they are longest in the large pyramid of d5 Mn2þ (a dx2y2-stabilizing Jahn–Teller distortion), and make an alternating pattern of small and large squares on the ab plane. All basal bonds expand linearly with temperature. The two ionic charges clearly remain ordered at all temperatures investigated. Fig. 3 shows thermal evolution of apical and basal Mn–O bond dis- tances in the two different Mn coordination square pyramids. Whereas the four equal basal Mn–O distances are the shortest bonds in the small pyramid of d4 Mn3þ (a dz2-stabilizing Jahn–Teller distortion), they are longest in the large pyramid of d5 Mn2þ (a dx2y2-stabilizing Jahn–Teller distortion), and make an alternating pattern of small and large squares on the ab plane. All basal bonds expand linearly with temperature. The two ionic charges clearly remain ordered at all temperatures investigated. Table 1 The primary collimation was removed from the setup in order to achieve higher in- tensity at the expense of resolution. The slit was set to 100/100 so that the beam size at the sample was 2 5 cm2. The counting limit was 1.6105 counts per step of 0.05 of the angle 2θ. The angular range was 5–160 (2θ). lower composition limit of which is the YBaMn2O5.5 reported in Ref. [9] to coexist in equilibrium with YBaMn2O5. The crystal structure of YBaMn2O5.006 was reﬁned in the space-group P4/nmm, suggested in Ref. [8] for the three-dimensional checkerboard order of Mn2þ and Mn3þ (Fig. 1). The minute amount of non- stoichiometric oxygen is located in the yttrium layer between the bases of the square pyramids, and reﬁnements of its occupancy yielded values between 0.008 and 0.014, in good agreement with the 0.006 oxygen per formula obtained by chemical analysis after the sample synthesis. The reﬁnement shows that the crystal structure keeps the P4/nmm space-group symmetry within the temperature range investigated, as did TbBaMn2O5 in the NPD study of the two-phase sample in Ref. [9]. Nu- merical overview of the structural evolution, with reﬁnement statistics and the reﬁned unit cell and atomic parameters is in Table 2. Rietveld reﬁnements. Crystal-structure reﬁnements in the GSAS soft- ware suite [13] used the Chebyshev function of 18 parameters to model the background. The NPD peak proﬁles were ﬁtted by Simpson integra- tion of the Thompson–Cox–Hastings pseudo-Voigt proﬁle with two Gaussian and one Lorentzian proﬁle parameters reﬁned together with the zero point, transparency and asymmetry. Fig. 2 illustrates the practically linear and continuous thermal expansion of this structure on unit-cell parameters per single-perovskite subcell. This suggests absence of structural or spin-state transitions in the investigated range of temperatures. Orbital energy calculations. The molecular-orbital energy levels for isolated manganese–oxygen coordinations were calculated from struc- tural data with the CAESAR 2.0 (Crystal And Electronic Structure AnalyzeR) software package [14]. The basis-set coefﬁcients for this Extended-Hückel tight-binding method were listed previously [6]. Fig. 3 shows thermal evolution of apical and basal Mn–O bond dis- tances in the two different Mn coordination square pyramids. 1. Introduction After testing the optimal sintering conditions (Table 1), pellets of 8 mm in diameter were pressed at 150 bar and sintered for 24 h at 1200 C into a silvery YBaMn2O5.006 under ﬂowing atmosphere of 10% H2/Ar gas mixed with Ar in two steps and wetted in the saturated solution of KBr at The three solid phases of the A-site ordered double perovskite along the YBaMn2O5–5.5–6 line convert one to another via reversible oxygen absorption at moderate temperatures, and this wide composition span opens for applications as oxygen-scavenger or -storage materials, solid- oxide fuel-cell cathodes, or as catalytic oxidants [1]. The YBaMnIIM- nIIIO5 end phase [2] of Mn2þ and Mn3þ conﬁgurations d5 and d4 is one-step from d4 and d3 of LaCaMnIIIMnIVO6 [3] and other colossal-magnetoresistance perovskite manganites [4,5]. It is also one-step from d6 and d5 that leads to valence mixing in YBaFeIIFeIIIO5 [6] above the Verwey-type [7] transition temperature. There are notable differences between the Fe and Mn variants. While the dxz orbital order in YBaFe2O5 stabilizes charge ordering that deﬁes the electrostatic minimum for point charges by alternating chains of Fe2þ coordinations and chains of Fe3þ coordinations [6], the Mn2þ and Mn3þ in YBaMn2O5 comply with it and alternate in all three directions [8]. While iron atoms in YBaFeIIFeIIIO5 become equivalent above 335 K [6], YBaMnIIMnIIIO5 remains charge ordered even at 873 K, as observed in the neutron diffraction study [9] of TbBaMn2O5 in an equilibrium 1:1 mixture with TbBaMn2O5.5. Only the magnetic cooperative order is the same, as charge-ordered YBaFe2O5 [6], and somewhat surprisingly YBaMn2O5 at low temperatures [8], both adopt the G-type spin arrangement of Wollan–Koehler [3]. YBaFe2O5 does so with moments along b, YBaMn2O5 along c. In this study, synthesis conditions to obtain phase-pure YBaMnIIM- nIIIO5 of non-magnetic and redox-stable yttrium were established, and the crystal structure investigated by high-intensity neutron powder diffraction up to 873 K for signs of approaching valence mixing. The reﬁned structure parameters are used to calculate molecular-orbital en- ergies, from which it is argued how the magnetic ordering in YBaMn2O5 complies with the Goodenough's model [10] for semicovalent exchange and what it might take to mix the Mn valences. Journal of Solid State Chemistry 315 (2022) 123469 P. Karen Fig. 1. P4/nmm unit cell of the paramagnetic YBaMn2O5 at 298 K. Table 2 Table 2 Temperature evolution of the YBaMn2O5 structure parameters* and reﬁnement ﬁgures of merit. T (K) 298 373 473 573 673 773 873 Rwp 0.046 0.045 0.043 0.044 0.043 0.043 0.045 Rp 0.035 0.034 0.033 0.034 0.033 0.033 0.035 RF 2 0.033 0.038 0.041 0.049 0.054 0.058 0.070 χ2 3.258 3.162 3.002 3.094 3.083 3.153 3.461 a (Å) 5.54891(8) 5.55306(8) 5.55801(8) 5.56386(8) 5.56928(4) 5.57506(9) 5.58125(9) c (Å) 7.65836(13) 7.67326(13) 7.68955(12) 7.70642(13) 7.72131(13) 7.73706(13) 7.75332(13) V (Å3) 235.80(10) 236.62(10) 237.54(10) 238.56(10) 239.49(10) 240.48(10) 241.52(11) z Mn2þ 0.74624(54) 0.74571(54) 0.74429(55) 0.74384(58) 0.74445(61) 0.74590(61) 0.74641(68) z Mn3þ 0.27565(53) 0.27493(52) 0.27458(54) 0.27413(58) 0.27360(63) 0.27398(65) 0.27379(74) xO1 0.49138(18) 0.49135(18) 0.49163(19) 0.49174(20) 0.49227(23) 0.49235(25) 0.49287(31) zO1 0.31668(10) 0.31670(10) 0.31677(10) 0.31679(11) 0.31653(11) 0.31678(12) 0.31676(13) zO2 0.00724(58) 0.00833(55) 0.00950(53) 0.00988(57) 0.00983(62) 0.00910(67) 0.00841(79) O3 occ. 0.008(4) 0.014(4) 0.010(4) 0.010(4) 0.009(4) 0.008(4) 0.012(4) * Space group P4/nmm number 129 and origin choice 2, Wyckoff positions: Y 2b, Ba 2a, Mn2þ 2c, Mn3þ 2c, O1 8j, O2 2c, O3 2c of z ﬁxed to ½ and occupancy (occ.) reﬁned. Observed reﬂections: 166, reﬁned variables: 39. Table 2 Temperature evolution of the YBaMn2O5 structure parameters* and reﬁnement ﬁgures of merit. oichiometric LaMnO3, where it disappears at about 750 K [16,17]. In two Mn valences starts, demonstrated by resumed contraction of the z Mn3þ 0.27565(53) 0.27493(52) 0.27458(54) 0.27413(58) 0.27360(63) 0.27398(65) 0.27379(74) xO1 0.49138(18) 0.49135(18) 0.49163(19) 0.49174(20) 0.49227(23) 0.49235(25) 0.49287(31) zO1 0.31668(10) 0.31670(10) 0.31677(10) 0.31679(11) 0.31653(11) 0.31678(12) 0.31676(13) zO2 0.00724(58) 0.00833(55) 0.00950(53) 0.00988(57) 0.00983(62) 0.00910(67) 0.00841(79) O3 occ. 0.008(4) 0.014(4) 0.010(4) 0.010(4) 0.009(4) 0.008(4) 0.012(4) Space group P4/nmm number 129 and origin choice 2, Wyckoff positions: Y 2b, Ba 2a, Mn2þ 2c, Mn3þ 2c, O1 8j, O2 2c, O3 2c of z ﬁxed to ½ and occupancy (occ.) ﬁned. Observed reﬂections: 166, reﬁned variables: 39. Fig. 2. Thermal evolution of the unit-cell parameters per single-perovskite subcell. The values for 1.5 K are from Ref. [8]. g. 3. Thermal evolutions of bond lengths Mn2þ–O (magenta) and Mn3þ–O (brown) to the square-pyramidal basis (■) and to the apex (▴). The 1.5 K values are from ef. [8]. Lines are guides for eye. * Space group P4/nmm number 129 and origin choice 2, Wyckoff positions: Y 2b, Ba 2a, Mn2þ 2c, Mn3þ 2c, O1 8j, O2 2c, O3 2c of z ﬁxed to ½ and occupancy (occ.) reﬁned. Observed reﬂections: 166, reﬁned variables: 39. Fig. 2. Thermal evolution of the unit-cell parameters per single-perovskite subcell. 3. Results and discussion 0.008(4) 0.014(4) 0.010(4) 0.010(4) 0.009(4) 0.008(4) 0.012(4) * Space group P4/nmm number 129 and origin choice 2, Wyckoff positions: Y 2b, Ba 2a, Mn2þ 2c, Mn3þ 2c, O1 8j, O2 2c, O3 2c of z ﬁxed to ½ and occupancy (occ.) reﬁned. Observed reﬂections: 166, reﬁned variables: 39. Fig. 2. Thermal evolution of the unit-cell parameters per single-perovskite subcell. The values for 1.5 K are from Ref. [8]. P. Karen Journal of Solid State Chemistry 315 (2022) 123469 3. Results and discussion The synthesis conditions of the highly sintered YBaMn2O5.006(0) NPD sample of ~10 g are listed in Table 1. The iodometric titration is well reproducible, and standard deviations of the oxygen content at the fourth decimal are not unusual. The non-stoichiometry span of the synthesis tests in Table 1 is close to the formation limits of this YBaMn2O5-type structure as a single phase under given synthesis conditions, also in agreement with results in Ref. [15]. The window in terms of pO2 in the reaction atmosphere is narrow. As an example, at the highest tempera- ture tested, 1200 C, increasing the Ar/H2 mixing ratio to 9600 in the synthesis gas wetted at 22.8 C yielded a solid solution YBaMn2O5.67, the The thermal evolution of the apical Mn–O bond lengths is less straightforward. At low temperatures, the smaller Mn3þ pyramid has its apical bond expanded so much by the d4 Jahn–Teller distortion stabi- lizing the highest-occupied molecular orbital (HOMO) dz2 as less anti- bonding, that it is longer than the apical distance in the adjacent larger Mn2þ pyramid. Fig. 1 illustrates that this is achieved by having Mn3þ much closer to the base of its pyramid than Mn2þ is to its base. Upon increasing temperature, the length difference of these two apical bonds decreases (Fig. 3). This thermally induced decrease in the d4 Jahn–Teller distortion was also observed in other Mn3þ compounds such as 2 Journal of Solid State Chemistry 315 (2022) 123469 P. Karen Table 2 Temperature evolution of the YBaMn2O5 structure parameters* and reﬁnement ﬁgures of merit. T (K) 298 373 473 573 673 773 873 Rwp 0.046 0.045 0.043 0.044 0.043 0.043 0.045 Rp 0.035 0.034 0.033 0.034 0.033 0.033 0.035 RF 2 0.033 0.038 0.041 0.049 0.054 0.058 0.070 χ2 3.258 3.162 3.002 3.094 3.083 3.153 3.461 a (Å) 5.54891(8) 5.55306(8) 5.55801(8) 5.56386(8) 5.56928(4) 5.57506(9) 5.58125(9) c (Å) 7.65836(13) 7.67326(13) 7.68955(12) 7.70642(13) 7.72131(13) 7.73706(13) 7.75332(13) V (Å3) 235.80(10) 236.62(10) 237.54(10) 238.56(10) 239.49(10) 240.48(10) 241.52(11) z Mn2þ 0.74624(54) 0.74571(54) 0.74429(55) 0.74384(58) 0.74445(61) 0.74590(61) 0.74641(68) z Mn3þ 0.27565(53) 0.27493(52) 0.27458(54) 0.27413(58) 0.27360(63) 0.27398(65) 0.27379(74) xO1 0.49138(18) 0.49135(18) 0.49163(19) 0.49174(20) 0.49227(23) 0.49235(25) 0.49287(31) zO1 0.31668(10) 0.31670(10) 0.31677(10) 0.31679(11) 0.31653(11) 0.31678(12) 0.31676(13) zO2 0.00724(58) 0.00833(55) 0.00950(53) 0.00988(57) 0.00983(62) 0.00910(67) 0.00841(79) O3 occ. Table 2 Orbital symbols refer to local coordinate systems centered at Mn and oriented in the direction of the O atoms. The energies for 1.5 K are calculated from atomic coordinates in Ref. [8]. Fig. 5. Evolution of energies of essentially nonbonding Mn orbitals calculated for (Mn3þO5)7 (brown) and (Mn2þO5)8 (magenta). Orbital symbols refer to local coordinate systems centered at Mn and oriented in the direction of the O atoms. The energies for 1.5 K are calculated from atomic coordinates in Ref. [8]. 24] conﬁrm the YBaMn2O5 G-type spin order. Only YBaMn2O5 turns into a ferrimagnet upon cooling, though, since its checkerboard charge order coincides with the G-type spin order, which makes all Mn2þ moments to point up (along c [8]) and all Mn3þ down. manganese atoms, calculated with the average Mn–O single-bond length 1.75 Å according to Ref. [18]. As expected, the BVS values are close to þ2 and þ3, and they do start a mutual approach around 600 K. Extrapolation of the four highest-temperature points in Fig. 4 gives an upper estimate for how far YBaMn2O5 may be from valence mixing. Valence mixing via sharing the excess electron of Mn2þ by both manganese atoms that face each other in Fig. 1 would have to be accomplished via their dz2 orbitals, as in YBaFe2O5 [25]. Fig. 6 illustrates how much the energies of the dz2 orbital (in blue) for the electron of ﬁxed spin orientation differ between Mn2þ and Mn3þ. In contrast, in the charge-ordered YBaFe2O5 of Fig. 7, the minority-spin Fe2þ electron has only 0.8 eV to the Fe2þ dz2 orbital that is 0.2 eV below the dz2 of Fe3þ to mix with and thus couple these two Fe ferromagnetic (blue arrow) by this electron sharing across the Y layer [6]. The energies of the essentially nonbonding orbitals of the YBaMn2O5 high-spin [2,8] cations Mn2þ and Mn3þ in their ligand ﬁeld were calculated with CAESAR 2.0 and are plotted in Fig. 5 as a function of temperature. In the small Mn3þ coordination pyramid, the Jahn–Teller expansion of the apical bond upon contraction of basal bonds destabilizes the empty dx2y2 orbital by as much as 4.9 eV from dz2 at 300 K, and that makes the Mn3þ d4 conﬁguration behave as a half-ﬁlled orbital set. The apical oxide anion then mediates an antiferromagnetic semicovalent superexchange [10] with the truly half-ﬁlled spin-polarized orbital set of Mn2þ d5 (Fig. 6). Table 2 The values for 1.5 K are from Ref. [8]. Fig. 2. Thermal evolution of the unit-cell parameters per single-perovskite subcell. The values for 1.5 K are from Ref. [8] Fig. 3. Thermal evolutions of bond lengths Mn2þ–O (magenta) and Mn3þ–O (brown) to the square-pyramidal basis (■) and to the apex (▴). The 1.5 K values are from Ref. [8]. Lines are guides for eye. Fig. 3. Thermal evolutions of bond lengths Mn2þ–O (magenta) and Mn3þ–O (brown) to the square-pyramidal basis (■) and to the apex (▴). The 1.5 K values are from Ref. [8]. Lines are guides for eye. stoichiometric LaMnO3, where it disappears at about 750 K [16,17]. In YBaMn2O5, the apical and basal Mn–O distances in each pyramid become closest at about 600 K (Fig. 3) due to this thermal alleviation of the Jahn–Teller distortion. From that point up, a slow mutual approach of the stoichiometric LaMnO3, where it disappears at about 750 K [16,17]. In YBaMn2O5, the apical and basal Mn–O distances in each pyramid become closest at about 600 K (Fig. 3) due to this thermal alleviation of the Jahn–Teller distortion. From that point up, a slow mutual approach of the two Mn valences starts, demonstrated by resumed contraction of the apical distance in the larger Mn2þ pyramid versus expansion in the smaller Mn3þ one, both upon the overall thermal expansion. Fig. 4 shows the evolution of the bond-valence sums (BVS) of the two 3 Fig. 4. Thermal evolution of the bond-valence sums (BVS) at the two crystallographically different Mn atoms. The BVS for 1.5 K is calculated from data in Ref. [8]. P. Karen Journal of Solid State Chemistry 315 (2022) 123469 P. Karen Journal of Solid State Chemistry 315 (2022) 123469 Fig 4 Thermal evolution of the bond valence sums (BVS) at the two crystallographically different Mn atoms The BVS for 1 5 K is calculated from data in Ref [8] P. Karen Journal of Solid State Chemistry 315 (2022) 123469 Fig. 4. Thermal evolution of the bond-valence sums (BVS) at the two crystallographically different Mn atoms. The BVS for 1.5 K is calculated from data in Ref. [8]. Fig. 4. Thermal evolution of the bond-valence sums (BVS) at the two crystallographically different Mn atoms. The BVS fo Fig. 5. Evolution of energies of essentially nonbonding Mn orbitals calculated for (Mn3þO5)7 (brown) and (Mn2þO5)8 (magenta). Table 2 This is what yields below TN ¼ 167 K [8,19] the observed [8] G-type [3] coupling of all manganese spins. The basal ox- ygens of the pyramids mediate likewise. Their buckling angle Mn2þ–O–Mn3þ in YBaMn2O5 (157.20(10) at 298 K versus 156.75(14) at 873 K) is the same as in the charge-ordered phases YBaFe2O5 of 158.59(31) at 20 K [6] or YBaCo2O5 of 156.30(88) at 50 K [20], where the G-type coupling was identiﬁed by NPD in their charge-ordered state (on HoBaFe2O5 [21] and HoBaCo2O5 [22]). Also DFT calculations [23, 4. Conclusions Synthesis conditions to obtain single-phase YBaMnIIMnIIIO5 by sin- tering at high-temperatures make a narrow window of oxygen partial pressures in the reaction atmosphere (Table 1). Likewise narrow is the range in temperatures. Whereas sintering at 1000 C yields porosity of 60%, the silvery product at 1200 C has 6%. The crystal structure in the paramagnetic state, reﬁned from neutron powder diffraction data 4 P. Karen Journal of Solid State Chemistry 315 (2022) 123469 Fig. 6. Charge-ordered YBaMn2O5. Semicovalent linear superexchange interaction of effectively half-ﬁlled d4 Mn3þ and half-ﬁlled d5 Mn2þ cations. The spin-pairing energy UC is drawn to permit the observed [8] Mn2þ high-spin state. The two dz2 orbitals to eventually share the valence-mixing electron at high temperature are marked in blue. Fig. 6. Charge-ordered YBaMn2O5. Semicovalent linear superexchange interaction of effectively half-ﬁlled d4 Mn3þ and half-ﬁlled d5 Mn2þ cations. The spin-pairing energy UC is drawn to permit the observed [8] Mn2þ high-spin state. The two dz2 orbitals to eventually share the valence-mixing electron at high temperature are marked in blue. Fig. 7. Charge-ordered YBaFe2O5 on the scale of Fig. 6. Semicovalent linear superexchange interaction of half-ﬁlled d5 Fe3þ and of d6 Fe2þ cations. The spin- pairing energy UC is drawn to permit the observed [6] Fe2þ high-spin state. In blue: Upon demise of the charge ordering, the minority-spin Fe2þ electron in the ordered dxz orbital will mix via dz2 orbitals (blue shapes) of the two Fe atoms facing each other across their pyramidal bases and couple them ferromagnetic (blue double arrow) in the still antiferromagnetic crystal [6]. Fig. 7. Charge-ordered YBaFe2O5 on the scale of Fig. 6. Semicovalent linear superexchange interaction of half-ﬁlled d5 Fe3þ and of d6 Fe2þ cations. The spin- pairing energy UC is drawn to permit the observed [6] Fe2þ high-spin state. In blue: Upon demise of the charge ordering, the minority-spin Fe2þ electron in the ordered dxz orbital will mix via dz2 orbitals (blue shapes) of the two Fe atoms facing each other across their pyramidal bases and couple them ferromagnetic (blue double arrow) in the still antiferromagnetic crystal [6]. collected between 25 and 600 C, maintains Mn2þ and Mn3þ ordered in a three-dimensional checkerboard pattern of electrostatic minimization; a pattern that in YBaFe2O5 is modiﬁed by orbital ordering [6]. electron conﬁgurations via the intervening oxygen then yields antifer- romagnetic coupling of the two unequal-spin manganese atoms. 4. Conclusions Furthermore, the calculations show that the energies of the dz2 electron of the same spin orientation differ profoundly between Mn2þ and Mn3þ, and this is what makes it hard to mix thermally the Mn2þ excess electron with Mn3þ in YBaMn2O5 when compared with the easy mixing in YBaFe2O5. At ambient temperature, the presence of one electron in the Mn3þ HOMO dz2 expands the apical Mn–O distance of this small coordination pyramid due to Jahn–Teller distortion. The apical distance of the larger Mn2þ square pyramid is contracted due to corresponding expansion of its basal Mn–O distances that stabilizes the Mn2þ HOMO dx2y2. Upon warming to ~600 K, the apical and basal Mn–O distances become closest both in the larger Mn2þ and in the smaller Mn3þ pyramid (Fig. 3), due to thermal alleviation of the Jahn–Teller distortion. Above this tempera- ture, the Mn2þ–O apical distance begins to contract and Mn3þ–O expand upon the overall thermal expansion. From this point, the BVS at Mn2þ and Mn3þ start slowly approaching one another as a ﬁrst sign of a possible valence mixing at a higher temperature. Interestingly, combining Mn and Co in the prototype double-cell perovskite structure does not simulate YBaFe2O5. There is no charge ordering, no valence mixing, just a Co2þ and Mn3þ solid solution of d7–d4 conﬁgurations [26]. The reason is redox chemistry. If the product were YBaCoIIIMnIIO5, the Co3þ and Mn2þ d6–d5 conﬁguration would have behaved as it does in YBaFeIIFeIIIO5. CRediT authorship contribution statement Extended-Hückel calculations of the orbital energies for the isolated square-pyramidal coordinations around Mn3þ and Mn2þ bring further details. The very high energy of the Mn3þ LUMO dx2y2 makes this electron conﬁguration effectively half-ﬁlled. The semicovalent super- exchange interaction [10] of such d4 Mn3þ and d5 Mn2þ half-ﬁlled Pavel Karen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. 5 P. Karen Journal of Solid State Chemistry 315 (2022) 123469 Data availability [12] P. 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https://openalex.org/W2949927737	https://europepmc.org/articles/pmc6628327?pdf=render	English	null	Circulating miRNAs in Untreated Breast Cancer: An Exploratory Multimodality Morpho-Functional Study	Cancers	2,019	cc-by	14,993	Circulating miRNAs in Untreated Breast Cancer: An Exploratory Multimodality Morpho-Functional Study 1 IRCCS SDN, 80143 Naples, Italy; agrimaldi@sdn-napoli.it (A.M.G.); pmirabelli@sdn-napoli.it (P.M.); ccavaliere@sdn-napoli.it (C.C.); caparente@sdn-napoli.it (C.A.P.); mfranzese@sdn-napoli.it (M.F.); soricelli@uniparthenope.it (A.S.); direzionescientiﬁca@sdn-napoli.it (M.S.) p , y, p , y; staibano@unina.it (S.S.); gennaro.ilardi@gmail.com (G.I.); danielarusso83@yahoo.it (D.R.) 3 Department of Motor Sciences & Wellness, University of Naples Parthenope, 80133 Naples, Italy 4 Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts Gener Hospital, Harvard Medical School, Charlestown, MA 02129, USA * Correspondence: mincoronato@sdn-napoli.it (M.I.); onofriocatalano@yahoo.it (O.A.C.) cancers cancers cancers Cancers 2019, 11, 876; doi:10.3390/cancers11060876 Received: 24 May 2019; Accepted: 20 June 2019; Published: 22 June 2019 Abstract: The aim of this study was to identify new disease-related circulating miRNAs with high diagnostic accuracy for breast cancer (BC) and to correlate their deregulation with the morpho-functional characteristics of the tumour, as assessed in vivo by positron emission tomography/magnetic resonance (PET/MR) imaging. A total of 77 untreated female BC patients underwent same-day PET/MR and blood collection, and 78 healthy donors were recruited as negative controls. The expression proﬁle of 84 human miRNAs was screened by using miRNA PCR arrays and validated by real-time PCR. The validated miRNAs were correlated with the quantitative imaging parameters extracted from the primary BC samples. Circulating miR-125b-5p and miR-143-3p were upregulated in BC plasma and able to discriminate BC patients from healthy subjects (miR-125-5p area under the receiver operating characteristic ROC curve (AUC) = 0.85 and miR-143-3p AUC = 0.80). Circulating CA15-3, a soluble form of the transmembrane glycoprotein Mucin 1 (MUC-1) that is upregulated in epithelial cancer cells of diﬀerent origins, was combined with miR-125b-5p and improved the diagnostic accuracy from 70% (CA15-3 alone) to 89% (CA15-3 plus miR-125b-5p). MiR-143-3p showed a strong and signiﬁcant correlation with the stage of the disease, apparent diﬀusion coeﬃcient (ADCmean), reverse eﬄux volume transfer constant (Kepmean) and maximum standardized uptake value (SUVmax), and it might represent a biomarker of tumour aggressiveness. Similarly, miR-125b-5p was correlated with stage and grade 2 but inversely correlated with the forward volume transfer constant (Ktransmean) and proliferation index (Ki67), suggesting a potential role as a biomarker of a relatively more favourable prognosis. In situ hybridization (ISH) experiments revealed that miR-143-3p was expressed in endothelial tumour cells, miR-125-5p in cancer-associated ﬁbroblasts, and neither in epithelial tumour cells. Our results suggested that miR-125-5p and miR-143-3p are potential biomarkers for the risk stratiﬁcation of BC, and Kaplan-Maier plots conﬁrmed this hypothesis. In addition, the combined use of miR-125-b-5p and CA15-3 enhanced the diagnostic accuracy up to 89%. This is the ﬁrst study that correlates circulating miRNAs with in vivo quantiﬁed tumour biology through PET/MR biomarkers. This integration elucidates the link between the plasmatic increase in these two potential circulating biomarkers and the biology of untreated BC. In conclusion, while miR-143-3b and miR-125b-5p provide valuable information for prognosis, a combination of miR-125b-5p with the tumour marker CA15-3 improves sensitivity for BC detection, which warrants consideration by further validation studies. 1. Introduction According to GLOBOCAN 2018, a project of the International Agency for Research on Cancer (IARC), approximately 2.1 million women (1.7 million in 2012) were diagnosed with breast cancer (BC) worldwide, and there were approximately 6.9 million women (6.3 million in 2012) who had been diagnosed with BC in the previous ﬁve years [1]. BC is also the most common cause of cancer death among women, with 627,000 deaths in 2018. Early diagnosis plays a major role in ﬁghting BC. However, on a worldwide scale, breast imaging techniques such as screening mammography and ultrasound have intrinsic limitations related to economic costs, limited technology availability in underdeveloped countries, the necessity of well-trained radiologists, and an overall unsatisfactory performance regarding the images. Screening mammography performance indexes vary considerably for sensitivity and speciﬁcity, inﬂuencing the ability of mammography to reach its full potential for decreasing BC mortality [2]; moreover, ultrasound plus mammography can indicate cancers in high-risk women, but the number of false positives are increased [3]. Circulating biomarkers are biomolecules released into the bloodstream by both tumour cells and other types of neighbouring cells [4]. Their quantiﬁcation and identiﬁcation are considered an economical, non-invasive diagnostic method that provides information on the presence and/or absence of the disease as well as its evolution. To date, the carcinoembryonic antigen (CEA) [5] and the soluble form of Mucin 1 (MUC-1) protein (CA15-3) [6] are the most commonly used serum biomarkers for the clinical monitoring of BC patients. Although these biomarkers have been recognized from the international healthcare system as molecules used for the management of BC patients, current guidelines suggest that they fail in the early diagnosis of the disease, having a clinical role only during follow-up [7]. Therefore, there is a compelling need to discover potential biomarkers that might be useful for diagnosing BC. The ideal tumour marker should have high speciﬁcity, sensitivity and predictive value and be detectable by an accurate, rapid, simple and inexpensive method that might also be used in countries with less access to expensive technology. In recent decades, non-coding RNA molecules (miRNAs) have emerged as a new class of regulatory genes involved in many cellular processes, such as development, diﬀerentiation, proliferation, apoptosis and the response to stress. In their mature form, these single-stranded small molecules are 19 to 25 nucleotides in length and are able to silence gene expression at the post-transcriptional level, thus inhibiting protein translation. Keywords: circulating miRNAs; breast cancer; imaging parameters; PET/MRI; biomarkers Keywords: circulating miRNAs; breast cancer; imaging parameters; PET/MRI; biomarkers Received: 24 May 2019; Accepted: 20 June 2019; Published: 22 June 2019 Abstract: The aim of this study was to identify new disease-related circulating miRNAs with high diagnostic accuracy for breast cancer (BC) and to correlate their deregulation with the morpho-functional characteristics of the tumour, as assessed in vivo by positron emission tomography/magnetic resonance (PET/MR) imaging. A total of 77 untreated female BC patients underwent same-day PET/MR and blood collection, and 78 healthy donors were recruited as negative controls. The expression proﬁle of 84 human miRNAs was screened by using miRNA PCR arrays and validated by real-time PCR. The validated miRNAs were correlated with the quantitative imaging parameters extracted from the primary BC samples. Circulating miR-125b-5p and miR-143-3p were upregulated in BC plasma and able to discriminate BC patients from healthy subjects (miR-125-5p area under the receiver operating characteristic ROC curve (AUC) = 0.85 and miR-143-3p AUC = 0.80). Cancers 2019, 11, 876; doi:10.3390/cancers11060876 www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers 2 of 22 Cancers 2019, 11, 876 1. Introduction They act by recognizing a sequence within the 3’ and/or 5’ untranslated region (UTR) of their mRNA target [8]. miRNAs can exert their action in cancers through both tumour suppression and oncogenic mechanisms [9,10]. Due to their structural characteristics, functions and tissue speciﬁcity, miRNAs have been proposed as a new class of biomarkers for the screening of diﬀerent tumours. Recent discoveries have shown that miRNAs are also present in biological ﬂuids, such as blood [11], urine [12], sputum [13] and saliva [14]; moreover, they were found to be aberrantly expressed in diﬀerent human cancers and feature unprecedented levels of diagnostic speciﬁcity and sensitivity that are associated with tumour burden and malignant progression [15,16]. Additionally, circulating miRNAs are extremely stable against degradation by RNase, easily collected through non-invasive methods (blood sampling), resistant to repeated freeze/thaw cycles and easily identiﬁed by nucleic acid ampliﬁcation techniques (real-time PCR). These factors also make their collection possible in third-world countries and underserved areas. Taken together, these ﬁndings highlight the potential usefulness of these molecules as promising biomarkers. To date, many studies have identiﬁed diﬀerent circulating miRNAs as new biomarkers for BC detection; however, the results are discordant [17]. Moreover, they have not been assessed in conjunction with diagnostic imaging, which is used for quantifying the extent of the disease and provides information related to tumour aggressiveness representing an obligatory step for treatment planning. The use of diagnostic imaging to extract 3 of 22 Cancers 2019, 11, 876 quantitative parameters related to the morphology, metabolism and functionality of tumours, as well as to correlate speciﬁc imaging parameters with tissue biomarkers, is an emerging research topic [18,19]. The aim of our study was to identify potential new disease-related circulating miRNAs with high diagnostic accuracy for BC and to elucidate the relationship between their circulating deregulation and tumour characteristics, including stage at presentation and the functional quantiﬁcation of tumour metabolism and perfusion, by using same-day positron emission tomography/magnetic resonance (PET/MR) imaging. Since PET/MR scanners have been approved for use less than 8 years ago, with very few installed worldwide, PET/MR is currently clinically used for BC diagnosis and treatment planning in only an extremely limited number of highly specialized centres that have both the expertise of scanner use and a strong oncologic breast programme. 1. Introduction In these centres, PET/MR is used for the whole-body and local staging of newly diagnosed BC cases that are amenable to curative resection, either without or after neoadjuvant treatments, and as problem-solving technology in complicated, more advanced cases. PET/MR is currently not mentioned in the National Comprehensive Cancer Network (NCCN) guidelines or in any other scientiﬁc organization/society guidelines despite its higher performances than those of any other non-invasive imaging modality. In fact, the accuracy of PET/MR staging outperformed that of PET/computed tomography (CT) (the most accurate of the routinely clinically approved imaging modalities) on a per patient analysis (98% versus 75%) and was also capable of demonstrating bony metastases not detected by same-day PET/CT in up to 12% of the cases [20,21]. These results have been conﬁrmed by other authors who showed that, in a lesion-per-lesion analysis, PET/MR had higher sensitivity than PET/CT in detecting bony lesions (0.924 and 0.6923, respectively). Moreover, PET/MR also had a better sensitivity than PET/CT in detecting contralateral tumours (1 and 0.25, respectively) and all lesions together (0.89 and 0.77, respectively) [22]. To the best of our knowledge, this is the ﬁrst study that aimed to correlate the deregulated expression of disease-related circulating miRNAs with the morphological, metabolic and functional parameters of BC lesions (tumour metabolism, cellular density and vascularisation) extracted through a hybrid PET/MR scanner. The plasma samples of BC patients and healthy donors were analysed for miRNA expression, and for each BC patient, PET/MR was used to ascertain the stage of the disease as well as the metabolic, diﬀusion and perfusion characteristics of the primary cancer. In silico studies and in situ hybridization (ISH) experiments were also performed to better understand miRNA expression in tissue and their cellular localization. Last, we correlated the expression levels of the validated miRNAs with immunohistochemical markers of BC, stage of disease and PET/MR-derived functional and metabolic biomarkers of primary cancer. 2. Results 2.1. Identiﬁcation and Validation of a Plasma miRNA Signature for Breast Cancer Detection 2.1. Identiﬁcation and Validation of a Plasma miRNA Signature for Breast Cancer Detection The study design is illustrated in Figure 1A. The healthy and BC subjects were age-matched (p-value = 0.157), and the clinicopathological characteristics of the study participants are reported in Table 1. By using the miScript miRNA PCR Array, we found that ﬁve (miR-125b-5p, miR-143-3p, miR-145-5p, miR-100-5p and miR-23a-3p; Figure 1B–D) out of the 84 miRNAs analysed were signiﬁcantly (p-value < 0.05) upregulated in the plasma samples of 27 BC patients vs. 14 healthy donors, with a fold change ≥1.5. RT-PCR was conducted to conﬁrm the array results in a study population of 46 healthy donors and 64 naïve BC patients (Supplementary Figure S1). Then, a logistic regression model (Supplementary Table S1A) identiﬁed miR-125b-5p and miR-143-3p as the circulating miRNAs able to eﬀectively discriminate aﬀected from unaﬀected subjects. A logistic stepwise regression model conﬁrmed these results (Supplementary Table S1B). Moreover, the results were corroborated by expanding the case studies to 78 healthy donors and 77 naïve BC patients (Figure 1A, Validation II). In fact, as shown in Figure 2A,B, we conﬁrmed that miR-125b-5p and miR-143-3p were signiﬁcantly upregulated in the plasma of BC patients with a fold change of 3.3 and 3.2, respectively. These results were conﬁrmed by logistic stepwise regression analysis (Figure 2C). 4 of 22 Cancers 2019, 11, 876 Table 1. Detailed clinicopathological characteristics of the study participants. Healthy Control Samples (n = 78) Age range 28–84 years Mean ± S.D. 51.05 ± 11.26 Breast Cancer Samples (n = 77) Age range 28–82 years Mean ± S.D. 53.68 ± 12.02 Subtype Luminal A 16 Luminal B 50 HER2+ 7 Triple negative 4 Ki67 Low (1–20%) 21 High (21–100%) 56 Grade G1 1 G2 41 G3 35 Stage Stage I 1 Stage II 12 Stage III 42 Stage IV 22 S.D.: standard deviation; HER2+: Human epidermal growth factor receptor positive. , x FOR PEER REVIEW Table 1. Detailed clinicopathological characteristics of the study participants. Healthy Control Samples (n = 78) Age range 28–84 years Mean ± S.D. 51.05 ± 11.26 Breast Cancer Samples (n = 77) Age range 28–82 years Mean ± S.D. 2. Results 53.68 ± 12.02 Subtype Luminal A 16 Luminal B 50 HER2+ 7 Triple negative 4 Ki67 Low (1–20%) 21 High (21–100%) 56 Grade G1 1 G2 41 G3 35 Stage Stage I 1 Stage II 12 Stage III 42 Stage IV 22 S D : standard deviation; HER2+: Human epidermal growth factor receptor posit Table 1. Detailed clinicopathological characteristics of the study participants. Healthy Control Samples (n = 78) Age range 28–84 years Mean ± S.D. 51.05 ± 11.26 Breast Cancer Samples (n = 77) Age range 28–82 years Mean ± S.D. 53.68 ± 12.02 Subtype Luminal A 16 Luminal B 50 HER2+ 7 Triple negative 4 Ki67 Low (1–20%) 21 High (21–100%) 56 Grade G1 1 G2 41 G3 35 Stage Stage I 1 Stage II 12 Stage III 42 Stage IV 22 S.D.: standard deviation; HER2+: Human epidermal growth factor receptor positive. , x FOR PEER REVIEW Table 1. Detailed clinicopathological characteristics of the study participants Healthy Control Samples (n = 78) Age range 28–84 years Mean ± S.D. 51.05 ± 11.26 Breast Cancer Samples (n = 77) Age range 28–82 years Mean ± S.D. 53.68 ± 12.02 Subtype Luminal A 16 Luminal B 50 HER2+ 7 Triple negative 4 Ki67 Low (1–20%) 21 High (21–100%) 56 Grade G1 1 G2 41 G3 35 Stage Stage I 1 Stage II 12 Stage III 42 Stage IV 22 S D : standard deviation; HER2+: Human epidermal growth factor receptor posi Table 1. Detailed clinicopathological characteristics of the study participants. x FOR PEER REVIEW S.D.: standard deviation; HER2+: Human epidermal growth factor receptor positive. Stage IV 22 Figure 1. Cont. Figure 1. Cont. 5 of 22 Cancers 2019, 11, 876 Figure 1. Study design for the identification of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). miRNAs significantly upregulated (p < 0.05) in BC patients h l h d (C D) Figure 1. Study design for the identiﬁcation of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. 2. Results Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). miRNAs signiﬁcantly upregulated (p < 0.05) in BC patients vs. healthy donors (C,D). Figure 1. Study design for the identification of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). miRNAs significantly upregulated (p < 0.05) in BC patients vs healthy donors (C,D). Figure 1. Study design for the identification of circulating miRNAs and protein biomarkers able t discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a se population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by usin a 96-well miScript miRNA PCR Array (B). miRNAs significantly upregulated (p < 0.05) in BC patient Figure 1. Study design for the identiﬁcation of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). * miRNAs signiﬁcantly upregulated (p < 0.05) in BC patients vs. healthy donors (C,D). Figure 1. Study design for the identification of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). miRNAs significantly upregulated (p < 0.05) in BC patients vs healthy donors (C,D). vs healthy donors (C,D). Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 health donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relativ expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. B patients. Logistic stepwise regression analysis (C). miRNAs that significantly (p < 0.05) estimate th b b l f h C Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. 2. Results Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients. Logistic stepwise regression analysis (C). miRNAs that significantly (p < 0.05) estimate the probability of having BC. Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients. Logistic stepwise regression analysis (C). miRNAs that signiﬁcantly (p < 0.05) estimate the probability of having BC. vs healthy donors (C,D). vs he Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients Logistic stepwise regression analysis (C) miRNAs that significantly (p < 0 05) estimate the Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients. Logistic stepwise regression analysis (C). miRNAs that significantly (p < 0.05) estimate the probability of having BC. Figure 2. Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients. Logistic stepwise regression analysis (C). * miRNAs that signiﬁcantly (p < 0.05) estimate the probability of having BC. Cancers 2019, 11, 876 6 of 22 2.2. Diagnostic miRNA Signature-Based Model Compared with Established BC Markers Cancers 2019, 11, x FOR PEER REVIEW 2.2. Diagnostic miRNA Signature-Based Model Compared with Established BC Markers Cancers 2019, 11, x FOR PEER REVIEW Receiver operating characteristic (ROC) curve analyses showed that miR-125b-5p (area under the ROC curve (AUC) = 0.85) and miR-143-3p (AUC = 0.80) were able to discriminate BC patients from healthy donors (Figure 3A,B). 2. Results Then, we compared the AUC value of the selected miRNAs with the AUC value of the traditional BC biomarkers CA15-3 and CEA. As reported in Figure 3C,D, the AUC values of CA15-3 (0.70) and CEA (0.68) were lower than those calculated for miR-125b-5p and miR-143-3p. Based on these results, a logistic stepwise regression analysis was performed considering the miR-125b-5p, miR-143-3p, CA15-3 and CEA molecules as independent variables (Figure 1A, Validation III). According to Table 2, miR-125b-5p, miR-143-3p and CA15-3 were signiﬁcantly correlated with the disease, although the statistical signiﬁcance of CA15-3 was lower than that of the miRNAs. Based on these ﬁndings, the diagnostic accuracy of each biomarker was evaluated. To this end, the cut-oﬀvalues of miR-125b-5p and miR-143-3p were calculated, and the clinical cut-oﬀvalues of CA15-3 and CEA were already known (CA15-3 = 35 UI/mL and CEA = 3 UI/mL). Using the “closest to top-left” method, we deﬁned the cut-oﬀvalue of miR-125b-5p as 0.0090 and that of miR-143-3p as 0.0064, both related to relative expression. Among the analysed molecules, miR-125b-5p showed the highest diagnostic accuracy (Table 3). 2.2. Diagnostic miRNA Signature-Based Model Compared with Established BC Markers Receiver operating characteristic (ROC) curve analyses showed that miR-125b-5p (area under the ROC curve (AUC) = 0.85) and miR-143-3p (AUC = 0.80) were able to discriminate BC patients from healthy donors (Figure 3A,B). Then, we compared the AUC value of the selected miRNAs with the AUC value of the traditional BC biomarkers CA15-3 and CEA. As reported in Figure 3C,D, the AUC values of CA15-3 (0.70) and CEA (0.68) were lower than those calculated for miR-125b-5p and miR-143-3p. Based on these results, a logistic stepwise regression analysis was performed considering the miR-125b-5p, miR-143-3p, CA15-3 and CEA molecules as independent variables (Figure 1A, Valida- tion III). According to Table 2, miR-125b-5p, miR-143-3p and CA15-3 were significantly correlated with the disease, although the statistical significance of CA15-3 was lower than that of the miRNAs. Based on these findings, the diagnostic accuracy of each biomarker was evaluated. To this end, the cut-off values of miR-125b-5p and miR-143-3p were calculated, and the clinical cut-off values of CA15-3 and CEA were already known (CA15-3 = 35 UI/mL and CEA = 3 UI/mL). Using the “closest to top-left” method, we defined the cut-off value of miR-125b-5p as 0.0090 and that of miR-143-3p as 0.0064, both related to relative expression. 2. Results Among the analysed molecules, miR-125b-5p showed the highest di- agnostic accuracy (Table 3). Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that signiﬁcantly (p < 0.05) estimate the probability of having a tumour. Circulating Biomarkers Estimate Std. Error p-Value miR-125b-5p * 9.018 2.401 0.000248 miR-143-3p * 1.017 × 101 2.644 0.000179 CA15-3 * 2.123 × 10−3 8.984 × 10−4 0.01941 CEA 4.866 × 10−3 7.095 × 10−3 0.4939 Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that significantly (p < 0.05) estimate the probability of having a tu- mour. Circulating Biomarkers Estimate Std. Error p-value miR-125b-5p * 9.018 2.401 0.000248 miR-143-3p * 1.017e+01 2.644 0.000179 CA15-3 * 2.123e-03 8.984e-04 0.01941 CEA 4.866e-03 7.095e-03 0.4939 Figure 3. ROC curve analyses and AUC values of miR-125-5p (A), miR-143-3p (B), CA15-3 (C), and CEA (D) for discriminating BC patients from healthy controls in 76 BC patients and 78 healthy donors. Figure 3. ROC curve analyses and AUC values of miR-125-5p (A), miR-143-3p (B), CA15-3 (C), and CEA (D) for discriminating BC patients from healthy controls in 76 BC patients and 78 healthy donors. ROC: Receiver operating characteristic; AUC: area under the ROC curve; CEA: carcinoembryonic antigen. Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that signiﬁcantly (p < 0.05) estimate the probability of having a tumour. Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that significantly (p < 0.05) estimate the probability of having a tu- mour. Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that signiﬁcantly (p < 0.05) estimate the probability of having a tumour. Table 2. Validation III. Logistic stepwise regression on the indicated molecules in a cohort of 78 CTR and 77 BC subjects. * Molecules that significantly (p < 0.05) estimate the probability of having a tu- mour. Circulating Biomarkers Estimate Std. 2. Results Error p-Value miR-125b-5p * 9.018 2.401 0.000248 miR-143-3p * 1.017 × 101 2.644 0.000179 CA15-3 * 2.123 × 10−3 8.984 × 10−4 0.01941 CEA 4.866 × 10−3 7.095 × 10−3 0.4939 Circulating Biomarkers Estimate Std. Error p-value miR-125b-5p * 9.018 2.401 0.000248 miR-143-3p * 1.017e+01 2.644 0.000179 CA15-3 * 2.123e-03 8.984e-04 0.01941 CEA 4.866e-03 7.095e-03 0.4939 Figure 3. ROC curve analyses and AUC values of miR-125-5p (A), miR-143-3p (B), CA15-3 (C), and CEA (D) for discriminating BC patients from healthy controls in 76 BC patients and 78 healthy donors. Figure 3. ROC curve analyses and AUC values of miR-125-5p (A), miR-143-3p (B), CA15-3 (C), and CEA (D) for discriminating BC patients from healthy controls in 76 BC patients and 78 healthy donors. ROC: Receiver operating characteristic; AUC: area under the ROC curve; CEA: carcinoembryonic antigen. Cancers 2019, 11, 876 7 of 22 Table 3. Diagnostic accuracy of the indicated circulating biomarkers. g y g miR-125b-5p Disease + Disease − Total Sensitivity Speciﬁcity Diagnostic Accuracy Test + 61 (TP) 16 (FP) 77 0.79 (CI 0.68–0.88) 0.79 (CI 0.69–0.88) 0.79 (CI 0.72–0.85) Test − 16 (FN) 62 (TN) 78 Total 77 78 155 miR-143-3p Disease + Disease − Total Sensitivity Speciﬁcity Diagnostic accuracy Test + 53 (TP) 20 (FP) 73 0.69 (CI 0.57–0.79) 0.74 (CI 0.63–0.84) 0.72 (CI 0.64–0.79) Test − 24 (FN) 58 (TN) 82 Total 77 78 155 CA15-3 Disease + Disease − Total Sensitivity Speciﬁcity Diagnostic accuracy Test + 29 (TP) 1 (FP) 30 0.38 (CI 0.27–0.50) 0.99 (CI 0.93–1.00) 0.68 (CI 0.60–0.76) Test − 47 (FN) 75 (TN) 122 Total 76 76 152 CEA Disease + Disease − Total Sensitivity Speciﬁcity Diagnostic accuracy Test + 25 (TP) 8 (FP) 73 0.33 (CI 0.23–0.45) 0.89 (CI 0.80–0.95) 0.61 (CI 0.53–0.69) Test − 51 (FN) 68 (TN) 82 Total 76 76 152 CI: conﬁdence interval 95%; Disease +: BC patients, Disease −: healthy donors, TP: True Positive TP, FP: False Positive, FN: False Negative and TN: True Negative. CEA: carcinoembryonic antigen and BC: breast cancer. 2.3. Co-Expression Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p and Their Association with Immunohistochemical Markers of BC 2.3. Co-Expression Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p and Their Association with Immunohistochemical Markers of BC Although miR-145-5p was not able to estimate the probability of pathology (Supplementary Table S1), it is transcribed from a putative miR-143/145 cluster on chromosome 5 in humans (5q33). 2. Results In addition, The BC patients enrolled in this study underwent same-day contrast-enhanced ﬂuorodeoxyglucose (CE-FDG)-PET/MR and blood collection to better correlate circulating miR-125-5p and miR-143-3p with the morphological, metabolic and functional imaging features of BC (Figure 4). No signiﬁcant correlations were found between the miRNA expression levels and lesion size (Supplementary Table S2). The plasma levels of miR-125b-5p increased signiﬁcantly at stage II, reaching higher expression levels at stage III and remained unchanged at stage IV compared to stage III (Figure 4D,G). This trend conﬁrmed that the expression levels of miR-125b-5p were variable and depended on the severity of the disease. Additionally, the expression levels of miR-143-3p (Figure 4E,G) became signiﬁcantly higher in stage III and drastically decreased in stage IV, reaching expression values close to those of healthy donors. This result suggests that at stage IV, this molecule is not required for the maintenance of the pathology. It is known that CA15-3 levels increase in the late stage of the disease, so its relationship with clinical staging was also evaluated. As reported in Figure 4F,G, in our cohort, CA15-3 was not able to diagnose stage II, but its levels increased signiﬁcantly at stage III, reaching the maximum concentration at stage IV. This was in agreement with the known performance of CA15-3. In addition, no correlations were found between CA15-3 and grade (p-value = 0.761) or subtypes (p-value = 0.450). no correlations were found between CA15-3 and grade (p-value = 0.761) or subtypes (p-value = 0.450). Correlation analyses between miR-125b-5p and miR-143-3p levels and PET/MR-based tumour biomarkers were performed by grouping the data according to staging. Only the statistically signifi- cant results were reported (Table 4); we found a strong and significant correlation between circulating miR-143-3p and the mean initial area under the concentration curve (iAUCmean) and the mean reverse efflux volume transfer constant (Kepmean) (for both, ρ: 0.943, p-value = 0.005) in the stage II group. Perfusion parameters such as iAUC and Kep are generally correlated with tumour vascularisation [23,24], suggesting that the plasmatic overexpression of miR-143-3p in BC patients might correlate with angiogenesis. The correlation between iAUC and miR-143-3p persisted at stage III but was lost at stage IV, where its relative expression drastically decreased (see Figure 4B). Notably, there was a strong correlation index between miR-143-3p and the maximum standardized uptake value (SUVmax) (ρ: 0.829 and p-value = 0.042) at stage II, although the statistical significance was borderline. 2. Results Expression levels of miR-125b-5p vs Ki67 (low <20%; high ≥20%) (B), and grade (C). Expression levels of miR-125b-5p (D), miR-143-3p (E), and CA15-3 (F) vs. stage. Statistical significance (G). Non- significant results are reported in bold. Figure 4. Correlation studies. Co-expression analyses of miR-125b-5p, miR-143-3p and miR-145-5p (A). Expression levels of miR-125b-5p vs. Ki67 (low <20%; high ≥20%) (B), and grade (C). Expression levels of miR-125b-5p (D), miR-143-3p (E), and CA15-3 (F) vs. stage. Statistical signiﬁcance (G). Non-signiﬁcant results are reported in bold. 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imag- 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imaging Parameters of Tumour Lesions 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imag- 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imaging Parameters of Tumour Lesions 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imag- 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imaging Parameters of Tumour Lesions ing Parameters of Tumour Lesions The BC patients enrolled in this study underwent same-day contrast-enhanced fluorodeoxyglu- cose (CE-FDG)-PET/MR and blood collection to better correlate circulating miR-125-5p and miR-143- 3p with the morphological, metabolic and functional imaging features of BC (Figure 4). No significant correlations were found between the miRNA expression levels and lesion size (Supplementary Table S2). The plasma levels of miR-125b-5p increased significantly at stage II, reaching higher expression levels at stage III and remained unchanged at stage IV compared to stage III (Figure 4D,G). This trend con- firmed that the expression levels of miR-125b-5p were variable and depended on the severity of the disease. Additionally, the expression levels of miR-143-3p (Figure 4E,G) became significantly higher in stage III and drastically decreased in stage IV, reaching expression values close to those of healthy donors. This result suggests that at stage IV, this molecule is not required for the maintenance of the pathology. It is known that CA15-3 levels increase in the late stage of the disease, so its relationship with clinical staging was also evaluated. As reported in Figure 4F,G, in our cohort, CA15-3 was not able to diagnose stage II, but its levels increased significantly at stage III, reaching the maximum concentration at stage IV. This was in agreement with the known performance of CA15-3. 2. Results Therefore, co-expression analyses by Spearman’s test were performed not only for miR-125b-5p and miR-143-3p but also for miR-145-5p. As reported in Figure 4A, we found that miR-143-3p and miR-145-5p were strongly co-expressed (ρ: 0.93; p-value < 2 × 10−16) in the plasma samples of BC patients, and the expression levels of miR-125b-5p were signiﬁcantly correlated with those of miR-143-3p (ρ: 0.57; p-value = 2 × 10−4) and miR-145-5p (ρ: 0.56; p-value = 6 × 10−6), although to a limited extent. The immunohistochemical (IHC) reports of the enrolled BC patients were used to determine the receptor status (oestrogen, progesterone, and human epidermal growth factor receptor 2), cellular diﬀerentiation status (grade) and proliferation index (Ki67) of the tumour lesions. No association was found between the expression levels of miR-125-5p, miR-143-3p and miR-145-5p and the hormonal receptor status of the lesions (Estrogen Receptor positive or negative: ER+/−, Progesteron receptor positive or negative: PR +/−and Human epidermal growth factor receptor positive or negative: HER2 +/−; Supplementary Table S2) or with tumour subtypes (all p-values > 0.05). Nevertheless, only miR-125b-5p was signiﬁcantly associated with the Ki67 proliferation index (Figure 4B, p-value = 0.038) and the diﬀerentiation status of tumour cells (Figure 4C, p-value = 0.003). Speciﬁcally, we found that in the presence of a low Ki67 index and a low grade, the expression levels of miR-125b-5p were increased, suggesting its inverse correlation with the aggressiveness of the disease. Cancers 2019, 11, 876 C 2019 11 FO 8 of 22 8 f 21 Figure 4. Correlation studies. Co-expression analyses of miR-125b-5p, miR-143-3p and miR-145-5p (A). Expression levels of miR-125b-5p vs Ki67 (low <20%; high ≥20%) (B), and grade (C). Expression levels of miR-125b-5p (D), miR-143-3p (E), and CA15-3 (F) vs. stage. Statistical significance (G). Non- significant results are reported in bold. 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imag- ing Parameters of Tumour Lesions Figure 4. Correlation studies. Co-expression analyses of miR-125b-5p, miR-143-3p and miR-145-5p (A). Expression levels of miR-125b-5p vs. Ki67 (low <20%; high ≥20%) (B), and grade (C). Expression levels of miR-125b-5p (D), miR-143-3p (E), and CA15-3 (F) vs. stage. Statistical signiﬁcance (G). Non-signiﬁcant results are reported in bold. 2.4. Correlation Analysis of Circulating miR-125b-5p, miR-143-3p and miR-145-5p with Quantitative Imaging Parameters of Tumour Lesions Figure 4. Correlation studies. Co-expression analyses of miR-125b-5p, miR-143-3p and miR-145-5p (A). 2. Results SUV is a Correlation analyses between miR-125b-5p and miR-143-3p levels and PET/MR-based tumour biomarkers were performed by grouping the data according to staging. Only the statistically significant results were reported (Table 4); we found a strong and significant correlation between circulating miR-143-3p and the mean initial area under the concentration curve (iAUCmean) and the mean reverse efflux volume transfer constant (Kepmean) (for both, ρ: 0.943, p-value = 0.005) in the stage II group. Perfusion parameters such as iAUC and Kep are generally correlated with tumour vascularisation [23,24], suggesting that the plasmatic overexpression of miR-143-3p in BC patients might correlate with angiogenesis. The correlation between iAUC and miR-143-3p persisted at stage III but was lost at stage IV, where its relative expression drastically decreased (see Figure 4B). Notably, there was a strong correlation index between miR-143-3p and the maximum standardized uptake value (SUVmax) (ρ: 0.829 and p-value = 0.042) at stage II, although the statistical signiﬁcance was borderline. SUV is a biomarker of tumour metabolism. This result together with those reported above suggest that 9 of 22 Cancers 2019, 11, 876 miR-143-3p could be correlated with tumour aggressiveness. miR-125-5p was signiﬁcantly inversely correlated with the mean forward volume transfer constant (Ktransmean) and the proliferation index Ki67 at stage IV (Table 4). Ktransmean, like iAUC and Kep, is a perfusion parameter linked to tumour vascularisation [24]. Since the highest plasma concentration of miR-125b-5p was associated with a low Ki67 index (Figure 4B) and grade G2 (Figure 4C) and inversely correlated with Ktransmean (Table 4), these results suggest that the highest plasma values of miR-125b-5p might predict a better prognosis. Table 4. Correlation studies among the miRNAs selected and the imaging parameters extracted from tumour lesions. The table reports ρ and p-values (p < 0.05 was considered statistically signiﬁcant). Stage II Stage III Stage IV iAUCmean Ki67 Kepmean SUVmax iAUCmean Ki67 Ki67 Ktransmean miR-125-5p ns ns ns ns ns ns −0.513 p-value 0.029 −0.421 p-value 0.040 miR-143-3p 0.943 p-value 0.005 ns 0.943 p-value 0.005 0.829 p-value 0.042 0.938 p-value 0.044 ns ns ns ns indicates non-signiﬁcant results. iAUCmean: area under the concentration curve; SUVmax: maximum standardized uptake value; Kepmean: mean reverse eﬄux volume transfer constant; Ktransmean: mean forward volume transfer constantin. Table 4. Correlation studies among the miRNAs selected and the imaging parameters extracted from tumour lesions. The table reports ρ and p-values (p < 0.05 was considered statistically signiﬁcant). 2.5. In Silico Studies Large publicly funded projects have generated extensive and freely available multi-assay data resources. To date, The Cancer Genome Atlas (TCGA) represents the largest available resource for multi-assay cancer genomics data that includes ~30000 cases of major primary cancers, including BC (TCGA-BRCA) [25]. To evaluate whether the upregulation of circulating miR-125b-5p, miR-143-3p and miR-145-5p in the plasma samples of BC patients reﬂected their tumour tissue expression, we performed in silico studies by using the TCGA-BRCA database. We also included miR-145-5p in order to perform co-expression analyses. We found that in BC tissue specimens, the expression of miR-125b-5p, miR-143-3p and miR-145-5p was signiﬁcantly lower compared to that in normal tissues (Supplementary Figure S2A) and that their downregulation was not correlated with the stage of the disease (Supplementary Figure S2B). 10 of 22 ge of the 10 of 22 ge of the Cancers 2019, 11, 876 (Supplementary F disease (Supplem Figure 5. Kaplan-Meier plots, which are based on breast cancer data from the METABRIC database, illustrate the survival probability for patients with low or high miR-125b (upper panel) and miR-143 (lower panel) expression levels in breast cancer. Hazard ratios (HRs) and p-values (log-rank test) were generated Figure 5. Kaplan-Meier plots, which are based on breast cancer data from the METABRIC database, illustrate the survival probability for patients with low or high miR-125b (upper panel) and miR-143 (lower panel) expression levels in breast cancer. Hazard ratios (HRs) and p-values (log-rank test) were generated. Figure 5. Kaplan-Meier plots, which are based on breast cancer data from the METABRIC database, illustrate the survival probability for patients with low or high miR-125b (upper panel) and miR-143 (lower panel) expression levels in breast cancer. Hazard ratios (HRs) and p-values (log-rank test) were generated Figure 5. Kaplan-Meier plots, which are based on breast cancer data from the METABRIC database, illustrate the survival probability for patients with low or high miR-125b (upper panel) and miR-143 (lower panel) expression levels in breast cancer. Hazard ratios (HRs) and p-values (log-rank test) were generated. In addition, we found that the downregulation of miR-143-3p and miR-145-5p in BC tissues (Supplementary Figure S2C) showed a lower correlation index compared to that we found in the plasma samples of BC patients (compare Figure 4A with Supplementary Figure S2C: ρ: 0.93 vs. ρ: 0.28). Taken together, these results showed an opposite expression trend of miR-125-5p, miR-143-3p and miR-145-5p in cancer tissue versus plasma. 2.5. In Silico Studies Since we were unable to monitor patients during follow-up, a survival analysis was performed for both miR-125b-5p and miR-143-3p by using the miRpower software (http://kmplot.com/analysis/index. php?p=service&cancer=breast_mirna). This software uses publicly available BC data from diﬀerent databases, including data from large databases such as TCGA and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). To generate Kaplan-Meier curves, we selected the option to auto select the best cut-oﬀfor deﬁning high and low groups. The algorithm computes all possible cut-oﬀvalues between the lower and upper quartiles and uses the best performing threshold. The hazard ratios (HRs) and p-values (log-rank test) were calculated. By using the TCGA dataset, the Kaplan-Meier plots were not statistically signiﬁcant for miR-125b-5p or miR-143-3p (data not shown), so the METABRIC dataset was used. As reported in Figure 5, we found that high tissue expression levels of miR-125b-5p were associated with the best prognosis (upper panel), and this result was in agreement with those obtained correlating this miRNA with grade, Ki67 and Ktransmean. The eﬀect of high expression levels of miR-143 on the survival of BC patients varied with age, and higher values contributed to worse prognoses starting at 250 months (lower panel). This result was in part in agreement with those obtained correlating this miRNA with the imaging parameters linked with tumour aggressiveness. It is necessary to point out that the miRpower software did not discriminate miR-143-3p from miR-143-5p, grouping them in miR-143, and miR-125b-5p from miR-125b-3p, grouping them in miR-125b. .6. Cellular Origin of miR-125b-5p, miR-143-3p and miR-145-5p in Normal and BC Tissues To corroborate the obtained results and conﬁrm the in silico data, we performed ISH experiments. We hypothesized that release of miR-125b-5p, miR-143-3p and miR-145-5p into the bloodstream did not originate from tumour cells. As reported in Figure 6, the results conﬁrmed that the expression of miR-143-3p (B) and miR-145-5p (C) was low or absent in epithelial tumour tissue (right) but high in endothelial cells, highlighting their vascular origin, as anticipated by correlation studies 11 of 22 Cancers 2019, 11, 876 among miRNAs with image biomarkers. In addition, in benign breast tissue, miR-143-3p stained in glandular epithelial and endothelial cells (Figure 6B, left), and miR-145-5p was expressed with the strongest staining intensity in myoepithelial cells and endothelial cells (Figure 6C, left panel). Moreover, miR-125b-5p was weakly expressed in normal tissue (Figure 6D, left) and low-to-absent in neoplastic cells (Figure 6D, right). This supported the hypothesis that miR-125b-5p could have a stromal origin. This hypothesis was further conﬁrmed by the result shown in Figure 6E, in which miR-125b-5p expression occurred in cancer-associated ﬁbroblasts. Taken together, these results conﬁrmed that the BC plasma upregulation of miR-125-5p and miR-143-3p could be related to their overexpression in endothelial and ﬁbroblast cells and not in tumour epithelial cells. Cancers 2019, 11, x FOR PEER REVIEW 11 of 21 Figure 6. In situ hybridization staining patterns in benign and malignant breast tissues. Scrambled miR negative control probe and U6 positive control probe (A). miR-143-3p in normal (left) and BC tissues (right) (B). miR-145-5p in normal (left) and BC tissues (right) (C). miR-125b-5p in normal (left) and BC tissues (right) (D). miR-125b-5p in stromal tumour tissue (E). Figure 6. In situ hybridization staining patterns in benign and malignant breast tissues. Scrambled miR negative control probe and U6 positive control probe (A). miR-143-3p in normal (left) and BC tissues (right) (B). miR-145-5p in normal (left) and BC tissues (right) (C). miR-125b-5p in normal (left) and BC tissues (right) (D). miR-125b-5p in stromal tumour tissue (E). BC: breast cancer. igure 6. In situ hybridization staining patterns in benign and malignant breast tissues. Scrambled miR negative control probe and U6 positive control probe (A). miR-143-3p in normal (left) and BC ssues (right) (B). miR-145-5p in normal (left) and BC tissues (right) (C). miR-125b-5p in normal (left) nd BC tissues (right) (D). miR-125b-5p in stromal tumour tissue (E). Figure 6. In situ hybridization staining patterns in benign and malignant breast tissues. .6. Cellular Origin of miR-125b-5p, miR-143-3p and miR-145-5p in Normal and BC Tissues Scrambled miR negative control probe and U6 positive control probe (A). miR-143-3p in normal (left) and BC tissues (right) (B). miR-145-5p in normal (left) and BC tissues (right) (C). miR-125b-5p in normal (left) and BC tissues (right) (D). miR-125b-5p in stromal tumour tissue (E). BC: breast cancer. ( g ) ( ) p 7 C bi i Di ti t t R lt t I A 2.7. Combining Diagnostic test Results to Increase Accuracy 3. Discussion 3. Discussion The detection of BC is pivotal for proper patient management, especially at an early stage. However, the currently utilized technology, which is heavily based on screening mammography and ultrasound (in speciﬁc patient populations), is imperfect, with limited accessibility in underdeveloped/underserved areas and might be economically challenging. Therefore, there is a compelling need to identify new biomarkers that might be diagnostically advantageous. Due to their structural and functional characteristics, microRNAs have been proposed as a new class of biomarkers for cancer screening; in fact, they are stable, deregulated, and found in diﬀerent biological ﬂuids, including blood that can be easily withdrawn in the ﬁeld and subsequently processed in centralized laboratories. To date, circulating biomarkers that are able to diagnose BC with high speciﬁcity and sensitivity are lacking, so our study aimed to (i) identify new circulating miRNAs for the diagnosis of BC; (ii) to elucidate the relationship between their circulating deregulation and the functional characteristics of tumour lesions for in vivo investigation through PET/MR; and (iii) understand the link between circulating miRNA deregulation and their origin in the primary tumour. By using the miScript miRNA PCR Array, we found that ﬁve miRNAs (miR-125b-5p, miR-143-3p, miR-145-5p, miR-100-5p and miR-23a-3p) were signiﬁcantly (p ≤0.0005) overexpressed in the plasma samples of BC patients compared to healthy subjects and deﬁned miR-125b-5p and miR-143-3p as the best molecules able to estimate the probability of having the disease. The circulating levels of miR-125b-5p and miR-143-3p are of intense debate in the scientiﬁc literature, especially in BC. Matamala et al. [26] found that circulating miR-143 was upregulated in BC patients; on the contrary, some studies showed a downregulation of circulating miR-143 in BC patients [27–30], and others found no signiﬁcant deregulation of circulating miR-125b and miR-143 in BC patients [30]. These discrepancies could be explained by the usage of serum rather than plasma [31]. Regardless of whether the choice of the biological sample can aﬀect the result, diﬀerent studies reported the upregulation of miR-125b in the serum samples of BC patients [32–34]. These discrepancies highlight the need to standardize protocols, including sample collection, storage, choice of the starting biological material, and processing. ( g ) ( ) p 2 7 Combining Diagnostic test Results to Increase Accuracy 2.7. Combining Diagnostic test Results to Increase Accuracy .7. Combining Diagnostic test Results to Increase Accuracy Since the relationship between upregulated circulating miRNA expression and the functional haracteristics of tumour lesions was clarified, we next sought to optimize their diagnostic role by evaluating a combined detection of miR-125b-5p and miR-143-3p with CA15-3. As reported in Figure 7A, we found that combining the diagnostic test results of miR-125b-5p and CA15-3 improved the accuracy (AUC = 0.89) compared to the same molecules analysed individually (see Figure 3: miR-125- 5p, AUC = 0.85; CA15-3, AUC = 0.68). In contrast, combining miR-143-3p and CA15-3 reduced the diagnostic accuracy (data not shown). Additionally, the combined analysis of miR-125b-5p and miR- 43-3p did not improve the accuracy (Figure 7B, AUC = 0.85) in comparison to the same molecules analysed individually (see Figure 3: miR 125 5p AUC = 0 85; miR 143 3p AUC = 0 80) Since the relationship between upregulated circulating miRNA expression and the functional characteristics of tumour lesions was clariﬁed, we next sought to optimize their diagnostic role by evaluating a combined detection of miR-125b-5p and miR-143-3p with CA15-3. As reported in Figure 7A, we found that combining the diagnostic test results of miR-125b-5p and CA15-3 improved the accuracy (AUC = 0.89) compared to the same molecules analysed individually (see Figure 3: miR-125-5p, AUC = 0.85; CA15-3, AUC = 0.68). In contrast, combining miR-143-3p and CA15-3 reduced the diagnostic accuracy (data not shown). Additionally, the combined analysis of miR-125b-5p and miR-143-3p did not improve the accuracy (Figure 7B, AUC = 0.85) in comparison to the same molecules analysed individually (see Figure 3: miR-125-5p, AUC = 0.85; miR-143-3p, AUC = 0.80). 12 of 22 molecules 12 of 22 molecules Cancers 2019, 11, 876 143-3p did not im a aly ed i di id Figure 7. ROC curve analyses and AUC values combining the profiles of miR-125b-5p and CA15-3 (A) and miR-125-5p and miR-143-3p (B) for discriminating BC patients from healthy patients. Figure 7. ROC curve analyses and AUC values combining the proﬁles of miR-125b-5p and CA15-3 (A) and miR-125-5p and miR-143-3p (B) for discriminating BC patients from healthy patients. ROC: Receiver operating characteristic; AUC: area under the ROC curve; BC: breast cancer. Figure 7. ROC curve analyses and AUC values combining the profiles of miR-125b-5p and CA15-3 (A) and miR-125-5p and miR-143-3p (B) for discriminating BC patients from healthy patients. Figure 7. ( g ) ( ) p 2 7 Combining Diagnostic test Results to Increase Accuracy 2.7. Combining Diagnostic test Results to Increase Accuracy ROC curve analyses and AUC values combining the proﬁles of miR-125b-5p and CA15-3 (A) and miR-125-5p and miR-143-3p (B) for discriminating BC patients from healthy patients. ROC: Receiver operating characteristic; AUC: area under the ROC curve; BC: breast cancer. 3. Discussion 3. Discussion We concluded that miR-125b-5p was inversely associated with the proliferation index Ki67 (p-value = 0.038), grading (p-value = 0.003) and the perfusion imaging parameter Ktransmean (ρ: −0.421, p-value = 0.040) and that its expression occurred in cancer-associated ﬁbroblasts. These ﬁndings suggest that the highest concentration of miR-125b-5p could be correlated with a better prognosis, and the survival analysis conﬁrmed this hypothesis. This is in agreement with the “seed and soil” hypothesis, which postulates that diﬀerent components of the tumour microenvironment (the soil) may have stimulatory or inhibitory eﬀects on tumour progression (the seed) [37], and with recent studies showing that miR-125b-5p overexpression in BC cell lines could inhibit cell proliferation, migration and invasion [38,39]. One of the limitations of this study is the sample size (n = 77 BC patients). In fact, the heterogeneity and complexity of BC make it challenging to ﬁnd a unique circulating signature of pathology with high diagnostic accuracy. Each circulating biomarker released in the blood of aﬀected patients exists as a consequence of genetic and/or epigenetic alterations linked to the pathology. Breast carcinoma is divided into diﬀerent subtypes with diﬀerent genetic characteristics [40,41], which can aﬀect the release of speciﬁc miRNAs in the blood. Moreover, the variety of biomarkers and the diﬀerence in concentration in the blood might be inﬂuenced by the stage, so the analysis of a cohort not homogeneous for staging could be a limitation, and the BC patients included in this cohort are lacking stage I and grade 1 (one patient for each condition). Therefore, a limitation of our study is the paucity of stage I and II cancers. However, this is unavoidable in studies that recruit clinically justiﬁed PET acquisition since, as per the Clinical Practice Guidelines in Oncology (NCCN guidelines), PET is not indicated in the initial work-up of clinical stage I, II, or operable stage III (NCCN Guidelines Version 1.2019, accessed on 22th May 2019). This staging group heterogeneity might lead to diﬀerent results in diﬀerent cohorts. Another limitation was the inability of monitoring patients during follow-up to better understand the prognostic and predictive role of miR-125b-5p and miR-143-3p. Nevertheless, the strengths of this study included the analysis performed on naïve patients as well as the availability of the same-day imaging parameters that provided information related to the morpho-functional characteristics of the primary cancers to correlate circulating miRNAs with in vivo quantiﬁed tumour biology. 3. Discussion 3. Discussion To better understand the biological basis and clinical implications of miR-125b-5p and miR-143-3p, we performed a correlation analysis between the circulating expression of the deregulated disease-related miRNAs and the metabolic and functional characteristics of the primary tumour, also taking into consideration the TNM (Tumour Lymph Nodes Metastasis) classiﬁcation of malignant tumours stage of the disease. For this purpose, we employed biomarkers extracted from a hybrid clinically approved PET/MR scanner. We decided to use PET/MR since it couples two modalities (FDG-PET and MR) capable of sampling diﬀerent biological features of cancers in vivo, speciﬁcally glucose metabolism through PET and cellular density plus tissue perfusion through MR. The advantages arising from 13 of 22 Cancers 2019, 11, 876 the simultaneous acquisition of PET- and MR-derived biomarkers in investigating BC biology have been shown by some exploratory studies that demonstrated the capability of PET/MR to predict BC subtypes, including those with a better prognosis [19,35]. In this study, we found that the expression of circulating miR-143-3p was strongly and signiﬁcantly correlated with iAUCmean at stage II (ρ: 0.943, p-value = 0.005) and at stage III (ρ: 0.938, p-value = 0.044) and with Kepmean at stage II (ρ: 0.943, p-value = 0.005). Both iAUCmean and Kepmean are perfusion parameters that provide information on tumour vascularisation, suggesting that miR-143-3p overexpression in the plasma samples of BC patients could be linked to tumour angiogenesis. Our hypothesis was in agreement with the editorial comment of Almeida and Calin [36] that argued the possible critical role of these miRNAs in the neoangiogenesis of lung cancer. To conﬁrm this hypothesis and to further validate our results, we performed ISH experiments on BC and normal breast tissue specimens and found that miR-143-3p had a clear vascular endothelial origin. In addition, we also found that miR-143-3p was correlated with SUVmax at stage II (ρ: 0.829, p-value = 0.042). The SUV parameter reﬂects the metabolic activity of tumours, so we speculate that miR-143-3p could have a role in neoangiogenesis and that its overexpression in the plasma samples of BC patients could be correlated with the aggressiveness of the disease, both in terms of tumour vascularisation and metabolic activity. This hypothesis was in part corroborated by the survival analysis performed in silico by using the miRpower software. Another interesting result correlated the expression levels of miR-125b-5p with IHC biomarkers and imaging parameters. 4.1. Participants and Study Design This Health Insurance Portability and Accountability Act–compliant prospective study was approved by the institutional review board. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the principles of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. For study enrolment, all participants provided written informed consent before undergoing imaging PET/MR and blood collection. Between September 2012 and November 2015 at the IRCCS SDN Institute, 221 female Caucasian patients with BC who underwent a clinically indicated same-day CE-FDG-PET/MR and blood collection were evaluated for inclusion in this study. Plasma haemolysation was evaluated as described by Kirschner MB and colleagues [47]. The inclusion criteria for BC patients were as follows: (i) diagnosis of BC by core biopsy report; (ii) absence of any prior surgical or pharmacological treatment for BC (naïve); (iii) negative previous personal oncological history; (iv) age > 18 years; (v) CE-FDG-PET/MR and blood collection were performed on the same day; and (vi) fasting for at least 8 hours. The exclusion criteria for BC patients were as follows: (i) pregnancy; (ii) blood glucose levels > 140 mg/dL (7.77 mmol/L); (iii) artefacts aﬀecting PET/MR images; (iv) standard contraindications for MR; (v) performance of CE-FDG-PET/MR and blood collection on diﬀerent days; and (vi) plasma haemolysation. Blood samples of age-matched healthy donors were collected; their inclusion criteria were as follows: (i) >18 years of age; (ii) negative previous personal oncological history; (iii) absence of suspected BC as conﬁrmed by ultrasound and/or mammography during the last 6 months from enrolment; iv) absence of a family history of BC; and (v) fasting for at least 8 hours. The exclusion criteria for healthy donors were the presence of one or more of the criteria reported above, including haemolysed plasma. We recruited 77 BC patients and 78 healthy donors for a total of 155 plasma/serum samples. The clinicopathological characteristics of the enrolled patients are reported in Table 1, and the whole study design is shown in Figure 1A. This study was approved by the institutional Ethics Committee (Protocol Number: Prot2-11, approved 06/07/2011 by Ethical Committee IRCCS Fondazione SDN). This article does not describe any studies with animals performed by any of the authors 3. Discussion 3. Discussion We performed a combined detection of miR-125b-5p and miR-143-3p with CA15-3 to improve the diagnostic accuracy. As suggested by guidelines, CA15-3 is not recommended for BC diagnosis [42,43], and our result (CA15-3AUC = 0.70) was in agreement with those of Liu and colleagues (CA15-3AUC = 0.71 [44]), Zaleski and colleagues (CA15-3AUC = 0.72 [45]) and Zajkowska and colleagues (CA15-3AUC = 0.70 [46]). Nevertheless, the 14 of 22 14 of 22 Cancers 2019, 11, 876 coupled analysis of miR-125b-5p and CA15-3 increased the diagnostic accuracy up to AUC = 0.89. To the best of our knowledge, this is one of the best results published so far and the ﬁrst study that combines the expression of a circulating miRNA with the expression of an established BC tumour marker. The combined detection of plasma miR-125b-5p and serum CA15-3 improved the diagnostic accuracy of BC detection up to ~90%, suggesting that this signature could be used for the diagnosis of BC. In addition, for stage II, the expression levels of CA15-3 were not correlated with the pathology (p-value = 0.658), whereas those of miR-125b-5p were (p-value = 0.002). The combined analysis of miR-125b-5p and CA 15-3 not only improved diagnostic accuracy in its entirety but also provided additional diagnostic information at less advanced pathological stages, suggesting the use of miRNAs as potential diagnostic circulating biomarkers that warrants consideration by further validation studies. 4.3. Data Processing and Multiparametric Analysis The PET data obtained from the PET/MR examinations were processed with comparable reconstruction and correction algorithms. Emission data were corrected for randomness, dead time, scatter, and attenuation. Lymph node involvement and distant metastases were assessed on the entire dataset of PET/MR sequences for the purpose of NM staging. The primary tumour size and inﬁltration of neighbouring structures were evaluated on the dedicated breast PET/MR protocol for T staging [50]. The dynamic contrast-enhanced (DCE)-MRI images were processed through commercially available and clinically approved software for estimating tissue perfusion (Tissue 4D, Siemens Healthiness) according to the same established procedures provided by the manufacturer manual and widely used in the published literature. The pharmacokinetic modelling is based on a two-compartment Toft’s model that allows for the calculation of the following: the transfer constant between vascular, extravascular, and extracellular space (EES) (Ktrans); volume of EES (Ve); constant reﬂux between EES and blood plasma (kep); initial area under the concentration curve (iAUC) [51,52]. Ktrans is a parameter related to vessel permeability and tissue blood ﬂow. The volume of the extravascular extracellular space Ve is a marker of cell density, kep is a transfer constant from the extracellular/extravascular space to plasma, and iAUC is related to the blood volume in the tissue of interest [51]. After automatic motion correction and registration of the pre- and post-contrast acquisitions, T1 mapping was automatically obtained from the multiple ﬂip angle pre-contrast part of the DCE-MRI sequence. Arterial input function (AIF) was related to the gadolinium dose injected and automatically modelled by a bi-exponential function using an intermediate population-derived mode provided by the software. On each single slice, a region-of-interest (ROI) was manually plotted around the tumour excluding the neighbouring vessels (internal mammary arteries and heart chambers) to compute the MR perfusion biomarker maps that were stored in the system. For the subsequent tumour ROI analysis, PET/MR datasets (PET acquisition, dynamic axial T1 weighted post-contrast, dynamic axial T1 weighted subtracted contrast-enhanced images, axial T2 weighted sequences, axial apparent diﬀusion coeﬃcient (ADC) map, and perfusion maps for Ktrans, Ve, kep and iAUC) were simultaneously evaluated on a clinically approved hybrid imaging workstation (Syngo.via, Siemens Healthiness) allowing the visual and quantitative comparison of the multiparametric data. The maximum standardized uptake value (SUVmax) was automatically calculated by using an iso-contour automatic volume-of-interest (VOI) method of tissue delineation that selected a ﬁxed threshold fraction (40%) of the peak activity in the tumour. 4.2. PET/MR Data Acquisition PET/MR was performed with a Biograph mMR imager (Siemens Healthiness, Erlangen, Germany). First, a whole-body protocol was used to assess the overall extent of cancer and provide the NM stage. Thereafter, a dedicated breast protocol to provide both metabolic and functional biomarkers of primary cancer and its T stage was acquired. For total body acquisition, we used a 16-channel head-neck coil and three or four 12-channel body coils depending on the patient’s height; for breast acquisition, a 4-channel breast coil was employed. Acquisitions were performed according to previously described protocols [19,20,35,48,49]. All patients fasted for at least 8 h before the procedure. They received 15 of 22 Cancers 2019, 11, 876 401 ± 32 MBq (mean ± standard deviation) of 18F-FDG intravenously. After a 60-min incubation time, PET and MR data were acquired simultaneously. The mean total time for the PET/MR examination was 107.87 ± 18.92 min [19]. 4.5. miRNA Microarray and Validation by Real-Time PCR (qRT-PCR) By using the miScript miRNA PCR Array (Qiagen; MIHS-106Z), the expression proﬁle of 84 human miRNAs (Supplementary Table S3) from the plasma samples of naïve BC patients and healthy donors were screened according to the manufacturer’s protocol. The miScript miRNA PCR Array proﬁles the expression of 84 human miRNAs known to be deregulated in diﬀerent cancers and are best characterized in miRBase. miRNA proﬁles were initially checked against a set population of 41 plasma samples (14 plasma samples from healthy donors and 27 from BC patients at diagnosis) on a MyiQ PCR system (Bio-Rad, Hercules, CA, USA). To identify interindividual variability, we did not pool plasma samples from each group (BC patients and healthy donors), but each sample was individually analysed by miRNA PCR arrays. The 96-well array also included n = 2 miRNA isolation controls, n = 6 “Housekeeping” snRNAs, n = 2 miRNA Reverse Transcription Controls (miRTC) and n = 2 Positive PCR Controls (PPC). Brieﬂy, 200 µL of RNase-free water was added to each 20 µL of reverse transcription reaction, and 100 µL of diluted cDNA was used to prepare a reaction mix according to the manufacturer’s protocol. For data analysis, the Baseline and Threshold settings, which were the same across all PCR runs, were from cycle 2 to cycle 15 for Baseline and 20 for Threshold. By using a data analysis tool (GeneGlobe Data Analysis Center, Qiagen, Hilden, Germany), the threshold cycle (CT) values were exported, and the steps performed by the software were as follows: any CT value ≥33 was considered negative; if the RNA sample was of high quality, the cycling programme was correctly run and the threshold was correctly deﬁned, the value of the positive PCR control wells from the CT samples (CTPPC) should be 19 ± 2 or 15 ± 2; if the CT values of the reverse transcription control are less than 7, no inhibition of the reverse transcription reaction is apparent; the ∆CT value for each mature miRNA proﬁled is calculated using the formula ∆CT = CTmiRNA - CTnormalizer. The relative expression for each miRNA was calculated as 2−∆CT, and the criteria for the selection of miRNAs diﬀerentially expressed between the two populations with a statistical signiﬁcance were a fold change ≥1.5 and a p-value < 0.05. Data validations were performed by qRT-PCR screening for only the following molecules: miR-125b-5p, miR-143-3p, miR-145-5p, miR-100-5p and miR-23a-3p. 4.3. Data Processing and Multiparametric Analysis To obtain Ktrans, Ve, Kep and iAUC, the subtracted dynamic post-contrast T1-weighted series that best visualized the tumour was chosen. ROIs were manually drawn around the tumour on each image of that series, copied and then pasted on the corresponding Ktrans, Ve, Kep and iAUC maps. The values of all the ROIs were averaged. To obtain the ADC values, the ROIs were drawn on each image of the high-b value (800 s/mm2) diﬀusion-weighted imaging (DWI), copied and then pasted on the ADC maps. The values of all the ROIs were averaged. While the ROIs were drawn around the tumours, care was taken to avoid including large feeding vessels and areas of frank necrosis, as seen on T2 weighted and subtracted dynamic post-contrast T1-weighted images. The maximal diameter of the tumour was measured on every single slice of the reference subtracted dynamic post-contrast T1-weighted images. The measurements were performed by a radiologist and a nuclear medicine physician with more than 5 years of experience. The whole-body and breast PET/MR protocols were derived from the published literature [20,35]. Cancers 2019, 11, 876 16 of 22 16 of 22 4.4. Plasma Sampling, RNA Extraction and Reverse Transcription 4.4. Plasma Sampling, RNA Extraction and Reverse Transcription Blood samples were collected immediately before FDG injection. For each patient, a total of 10.5 mL of venous blood was collected in BD Vacutainer® 3 mL ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dikinson, Franklin Lakes, NJ, USA) and 7.5 mL serum separating tubes. Blood was processed within 1 h of harvest. Plasma and serum were obtained from the whole blood samples by centrifugation at 1900× g for 10 min at 4 ◦C. The supernatant was further centrifuged at 16,000× g for 10 min at 4 ◦C and stored in aliquots of 0.5 mL at −80 ◦C until analysis. The samples were stored at −80 ◦C at the SDN Biobank (IRCCS DSN, Naples, Italy) [53]. The extraction of total RNA from 200 µL of plasma was performed within 1 year of storage at −80 ◦C using an miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Brieﬂy, 3.5 µL of Caenorhabditis elegans miR-39 (cel-miR-39, 1.6 × 108 copies/µL) was added as the spike-in control after the denaturing solution. Total RNA (including miRNAs) was eluted in 14 µL of RNase-free water. Reverse transcription was performed using the miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Because extracellular miRNAs are less than 1% of the total RNA recovered [54] their concentration is often under the detection limits of spectrophotometric devices, so we decided to use a ﬁxed volume rather than a ﬁxed miRNA amount for qRT-PCR. To this aim, 1.5 µL of total RNA was added to the HiSpect Buﬀer (Qiagen, Hilden, Germany), nucleic acid mix and reverse transcriptase in a ﬁnal volume of 20 µL. Reactions were incubated for 60 min at 37 ◦C and then for 5 min at 95 ◦C to inactivate the miScript Reverse Transcriptase mix (Qiagen, Hilden, Germany). At the end of the reactions, the resulting cDNAs were stored undiluted at −20 ◦C. The cycling conditions for real-time PCR were as follows: 15 min at 95 ◦C as the initial activation step, followed by 3-step cycling including denaturation (15 s at 94 ◦C), annealing (30 s at 55 ◦C) and extension (30 s at 70 ◦C) for a total of 40 cycles. 4.5. miRNA Microarray and Validation by Real-Time PCR (qRT-PCR) The Qiagen miScript SYBR Green PCR Kit was used for qPCR according to the manufacturer’s protocol on the 17 of 22 Cancers 2019, 11, 876 Bio-Rad MyiQ PCR system. To determine the best standard genes to normalize our data, BestKeeper software was used [55]. By analysing n = 41 plasma samples, we found that the best normalizer included in the miScript miRNA PCR Array was the exogenous cel-mir 39. The MiScript Primers Assay was purchased from Qiagen (Qiagen, Hilden, Germany). 4.6. TCGA Data Processing TCGA Breast Invasive Carcinoma (TCGA-BRCA project, February 2018) transcriptome proﬁling data (miRNA Expression Quantiﬁcation) and clinical metadata were extracted from the Genomic Data Commons (GDC) portal (https://portal.gdc.cancer.gov/) for primary tumour (n = 1096 ﬁles for 1078 BC cases) and normal solid tissue (n = 104 ﬁles for 104 normal cases) using the open source R software and the TCGAbiolinks R package [56]. For the miRNA datasets, we selected the level-3 miRNA-Seq data, which were produced on Illumina HiSeq 2000 sequencers (Illumina Ventures, San Diego, CA, USA) The miRNA-Seq expression level-3 data contains raw read counts (n = 1881 miRNAs). The miRNA raw counts were ﬁltered as follows: we calculated the mean value for duplicated BC cases and ﬁltered out miRNA counts with null expression values on both conditions (tumour/normal) for a total of 1625 ﬁltered counts. We considered only 1625 ﬁltered miRNAs for the normalization step. Filtered raw counts were normalized using the Upper Quartile approach, and the patients with “−Inf” normalized values were removed for a total of 1076 tumours. The normalized counts were used to evaluate miR-125b-5p, miR-143-3p and miR-145-5p expression levels (tumour vs. normal) as log2-transformed relative expression values. We performed a Wilcoxon test to assess the statistical signiﬁcance between tumour and normal conditions and calculated the adjusted p-value for each selected miRNA proﬁle. We also reported miRNA tumour expression proﬁles based on the American Joint Committee on Cancer (AJCC) TNM staging. We grouped the clinical stages into four categories: (1) stage I; (2) stage II; (3) stage III; and (4) stage IV. We ﬁltered out data with unassessed stage (stage x) or unreported information. Finally, we carried out a Spearman correlation analysis among the selected miRNA proﬁles and reported the associated p-values. 4.7. In Situ Hybridization ISH was performed on formalin-ﬁxed and paraﬃn-embedded tissue specimens surgically resected from patients diagnosed with BC. For each case analysed (a total of 22 breast tissues), we selected a block of tissue ﬁxed in formalin and embedded in paraﬃn representative of the tumour and used it to obtain 4 µm-thick sections mounted on Superfrost Plus slides (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The paraﬃn sections were placed in an oven at 60 ◦C for 45 min the day before ISH was performed and were stored overnight at 4 ◦C. Double digoxigenin (DIG)-labelled miRCURY LNA™ microRNA detection probes (Exiqon, Vedbæk, Denmark) were employed in this study. Speciﬁcally, we used 60 nM miR-145-5p target probe (Exiqon, Cat #619865-360), 60 nM miR-143-3p target probe (Exiqon, Cat #619872-360), 60 nM miR-125-5p target probe (Exiqon, Cat #611756-360), 40 nM scramble miR negative control probe (Exiqon, Cat #90-001) and 10 nM U6 positive control probe (Exiqon, Cat #90-002). The detection of microRNA by ISH was performed using the Enhanced miRCURY LNA microRNA ISH Optimization Kit (Exiqon, Vedbæk, Denmark) according to the manufacturer’s protocol. Brieﬂy, formalin-ﬁxed paraﬃn-embedded sections were deparaﬃnized in xylene, rehydrated through solutions of ethanol to phosphate-buﬀered saline (PBS, pH 7.4) and pre-treated by enzymatic digestion (Proteinase K at 20 µg/mL for 30 min at 37 ◦C). Hybridization was performed by adding 60 nM LNA detection probes diluted in Exiqon hybridization buﬀer (Exiqon, Vedbæk, Denmark, Cat #90000) to each slide and incubating for 1 h at 60 ◦C in a hybridizer oven. After hybridization, the sections were washed under stringent conditions. Stringent washes were performed in pre-heated saline-sodium citrate (SSC) buﬀers in 5-min washes at 60 ◦C: once in 5× SSC, twice in 1× SSC and twice in 0.2× SSC. Then, the slides were placed in 0.2× SSC at room temperature and washed in PBS with 0.1% Tween-20. The sections were blocked against the unspeciﬁc binding of the detection antibody using a blocking solution according to the manufacturer’s recommendations at room temperature for 15 min. The chromogenic detection 18 of 22 Cancers 2019, 11, 876 of the miRNA LNA-ISH probe was performed with anti- anti-digoxygenin -alkaline phosphatase conjugate (1:800 dilution of the conjugate in blocking reagent, Roche Diagnostics GmbH, Mannheim, Germany) and incubated at room temperature for 1 h. 4.7. In Situ Hybridization Enzymatic development was performed by incubating the slides with 4-nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3′-indolyl phosphate (BCIP) substrate (Roche) at 30 ◦C overnight to allow the formation of dark-blue 4-nitro-blue tetrazolium formazan precipitate. The following day, the sections were washed twice for 5 min in KTBT buﬀer (50 mM Tris–HCl, 150 mM NaCl, 10 mM KCl) and then shortly twice in water. Counterstaining using ISH nuclear fast red was performed for 1 min at room temperature. The sections were rinsed in tap water for 10 min, dehydrated through an increasing gradient of ethanol solutions and mounted with Eukitt mounting medium (VWR, Herlev, Denmark). A scrambled probe and U6 small nuclear RNA-speciﬁc probe were used as controls. 4.9. Statistical Analysis Statistical analyses were performed using the open source R Statistical Software. All p-values were two-sided, and p < 0.05 was considered statistically signiﬁcant. We used Shapiro–Wilk analysis and the Levene test to assess the normality and homoscedasticity of the distribution of the data, respectively. We performed the Mann-Whitney non-parametric test to compare the expression of the selected miRNAs between the two groups (BC and healthy controls). Furthermore, stepwise logistic regression was implemented to assess the association between miRNA expression and the probability of pathology. ROC curves were constructed for each miRNA, CA15-3 and CEA, and AUC values with 95% conﬁdence intervals were calculated to evaluate the predictive power of the selected molecules for detecting BC. We evaluated the diagnostic accuracy of each circulating miRNA, CA15-3 and CEA, and their combination. The non-parametric Spearman’s rank-order correlation and Mann-Whitney U test were performed to verify the correlations/associations among the biological markers and imaging parameters. To assess the association between the molecular and imaging parameters, the data were grouped according to positive or negative expression of ER, PR and HER2 receptors, low or high expression of Ki67 (low ≤20% and high >20%), grading, and staging as I, II, III, and IV, according to BC guidelines. 4.8. Measurement of CA 15-3 The CA15-3 serology test was performed in accordance with the manufacturer’s protocols and reference intervals. Speciﬁcally, this tumour marker was measured in 155 serum samples (n = 78 healthy donors and n = 77 BC patients) on a Dimension Vista®1500 System (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA) that uses the LOCI method (Siemens Healthcare Diagnostics, Eschborn, Germany), a homogeneous sandwich chemiluminescent immunoassay-based LOCI® technology (Siemens Healthcare Diagnostics, Eschborn, Germany). The threshold value provided by the supplying company for CA15-3 was 35 UI/mL. For serum tumour marker determination, when its level exceeded a threshold value, the samples were considered to be positive; otherwise, they were considered to be negative. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/6/876/s1: Table S1: Statistical analyses, Table S2: Correlation analysis among the indicated miRNAs and hormonal receptor status of the lesions in a cohort of 77 BC patients, Table S3: Array Layout, Figure S1: Validation I: Relative expression of the selected miRNAs in a study population of 46 healthy donors (CTR) and 64 BC subjects, Figure S2: Expression analysis of miR-125b-5p, miR-143-3p and miR-145-5p by using the TCGA database. 5. Conclusions Our study highlighted that the addition of PET/MR biomarkers led to a better understanding of the relationships between circulating miRNAs and tumour biology in our BC population. Nevertheless, further studies with larger samples and a more homogeneous distribution of tumour subtypes and staging are needed to conﬁrm our results. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/6/876/s1: Table S1: Statistical analyses, Table S2: Correlation analysis among the indicated miRNAs and hormonal receptor status of the lesions in a cohort of 77 BC patients, Table S3: Array Layout, Figure S1: Validation I: Relative expression of the selected miRNAs in a study population of 46 healthy donors (CTR) and 64 BC subjects, Figure S2: Expression analysis of miR-125b-5p, miR-143-3p and miR-145-5p by using the TCGA database. 19 of 22 Cancers 2019, 11, 876 19 of 22 Author Contributions: Conception and design: M.I. and O.A.C.; development of methodology: M.I., O.A.C., A.S. and S.S.; acquisition of data: M.I., O.A.C., P.M., C.C., G.I. and D.R.; analysis and interpretation of data: M.I., A.M.G., C.C., C.A.P. and M.F.; writing, review, and/or revision of the manuscript: M.I., A.M.G., O.A.C., C.C. and P.M.; study supervision: M.S. Author Contributions: Conception and design: M.I. and O.A.C.; development of methodology: M.I., O.A.C., A.S. and S.S.; acquisition of data: M.I., O.A.C., P.M., C.C., G.I. and D.R.; analysis and interpretation of data: M.I., A.M.G., C.C., C.A.P. and M.F.; writing, review, and/or revision of the manuscript: M.I., A.M.G., O.A.C., C.C. and P.M.; study supervision: M.S. Funding: This work was supported by the Ministry of Health under contract “Ricerca Corrente RRC-2019-2366651” and in part under contract “5 per mille 2014 5M-2353583”. Funding: This work was supported by the Ministry of Health under contract “Ricerca Corrente RRC-2019-2366651” and in part under contract “5 per mille 2014 5M-2353583”. Acknowledgments: We would like to thank Silvia Varricchio for technical support on the ISH assay. Acknowledgments: We would like to thank Silvia Varricchio for technical support on the ISH assay. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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